Department Environmental Microbiology

The ability of gut bacterial pathogens to escape immunity by antigenic variation—particularly via changes to surface-exposed antigens—is a major barrier to immune clearance¹. However, not all variants are equally fit in all environments^2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation^4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

We describe moral cognition as a process occurring in a distinctive cognitive space, wherein moral relationships are defined along several morally relevant dimensions. After identifying candidate dimensions, we show how moral judgments can emerge in this space directly from object perception, without any appeal to moral rules or abstract values. Our reductive "minimal model" (Batterman & Rice, 2014) elaborates Beal's (2020) claim that moral cognition is determined, at the most basic level, by "ontological frames" defining subjects, objects, and the proper relation between them. We expand this claim into a set of formal hypotheses that predict moral judgments based on how objects are "framed" in the relevant dimensions of "moral space."

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ’s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

In der Praxis werden Legionellen primär mittels kultivierungsbasierter Methoden im Wasser nachgewiesen und quantifiziert. Nun rücken neue Entwicklungen vermehrt auch molekularbiologische Methoden in den Fokus, die bisher vor allem im Forschungskontext eingesetzt wurden. Sie bergen das Potenzial, einige Aspekte der Kultivierungsmethoden zu ergänzen, vervollständigen oder allenfalls sogar zu ersetzen. Zudem ermöglichen molekularbiologische Methoden die Ökologie von Legionellen in Trinkwassersystemen besser zu verstehen.

Microalgal cell attaching and biofilm formation are critical in the application of microalgal biocathode, which severs as one of the hopeful candidates to an original cathode in bioelectrochemical systems. Many efforts have been put in biofilm formation and bioelectrochemical systems for years, but the predominant factors shaping microalgal biocathode formation are sketchy. We launched a pair of researches to investigate microalgal attachment and biofilm formation in the presence/absence of applied voltages using Chlamydomonas microsphaera as a model unicellular motile microalga. In this study, we presented how microalga attached and biofilm formed on a carbon felt surface without applied voltages and try to manifest the most important aspects in this process. Results showed that while nutrient sources did not directly regulate cell attachment onto the carbon felt, limited initial nutrient concentration nevertheless promoted cell attachment. Specifically, nutrient availability did not influence the early stage (20–60 min) of microalgal cell attachment but did significantly impact cell attachment during later stages (240–720 min). Further analysis revealed that nutrient availability-mediated chemotactic movements and zeta potential are crucial to facilitate the initial attachment and subsequent biofilm formation of C. microsphaera onto the surfaces, serving as an important factor controlling microalgal surface attachment. Our results demonstrate that nutrient availability is a dominant factor controlling microalgal surface attachment and subsequent biofilm formation processes. This study provides a mechanistic understanding of microalgal surface attachment and biofilm formation processes on carbon felts surfaces in the absence of applied voltages.

Background: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40–80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021).
Aim: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland.
Methods: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale.
Results: We estimate B.1.1.7 had a transmission fitness advantage of 43–52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07–1.41] from 01 January until 17 January 2021 and 1.18 [1.06–1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00–1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021.
Conclusion: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2–3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

In 2020 the world was hit by the COVID-19 pandemic putting entire governments and civil societies in crisis mode. Around the globe unprecedented shortages of equipment and qualified personnel were reported in hospitals and diagnostic laboratories. When a crisis is global, supply chains are strained worldwide and external help may not be readily available. In Switzerland, as part of the efforts of the Swiss National COVID-19 Science Task Force, we developed a tailor-made web-based tool where needs and offers for critical laboratory equipment and expertise can be brought together, coordinated, prioritized, and validated. This Academic Resources for COVID-19 (ARC) Platform presents the specialized needs of diagnostic laboratories to academic research groups at universities, allowing the sourcing of said needs from unconventional supply channels, while keeping the entities tasked with coordination of the crisis response in control of each part of the process. An instance of the ARC Platform is operated in Switzerland (arc.epfl.ch) catering to the diagnostic efforts in Switzerland and sourcing from the Swiss academic sector. The underlying technology has been released as open source so that others can adopt the customizable web-platform for need/supply match-making in their own relief efforts, during the COVID-19 pandemic or any future disaster.

Interspecific interactions are thought to govern the stability and functioning of microbial communities, but the influence of the spatial environment and its structural connectivity on the potential of such interactions to unfold remain largely unknown. Here we studied the effects on community growth and microbial diversity as a function of environmental connectivity, where we define environmental connectivity as the degree of habitat fragmentation preventing microbial cells from living together. We quantitatively compared growth of a naturally-derived high microbial diversity community from soil in a completely mixed liquid suspension (high connectivity) to growth in a massively fragmented and poorly connected environment (low connectivity). The low connectivity environment consisted of homogenously-sized miniature agarose beads containing random single or paired founder cells. We found that overall community growth was the same in both environments, but the low connectivity environment dramatically reduced global community-level diversity compared to the high connectivity environment. Experimental observations were supported by community growth modeling. The model predicts a loss of diversity in the low connectivity environment as a result of negative interspecific interactions becoming more dominant at small founder species numbers. Counterintuitively for the low connectivity environment, growth of isolated single genotypes was less productive than that of random founder genotype cell pairs, suggesting that the community as a whole profited from emerging positive interspecific interactions. Our work demonstrates the importance of environmental connectivity for growth of natural soil microbial communities, which aids future efforts to intervene in or restore community composition to achieve engineering and biotechnological objectives.

Microbial populations often experience fluctuations in nutrient complexity in their natural environment such as between high molecular weight polysaccharides and simple monosaccharides. However, it is unclear if cells can adopt growth behaviors that allow individuals to optimally respond to differences in nutrient complexity. Here, we directly control nutrient complexity and use quantitative single-cell analysis to study the growth dynamics of individuals within populations of the aquatic bacterium Caulobacter crescentus. We show that cells form clonal microcolonies when growing on the polysaccharide xylan, which is abundant in nature and degraded using extracellular cell-linked enzymes; and disperse to solitary growth modes when the corresponding monosaccharide xylose becomes available or nutrients are exhausted. We find that the cellular density required to achieve maximal growth rates is four-fold higher on xylan than on xylose, indicating that aggregating is advantageous on polysaccharides. When collectives on xylan are transitioned to xylose, cells start dispersing, indicating that colony formation is no longer beneficial and solitary behaviors might serve to reduce intercellular competition. Our study demonstrates that cells can dynamically tune their behaviors when nutrient complexity fluctuates, elucidates the quantitative advantages of distinct growth behaviors for individual cells and indicates why collective growth modes are prevalent in microbial populations.

Wastewater-based epidemiology (WBE) has been shown to coincide with, or anticipate, confirmed COVID-19 case numbers. During periods with high test positivity rates, however, case numbers may be underreported, whereas wastewater does not suffer from this limitation. Here we investigated how the dynamics of new COVID-19 infections estimated based on wastewater monitoring or confirmed cases compare to true COVID-19 incidence dynamics. We focused on the first pandemic wave in Switzerland (February to April, 2020), when test positivity ranged up to 26%. SARS-CoV-2 RNA loads were determined 2–4 times per week in three Swiss wastewater treatment plants (Lugano, Lausanne and Zurich). Wastewater and case data were combined with a shedding load distribution and an infection-to-case confirmation delay distribution, respectively, to estimate infection incidence dynamics. Finally, the estimates were compared to reference incidence dynamics determined by a validated compartmental model. Incidence dynamics estimated based on wastewater data were found to better track the timing and shape of the reference infection peak compared to estimates based on confirmed cases. In contrast, case confirmations provided a better estimate of the subsequent decline in infections. Under a regime of high-test positivity rates, WBE thus provides critical information that is complementary to clinical data to monitor the pandemic trajectory.

Background: Infectious diseases can be transmitted via fomites (contaminated surfaces/objects); disinfection can interrupt this transmission route. However, disinfection guidelines for low-resource outbreak settings are inconsistent and not evidence-based.
Methods: A systematic review of surface disinfection efficacy studies was conducted to inform low-resource outbreak guideline development. Due to variation in experimental procedures, outcomes were synthesized in a narrative summary focusing on chlorine-based disinfection against seven pathogens with potential to produce outbreaks in low-resource settings (Mycobacterium tuberculosis, Vibrio cholerae, Salmonella spp., hepatitis A virus, rotavirus, norovirus, Ebola virus).
Results: Data were extracted from 89 laboratory studies and made available, including 20 studies on relevant pathogens used in combination with surrogate data to determine minimum target concentration × time ("CT") factors. Stainless steel (68%) and chlorine-based disinfectants (56%) were most commonly tested. No consistent trend was seen in the influence of chlorine concentration and exposure time on disinfection efficacy. Disinfectant application mode; soil load; and surface type were frequently identified as influential factors in included studies.
Conclusions: This review highlights that surface disinfection efficacy estimates are strongly influenced by each study's experimental conditions. We therefore recommend laboratory testing to be followed by field-based testing/monitoring to ensure effectiveness is achieved in situ.

Objective: The Reducing Enteropathy, Undernutrition, and Contamination in the Environment (REDUCE) program focuses on identifying exposure pathways to faecal pathogens for young children in the Democratic Republic of the Congo (DRC) and on developing scalable interventions to reduce faecal contamination from these pathways. Methods: A prospective cohort study of 690 participants was conducted to investigate the association between hand, food, and environmental faecal contamination and diarrhoeal disease prevalence among young children in Walungu Territory, South Kivu, DRC. A total of 1923 hand rinse, soil, food, object, surface, stored water and water source samples were collected during unannounced spot checks after baseline enrolment and analysed for Escherichia coli. Caregiver reports of diarrhoea were obtained from children < 5 years at a 6-month follow-up. Results: E.coli was detected in 73% of child and caregiver hand-rinse samples, 69% of soil samples from child play spaces, 54% of child food samples, 38% of objects and surfaces children were observed putting in their mouths, 74% of stored water samples, and 40% of source water samples. Children < 5 years with E. coli on their hands had significantly higher odds of diarrhoea at the 6-month follow-up (odds ratio: 2.03 (95% confidence interval: 1.05, 3.92)). Conclusion: The cohort study findings from the REDUCE program have shown that child hand contamination is associated with diarrhoeal disease in rural DRC, and that there is high faecal contamination in child plays spaces and food. These findings provide evidence demonstrating the urgent need to provide clean play spaces for young children and interventions targeting hand hygiene to reduce paediatric exposure to faecal pathogens.

Surface-attached microbial communities constitute a vast amount of life on our planet. They contribute to all major biogeochemical cycles, provide essential services to our society and environment, and have important effects on human health and disease. They typically consist of different interacting genotypes that arrange themselves non-randomly across space (referred to hereafter as spatial self-organization). While spatial self-organization is important for the functioning, ecology, and evolution of these communities, the underlying determinants of spatial self-organization remain unclear. Here, we performed a combination of experiments, statistical modeling, and mathematical simulations with a synthetic cross-feeding microbial community consisting of two isogenic strains. We found that two different patterns of spatial self-organization emerged at the same length and time scales, thus demonstrating pattern diversification. This pattern diversification was not caused by initial environmental heterogeneity or by genetic heterogeneity within populations. Instead, it was caused by nongenetic heterogeneity within populations, and we provide evidence that the source of this nongenetic heterogeneity is local differences in the initial spatial positionings of individuals. We further demonstrate that the different patterns exhibit different community-level properties; namely, they have different expansion speeds. Together, our results demonstrate that pattern diversification can emerge in the absence of initial environmental heterogeneity or genetic heterogeneity within populations and can affect community-level properties, thus providing novel insights into the causes and consequences of microbial spatial self-organization.

The importance of interference competition, where individuals compete through antagonistic traits such as the production of toxins, has long been recognized by ecologists, yet understanding how these types of interactions evolve remains limited. Toxin production is thought to be beneficial when competing with a competitor. Here, we explore if antagonism can evolve by long-term selection of the toxin (pyocin) producing strain Pseudomonas aeruginosa PAO1 in the presence (or absence) of one of three clinical isolates of the same species (Recipient) over ten serial transfers. We find that inhibition decreases in the absence of a recipient. In the presence of a recipient, antagonism evolved to be different depending on the recipient used. Our study shows that the evolution of interference competition by toxins can decrease or increase, experimentally demonstrating the importance of this type of interaction for the evolution of species interactions.

Environmental surveillance of surface contamination is an unexplored tool for understanding transmission of SARS-CoV-2 in community settings. We conducted longitudinal swab sampling of high-touch non-porous surfaces in a Massachusetts town during a COVID-19 outbreak from April to June 2020. Twenty-nine of 348 (8.3%) surface samples were positive for SARS-CoV-2 RNA, including crosswalk buttons, trash can handles, and door handles of essential business entrances (grocery store, liquor store, bank, and gas station). The estimated risk of infection from touching a contaminated surface was low (less than 5 in 10,000) by quantitative microbial risk assessment, suggesting fomites play a minimal role in SARS-CoV-2 community transmission. The weekly percentage of positive samples (out of n = 33 unique surfaces per week) best predicted variation in city-level COVID-19 cases with a 7-day lead time. Environmental surveillance of SARS-CoV-2 RNA on high-touch surfaces may be a useful tool to provide early warning of COVID-19 case trends.

Hexabromocyclododecanes (HBCDs) were used as flame-retardants until their ban in 2013. Among the 16 stereoisomers known, ε-HBCD has the highest symmetry. This makes ε-HBCD an interesting substrate to study the selectivity of biotransformations. We expressed three LinA dehydrohalogenase enzymes in E. coli bacteria, two wild-type, originating from Sphingobium indicum B90A bacteria and LinATM, a triple mutant of LinA2, with mutations of L96C, F113Y and T133 M. These enzymes are involved in the hexachlorocyclohexane (HCH) metabolism, specifically of the insecticide γ-HCH (Lindane). We studied the reactivity of those eight HBCD stereoisomers found in technical HBCD. Furthermore, we compared kinetics and selectivity of these LinA variants with respect to ε-HBCD. LC-MS data indicate that all enzymes converted ε-HBCD to pentabromocyclododecenes (PBCDens). Transformations followed Michaelis-Menten kinetics. Rate constants k_cat and enzyme specificities k_cat/K_M indicate that ε-HBCD conversion was fastest and most specific with LinA2. Only one PBCDen stereoisomer was formed by LinA2, while LinA1 and LinATM produced mixtures of two PBCDE enantiomers at three times lower rates than LinA2. In analogy to the biotransformation of (−)β-HBCD, with selective conversion of dibromides in R-S-configuration, we assume that 1E,5S,6R,9S,10R-PBCDen is the ε-HBCD transformation product from LinA2. Implementing three amino acids of the LinA1 substrate-binding site into LinA2 resulted in a triple mutant with similar kinetics and product specificity like LinA1. Thus, point-directed mutagenesis is an interesting tool to modify the substrate- and product-specificity of LinA enzymes and enlarge their scope to metabolize other halogenated persistent organic pollutants regulated under the Stockholm Convention.

A key characteristic of decentralized greywater treatment and reuse is high variability in both nutrient concentrations and flow. This variability in flow leads to stagnant water in the system and causes short-term fluctuations in the effluent water quality. Automated monitoring tools provide data to understand the mechanisms underlying the dynamics and to adapt control strategies accordingly. We investigated the fluctuations in a building-scale greywater treatment system comprising a membrane bioreactor followed by a biological activated carbon filter. Short-term dynamics in the effluent of the biological activated carbon filter were monitored with automated flow cytometry and turbidity, and the impact of these fluctuations on various hygiene-relevant parameters in the reuse water was evaluated. Continuous biofilm detachment into the stagnant water in the biological activated carbon filter led to temporarily increased turbidity and cell concentrations in the effluent after periods of stagnation. The fluctuations in cell concentrations were consistent with a model assuming higher detachment rates during flow than during times with stagnant water. For this system, total cell concentration and turbidity were strongly correlated. We also showed that the observed increase in cell concentration was not related to either an increase of organic carbon concentration or the concentration of two opportunistic pathogens, P. aeruginosa and L. pneumophila. Our findings demonstrate that turbidity measurements are sensitive to changes in the effluent water quality and can be used to monitor the fluctuations caused by intermittent flow. Intermittent flow did not lead to an increase in opportunistic pathogens, and this study provides no indications that stagnant water in biological activated carbon filters need be prevented.

Salmonella spp. express Salmonella pathogenicity island 1 Type III Secretion System 1 (T3SS-1) genes to mediate the initial phase of interaction with their host. Prior studies indicate short-chain fatty acids, microbial metabolites at high concentrations in the gastrointestinal tract, limit population-level T3SS-1 gene expression. However, only a subset of Salmonella cells in a population express these genes, suggesting short-chain fatty acids could decrease T3SS-1 population-level expression by acting on per-cell expression or the proportion of expressing cells. Here, we combine single-cell, theoretical, and molecular approaches to address the effect of short-chain fatty acids on T3SS-1 expression. Our in vitro results show short-chain fatty acids do not repress T3SS-1 expression by individual cells. Rather, these compounds act to selectively slow the growth of T3SS-1–expressing cells, ultimately decreasing their frequency in the population. Further experiments indicate slowed growth arises from short-chain fatty acid–mediated depletion of the proton motive force. By influencing the T3SS-1 cell-type proportions, our findings imply gut microbial metabolites act on cooperation between the two cell types and ultimately influence Salmonella’s capacity to establish within a host.

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

Structure, reactivity and physico-chemical properties of polyhalogenated compounds determine their up-take, transport, bio-accumulation, transformation and toxicity and their environmental fate. In technical mixtures of chlorinated paraffins (CPs), these properties are distributed due to the presence of thousands of homologues. We hypothesized that roles of CP dehalogenation reactions, catalyzed by the haloalkane dehalogenase LinB, depend on structural properties of the substrates, e.g. chlorination degree and carbon-chain length. We exposed mixtures of chlorinated undecanes, dodecanes and tridecanes in-vitro to LinB from Sphingobium Indicum bacteria. These single-chain CP-materials also contain small amounts of chlorinated olefins (COs), which can be distinct by mathematical deconvolution of respective mass-spectra. With this procedure, we obtained homologue-specific transformation kinetics of substrates differing in saturation degree, chlorination degree and carbon chain-length. For all homologues, two-stage first-order kinetic models were established, which described the faster conversion of reactive material and the slower transformation of more persistent material. Half-lifes of 0.5–3.2 h and 56–162 h were determined for more reactive and more persistent CP-material. Proportions of persistent material increased steadily from 18 to 67% for lower (Cl₆) to higher (Cl₁₁) chlorinated paraffins and olefins. Conversion efficiencies decreased with increasing chlorination degree from 97 to 70%. Carbon-chain length had only minor effects on transformation rates. Hence, the conversion was faster and more efficient for lower-chlorinated material, and slower for higher-chlorinated and longer-chained CPs and COs. Current legislation has banned short-chain chlorinated paraffins (SCCPs) and forced a transition to longer-chain CPs. This may be counterproductive with regard to enzymatic transformation with LinB.

Short-chain chlorinated paraffins (SCCPs) are listed as persistent organic pollutants (POPs) under the Stockholm Convention. Such substances are toxic, bioaccumulating, transported over long distances and degrade slowly in the environment. Certain bacterial strains of the Sphingomonadacea family are able to degrade POPs, such as hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). The haloalkane dehalogenase LinB, expressed in certain Sphingomonadacea, is able to catalyze the transformation of haloalkanes to hydroxylated compounds. Therefore, LinB is a promising candidate for conversion of SCCPs. Hence, a mixture of chlorinated tridecanes was exposed in vitro to LinB, which was obtained through heterologous expression in Escherichia coli. Liquid chromatography mass spectrometry (LC-MS) was used to analyze chlorinated tridecanes and their transformation products. A chloride-enhanced soft ionization method, which favors the formation of chloride adducts [M+Cl]^- without fragmentation, was applied. Mathematical deconvolution was used to distinguish interfering mass spectra of paraffinic, mono-olefinic and di-olefinic compounds. Several mono- and di-hydroxylated products including paraffinic, mono-olefinic and di-olefinic compounds were found after LinB exposure. Mono- (rt = 5.9–6.9 min) and di-hydroxylated (rt = 3.2–4.5 min) compounds were separated from starting material (rt = 7.7–8.5 min) by reversed phase LC. Chlorination degrees of chlorinated tridecanes increased during LinB-exposure from n_Cl = 8.80 to 9.07, indicating a preferential transformation of lower chlorinated (Cl_<9) tridecanes. Thus, LinB indeed catalyzed a dehalohydroxylation of chlorinated tridecanes, tridecenes and tridecadienes. The observed hydroxylated compounds are relevant CP transformation products whose environmental and toxicological effects should be further investigated.

Bacterial inactivation efficiency of chlorine varies with organisms and environmental conditions. The comparability of different samples/studies, especially comparing indigenous bacterial communities with pure cultures, is impeded by inconsistent experimental conditions and analytical methods used in various studies. We tested a novel 96-well plate FCM experimental and automated analytical approach, where bacterial communities and pure cultures were suspended in the same natural water matrix prior to chlorination directly in the plate. We demonstrated the ability to rapidly monitor the efficiency of 32 different combinations of chlorine concentration and time (i.e. chlorine exposure) on bacterial pure cultures and indigenous aquatic communities, which enabled correct comparison of the data from different samples under the exact same experimental conditions. In this study, the 96-well plate automated FCM approach enabled large sets (896) of independent chlorination experiments to be carried out in a short time period. To our knowledge, this is the largest dataset of chlorination experiments which consumed least time (within 18 h after sampling) until now. Staining with SYBR Green I (SG) and SG combined with propidium iodide (SGPI) was used to assess cellular damage during chlorination. The results showed that with the same chlorine exposure, a higher chlorine concentration with a shorter contact time is favorable for inactivation of bacteria. Our research provides a promising framework to compare disinfection characteristics of various microorganism and can be further developed to diagnose effect of antimicrobial products.

Les légionelles, des pathogènes opportunistes de l’être humain, peuvent contaminer les installations domestiques d’eau potable et d’eau de douche. Dans cette étude de cas, nous analysons l’effet d’interventions techniques successives au niveau du réglage de la température du chauffe-eau sur la concentration de la bactérie dans un bâtiment contaminé. Les résultats de cette étude démontrent qu’une température élevée (60 °C) dans le chauffe-eau permet de contrôler efficacement la prolifération du pathogène, tandis qu’une température plus modérée (45 °C) accompagnée de cycles de surchauffe ne permet pas d’assurer le contrôle des légionelles.

Nutrient limitation is one of the most common triggers of antibiotic tolerance and persistence. Here, we present two microfluidic setups to study how spatial and temporal variation in nutrient availability lead to increased survival of bacteria to antibiotics. The first setup is designed to mimic the growth dynamics of bacteria in spatially structured populations (e.g., biofilms) and can be used to study how spatial gradients in nutrient availability, created by the collective metabolic activity of a population, increase antibiotic tolerance. The second setup captures the dynamics of feast-and-famine cycles that bacteria recurrently encounter in nature, and can be used to study how phenotypic heterogeneity in growth resumption after starvation increases survival of clonal bacterial populations. In both setups, the growth rates and metabolic activity of bacteria can be measured at the single-cell level. This is useful to build a mechanistic understanding of how spatiotemporal variation in nutrient availability triggers bacteria to enter phenotypic states that increase their tolerance to antibiotics.

It has long been known that bacteria coordinate their physiology with their nutrient environment, yet our current understanding offers little intuition for how bacteria respond to the second-to-minute scale fluctuations in nutrient concentration characteristic of many microbial habitats. To investigate the effects of rapid nutrient fluctuations on bacterial growth, we couple custom microfluidics with single-cell microscopy to quantify the growth rate of E. coli experiencing 30 s to 60 min nutrient fluctuations. Compared to steady environments of equal average concentration, fluctuating environments reduce growth rate by up to 50%. However, measured reductions in growth rate are only 38% of the growth loss predicted from single nutrient shifts. This enhancement derives from the distinct growth response of cells grown in environments that fluctuate rather than shift once. We report an unexpected physiology adapted for growth in nutrient fluctuations and implicate nutrient timescale as a critical environmental parameter beyond nutrient identity and concentration.

Hurricane Hurricane Harvey made landfall on the Texas Gulf Coast on August 25, 2017, as a Category 4 hurricane and caused widespread flooding. We explored spatial and temporal distributions of well testing and contamination rates; relationships between contamination and system characteristics and recovery behaviors; and efficacy of mitigation strategies. We estimated that over 500 000 well users (∼130 000 to 260 000 wells) may have been affected, but only around 15 000 well users (∼3800 to 7500 wells) had inundated systems based on inundation maps. Local health departments and our team sampled 8822 wells in 44 counties in the 10 months that followed. Total coliform occurrence was 1.5 times and Escherichia coli was 2.8 times higher after Hurricane Harvey compared to baseline levels. Microbial contamination was more likely (1.7–2.5 times higher) when wells were inundated and/or residents felt their water was unsafe. Although more wells in urban counties were affected, E. coli rates were higher in wells in rural counties. Disinfection did not always eliminate contamination, highlighting concerns about the implementation and efficacy of chlorination procedures. Despite this extensive well testing conducted after Hurricane Harvey, we estimate that only 4.1% of potentially affected wells were tested, underscoring the magnitude of recovery assistance needed to assist well users after flooding events.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is perceived to be primarily transmitted via person-to-person contact through droplets produced while talking, coughing, and sneezing. Transmission may also occur through other routes, including contaminated surfaces; nevertheless, the role that surfaces have on the spread of the disease remains contested. Here, we use the Quantitative Microbial Risk Assessment framework to examine the risks of community transmission of SARS-CoV-2 through surfaces and to evaluate the effectiveness of hand and surface disinfection as potential interventions. Using conservative assumptions on input parameters of the model (e.g., dose-response relationship, ratio of genome copies to infective virus), the average of the median risks for single hand-to-surface contact followed by hand-to-face contact range from 1.6 × 10-4 to 5.6 × 10-9 for modeled prevalence rates of 0.2%-5%. For observed prevalence rates (0.2%, 1%), this corresponds to a low risk of infection (<10-6). Hand disinfection substantially reduces risks of transmission independently of the disease's prevalence and contact frequency. In contrast, the effectiveness of surface disinfection is highly dependent on the prevalence and the frequency of contacts. The work supports the current perception that contaminated surfaces are not a primary mode of transmission of SARS-CoV-2 and affirms the benefits of making hand disinfectants widely available.

Spatial and short-term temporal changes in water quality as a result of water age and fluctuating hydraulic conditions were investigated in a drinking water distribution system. Online measurements of total and intracellular adenosine tri-phosphate (ATP), total and intact cell concentrations measured with flow cytometry (FCM), turbidity, and particle counts were performed over five weeks at five subsequent locations of the distribution system. The high number of parallel FCM and ATP measurements revealed the combined effect of water age and final disinfection on spatial changes in microbiology in the system. The results underlined that regular daily dynamics in flow velocities are normal and inevitable in drinking water distribution systems, and significantly impact particle counts and turbidity. However, hydraulic conditions had no detectable impact on the concentration of suspended microbial cells. A weak correlation between flow velocity and ATP concentrations suggests incidental resuspension of particle-bound bacteria, presumably caused by either biofilm detachment or resuspension from sediment when flow velocities increase. The highly dynamic hydraulic conditions highlight the value of online monitoring tools for the meaningful description of short-term dynamics (day-scale) in drinking water distribution systems.

Microbial community composition and stability affect pollutant removal for biological/granular activated carbon (BAC/GAC) processes. Here, we pre-loaded the organic carbon substrates sucrose, lactose, and Lysogeny Broth (LB) medium onto new GAC prior to use and then tested whether this substrate pre-loading promoted development of biofilms with high coverage that remained stable for prolonged operational periods. Temporal dynamics of the biomass and microbial community on the GAC were monitored via flow cytometry (FCM) and sequencing, respectively, in both batch and continuous-flow experiments. In comparison with the non-loaded GAC (control), the initial biofilm biomass on substrate-loaded GAC was 3–114 times higher, but the initial richness was considerably lower (only accounting for 13–28% of the control). The initial community compositions were significantly different between batch and continuous-flow column experiments, even when loaded with the same substrates. In the continuous-flow column experiments, both biomass and microbial community composition became remarkably similar to the control filters after 64 days of operation. From these findings, we conclude that substrate-loaded GAC could enhance initial colonization, affecting both biomass and microbial community composition. However, the biomass and composition did not remain stable during long-term operation due to continuous dispersal and competition from influent bacteria.

Freshwater ecosystems are considered hotspots of biodiversity in Antarctic polar deserts. Anticipated warming is expected to change the hydrology of these systems due to increased meltwater and reduction of ice cover, with implications for environmental conditions and physical connectivity between habitats. Using 16S rRNA gene sequencing, we evaluated microbial mat and planktonic communities within a connected freshwater system in the McMurdo Wright Valley, Antarctica, to determine the roles of connectivity and habitat conditions in controlling microbial assemblage composition. We examined communities from glacial Lake Brownworth, the perennially ice-covered Lake Vanda and the Onyx River, which connects the two. In Lake Vanda, we found distinct microbial assemblages occupying sub-habitats at different lake depths, while the communities from Lake Brownworth and Onyx River were structurally similar. Despite the higher physical connectivity and dispersal opportunities between bacterial communities in the shallow parts of the system, environmental abiotic conditions dominated over dispersal in driving community structure. Functional metabolic pathway predictions suggested differences in the functional gene potential between the microbial mat communities located in shallower and deeper water depths. The findings suggest that increasing temperatures and meltwater due to future climate change will affect bacterial diversity and functioning in Antarctic freshwater ecosystems.

1. Soil microbial community coalescence, where entire microbial communities mix and interact under new conditions, is a widespread phenomenon whose applicability for targeted root microbiome assembly has not been studied. Whether soil mixing can lead to predictable outcomes for community assembly and functioning of specific functional groups, for example, N₂-fixing rhizobia, remains unknown.
2. Using a legume shrub adapted to nutrient-poor soils, we tested the effects of community coalescence on plant nutrition and growth, and its influence on the rhizobial root nodule symbiosis. We grew seedlings of rooibos [Aspalathus linearis (Burm.f.) Dahlg.] in individual rhizosphere soils collected from cultivated and wild rooibos plants and their mixtures, and included a fertilization treatment. Portions of the taxonomic gyrB and the symbiotic nodA gene makers were sequenced to characterize rhizobial communities present in rooibos root nodules under the different soil conditions.
3. Overall, community coalescence by soil mixing had positive effects on plant nutrition and growth, and interacted with fertilizer addition to concurrently change rhizobial taxonomic evenness and promote higher relative N₂ fixation. We identified particular rhizobia preferentially associated with rooibos plants that showed higher N fractions from N₂ fixation, raised in mixed fertilized soils.
4. These findings indicate that soil bacterial community coalescence and fertilization can have synergistic effects on plant performance, while promoting the assembly of alternative symbiotic rhizobial communities that provide improved nutritional benefits to host plants.
5. Synthesis and applications. The combination of soil mixing and fertilizer addition may be an important, but hitherto overlooked measure to improve the functioning of rhizobium symbioses in legume crops. Microbial community coalescence should gain recognition as a potentially effective mechanism to improve the functioning of plant microbiomes.

While "avoiding stagnation" has been the mantra of many building water quality experts, the foundational support for how this terminology is used in peer-reviewed publications and building water management guidelines is not overly convincing. With COVID-19 lockdowns and subsequent reopening, the concern that extended stagnation will trigger increased incidence (or even an epidemic) of legionellosis has been widely raised in news and social media. Here, we provide a perspective on four simple questions to a complex topic, with hope that it encourages a broader dialogue and more critical assessment of assumptions and communication strategies surrounding stagnation and Legionella growth.

We present the results of three field tests and three laboratory tests of a new physical-chemical urine treatment system, which can recover all nutrients, while pathogens are inactivated. The system consists of two steps. In the first reactor, biological processes including urea hydrolysis are prevented by mixing fresh urine with calcium hydroxide (Ca(OH)₂). Due to the high pH value and the high availability of calcium, phosphate can be recovered by precipitation. The high pH value also fosters the inactivation of microorganisms, including pathogens. In the second reactor, water is evaporated at low energy consumption by blowing unheated ambient air over the stabilized urine. Stabilization in the first reactor was successful in all field and laboratory tests. The pH value remained between 12 and 13, except for short dips due to shortages of Ca(OH)₂. Nearly all phosphorus (92-96%) precipitated and could be recovered as calcium phosphate in the first reactor, while nitrogen and potassium overflowed with the urine into the evaporation reactor. The efficiency of the second treatment step was very different for field and laboratory experiments and depended on the duration of the experiment. During a four-day laboratory test, nitrogen recovery was 98%. In contrast, nitrogen recovery was only around 20% in the long-term field experiments. The high nitrogen losses occurred, because biological urea hydrolysis was not inhibited anymore, when the pH value in the second reactor decreased due to the dissolution of high amounts of carbon dioxide from the ambient air. Potassium was not subject to any significant loss, and the measured recovery in the solid evaporation product was 98%. Evaporation rates ranged between 50 g m^-2 h^-1 (RH = 82±13%, T = 12±6°C) and 130 g m^-2 h^-1 (RH = 60±19%, T = 24±5°C) in the three field tests. Apart from some disturbances due to low supply of Ca(OH)₂, the urine module functioned without any substantial failures and was simple to maintain. The minimum consumption of Ca(OH)₂ at full capacity was 6 g·L^-1 urine and the electricity demand was 150 Wh kg^-1 water evaporated from urine, resulting in operational costs of 0.05 EUR pers^-1 d^-1.

Positive species interactions underlie the functioning of ecosystems. Given their importance, it is crucial to understand the stability of positive interactions over evolutionary timescales, in both constant and fluctuating environments; e.g., environments interrupted with periods of competition. We addressed this question using a two-species microbial system in which we modulated interactions according to the nutrient provided. We evolved in parallel four experimental replicates of species growing in isolation or together in consortia for 200 generations in both a constant and fluctuating environment with daily changes between commensalism and competition. We sequenced full genomes of single clones isolated at different time points during the experiment. We found that the two species coexisted over 200 generations in the constant commensal environment. In contrast, in the fluctuating environment, coexistence broke down when one of the species went extinct in two out of four cases. We showed that extinction was highly deterministic: when we replayed the evolution experiment from an intermediate time point we repeatably reproduced species extinction. We further show that these dynamics were driven by adaptive mutations in a small set of genes. In conclusion, in a fluctuating environment, rapid evolution destabilizes the long-term stability of positive pairwise interactions.

Conjugative plasmids bestow important traits to microbial communities, such as virulence, antibiotic resistance, pollutant biotransformation, and biotechnology-relevant functions. While the biological mechanisms and determinants of plasmid conjugation are well established, the underlying physical and ecological driving forces remain unclear. Microbial communities often inhabit unsaturated environments, such as soils and host surfaces (e.g., skin, teeth, leaves, roots), where water evaporation and associated small-scale hydrodynamic processes frequently occur at numerous air-water and solid-water interfaces. Here, we hypothesized that evaporation can induce water flows with profound effects on the spatial distribution and surface deposition of cells, and consequently on the extent of plasmid conjugation. Using droplet experiments with an antibiotic resistance-encoding plasmid, we show that evaporation-induced water flows reduce cell-cell distances and significantly increase the extent of plasmid conjugation. Counterintuitively, we found that evaporation results in lower expression levels of conjugation-related genes. This negative relationship between the extent of plasmid conjugation and the expression of conjugation-related genes could be attributed to increased conjugation efficiency during evaporation. This study provides new insights into the physical and ecological determinants of plasmid conjugation, with important implications for understanding the spread and proliferation of plasmid-encoded traits.

A new Bacillus cereus phage, SWEP1, was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins, 103 of which were assigned functionally. No tRNA genes were found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggested that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.

Anomaly detection is the process of identifying unexpected data samples in datasets. Automated anomaly detection is either performed using supervised machine learning models, which require a labelled dataset for their calibration, or unsupervised models, which do not require labels. While academic research has produced a vast array of tools and machine learning models for automated anomaly detection, the research community focused on environmental systems still lacks a comparative analysis that is simultaneously comprehensive, objective, and systematic. This knowledge gap is addressed for the first time in this study, where 15 different supervised and unsupervised anomaly detection models are evaluated on 5 different environmental datasets from engineered and natural aquatic systems. To this end, anomaly detection performance, labelling efforts, as well as the impact of model and algorithm tuning are taken into account. As a result, our analysis reveals the relative strengths and weaknesses of the different approaches in an objective manner without bias for any particular paradigm in machine learning. Most importantly, our results show that expert-based data annotation is extremely valuable for anomaly detection based on machine learning.

Copper (Cu) is a promising antimicrobial for premise plumbing, where ions can be dosed directly via copper silver ionization or released naturally via corrosion of Cu pipes, but Cu sometimes inhibits and other times stimulates Legionella growth. Our overarching hypothesis was that water chemistry and growth phase control the net effect of Cu on Legionella. The combined effects of pH, phosphate concentration, and natural organic matter (NOM) were comprehensively examined over a range of conditions relevant to drinking water in bench-scale pure culture experiments, illuminating the effects of Cu speciation and precipitation. It was found that cupric ions (Cu²⁺) were drastically reduced at pH > 7.0 or in the presence of ligand-forming phosphates or NOM. Further, exponential phase L. pneumophila were 2.5× more susceptible to Cu toxicity relative to early stationary phase cultures. While Cu²⁺ ion was the most effective biocidal form of Cu, other inorganic ligands also had some biocidal impacts. A comparison of 33 large drinking water utilities' field-data from 1990 and 2018 showed that Cu²⁺ levels likely decreased more dramatically (>10×) than did the total or soluble Cu (2×) over recent decades. The overall findings aid in improving the efficacy of Cu as an actively dosed or passively released antimicrobial against L. pneumophila.

The macroevolutionary transition from terra firma to obligatory inhabitance of the marine hydrosphere has occurred twice in the history of Mammalia: Cetacea and Sirenia. In the case of Cetacea (whales, dolphins, and porpoises), molecular phylogenies provide unambiguous evidence that fully aquatic cetaceans and semiaquatic hippopotamids (hippos) are each other’s closest living relatives. Ancestral reconstructions suggest that some adaptations to the aquatic realm evolved in the common ancestor of Cetancodonta (Cetacea + Hippopotamidae). An alternative hypothesis is that these adaptations evolved independently in cetaceans and hippos. Here, we focus on the integumentary system and evaluate these hypotheses by integrating new histological data for cetaceans and hippos, the first genome-scale data for pygmy hippopotamus, and comprehensive genomic screens and molecular evolutionary analyses for protein-coding genes that have been inactivated in hippos and cetaceans. We identified eight skin-related genes that are inactivated in both cetaceans and hippos, including genes that are related to sebaceous glands, hair follicles, and epidermal differentiation. However, none of these genes exhibit inactivating mutations that are shared by cetaceans and hippos. Mean dates for the inactivation of skin genes in these two clades serve as proxies for phenotypic changes and suggest that hair reduction/loss, the loss of sebaceous glands, and changes to the keratinization program occurred ∼16 Ma earlier in cetaceans (∼46.5 Ma) than in hippos (∼30.5 Ma). These results, together with histological differences in the integument and prior analyses of oxygen isotopes from stem hippopotamids (“anthracotheres”), support the hypothesis that aquatic skin adaptations evolved independently in hippos and cetaceans.

Opportunistic pathogens can linger on surfaces in hospital and building plumbing environments, leading to infections in at-risk populations. Furthermore, biofilm-associated bacteria are protected from removal and inactivation protocols such as disinfection. Bacteriophages show promise as tools to treat antibiotic-resistant infections. As such, phages may also be useful in environmental applications to prevent newly acquired infections. In the current study, the potential of synergies between bacteriophage and chemical disinfection against the opportunistic pathogen Pseudomonas aeruginosa was assessed under various conditions. Specifically, surface-associated P. aeruginosa was treated with various concentrations of phages (P1 or JG004), chemical disinfectants (sodium hypochlorite or benzalkonium chloride), or combined sequential treatments under three distinct attachment models (spot inoculations, dry biofilms, and wet biofilms). Phages were very effective at removing bacteria in spot inoculations (>3.2 log₁₀ removal) and wet biofilms (up to 2.6 log₁₀ removal), while phages prevented the regrowth of dry biofilms in the application time. In addition, phage treatment followed by chemical disinfection inactivated P. aeruginosa cells under wet biofilm conditions better than either treatment alone. This effect was hindered when chemical disinfection was applied first, followed by phage treatment, suggesting that the additive benefits of combination treatments are lost when phage is applied last. Furthermore, we confirm previous evidence of greater phage tolerance to benzalkonium chloride than to sodium hypochlorite, informing choices for combination phage-disinfectant approaches. Overall, this paper further supports the potential of using combination phage and chemical disinfectant treatments to improve the inactivation of surface-associated P. aeruginosa.

The emergence and spread of antibiotic resistance is a major societal challenge and new antibiotics are needed to successfully fight bacterial infections. Because the release of antibiotics into wastewater and downstream environments is expected to contribute to the problem of antibiotic resistance, it would be beneficial to consider the environmental fate of antibiotics in the development of novel antibiotics. In this article, we discuss the possibility of designing peptide-based antibiotics that are stable during treatment (e.g. in human blood), but rapidly inactivated through hydrolysis by peptidases after their secretion into wastewater. In the first part, we review studies on the biotransformation of peptide-based antibiotics during biological wastewater treatment and on the specificity of dissolved extracellular peptidases derived from wastewater. In the second part, we present first results of our endeavour to identify peptide bonds that are stable in human blood plasma and susceptible to hydrolysis by the industrially produced peptidase Subtilisin A.

In bacterial communities, cells often communicate by the release and detection of small diffusible molecules, a process termed quorum-sensing. Signal molecules are thought to broadly diffuse in space; however, they often regulate traits such as conjugative transfer that strictly depend on the local community composition. This raises the question how nearby cells within the community can be detected. Here, we compare the range of communication of different quorum-sensing systems. While some systems support long-range communication, we show that others support a form of highly localized communication. In these systems, signal molecules propagate no more than a few microns away from signaling cells, due to the irreversible uptake of the signal molecules from the environment. This enables cells to accurately detect micron scale changes in the community composition. Several mobile genetic elements, including conjugative elements and phages, employ short-range communication to assess the fraction of susceptible host cells in their vicinity and adaptively trigger horizontal gene transfer in response. Our results underscore the complex spatial biology of bacteria, which can communicate and interact at widely different spatial scales.

Biotransformation of chemical contaminants is of importance in various natural and engineered systems. However, in complex microbial communities and with chemical contaminants at low concentrations, our current understanding of biotransformation at the level of enzyme-chemical interactions is limited. Here, we explored an approach to identify associations between micropollutant biotransformation and specific gene products in complex microbial communities, using association mining between chemical and metatranscriptomic data obtained from experiments with activated sludge grown at different solid retention times. We successfully demonstrate proportional relationships between the measured rate constants and associated gene transcripts for nitrification as a major community function, but also for the biotransformation of two nitrile-containing micropollutants (bromoxynil and acetamiprid) and transcripts of nitrile hydratases, a class of enzymes that we experimentally confirmed to produce the detected amide transformation products. As these results suggest that metatranscriptomic information can indeed be quantitatively correlated with low abundant community functions such as micropollutant biotransformation in complex microbial communities, we proceeded to explore the potential of association mining to highlight enzymes likely involved in catalyzing less well-understood micropollutant biotransformation reactions. Specifically, we use the cases of nitrile hydration and oxidative biotransformation reactions to show that the consideration of additional experimental evidence (such as information on biotransformation pathways) increases the likelihood of detecting plausible novel enzyme-chemical relationships. Finally, we identify a cluster of mono- and dioxygenase fourth-level enzyme classes that most strongly correlate with oxidative micropollutant biotransformation reactions in activated sludge.

Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O₂, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O₂ uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O₂ activation to substrate hydroxylation, depending on the extent of O-O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O₂ uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.

Evidence suggests that bacterial community spatial organization affects their ecological function, yet details of the mechanisms that promote spatial patterns remain difficult to resolve experimentally. In contrast to bacterial communities in liquid cultures, surface-attached range expansion fosters genetic segregation of the growing population with preferential access to nutrients and reduced mechanical restrictions for cells at the expanding periphery. Here we elucidate how localized conditions in cross-feeding bacterial communities shape community spatial organization. We combine experiments with an individual based mathematical model to resolve how trophic dependencies affect localized growth rates and nucleate successful cell lineages. The model tracks individual cell lineages and attributes these with trophic dependencies that promote counterintuitive reproductive advantages and result in lasting influences on the community structure, and potentially, on its functioning. We examine persistence of lucky lineages in structured habitats where expansion is interrupted by physical obstacles to gain insights into patterns in porous domains.

Background: Recent experimental work has shown that the evolutionary dynamics of bacteria expanding across space can differ dramatically from what we expect under well-mixed conditions. During spatial expansion, deleterious mutations can accumulate due to inefficient selection on the expansion front, potentially interfering with and modifying adaptive evolutionary processes.
Results: We used whole genome sequencing to follow the genomic evolution of 10 mutator Escherichia coli lines during 39 days ( ~ 1650 generations) of a spatial expansion, which allowed us to gain a temporal perspective on the interaction of adaptive and non-Adaptive evolutionary processes during range expansions. We used elastic net regression to infer the positive or negative effects of mutations on colony growth. The colony size, measured after three day of growth, decreased at the end of the experiment in all 10 lines, and mutations accumulated at a nearly constant rate over the whole experiment. We find evidence that beneficial mutations accumulate primarily at an early stage of the experiment, leading to a non-linear change of colony size over time. Indeed, the rate of colony size expansion remains almost constant at the beginning of the experiment and then decreases after ~ 12 days of evolution. We also find that beneficial mutations are enriched in genes encoding transport proteins, and genes coding for the membrane structure, whereas deleterious mutations show no enrichment for any biological process.
Conclusions: Our experiment shows that beneficial mutations target specific biological functions mostly involved in inter or extra membrane processes, whereas deleterious mutations are randomly distributed over the whole genome. It thus appears that the interaction between genetic drift and the availability or depletion of beneficial mutations determines the change in fitness of bacterial populations during range expansion.

Sunlight, temperature, and microbial grazing are among the environmental factors promoting the inactivation of viral pathogens in surface waters. Globally, these factors vary across time and space. The persistence of viral pathogens, and ultimately their ecology and dispersion, hinges on their ability to withstand the environmental conditions encountered. To understand how virus populations evolve under changing environmental conditions, we experimentally adapted echovirus 11 (E11) to four climate regimes. Specifically, we incubated E11 in lake water at 10 and 30 °C and in the presence and absence of sunlight. Temperature was the main driver of adaptation, resulting in an increased thermotolerance of the 30 °C adapted populations, whereas the 10 °C adapted strains were rapidly inactivated at higher temperatures. This finding is consistent with a source-sink model in which strains emerging in warm climates can persist in temperate regions, but not vice versa. A microbial risk assessment revealed that the enhanced thermotolerance increases the length of time in which there is an elevated probability of illness associated with swimming in contaminated water. Notably, 30 °C-adapted viruses also exhibited an increased tolerance toward disinfection by free chlorine. Viruses adapting to warm environments may thus become harder to eliminate by common disinfection strategies.

Surface-attached microbial communities consist of different cell types that, at least to some degree, organize themselves non-randomly across space (referred to as spatial self-organization). While spatial self-organization can have important effects on the functioning, ecology and evolution of communities, the underlying determinants of spatial self-organization remain unclear. Here, we hypothesize that the presence of physical objects across a surface can have important effects on spatial self-organization. Using pairs of isogenic strains of Pseudomonas stutzeri, we performed range expansion experiments in the absence or presence of physical objects and quantified the effects on spatial self-organization. We demonstrate that physical objects create local deformities along the expansion frontier, and these deformities increase in magnitude during range expansion. The deformities affect the densities of interspecific boundaries and diversity along the expansion frontier, and thus affect spatial self-organization, but the effects are interaction-dependent. For competitive interactions that promote sectorized patterns of spatial self-organization, physical objects increase the density of interspecific boundaries and diversity. By contrast, for cross-feeding interactions that promote dendritic patterns, they decrease the density of interspecific boundaries and diversity. These qualitatively different outcomes are probably caused by fundamental differences in the orientations of the interspecific boundaries. Thus, in order to predict the effects of physical objects on spatial self-organization, information is needed regarding the interactions present within a community and the general geometric shapes of spatial self-organization that emerge from those interactions.
This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Microbial cells in nature live within dense multispecies conglomerates, forming a self-organizing ecosystem. In such assemblies, genotypes interact with each other in a myriad of ways, driving community dynamics and functionalities. The role of interactions between genotypes and their consequences for spatial structure and functional outcomes are being increasingly studied to understand the ecology and evolution of microbial communities. An increasing body of work with simple microbial populations has elucidated that phenotypic variation, that is, differences within isogenic cells can have important consequences for population dynamics and evolution. However, the role of individual level behavioral differences for community level dynamics is relatively unknown. I argue that it is necessary to study phenotypic variation and microscale processes in order to understand the emergence and consequences of interactions within microbial communities. I highlight possible explanations that can explain the emergence of variation in multi-genotypic assemblages and propose possible consequences on community dynamics.

Communities of interacting microorganisms play important roles across all habitats on Earth. These communities typically consist of a large number of species that perform different metabolic processes. The functions of microbial communities ultimately emerge from interactions between these different microorganisms. To understand the dynamics and functions of microbial communities, we thus need to know the nature and strength of these interactions. Here, we quantified the interaction strength between individual cells in microbial communities. We worked with synthetic communities of Escherichia coli bacteria that exchange metabolites to grow. We combined single-cell growth rate measurements with mathematical modelling to quantify metabolic interactions between individual cells and to map the spatial interaction network in these communities. We found that cells only interact with other cells in their immediate neighbourhood. This short interaction range limits the coupling between different species and reduces their ability to perform metabolic processes collectively. Our experiments and models demonstrate that the spatial scale of biotic interaction plays a fundamental role in shaping the ecological dynamics of communities and the functioning of ecosystems.

Both multi-walled carbon nanotubes (MWCNTs) and metal or metal oxides have demonstrated virus removal efficacy in drinking water applications. In this study, MWCNTs were coated with copper(I) oxide (Cu₂O) using three distinct synthesis procedures (copper ion attachment, copper hydroxide precipitation, and [Cu(NH₃)₄]²⁺ complex attachment) and virus removal efficacy (using MS2 bacteriophages) was evaluated. All synthesis procedures resulted in the presence of adsorbed, nanosized Cu₂O particles on the MWCNTs, shown using X-ray diffraction. Further, transmission electron microscopy confirmed uniform copper(I) oxide distribution along the MWCNTs for all three materials. Virus removal efficacy was assessed for all three synthesised composites both before and after material conditioning (filtering for at least 24 h/280 mL/h), and accounting for additional MS2 inactivation in the permeate due to continued copper inactivation from dissolved/desorbed copper in permeate (time-control). Material conditioning influenced virus removal, with the first litres of water containing higher concentrations of copper than the sixth litres of water, suggesting excess or non-bonded copper species dissolve from filters. Higher copper dissolution was observed for water at pH 5 than at pH 7, which decreased with time. Copper dissolution most likely caused an associated decrease in copper adsorbed to MWCNTs in the filters, which may explain the observed lower MS2 removal efficacy after conditioning. Additionally, the time-control study (immediately after filtration as compared to 2 h after filtration) highlighted continued MS2 inactivation in the permeate over time. The obtained results indicate that the synthesis procedure influences virus removal efficacy for MWCNTs coated with copper oxides and that virus removal is likely due to not only virus electrostatic adsorption to the coated MWCNTs, but also through antiviral properties of copper which continues to act in the permeate. In conclusion, it is highly important to revise the methods of testing filter materials for virus removal, as well as procedure for virus concentration evaluation.

Microbial communities are complex multi-species assemblages that are characterized by a multitude of interspecies interactions, which can range from mutualism to competition. The overall sign and strength of interspecies interactions have important consequences for emergent community-level properties such as productivity and stability. It is not well understood how interspecies interactions change over evolutionary timescales. Here, we review the empirical evidence that evolution is an important driver of microbial community properties and dynamics on timescales that have traditionally been regarded as purely ecological. Next, we briefly discuss different modelling approaches to study evolution of communities, emphasizing the similarities and differences between evolutionary and ecological perspectives. We then propose a simple conceptual model for the evolution of interspecies interactions in communities. Specifically, we propose that to understand the evolution of interspecies interactions, it is important to distinguish between direct and indirect fitness effects of a mutation. We predict that in well-mixed environments, traits will be selected exclusively for their direct fitness effects, while in spatially structured environments, traits may also be selected for their indirect fitness effects. Selection of indirectly beneficial traits should result in an increase in interaction strength over time, while selection of directly beneficial traits should not have such a systematic effect. We tested our intuitions using a simple quantitative model and found support for our hypotheses. The next step will be to test these hypotheses experimentally and provide input for a more refined version of the model in turn, thus closing the scientific cycle of models and experiments.
This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.

A leading challenge in drinking water treatment is to remove small-sized viruses from the water in a simple and efficient manner. Multi-walled carbon nanotubes (MWCNT) are new generation adsorbents with previously demonstrated potential as filter media to improve virus removal. This study therefore aimed to evaluate the field applicability of MWCNT-filters for virus removal in water containing natural organic matter (NOM) as co-solute to viruses, using batch equilibrium experiments. Contrary to previous studies, our results showed with MS2 bacteriophages single-solute systems that the affinity of MWCNT for MS2 was low, since after 3 h of equilibration only 4 log₁₀ reduction value (LRV) of MS2 (20 mL at an initial concentration of 10⁶ PFU MS2/mL) were reached. Single solute experiments with Suwannee river NOM (SRNOM) performed with environmentally-relevant concentrations showed MWCNT surface saturation at initial SRNOM concentrations between 10 and 15 mgC/L, for water pH between 5.2 and 8.7. These results suggested that at NOM:virus ratios found in natural waters, the NOM would competitively suppress virus adsorption onto MWCNT, even at low NOM concentrations. We confirmed this expectation with SRNOM-MS2 co-solute experiments, which showed an exponential decrease of the MS2 LRV by MWCNT with an increase in the initial SRNOM concentration. More interestingly, we showed that pre-equilibrating MWCNT with a SRNOM solution at a concentration as low as 0.4 mgC/L resulted in a LRV decrease of 3 for MS2, due to the formation of a negatively charged SRNOM adlayer on the MWCNT surface. Complementary batch experiments with natural NOM-containing waters and competition experiments with SRNOM in the presence of CaCl₂ confirmed that the presence of NOM in waters challenges virus removal by MWCNT-filters, irrespective of the concentration and type of NOM and also in the presence of Ca²⁺. We therefore conclude that MWCNT-filters produced with commercially available pristine MWCNT cannot be considered as a viable technology for drinking water virus removal.

The importance of functional diversity for the functioning and behaviour of microbial communities is clear, yet the widespread incorporation of functional diversity measurements into environmental microbiology study designs remains surprisingly limited. This may, at least to some extent, be a consequence of the unique conceptual and methodological challenges to measuring functional diversity in microbial communities. To facilitate the increased incorporation of functional diversity measurements into environmental microbiology study designs, we review here the process and some key caveats for measuring functional diversity and provide specific examples. We highlight three main decision points and provide guidance to making these decisions based on the underlying mechanisms for how functional diversity relates to an ecosystem process or property of interest. We discuss the selection of an appropriate type of functional trait, selection of the specificity at which functional diversity will be measured, and selection of an appropriate metric for estimating functional diversity from quantitative measures of those traits. We further discuss decisions regarding the use of one- or multi-dimensional measures of functional diversity and how advances in the field of trait-based community ecology could be applied or adapted to address questions in environmental microbiology.

In the present study, triplicate rings of 360° pipe surfaces of an operational drinking water distribution pipe were swabbed. Each ring was equally divided into 16 parts for swabbing. The collected swabs were grouped into 3 sections and compared with the biofilm samples sampled by sonication of specimens from the same pipe. The results showed that the biofilm is unevenly distributed over the 16 parts and the 3 sections of the pipe surface. Both the active biomass and the number of observed OTUs increased as the measurements proceeded from the top to the bottom of the pipe. The bacterial community was dominated in all sections by Proteobacteria. At the genus level, Nitrospira spp., Terrimonas spp., and Hyphomicrobium spp. were dominant in all sections. Gaiella spp. and Vicinamibacter spp. dominated in S-I, Blastopirellula spp. and Pirellula spp. dominated in S-II, while Holophaga spp. and Phaeodactylibacter spp. dominated in S-III. When swabbing and pipe specimen sonication were compared, the results showed that the sampling strategy significantly influences the obtained biofilm bacterial community. A consistent multisectional swabbing strategy is proposed for future biofilm sampling; it involves collecting swabs from all sections and comparing the swabs from the same position/section across locations.

The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the "yeast machine", with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined and customizable spatial dimensions. With Saccharomyces cerevisiae as a model yeast system we used the "yeast machine" to demonstrate the emergence of glucose gradients by following expression of fluorescently labelled hexose transporters. We further quantified the expression spatial patterns with intra-colony growth rates and expression of other genes regulated by glucose availability. In addition to this, we showed that gradients of amino acids also form within a colony, potentially opening similar approaches to study spatiotemporal formation of gradients of many other nutrients and metabolic waste products. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development, and morphogenesis in other same species or more complex multi-species systems at single-cell resolution and timescales relevant to ecology and evolution.

Lakes and reservoirs contribute substantially to atmospheric concentrations of the potent greenhouse gas methane. Lake sediments produce large amounts of methane, which accumulate in the oxygen-depleted bottom waters of stratified lakes. Climate change and eutrophication may increase the number of lakes with methane storage in the future. Whether stored methane escapes to the atmosphere during annual lake overturn is a matter of controversy and depends critically on the response of the methanotroph assemblage. Here we show, by combining 16S rRNA gene and pmoA mRNA amplicon sequencing, qPCR, CARD-FISH and potential methane-oxidation rate measurements, that the methanotroph assemblage in a mixing lake underwent both a substantial bloom and ecological succession. As a result, methane oxidation kept pace with the methane supplied from methane-rich bottom water and most methane was oxidized. This aspect of freshwater methanotroph ecology represents an effective mechanism limiting methane transfer from lakes to the atmosphere.

Escherichia coli is present in multiple hosts and environmental compartments as a normal inhabitant, temporary or persistent colonizer, and as a pathogen. Transmission of E. coli between hosts and with the environment is considered to occur more often in areas with poor sanitation. We performed whole-genome comparative analyses on 60 E. coli isolates from soils and fecal sources (cattle, chickens, and humans) in households in rural Bangladesh. Isolates from household soils were in multiple branches of the reconstructed phylogeny, intermixed with isolates from fecal sources. Pairwise differences between all strain pairs were large (minimum, 189 single nucleotide polymorphisms [SNPs]), suggesting high diversity and heterogeneous origins of the isolates. The presence of multiple virulence and antibiotic resistance genes is indicative of the risk that E. coli from soil and feces represent for the transmission of variants that pose potential harm to people. Analysis of the accessory genomes of the Bangladeshi E. coli relative to E. coli genomes available in NCBI identified a common pool of accessory genes shared among E. coli isolates in this geographic area. Together, these findings indicate that in rural Bangladesh, a high level of E. coli in soil is likely driven by contributions from multiple and diverse E. coli sources (human and animal) that share an accessory gene pool relatively unique to previously published E. coli genomes. Thus, interventions to reduce environmental pathogen or antimicrobial resistance transmission should adopt integrated One Health approaches that consider heterogeneous origins and high diversity to improve effectiveness and reduce prevalence and transmission.

Many microorganisms face a fundamental trade-off between reproduction and survival: Rapid growth boosts population size but makes microorganisms sensitive to external stressors. Here, we show that starved bacteria encountering new resources can break this trade-off by evolving phenotypic heterogeneity in lag time. We quantify the distribution of single-cell lag times of populations of starved Escherichia coli and show that population growth after starvation is primarily determined by the cells with shortest lag due to the exponential nature of bacterial population dynamics. As a consequence, cells with long lag times have no substantial effect on population growth resumption. However, we observe that these cells provide tolerance to stressors such as antibiotics. This allows an isogenic population to break the trade-off between reproduction and survival. We support this argument with an evolutionary model which shows that bacteria evolve wide lag time distributions when both rapid growth resumption and survival under stressful conditions are under selection. Our results can explain the prevalence of antibiotic tolerance by lag and demonstrate that the benefits of phenotypic heterogeneity in fluctuating environments are particularly high when minorities with extreme phenotypes dominate population dynamics.

Antimicrobial resistance (AMR) is a growing public health challenge that is expected to disproportionately burden lower- and middle-income countries (LMICs) in the coming decades. Although the contributions of human and veterinary antibiotic misuse to this crisis are well-recognized, environmental transmission (via water, soil or food contaminated with human and animal faeces) has been given less attention as a global driver of AMR, especially in urban informal settlements in LMICs - commonly known as 'shanty towns' or 'slums'. These settlements may be unique hotspots for environmental AMR transmission given: (1) the high density of humans, livestock and vermin living in close proximity; (2) frequent antibiotic misuse; and (3) insufficient drinking water, drainage and sanitation infrastructure. Here, we highlight the need for strategies to disrupt environmental AMR transmission in urban informal settlements. We propose that water and waste infrastructure improvements tailored to these settings should be evaluated for their effectiveness in limiting environmental AMR dissemination, lowering the community-level burden of antimicrobial-resistant infections and preventing antibiotic misuse. We also suggest that additional research is directed towards developing economic and legal incentives for evaluating and implementing water and waste infrastructure in these settings. Given that almost 90% of urban population growth will occur in regions predicted to be most burdened by the AMR crisis, there is an urgent need to build effective, evidence-based policies that could influence massive investments in the built urban environment in LMICs over the next few decades.

The environmental conditions in building plumbing systems differ considerably from the larger distribution system and, as a consequence, uncontrolled changes in the drinking water microbiome through selective growth can occur. In this regard, synthetic polymeric plumbing materials are of particular relevance, since they leach assimilable organic carbon that can be utilized for bacterial growth. Here, we discuss the complexity of building plumbing in relation to microbial ecology, especially in the context of low-quality synthetic polymeric materials (i.e., plastics) and highlight the major knowledge gaps in the field. We furthermore show how knowledge on the interaction between material properties (e.g., carbon migration) and microbiology (e.g., growth rate) allows for the quantification of initial biofilm development in buildings. Hence, research towards a comprehensive understanding of these processes and interactions will enable the implementation of knowledge-based management strategies. We argue that the exclusive use of high-quality materials in new building plumbing systems poses a straightforward strategy towards managing the building plumbing microbiome. This can be achieved through comprehensive material testing and knowledge sharing between all stakeholders including architects, planners, plumbers, material producers, home owners, and scientists.

The frequent contact people have with liquids containing pathogenic microorganisms provides opportunities for disease transmission. In this work, we quantified the transfer of bacteria - using E. coli as a model- from liquid to skin, estimated liquid retention on the skin after different contact activities (hand immersion, wet-cloth and wet-surface contact), and estimated liquid transfer following hand-to-mouth contacts. The results of our study show that the number of E. coli transferred to the skin per surface area (n [E. coli/cm²]) can be modeled using n = C (10^-3.38+h), where C [E. coli/cm³] is the concentration of E. coli in the liquid, and h [cm] is the film thickness of the liquid retained on the skin. Findings from the E. coli transfer experiments reveal a significant difference between the transfer of E. coli from liquid to the skin and the previously reported transfer of viruses to the skin. Additionally, our results demonstrate that the time elapsed since the interaction significantly influences liquid retention, therefore modulating the risks associated with human interaction with contaminated liquids. The findings enhance our understanding of liquid-mediated disease transmission processes and provide quantitative estimates as inputs for microbial risk assessments.

On-site wastewater reuse can improve global access to clean water, sanitation and hygiene. We developed a treatment system (aerated bioreactor, ultrafiltration membrane, granular activated carbon and electrolysis for chlorine disinfection) that recycles hand washing and toilet flush water.
Three prototypes were field-tested in non-sewered areas, one in Switzerland (hand washing) and two in South Africa (hand washing, toilet flushing), over periods of 63, 74 and 94 days, respectively.
We demonstrated that the system is able to recycle sufficient quantities of safe and appealing hand washing and toilet flush water for domestic or public use in real-life applications. Chemical contaminants were effectively removed from the used water in all prototypes. Removal efficiencies were 99.7% for the chemical oxygen demand (COD), 98.5% for total nitrogen (TN) and 99.9% for phosphate in a prototype treating hand washing water, and 99.8% for COD, 95.7% for TN and 89.6% for phosphate in a prototype treating toilet flush water. While this system allowed for true recycling for the same application, most on-site wastewater reuse systems downcycle the treated water, i.e., reuse it for an application requiring lower water quality. An analysis of 18 selected wastewater reuse specifications revealed that at best these guidelines are only partially applicable to innovative recycling systems as they are focused on the downcycling of water to the environment (e.g., use for irrigation). We believe that a paradigm shift is necessary and advocate for the implementation of risk-based (and thus end-use dependent) system performance targets to evaluate water treatment systems, which recycle and not only downcycle water.

Die Beprobung von Gebäude-Trinkwasserinstallationen auf Legionellen ist komplex und stellt hohe Anforderungen an die Probenahme-Verantwortlichen. Wird die Beprobung nicht fachgerecht umgesetzt, kann dies zu einer falschen Einschätzung der Gefährdungssituation und in der Folge zu Krankheitsfällen führen. Zudem können unnötige Kosten aufgrund wenig zielführender Massnahmen generiert werden. Im Rahmen zweier Forschungsprojekte wurde deshalb ein Gesamtkonzept für die Probenahme auf Legionellen ausgearbeitet, einschliesslich verschiedener unterstützender Anleitungen, Entscheidungs- und  Dokumentationsvorlagen.

Microorganisms perform multiple metabolic functions that shape the global cycling of elements, health and disease of their host organisms, and biotechnological processes. The rates, at which different metabolic activities are performed by individual cells, can vary between genetically identical cells within clonal populations. While the molecular mechanisms that result in such metabolic heterogeneity have attracted considerable interest, the environmental conditions that shape heterogeneity and its consequences have received attention only in recent years. Here, we review the environmental drivers that lead to metabolic heterogeneity with a focus on nutrient limitation, temporal fluctuations and spatial structure, and the functional consequences of such heterogeneity. We highlight studies using single-cell methods that allow direct investigation of metabolic heterogeneity and discuss the relevance of metabolic heterogeneity in complex microbial communities.

Background: The increase in antimicrobial resistance is of worldwide concern. Surrogate tracers attempt to simulate microbial transmission by avoiding the infectious risks associated with live organisms. We evaluated silica nanoparticles with encapsulated DNA (SPED) as a new promising surrogate tracer in healthcare.
Methods: SPED and Escherichia coli were used to implement three experiments in simulation rooms and a microbiology laboratory in 2017-2018. Experiment 1 investigated the transmission behaviour of SPED in a predefined simulated patient-care scenario. SPED marked with 3 different DNA sequences (SPED1-SPED3) were introduced at 3 different points of the consecutive 13 touch sites of a patient-care scenario that was repeated 3 times, resulting in a total of 288 values. Experiment 2 evaluated SPED behaviour following hand cleaning with water and soap and alcohol-based handrub. Experiment 3 compared transfer dynamics of SPED versus E. coli in a laboratory using a gloved finger touching two consecutive sites on a laminate surface after a first purposefully contaminated site.
Results: Experiment 1: SPED adhesiveness on bare skin after a hand-to-surface exposure was high, leading to a dissemination of SPED1-3 on all consecutive surface materials with a trend of decreasing recovery rates, also reflecting touching patterns in concordance with contaminated fingers versus palms. Experiment 2: Hand washing with soap and water resulted in a SPED reduction of 96%, whereas hand disinfection led to dispersal of SPED from the palm to the back of the hand. Experiment 3: SPED and E. coli concentration decreased in parallel with each transmission step - with SPED showing a trend for less reduction and variability.
Conclusions: SPED represent a convenient and safe instrument to simulate pathogen spread by contact transmission simultaneously from an infinite number of sites. They can be further developed as a central asset for successful infection prevention in healthcare.

Bacteria can resist antibiotics by expressing enzymes that remove or deactivate drug molecules. Here, we study the effects of gene expression stochasticity on efflux and enzymatic resistance. We construct an agent-based model that stochastically simulates multiple biochemical processes in the cell and we observe the growth and survival dynamics of the cell population. Resistance-enhancing mutations are introduced by varying parameters that control the enzyme expression or efficacy. We find that stochastic gene expression can cause complex dynamics in terms of survival and extinction for these mutants. Regulatory mutations, which augment the frequency and duration of resistance gene transcription, can provide limited resistance by increasing mean expression. Structural mutations, which modify the enzyme or efflux efficacy, provide most resistance by improving the binding affinity of the resistance protein to the antibiotic; increasing the enzyme's catalytic rate alone may contribute to resistance if drug binding is not rate limiting. Overall, we identify conditions where regulatory mutations are selected over structural mutations, and vice versa. Our findings show that stochastic gene expression is a key factor underlying efflux and enzymatic resistances and should be taken into consideration in future antibiotic research.

Increasing incidence of antibiotic resistance in clinical and environmental settings calls for increased scalability in their surveillance. Current screening technologies are limited by the number of samples and genes that can easily be screened. We demonstrate here digital multiplex ligation assay (dMLA) as a low-cost targeted genomic detection workflow capable of highly-parallel screening of bacterial isolates for multiple target gene regions simultaneously. Here, dMLA is used for simultaneous detection of 1187 β-lactamase-encoding genes, including extended spectrum β-lactamase (ESBL) genes, in 74 bacterial isolates. We demonstrate dMLA as a light-weight and cost-efficient workflow which provides a highly scalable tool for antimicrobial resistance surveillance and is also adaptable to genetic screening applications beyond antibiotic resistance.

Enteric viruses, such as enterovirus, norovirus, and rotavirus are among the leading causes of disease outbreaks due to contaminated drinking and recreational water. Viruses are difficult to remove from water through filtration based on physical size exclusion - for example by gravity driven filters - due to their nanoscale size. To understand virus removal in drinking water treatments systems, the colloidal nanostructure of a model virus, the MS2 bacteriophage, has been investigated in relation to the effect of pH and natural organic matter in water. Dynamic light scattering, small angle X-ray scattering and cryogenic transmission electron microscopy demonstrated that the water pH has a major influence on the colloidal structure of the virus: The bacteriophage MS2’s structure in water in the range pH = 7.0 to 9.0 was found to be spherical with core-shell type structure with a total diameter of 27 nm and a core radius of around 8 nm. Reversible transformations from 27 nm particles at pH = 7.0 to micrometer sized aggregate at pH = 3.0 were observed. In addition, the presence of natural organic matter that simulates the organic components present in surface water was found to enhance repulsion between virus particles, reduce size of aggregates and promote disaggregation upon pH increase. These findings allow a better understanding of virus interactions in water and have implications for water treatment using filtration processes and coagulation. The results will further guide the comprehensive design of advanced virus filter materials.

Current microbial exposure models assume that microbial exchange follows a concentration gradient during hand-to-surface contacts. Our objectives were to evaluate this assumption using transfer efficiency experiments and to evaluate a model's ability to explain concentration changes using approximate Bayesian computation (ABC) on these experimental data. Experiments were conducted with two phages (MS2, ΦX174) simultaneously to study bidirectional transfer. Concentrations on the fingertip and surface were quantified before and after fingertip-to-surface contacts. Prior distributions for surface and fingertip swabbing efficiencies and transfer efficiency were used to estimate concentrations on the fingertip and surface post contact. To inform posterior distributions, Euclidean distances were calculated for predicted detectable concentrations (log₁₀ PFU cm^-2) on the fingertip and surface post contact in comparison with experimental values. To demonstrate the usefulness of posterior distributions in calibrated model applications, posterior transfer efficiencies were used to estimate rotavirus infection risks for a fingertip-to-surface and subsequent fingertip-to-mouth contact. Experimental findings supported the transfer gradient assumption. Through ABC, the model explained concentration changes more consistently when concentrations on the fingertip and surface were similar. Future studies evaluating microbial transfer should consider accounting for differing fingertip-to-surface and surface-to-fingertip transfer efficiencies and extend this work for other microbial types.

Slow ecological cycles, resulting in the recurrent dispersal of cells between resource patches, can drive the evolution of collectivity.

Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

Human exposure to pathogenic viruses in environmental waters results in a significant global disease burden. Current microbial water quality monitoring approaches, mainly based on fecal indicator bacteria, insufficiently capture human health impacts posed by pathogenic viruses in water. The emergence of the 'microbiome era' and high-throughput metagenome sequencing has led to the discovery of novel human-associated viruses, including both pathogenic and commensal viruses in the human microbiome. The discovery of novel human-associated viruses is often followed by their detection in wastewater, highlighting the great diversity of human-associated viruses potentially present in the water environment. Novel human-associated viruses provide a rich reservoir to develop viral water quality management tools with diverse applications, such as regulating wastewater reuse and monitoring agricultural and recreational waters. Here, we review the pathway from viral discovery to water quality monitoring tool, and highlight select human-associated viruses identified by metagenomics and subsequently detected in the water environment (namely Bocavirus, Cosavirus, CrAssphage, Klassevirus, and Pepper Mild Mottle Virus). We also discuss research needs to enable the application of recently discovered human-associated viruses in water quality monitoring, including investigating the geographic distribution, environmental fate, and viability of potential indicator viruses. Examples suggest that recently discovered human pathogens are likely to be less abundant in sewage, while other human-associated viruses (e.g., bacteriophages or viruses from food) are more abundant but less human-specific. The improved resolution of human-associated viral diversity enabled by metagenomic tools provides a significant opportunity for improved viral water quality management tools.

Container-based sanitation (CBS) within a comprehensive service system value chain offers a low-cost sanitation option with potential for revenue generation but may increase microbial health risks to sanitation service workers. This study assessed occupational exposure to rotavirus and Shigella spp. during CBS urine collection and subsequent struvite fertilizer production in eThekwini, South Africa. Primary data included high resolution sequences of hand-object contacts from annotated video and measurement of fecal contamination from urine and surfaces likely to be contacted. A stochastic model incorporated chronological surface contacts, pathogen concentrations in urine, and literature data on transfer efficiencies of pathogens to model pathogen concentrations on hands and risk of infection from hand-to-mouth contacts. The probability of infection was highest from exposure to rotavirus during urine collection (∼10^–1) and struvite production (∼10^–2), though risks from Shigella spp. during urine collection (∼10^–3) and struvite production (∼10^–4) were non-negligible. Notably, risk of infection was higher during urine collection than during struvite production due to contact with contaminated urine transport containers. In the scale-up of CBS, disinfection of urine transport containers is expected to reduce pathogen transmission. Exposure data from this study can be used to evaluate the effectiveness of measures to protect sanitation service workers.

Bacterial populations have been shown to accumulate deleterious mutations during spatial expansions that overall decrease their fitness and ability to grow. However, it is unclear if and how they can respond to selection in face of this mutation load. We examine here if artificial selection can counteract the negative effects of range expansions. We examined the molecular evolution of 20 mutator lines selected for fast expansions (SEL) and compared them to 20 other mutator lines freely expanding without artificial selection (CONTROL). We find that the colony size of all 20 SEL lines have increased relative to the ancestral lines, unlike CONTROL lines, showing that enough beneficial mutations are produced during spatial expansions to counteract the negative effect of expansion load. Importantly, SEL and CONTROL lines have similar numbers of mutations indicating that they evolved for the same number of generations and that increased fitness is not due to a purging of deleterious mutations. We find that loss of function mutations better explain the increased colony size of SEL lines than nonsynonymous mutations or a combination of the two. Interestingly, most loss of function mutations are found in simple sequence repeats (SSRs) located in genes involved in gene regulation and gene expression. We postulate that such potentially reversible mutations could play a major role in the rapid adaptation of bacteria to changing environmental conditions by shutting down expensive genes and adjusting gene expression.

In some species of myxobacteria, adjacent cells sufficiently similar at the adhesin protein TraA can exchange components of their outer membranes. The primary benefits of such outer membrane exchange (OME) in natural populations are unclear, but in some OME interactions, transferred OM content can include SitA toxins that kill OME participants lacking an appropriate immunity gene. Such OME-dependent toxin transfer across Myxococcus xanthus strains that differ only in their sitBAI toxin/antitoxin cassette can mediate inter-strain killing and generate colony-merger incompatibilities (CMIs)–inter-colony border phenotypes between distinct genotypes that differ from respective self-self colony interfaces. Here we ask whether OME-dependent toxin transfer is a common cause of prevalent CMIs and antagonisms between M. xanthus natural isolates identical at TraA. We disrupted traA in eleven isolates from a cm-scale soil population and assayed whether traA disruption eliminated or reduced CMIs between swarming colonies or antagonisms between strains in mixed cultures. Among 33 isolate pairs identical at traA that form clear CMIs, in no case did functional disruption of traA in one partner detectably alter CMI phenotypes. Further, traA disruption did not alleviate strong antagonisms observed during starvation-induced fruiting-body development in seven pairs of strains identical at traA. Collectively, our results suggest that most mechanisms of interference competition and inter-colony kin discrimination in natural populations of myxobacteria do not require OME. Finally, our experiments also indicate that several closely related laboratory reference strains kill some natural isolates by toxins delivered by a shared, OME-independent type VI secretion system (T6SS), suggesting that some antagonisms between sympatric natural isolates may also involve T6SS toxins.

Organized by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single cell analysis. It is an annual assembly of cytometry experts as well as novices in this field—all having one common denominator—passion for cytometry. This meeting is not only unique possibility to learn new, exciting cutting‐edge research directly from the source by attending the Frontiers and the State‐Of‐The‐Art lectures, research, and technology sessions, or Tutorials.
CYTO conferences are also the best possible live forum to discuss current and emerging challenges in cytometry associated sciences at the utmost interactive CYTO formats—the Workshops. The Workshops attract a lot of attention within and outside the cytometry community as they generally discuss where we stand in specific fields of interest, how problems can be addressed and solved, and what are the future development areas and demands. Due to limited time frame allocated to this format; however, the workshops are run in parallel so sometimes attendees cannot participate in all the workshops of their interest. Moreover, those who could have not join the CYTO conference miss the content entirely. Therefore, we decided to summarize all the workshops to disseminate the information provided and the state‐of‐the art view of the community. Thus, the main reason for creating such combined summary of reports was to keep the record of the live discussions and main conclusions as well as to propose eventual guidelines and make them available to the broader public.
This manuscript serves as a summary report of 16 workshops (WS) that were held at CYTO2018 in Prague. We present in concise form the current and the future challenges in cytometry identified by workshop organizers. The manuscript is divided into four topic chapters: Trends, Shared Resource Laboratory (SRL) Best Practices, Quality Assurance and Reproducibility, and Technology. Each chapter is a summary of four workshops. We intend to serve with this joint workshop report the global community involved in single cell analysis and cytometry.

Bacteria often live in spatially structured groups such as biofilms. In these groups, cells can collectively generate gradients through the uptake and release of compounds. In turn, individual cells adapt their activities to the environment shaped by the whole group. Here, we studied how these processes can generate phenotypic variation in clonal populations and how this variation contributes to the resilience of the population to antibiotics. We grew two-dimensional populations of Escherichia coli in microfluidic chambers where limiting amounts of glucose were supplied from one side. We found that the collective metabolic activity of cells created microscale gradients where nutrient concentration varied over a few cell lengths. As a result, growth rates and gene expression levels varied strongly between neighbouring cells. Furthermore, we found evidence for a metabolic cross-feeding interaction between glucose-fermenting and acetate-respiring subpopulations. Finally, we found that subpopulations of cells were able to survive an antibiotic pulse that was lethal in well-mixed conditions, likely due to the presence of a slow-growing subpopulation. Our work shows that emergent metabolic gradients can have important consequences for the functionality of bacterial populations as they create opportunities for metabolic interactions and increase the populations' tolerance to environmental stressors.
This article is part of a discussion meeting issue 'Single cell ecology'.

Microbes live in ever-changing environments where they need to adapt their metabolism to different nutrient conditions. Many studies have characterized the response of genetically identical cells to nutrient switches in homogeneous cultures; however, in nature, microbes often live in spatially structured groups such as biofilms where cells can create metabolic gradients by consuming and releasing nutrients. Consequently, cells experience different local microenvironments and vary in their phenotype. How does this phenotypic variation affect the ability of cells to cope with nutrient switches? Here, we address this question by growing dense populations of Escherichia coli in microfluidic chambers and studying a switch from glucose to acetate at the single-cell level. Before the switch, cells vary in their metabolic activity: some grow on glucose, while others cross-feed on acetate. After the switch, only few cells can resume growth after a period of lag. The probability to resume growth depends on a cells' phenotype prior to the switch: it is highest for cells cross-feeding on acetate, while it depends in a non-monotonic way on the growth rate for cells growing on glucose. Our results suggest that the strong phenotypic variation in spatially structured populations might enhance their ability to cope with fluctuating environments.

Biodegradable polyesters are being increasingly used to replace conventional, nondegradable polymers in agricultural applications such as plastic film for mulching. For many of these applications, poly(butylene adipate-co-terephthalate) (PBAT) is a promising biodegradable material. However, PBAT is also susceptible to photochemical transformations. To better understand how photochemistry affects the biodegradability of PBAT, we irradiated blown, nonstabilized, transparent PBAT films and studied their enzymatic hydrolysis, which is considered the rate-limiting step in polyester biodegradation. In parallel, we characterized the irradiated PBAT films by dynamic mechanical thermal analysis. The rate of enzymatic PBAT hydrolysis decreased when the density of light-induced cross-links within PBAT exceeded a certain threshold. Mass-spectrometric analysis of the enzymatic hydrolysis products of irradiated PBAT films provided evidence for radical-based cross-linking of two terephthalate units that resulted in the formation of benzophenone-like molecules. In a proof-of-principle experiment, we demonstrated that the addition of photostabilizers to PBAT films mitigated the negative effect of UV irradiation on the enzymatic hydrolyzability of PBAT. This work advances the understanding of light-induced changes on the enzyme-mediated hydrolysis of aliphatic-aromatic polyesters and will therefore have important implications for the development of biodegradable plastics.

Treatment failure in biofilm-associated bacterial infections is an important healthcare issue. In vitro studies and mouse models suggest that bacteria enter a slow-growing/non-growing state that results in transient tolerance to antibiotics in the absence of a specific resistance mechanism. However, little clinical confirmation of antibiotic tolerant bacteria in patients exists. In this study we investigate a Staphylococcus epidermidis pacemaker-associated endocarditis, in a patient who developed a break-through bacteremia despite taking antibiotics to which the S. epidermidis isolate is fully susceptible in vitro. Characterization of the clinical S. epidermidis isolates reveals in-host evolution over the 16-week infection period, resulting in increased antibiotic tolerance of the entire population due to a prolonged lag time until growth resumption and a reduced growth rate. Furthermore, we observe adaptation towards an increased biofilm formation capacity and genetic diversification of the S. epidermidis isolates within the patient.

Direct testing of water quality to promote drinking water safety contributes to the sustainable development goals, which call for universal access to safely-managed drinking water services by 2030. Enzyme–substrate tests offer a potentially simple and reliable approach for the detection and quantification of fecal indicator bacteria, including Escherichia coli (E. coli). The novel aquatest (AT) based on resorufin-β-D-glucuronide methyl ester (RUG™) (AT-RUG) is an enzyme–substrate test that overcomes several drawbacks of other established tests. In this study, AT-RUG was used to detect and quantify E. coli in water from temperate (Zurich, Switzerland) and tropical (Bushenyi, Uganda) regions. Quantitative results of AT-RUG were compared with IDEXX Colilert-18® (C-18), m-TEC and m-ColiBlue24®. In temperate waters, AT-RUG was found to be as sensitive as m-TEC (97.0%) and C-18 (98.5%) and showed strong agreement with the reference methods. The false-positive rate for E. coli detection in temperate waters using AT-RUG was 6%. AT-RUG performed well at incubation temperatures of 37 °C and 45 °C, but not at 24 °C. In tropical waters, AT-RUG sensitivity was 94.1% compared to m-ColiBlue24®. AT-RUG detected significantly more E. coli than m-ColiBlue24®, suggesting it is a more conservative estimate. At both field sites, AT-RUG was able to effectively indicate categorical concentrations of E. coli in water samples indicating the level of risks of fecal contamination of water supplies. This study indicates that AT-RUG is a reliable and accurate medium for the detection and quantification of E. coli in temperate and tropical waters.

Leptospirosis is a widespread and potentially life-threatening zoonotic disease caused by spirochaetes of the genus Leptospira. Humans become infected primarily via contact with environmental reservoirs contaminated by the urine of shedding mammalian hosts. Populations in high transmission settings, such as urban slums and subsistence farming communities, are exposed to low doses of Leptospira on a daily basis. Under these conditions, numerous factors determine whether infection occurs, including the route of exposure and inoculum dose. Skin wounds and abrasions are risk factors for leptospirosis, but it is not known whether broken skin is necessary for spillover, or if low-dose exposures to intact skin and mucous membranes can also cause infection. To establish a quantitative relationship between dose, route and probability of infection, we performed challenge experiments in hamsters and rats, developed mechanistic dose–response models representing the spatial dynamics of within-host infection and persistence, and fitted models to experimental data. Results show intact skin is a strong barrier against infection, and that broken skin is the predominant route by which low-dose environmental exposures cause infection. These results identify skin integrity as a bottleneck to spillover of Leptospira and underscore the importance of barrier interventions in the prevention of leptospirosis.
This article is part of the theme issue ‘Dynamic and integrative approaches to understanding pathogen spillover’.

Conventional biofiltration refers to filtration processes where the filter comprises porous material that is colonized by indigenous microbial communities and where these microorganisms perform at least one of the essential functions of the filtration process. Biofilters such as granular active carbon (GAC) filters, rapid sand filters (RSFs), and slow sand filters (SSFs) are key processes in the biotreatment of drinking water in both industrialized and developing countries. The main purpose of biofiltration is the removal of biodegradable organic carbon, but a wide variety of possible filter configurations, together with a broad range of different water types and pretreatment methods, contribute to extremely diverse biofiltration performance results reported in literature. The biological component of all biofilters comprises indigenous bacteria that proliferate in biofilms at low nutrient concentrations. This chapter specifically focuses on the state of the art of engineered biofiltration systems (e.g., GAC, RSF, SSF) and investigates the key differences between these filter types from a microbiological perspective. Different methodologies for the analysis of organic carbon, particularly the bioavailable fractions, as well as for the analysis of biomass on granulated materials are discussed and compared. The final section highlights open questions and future research opportunities of this field.

Short-chain chlorinated paraffins (SCCPs) are polyhalogenated hydrocarbons as are hexachlorocyclohexanes (HCHs) and hexabromocyclododecanes (HBCDs). They all have been classified as persistent organic pollutants (POPs) under the UN Stockholm Convention. Per se such compounds are transformed slowly in the environment, transported over long distances and accumulate in biota. Several Sphingomonadacea strains isolated from HCH dump sites have evolved to express enzymes that can transform HCHs and HBCDs. We hypothesized that LinA2, a dehydrohalogenase expressed in such bacteria, may also transform CPs to chlorinated olefins (COs). Three mixtures of penta- to deca-chlorinated undecanes (C₁₁), dodecanes (C₁₂) and tridecanes (C₁₃) were exposed to LinA2. High-resolution full-scan mass spectra (R~8'000) of CPs and COs were obtained applying a soft ionization method, enhancing chloride-adduct [M+Cl]^- formation. A mathematical deconvolution procedure was used to separate interfering spectra to verify that LinA2 indeed catalyzed the conversion of CPs to COs. About 20–40% of the material was transformed in 24 h, about 50–70% was converted in 200 h. A bimodal first-order kinetic model could describe transformations of reactive and persistent CPs. Under the given conditions reactive CPs (τ_1/2 = 1.4–6.9 h) were converted 30 to 190-times faster than the persistent ones (τ_1/2 = 150–260 h). Proportions of persistent isomers (p_p) varied from 60 to 80%. Lower chlorinated homologues contained higher proportions of persistent isomers. In conclusion, SCCP mixtures contain both, material that is readily converted by LinA2, and persistent material that is not or only slowly transformed.

The transition from land to water in whales and dolphins (cetaceans) was accompanied by remarkable adaptations. To reveal genomic changes that occurred during this transition, we screened for protein-coding genes that were inactivated in the ancestral cetacean lineage. We found 85 gene losses. Some of these were likely beneficial for cetaceans, for example, by reducing the risk of thrombus formation during diving (F12 and KLKB1), erroneous DNA damage repair (POLM), and oxidative stress-induced lung inflammation (MAP3K19). Additional gene losses may reflect other diving-related adaptations, such as enhanced vasoconstriction during the diving response (mediated by SLC6A18) and altered pulmonary surfactant composition (SEC14L3), while loss of SLC4A9 relates to a reduced need for saliva. Last, loss of melatonin synthesis and receptor genes (AANAT, ASMT, and MTNR1A/B) may have been a precondition for adopting unihemispheric sleep. Our findings suggest that some genes lost in ancestral cetaceans were likely involved in adapting to a fully aquatic lifestyle.

Third generation cephalosporins (3GC) are one of the main choices for treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Due to their overuse, an increasing trend of resistance to 3GC has been observed in developing countries. Here, we describe fecal colonization of 3GC-resistant (3GCr) Escherichia coli in healthy infants (1–12 months old) living in rural areas of Bangladesh. We found that stool samples of 82% of infants (n = 100) were positive for 3GCr E. coli with a mean ± standard deviation of 6.21 ± 1.32 log₁₀ CFU/g wet weight of stool. 3GCr E. coli encompasses an average one third (33%) of the total E. coli of stool. Almost 77% (n = 63) of these 3GCr E. coli were MDR (or resistant to ≥3 classes of antibiotics). Around 90% (n = 74) of 3GCr E. coli were extended spectrum beta-lactamase (ESBL)-producing in which bla_{CTX–M–group–1} was the predominant (96%, n = 71) ESBL-gene followed by bla_TEM (41%, n = 30) and bla_OXA–1 (11%, n = 8). A significant proportion (26.5%, n = 22) of 3GCr E. coli was pathogenic, comprising two types, enteroaggregative (EAEC, n = 19) and enteropathogenic (EPEC, n = 3). Colonization of 3GCr E. coli in infant guts was not associated with demographic characteristics such as age, sex, mode of delivery, maternal and infant antibiotic use, disease morbidity, and feeding practices. The high rate of colonization of 3GCr E. coli in infants’ guts is a serious public health concern which needs immediate attention and warrants further studies to explore the cause.

Wastewater treatment plants (WWTPs) are implicated as hotspots for the dissemination of antibacterial resistance into the environment. However, the in situ processes governing removal, persistence, and evolution of resistance genes during wastewater treatment remain poorly understood. Here, we used quantitative metagenomic and metatranscriptomic approaches to achieve a broad-spectrum view of the flow and expression of genes related to antibacterial resistance to over 20 classes of antibiotics, 65 biocides, and 22 metals. All compartments of 12 WWTPs share persistent resistance genes with detectable transcriptional activities that were comparatively higher in the secondary effluent, where mobility genes also show higher relative abundance and expression ratios. The richness and abundance of resistance genes vary greatly across metagenomes from different treatment compartments, and their relative and absolute abundances correlate with bacterial community composition and biomass concentration. No strong drivers of resistome composition could be identified among the chemical stressors analyzed, although the sub-inhibitory concentration (hundreds of ng/L) of macrolide antibiotics in wastewater correlates with macrolide and vancomycin resistance genes. Contig-based analysis shows considerable co-localization between resistance and mobility genes and implies a history of substantial horizontal resistance transfer involving human bacterial pathogens. Based on these findings, we propose future inclusion of mobility incidence (M%) and host pathogenicity of antibiotic resistance genes in their quantitative health risk ranking models with an ultimate goal to assess the biological significance of wastewater resistomes with regard to disease control in humans or domestic livestock.

Many microorganisms secrete molecules that interact with resources outside of the cell. This includes, for example, enzymes that degrade polymers like chitin, and chelators that bind trace metals like iron. In contrast to direct uptake via the cell surface, such release strategies entail the risk of losing the secreted molecules to environmental sinks, including ‘cheating’ genotypes. Nevertheless, such secretion strategies are widespread, even in the well-mixed marine environment. Here, we investigate the benefits of a release strategy whose efficiency has frequently been questioned: iron uptake in the ocean by secretion of iron chelators called siderophores. We asked the question whether the release itself is essential for the function of siderophores, which could explain why this risky release strategy is widespread. We developed a reaction–diffusion model to determine the impact of siderophore release on iron uptake from the predominant iron sources in marine environments, colloidal or particulate iron, formed due to poor iron solubility. We found that release of siderophores is essential to accelerate iron uptake, as secreted siderophores transform slowly diffusing large iron particles to small, quickly diffusing iron–siderophore complexes. In addition, we found that cells can synergistically share their siderophores, depending on their distance and the size of the iron sources. Our study helps understand why release of siderophores is so widespread: even though a large fraction of siderophores is lost, the solubilization of iron through secreted siderophores can efficiently increase iron uptake, especially if siderophores are produced cooperatively by several cells. Overall, resource uptake mediated via release of molecules transforming their substrate could be essential to overcome diffusion limitation specifically in the cases of large, aggregated resources. In addition, we find that including the reaction of the released molecule with the substrate can impact the result of cooperative and competitive interactions, making our model also relevant for release-based uptake of other substrates.

Background: Substrate cross-feeding occurs when one organism partially consumes a primary substrate into one or more metabolites while other organisms then consume the metabolites. While pervasive within microbial communities, our knowledge about the effects of substrate cross-feeding on microbial evolution remains limited. To address this knowledge gap, we experimentally evolved isogenic nitrite (NO₂⁻) cross-feeding microbial strains together for 700 generations, identified genetic changes that were acquired over the evolution experiment, and compared the results with an isogenic completely denitrifying strain that was evolved alone for 700 generations. We further investigated how the magnitude of interdependence between the nitrite cross-feeding strains affects the main outcomes. Our main objective was to quantify how substrate cross-feeding and the magnitude of interdependence affect the speed and trajectory of molecular evolution.
Results: We found that each nitrite (NO₂⁻) cross-feeding strain acquired fewer genetic changes than did the completely denitrifying strain. In contrast, pairs of nitrite cross-feeding strains together acquired more genetic changes than did the completely denitrifying strain. Moreover, nitrite cross-feeding promoted population diversification, as pairs of nitrite cross-feeding strains acquired a more varied set of genetic changes than did the completely denitrifying strain. These outcomes likely occurred because nitrite cross-feeding enabled the co-existence of two distinct microbial strains, thus increasing the amount of genetic variation for selection to act upon. Finally, the nitrite cross-feeding strains acquired different types of genetic changes than did the completely denitrifying strain, indicating that nitrite cross-feeding modulates the trajectory of molecular evolution.
Conclusions: Our results demonstrate that substrate cross-feeding can affect both the speed and trajectory of molecular evolution within microbial populations. Substrate cross-feeding can therefore have potentially important effects on the life histories of microorganisms.

The influence that coincidental carbon substrates (i.e., assimilable organic carbon, AOC) and indigenous microbial communities has on pesticide biotransformation by degrader strains in aquatic environments is poorly understood. We conducted batch experiments to investigate carbofuran biotransformation by Novosphingobium sp. KN65.2 using four environmentally derived water samples with varying amounts and types of AOC and indigenous microbial communities. We designed experimental scenarios to explore the influence of AOC and indigenous microbial communities on the growth of strain KN65.2 and the biotransformation of carbofuran. Relevant kinetic parameters were estimated from simpler experiments, and used to predict the growth of strain KN65.2 and the biotransformation of carbofuran in more complex experiments with an additive biokinetic model. We found that an additive biokinetic model adequately predicts the growth of strain KN65.2 and the rate of carbofuran biotransformation in natural waters that support the growth of strain KN65.2. However, our model over-predicts the growth of strain KN65.2 and the rate of carbofuran biotransformation in low-AOC environments. Overall, our results define the scope within which additive biokinetic models can be used to predict pesticide biotransformation in the presence of coincidental carbon substrates and indigenous microbial communities.

A remaining challenge within microbial ecology is to understand the determinants of richness and diversity observed in environmental microbial communities. In a range of systems, including activated sludge bioreactors, the microbial residence time (MRT) has been previously shown to shape the microbial community composition. However, the physiological and ecological mechanisms driving this influence have remained unclear. Here, this relationship is explored by analyzing an activated sludge system fed with municipal wastewater. Using a model designed in this study based on Monod-growth kinetics, longer MRTs were shown to increase the range of growth parameters that enable persistence, resulting in increased richness and diversity in the modeled community. In laboratory experiments, six sequencing batch reactors treating domestic wastewater were operated in parallel at MRTs between 1 and 15 days. The communities were characterized using both 16S ribosomal RNA and non-target messenger RNA sequencing (metatranscriptomic analysis), and model-predicted monotonic increases in richness were confirmed in both profiles. Accordingly, taxonomic Shannon diversity also increased with MRT. In contrast, the diversity in enzyme class annotations resulting from the metatranscriptomic analysis displayed a non-monotonic trend over the MRT gradient. Disproportionately high abundances of transcripts encoding for rarer enzymes occur at longer MRTs and lead to the disconnect between taxonomic and functional diversity profiles.

Microbial colonies are fascinating structures in which growth and internal organization reflect complex morphogenetic processes. Here, we generated a microfluidics device with arrays of long monolayer yeast colonies to further global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined boundary conditions. We observed the emergence of stable glucose gradients using fluorescently labeled hexose transporters and quantified the spatial correlations with intra-colony growth rates and expression of other genes regulated by glucose availability. These landscapes depended on the external glucose concentration as well as secondary gradients, for example amino acid availability. This work demonstrates the regulatory genetic networks governing cellular physiological adaptation are the key to internal structuration of cellular assemblies. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development and morphogenesis in more complex systems.

Cells are often considered input-output devices that maximize the transmission of information by converting extracellular stimuli (input) via signaling pathways (communication channel) to cell behavior (output). However, in biological systems outputs might feed back into inputs due to cell motility, and the biological channel can change by mutations during evolution. Here, we show that the conventional channel capacity obtained by optimizing the input distribution for a fixed channel may not reflect the global optimum. In a new approach we analytically identify both input distributions and input-output curves that optimally transmit information, given constraints from noise and the dynamic range of the channel. We find a universal optimal input distribution only depending on the input noise, and we generalize our formalism to multiple outputs (or inputs). Applying our formalism to Escherichia coli chemotaxis, we find that its pathway is compatible with optimal information transmission despite the ultrasensitive rotary motors.

Soil biodegradable mulch films composed of the polyester polybutylene adipate-co-terephthalate (PBAT) are being increasingly used in agriculture. Analytical methods to quantify PBAT in field soils are needed to assess its soil occurrence and fate. Here, we report an analytical method for PBAT in soils that couples Soxhlet extraction or accelerated solvent extraction (ASE) with quantitative protonnuclear magnetic resonance (q-¹H NMR) spectroscopy detection. The ¹H NMR peak areas of aromatic PBAT protons increased linearly with PBAT concentrations dissolved in deuterated chloroform (CDCl₃), demonstrating accurate quantitation of PBAT by q-¹H NMR. Spike-recovery experiments involving PBAT addition to model sorbents and soils showed increased PBAT extraction efficiencies into chloroform (CHCl₃) with methanol (MeOH) as cosolvent, consistent with MeOH competitively displacing PBAT from H-bond donating sites on mineral surfaces. Systematic variations in solvent composition and temperatures in ASE revealed quantitative PBAT extraction from soil with 90/10 volume % CHCl₃/MeOH at 110-120 °C. Both Soxhlet extraction and ASE resulted in the complete recovery of PBAT added to a total of seven agricultural soils covering a range of physicochemical properties, independent of whether PBAT was added to soils dissolved in CHCl₃, as film, or as particles. Recovery was also complete for PBAT added to soil in the form of a commercial soil biodegradable mulch film with coextractable polylactic acid (PLA). The presented analytical method enables accurate quantification and biodegradation monitoring of PBAT in agricultural field soils.

Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling (200 individual biofilm sections per hose) on varying scales (μm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real (i.e., uncontrolled) use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with <400 taxa each, and only three taxa accounting for 57%, respectively, 73% of the community. This low diversity was attributed to a strong selective pressure, originating in migrating carbon from the flexible hoses as major carbon source. High-resolution sampling strategy enabled detailed analysis of spatial heterogeneity within an individual drinking water biofilm. This study gives insight into biofilm structure and community composition on cm-to m-scale and is useful for decision-making on sampling strategies in biofilm research and monitoring.

In many low-income countries, the poor conditions of sanitation systems have been a significant cause of mortality since they accelerate waterborne disease transmission. Developing sanitation systems in these countries is a pressing concern in both the public and private sectors.
This research investigated a decentralized domestic wastewater treatment system using ultraviolet light-emitting diodes (UV-LEDs). Although UV-LED disinfection has become more widespread in recent years, it is a novel approach for domestic wastewater treatment. Domestic wastewater was pretreated by a low-cost pretreatment system with an inclined settler and a sand filter prior to feeding a novel flowthrough UV LED reactor. At an inlet flow rate of 30 L/h, the COD, TSS, and turbidity of the effluent were 17.7 mg/L, 3.0 mg/L, and 3.9 NTU, respectively. UV transmittance at 285 nm was enhanced from 29.1% to 70.4%, improving the influent quality for UV LED disinfection.
The flow-through UV LED reactor was operated at various flow rates from 10 to 50 mL/min, resulting in applied UV doses of 69.4 to 47.8 mJ/cm² respectively. These doses are sufficient for inactivating total coliforms in the wastewater to meet the water reuse guidelines for agriculture for both processed food crops and non-food crops.
Fouling, which was observed starting at 2 d of operation, decreased the disinfection efficacy to 27% after 25 days of continuous operation. Of the fouling layer, 67% was attributed to organic matter, in contrast to previous fouling studies with mercury UV lamps in which the fouling layer consisted primarily of inorganic compounds. The fouling was reversed by off-line citric acid cleaning for 4 h after every 400 h of continuous operation.

2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobacter sp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/β hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/β hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG.

Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing β-1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic.

Species interactions change when the external conditions change. How these changes affect microbial community properties is an open question. We address this question using a two-species consortium in which species interactions change from exploitation to competition depending on the carbon source provided. We built a mathematical model and calibrated it using single-species growth measurements. This model predicted that low frequencies of change between carbon sources lead to species loss, while intermediate and high frequencies of change maintained both species. We experimentally confirmed these predictions by growing co-cultures in fluctuating environments. These findings complement more established concepts of a diversity peak at intermediate disturbance frequencies. They also provide a mechanistic understanding for how the dynamics at the community level emerges from single-species behaviours and interspecific interactions. Our findings suggest that changes in species interactions can profoundly impact the ecological dynamics and properties of microbial systems.

To assess potential risks posed by plastic nanoparticles, we must study the way in which they transfer and transform in the environment. Using ¹³C-labelled nanoplastics could provide a safe and effective way to establish whether the plastic is mineralized or whether it persists in the environment.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 °C - 39 °C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 °C and 27 °C), and at high temperatures (39 °C) when the incubator set temperature was higher. This incubator prototype is low-cost (< 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

How unicellular organisms optimize the production of compounds is a fundamental biological question. While it is typically thought that production is optimized at the individual-cell level, secreted compounds could also allow for optimization at the group level, leading to a division of labor where a subset of cells produces and shares the compound with everyone. Using mathematical modeling, we show that the evolution of such division of labor depends on the cost function of compound production. Specifically, for any trait with saturating benefits, linear costs promote the evolution of uniform production levels across cells. Conversely, production costs that diminish with higher output levels favor the evolution of specialization–especially when compound shareability is high. When experimentally testing these predictions with pyoverdine, a secreted iron-scavenging compound produced by Pseudomonas aeruginosa, we found linear costs and, consistent with our model, detected uniform pyoverdine production levels across cells. We conclude that for shared compounds with saturating benefits, the evolution of division of labor is facilitated by a diminishing cost function. More generally, we note that shifts in the level of selection from individuals to groups do not solely require cooperation, but critically depend on mechanistic factors, including the distribution of compound synthesis costs.

Contamination of soils and sediments with the highly persistent hexachlorocyclohexanes (HCHs) continues to be a threat for humans and the environment. Despite the existence of bacteria capable of biodegradation and cometabolic transformation of HCH isomers, such processes occur over time scales of decades and are thus challenging to assess. Here, we explored the use of compound-specific isotope analysis (CSIA) to track the aerobic biodegradation and biotransformation pathways of the most prominent isomers, namely, (−)-α-, (+)-α-, β-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes. Dehydrochlorination of (+)-α-, γ-, and δ-HCH catalyzed by LinA2 was subject to substantial C and H isotope fraction with apparent ¹³C- and ²H-kinetic isotope effects (AKIEs) of up to 1.029 ± 0.001 and 6.7 ± 2.9, respectively, which are indicative of bimolecular eliminations. Hydrolytic dechlorination of δ-HCH by LinB exhibited even larger C but substantially smaller H isotope fractionation with ¹³C- and ²H-AKIEs of 1.073 ± 0.006 and 1.41 ± 0.04, respectively, which are typical for nucleophilic substitutions. The systematic evaluation of isomer-specific phenomena showed that, in addition to contaminant uptake limitations, diffusion-limited turnover ((−)-α-HCH), substrate dissolution (β-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.

Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers. Here, we investigated the enzyme specificity of apparent ¹³C- and ²H-kinetic isotope effects (AKIEs) associated with the dehydrochlorination of γ-HCH (lindane) by two variants of the lindane dehydrochlorinases LinA1 and LinA2. While LinA1 and LinA2 attack γ-HCH at different trans-1,2-diaxial H–C–C–Cl moieties, the observed C and H isotope fractionation was large, typical for bimolecular eliminations, and was not affected by conformational mobility. ¹³C-AKIEs for transformation by LinA1 and LinA2 were the same (1.024 ± 0.001 and 1.025 ± 0.001, respectively), whereas ²H-AKIEs showed minor differences (2.4 ± 0.1 and 2.6 ± 0.1). Variations of isotope effects between LinA1 and LinA2 are small and in the range reported for different degrees of C–H bond cleavage in transition states of dehydrochlorination reactions. The large C and H isotope fractionation reported here for experiments with pure enzymes contrasts with previous observations from whole cell experiments and suggests that specific uptake processes by HCH-degrading microorganisms might modulate the observable HCH isotope fractionation at contaminated sites.

Environments that receive fecal pollution are reservoirs of antibiotic resistance. Recent metagenomic observations suggest that the fecal pollution indicator crAssphage correlates with the occurrence of antibiotic resistance genes (ARGs) in the environment. Expanding the utility of crAssphage to represent the environmental occurrence of ARGs would potentially facilitate ARG management in environments contaminated with fecal pollution. In this study, we analyzed a suite of molecular indicators for ARGs and crAssphage over a 30 day sampling period in an urban stream that receives combined sewer overflows. The sampled stream showed high levels of ARGs and crAssphage with statistically significantly elevated levels during wet weather events. The observed correlation between crAssphage and ARG molecular detection was high when all were measured using digital droplet polymerase chain reaction (PCR). Quantitative PCR and digital droplet PCR quantifications of crAssphage showed only moderate agreement, emphasizing the importance of detection technology when making quantitative comparisons. Overall, this study demonstrates the potential of a crAssphage fecal indicator to correlate with ARG occurrence when employing a "toolbox" approach to fecal pollution management.

The evolution of multicellularity has given rise to a remarkable diversity of multicellular life cycles and life histories. Whereas some multicellular organisms are long-lived, grow through cell division, and repeatedly release single-celled propagules (for example, animals), others are short-lived, form by aggregation, and propagate only once, by generating large numbers of solitary cells (for example, cellular slime moulds). There are no systematic studies that explore how diverse multicellular life cycles can come about. Here, we focus on the origin of multicellularity and develop a mechanistic model to examine the primitive life cycles that emerge from a unicellular ancestor when an ancestral gene is co-opted for cell adhesion. Diverse life cycles readily emerge, depending on ecological conditions, group-forming mechanism, and ancestral constraints. Among these life cycles, we recapitulate both extremes of long-lived groups that propagate continuously and short-lived groups that propagate only once, with the latter type of life cycle being particularly favoured when groups can form by aggregation. Our results show how diverse life cycles and life histories can easily emerge at the origin of multicellularity, shaped by ancestral constraints and ecological conditions. Beyond multicellularity, this finding has similar implications for other major transitions, such as the evolution of sociality.

Microbial communities are inherently complex systems. To address this complexity, microbial ecologists are developing new, more elaborate laboratory models at an ever-increasing pace. These model microbial communities and habitats have opened up the exploration of new territories that lie between the simplicity and controllability of “synthetic” systems and the convolution and complexity of natural environments. Here, we discuss this classic methodological divide, we propose a conceptual perspective that integrates new research developments, and we sketch a 3-point possible roadmap to cross the divide between controllability and complexity in microbial ecology.

Sustainable Development Goal (SDG) Indicator 6.2.1 requires household handwashing facilities to have soap and water, but there are no guidelines for handwashing water quality. In contrast, drinking water quality guidelines are defined: water must be “free from contamination” to be defined as “safely managed” (SDG Indicator 6.1.1). We modeled the hypothesized mechanism of infection due to contaminated handwashing water to inform risk-based guidelines for microbial quality of handwashing water. We defined two scenarios that should not occur: (1) if handwashing caused fecal contamination, indicated using Escherichia coli, on a person’s hands to increase rather than decrease and (2) if hand-to-mouth contacts following handwashing caused an infection risk greater than an acceptable threshold. We found water containing <1000 E. coli colony-forming units (CFU) per 100 mL removes E. coli from hands with>99.9% probability. However, for the annual probability of infection to be <1:1000, handwashing water must contain <2 × 10⁻⁶ focus-forming units of rotavirus, <1 × 10⁻⁴ CFU of Vibrio cholerae, and <9 × 10⁻⁶ Cryptosporidium oocysts per 100 mL. Our model suggests that handwashing with nonpotable water will generally reduce fecal contamination on hands but may be unable to lower the annual probability of infection risks from hand-to-mouth contacts below 1:1000.

The efficacy of germicidal ultraviolet (UV-C) light emitting diodes (LEDs) was evaluated for inactivating human enteroviruses included on the United States Environmental Protection Agency (EPA)’s Contaminant Candidate List (CCL). A UV-C LED device, emitting at peaks of 260 nm and 280 nm and the combination of 260/280 nm together, was used to measure and compare potential synergistic effects of dual wavelengths for disinfecting viral organisms. The 260 nm LED proved to be the most effective at inactivating the CCL enteroviruses tested. To obtain 2-log₁₀ inactivation credit for the 260 nm LED, the fluences (UV doses) required are approximately 8 mJ/cm² for coxsackievirus A10 and poliovirus 1, 10 mJ/cm² for enterovirus 70, and 13 mJ/cm² for echovirus 30. No synergistic effect was detected when evaluating the log inactivation of enteroviruses irradiated by the dual-wavelength UV-C LEDs.

Microorganisms in wastewater treatment plants (WWTPs) are essential for water purification to protect public and environmental health. However, the diversity of microorganisms and the factors that control it are poorly understood. Using a systematic global-sampling effort, we analysed the 16S ribosomal RNA gene sequences from ~1,200 activated sludge samples taken from 269 WWTPs in 23 countries on 6 continents. Our analyses revealed that the global activated sludge bacterial communities contain ~1 billion bacterial phylotypes with a Poisson lognormal diversity distribution. Despite this high diversity, activated sludge has a small, global core bacterial community (n = 28 operational taxonomic units) that is strongly linked to activated sludge performance. Meta-analyses with global datasets associate the activated sludge microbiomes most closely to freshwater populations. In contrast to macroorganism diversity, activated sludge bacterial communities show no latitudinal gradient. Furthermore, their spatial turnover is scale-dependent and appears to be largely driven by stochastic processes (dispersal and drift), although deterministic factors (temperature and organic input) are also important. Our findings enhance our mechanistic understanding of the global diversity and biogeography of activated sludge bacterial communities within a theoretical ecology framework and have important implications for microbial ecology and wastewater treatment processes.

Freshwater lakes and impoundments are an important source of methane into the atmosphere. Methane is the second most important greenhouse gas after carbon dioxide and accounts for about 75% of the climate impact of lakes. The only process that reduces methane emissions from lakes is the oxidation by methanotrophic bacteria. This diverse group of bacteria has the unique ability to use methane and oxygen to gain energy and produce biomass.
Aim of this dissertation was to improve our understanding of the ecology of these bacteria and their importance to prevent methane emissions from freshwater lakes. From the outset, the interdisciplinary project was designed to complement field studies with modelling. The focus was in particular on the methane dynamics during the autumnal overturn of stratified lakes. During this critical phase, large amounts of methane that accumulate in the anoxic hypolimnion during stratification are transported to the surface. [...]

Microbes are exposed to changing environments, to which they can respond by adopting various lifestyles such as swimming, colony formation or dormancy. These lifestyles are often studied in isolation, thereby giving a fragmented view of the life cycle as a whole. Here, we study lifestyles in the context of this whole. We first use machine learning to reconstruct the expression changes underlying life cycle progression in the bacterium Bacillus subtilis, based on hundreds of previously acquired expression profiles. This yields a timeline that reveals the modular organization of the life cycle. By analysing over 380 Bacillales genomes, we then show that life cycle modularity gives rise to mosaic evolution in which life stages such as motility and sporulation are conserved and lost as discrete units. We postulate that this mosaic conservation pattern results from habitat changes that make these life stages obsolete or detrimental. Indeed, when evolving eight distinct Bacillales strains and species under laboratory conditions that favour colony growth, we observe rapid and parallel losses of the sporulation life stage across species, induced by mutations that affect the same global regulator. We conclude that a life cycle perspective is pivotal to understanding the causes and consequences of modularity in both regulation and evolution.

Stormwater biofilters are distributed stormwater control measures for managing urban runoff. Recent work has shown that adding biochar to biofilters can reduce stormwater contaminant concentrations, including fecal indicator bacteria (FIB). However, the potential of biochar-augmented biofilters to remove human pathogens from stormwater has not been investigated. In this study, we investigated the removal of bacterial pathogens Salmonella enterica serovar Typhimurium and Staphylococcus aureus, as well as bacterial and viral indicators Escherichia coli and MS2 coliphage in laboratory-scale biochar-amended biofilters. Biochar-amended biofilters performed better than sand biofilters in removing the microorganisms from stormwater and removal of pathogenic bacteria was greater than that of FIB. Biochar-augmented biofilters provided up to 3.9, 1.9, and 1.8 log₁₀ removal for pathogenic bacteria, E. coli, and MS2, respectively. We utilized colloid filtration theory to elucidate potential microbial removal mechanisms. In biochar-amended biofilters, electrostatic interactions between the virus and collector surfaces likely controlled bacteriophage removal whereas the electrostatic interactions likely played a minor role in bacterial removal. Bacterial removal in biochar-augmented biofilters was likely controlled by straining and hydrophobic interactions. The findings of this study inform the design of geomedia-amended biofilters to reduce stormwater-derived microbial contamination in receiving waters.

Adenovirus, a waterborne pathogen responsible for causing bronchitis, pneumonia, and gastrointestinal infections, is highly resistant to UV disinfection and therefore drives the virus disinfection regulations set by the U.S. Environmental Protection Agency. Polychromatic UV irradiation has been shown to be more effective at inactivating adenovirus and other viruses than traditional monochromatic irradiation emitted at 254 nm; the enhanced efficacy has been attributed to UV-induced damage to viral proteins. This research shows UV-induced damage to adenoviral proteins across the germicidal UV spectrum at wavelength intervals between 200 and 300 nm. A deuterium lamp with bandpass filters and UV light-emitting diodes (UV LEDs) isolated wavelengths in approximate 10 nm intervals. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and image densitometry were used to detect signatures for the hexon, penton, fiber, minor capsid, and core proteins. The greatest loss of protein signature, indicating damage to viral proteins, occurred below 240 nm. Hexon and penton proteins exposed to a dose of 28 mJ/cm² emitted at 214 nm were approximately 4 times as sensitive and fiber proteins approximately 3 times as sensitive as those exposed to a dose of 50 mJ/cm² emitted at 254 nm. At 220 nm, a dose of 38 mJ/cm² reduced the hexon and penton protein quantities to approximately 33% and 31% of the original amounts, respectively. In contrast, a much higher dose of 400 mJ/cm² emitted at 261 and 278 nm reduced the original protein quantity to between 66-89% and 80-93%, respectively. No significant damage was seen with a dose of 400 mJ/cm² at 254 nm. This research directly correlates enhanced inactivation at low wavelengths with adenoviral protein damage at those wavelengths, adding fundamental insight into the mechanisms of inactivation of polychromatic germicidal UV irradiation for improving UV water disinfection.

Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (−80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant-free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific.

Over the last decade, the genomic revolution has offered the possibility to generate tremendous amounts of data that contain valuable information on the genetic basis of phenotypic traits, such as those linked to human diseases or those that allow for species to adapt to a changing environment. Most ecologically relevant traits are controlled by a large number of genes with small individual effects on trait variation, but that are connected with one another through complex developmental, metabolic and biochemical networks. As a result, it has recently been suggested that most adaptation events in natural populations are reached via correlated changes at multiple genes at a time, for which the name polygenic adaptation has been coined. The current challenge is to develop methods to extract the relevant information from genomic data to detect the signature of polygenic evolutionary change. The symposium entitled "Detecting the Genomic Signal of Polygenic Adaptation and the Role of Epistasis in Evolution" held in 2017 at the University of Zürich aimed at reviewing our current state of knowledge. In this review, we use the talks of the invited speakers to summarize some of the most recent developments in this field.

Bacteria frequently exchange metabolites with other micro- and macro-organisms. In these often obligate cross-feeding interactions, primary metabolites such as vitamins, amino acids, nucleotides, or growth factors are exchanged. The widespread distribution of this type of metabolic interactions, however, is at odds with evolutionary theory: why should an organism invest costly resources to benefit other individuals rather than using these metabolites to maximize its own fitness? Recent empirical work has shown that bacterial genotypes can significantly benefit from trading metabolites with other bacteria relative to cells not engaging in such interactions. Here, we will provide a comprehensive overview over the ecological factors and evolutionary mechanisms that have been identified to explain the evolution and maintenance of metabolic mutualisms among microorganisms. Furthermore, we will highlight general principles that underlie the adaptive evolution of interconnected microbial metabolic networks as well as the evolutionary consequences that result for cells living in such communities.

While limiting organic carbon through biofiltration controls microbial regrowth in drinking water main distribution systems, little is known about its effectiveness for building water plumbing systems. Here we examined regrowth under eight plumbing conditions: pipe material (iron or cross-linked polyethylene (PEX)), corrosion control (magnesium rod, elevated pH), corrosion sediments (Fe(OH)₃), nitrification (ammonium addition), a combination of magnesium rod/iron pipe/ammonium, and a no-modification control using simulated water heaters (SWHs) fed with biofiltered or unfiltered water and incubated at 32 °C. While biofiltration did effectively limit total bacterial numbers (half-log less), it did not offer protection against Legionella pneumophila. By the end of study (day 448), some conditions acted synergistically with (PEX, ammonium) or independently of (magnesium rod, combination) influent biofiltration to increase L. pneumophila numbers. Illumina sequencing of 16S rRNA genes confirmed the enrichment of phylogenetic groups associated with expected functions (e.g., ammonium, iron, and hydrogen oxidation) under corresponding plumbing modifications and also revealed that biofiltration had an overarching influence on the microbial community structure in the control, Fe(OH)₃, and combination conditions. This study demonstrates that a variety of factors under warm water plumbing conditions can override potential influences of upstream biofiltration on L. pneumophila growth.

The emergence of subpopulations that perform distinct metabolic roles has been observed in populations of genetically identical bacteria.

Drinking water production in the vicinity of rivers not only requires the consideration of different spatiotemporal scales and settings of river-groundwater interaction processes, but also of local and regional scale groundwater regimes.
Selected case studies in combination with field-experiments and the setup of high-resolution groundwater flow models enabled the investigation of the spatiotemporal development of microbial (classical fecal indicator bacteria and total cell counts) and selected organic micropollutants in riverine and regional groundwater for different hydrological settings, including low and high flow conditions. Proxy indicators suitable as surrogates for the diverse contaminations in alluvial aquifers with different settings could be identified.
Based on the study results, the basic elements for both groundwater management and river restoration concepts are derived, which include the: (1) compilation and evaluation of the "current state" concerning hydrogeology, microbiology and contamination by organic micropollutants, (2) definition of field-experiments to qualitatively assess variability related to the "current state", and (3) quantitative assessment of groundwater regimes, including variability of groundwater components and inflow areas, by application of high-resolution groundwater flow models.
The validity and transferability of the concept and inferred controls (specifically drivers and controls of river-groundwater interaction) are tested by evaluations derived from hydraulic relationships to river sections with comparable settings and regional groundwater flow regimes in general.
The results of our investigations illustrate the influence of dynamic hydrologic boundary conditions on river-groundwater interaction and of regional scale groundwater flow regimes on the water composition of riverine groundwater systems. It is demonstrated how to identify river sections and their variations with intensified river-groundwater exchange processes and how to quantify the transient character of the different groundwater components that constitute the raw water quality of drinking water wells near rivers.

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement.
This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed.
Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample.
The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon.

Metabolic interactions between populations can influence patterns of spatial organization and diversity within microbial communities. Cross-feeding is one type of metabolic interaction that is pervasive within microbial communities, where one genotype consumes a resource into a metabolite while another genotype then consumes the metabolite. A typical feature of cross-feeding is that the metabolite may impose toxicity if it accumulates to sufficient concentrations. However, little is known about the effect of metabolite toxicity on spatial organization and local diversity within microbial communities. We addressed this knowledge gap by experimentally varying the toxicity of a single cross-fed metabolite and measuring the consequences on a synthetic microbial cross-feeding community. Our results demonstrate that metabolite toxicity slows demixing and thus slows local diversity loss of the metabolite-producing population. Using mathematical modeling, we show that this is because toxicity slows growth, which enables more cells to emigrate from the founding region and contribute towards population expansion. Our results show that metabolite toxicity is an important factor affecting local diversity within microbial communities and that spatial organization can be affected by non-intuitive mechanisms.

The fitness landscape is a concept that is widely used for understanding and predicting evolutionary adaptation. The topography of the fitness landscape depends critically on the environment, with potentially far-reaching consequences for evolution under changing conditions. However, few studies have assessed directly how empirical fitness landscapes change across conditions, or validated the predicted consequences of such change. We previously evolved replicate yeast populations in the presence of either gradually increasing, or constant high, concentrations of the heavy metals cadmium (Cd), nickel (Ni), and zinc (Zn), and analyzed their phenotypic and genomic changes. Here, we reconstructed the local fitness landscapes underlying adaptation to each metal by deleting all repeatedly mutated genes both by themselves and in combination. Fitness assays revealed that the height, and/or shape, of each local fitness landscape changed considerably across metal concentrations, with distinct qualitative differences between unconditionally (Cd) and conditionally toxic metals (Ni and Zn). This change in topography had particularly crucial consequences in the case of Ni, where a substantial part of the individual mutational fitness effects changed in sign across concentrations. Based on the Ni landscape analyses, we made several predictions about which mutations had been selected when during the evolution experiment. Deep sequencing of population samples from different time points generally confirmed these predictions, demonstrating the power of landscape reconstruction analyses for understanding and ultimately predicting evolutionary dynamics, even under complex scenarios of environmental change.

In physics, it is customary to represent the fluctuations of a stochastic system at steady state in terms of linear response to small random perturbations. Previous work has shown that the same framework describes effectively the trade-off between cell-to-cell variability and correction in the control of cell division of single E. coli cells. However, previous analyses were motivated by specific models and limited to a subset of the measured variables. For example, most analyses neglected the role of growth rate variability. Here, we take a comprehensive approach and consider several sets of available data from both microcolonies and microfluidic devices in different growth conditions. We evaluate all the coupling coefficients between the three main measured variables (interdivision times, cell sizes and individual-cell growth rates). The linear-response framework correctly predicts consistency relations between a priori independent experimental measurements, which confirms its validity. Additionally, the couplings between the cell-specific growth rate and the other variables are typically non zero. Finally, we use the framework to detect signatures of mechanisms in experimental data involving growth rate fluctuations, finding that (1) noise-generating coupling between size and growth rate is a consequence of inter-generation growth rate correlations and (2) the correlation patterns agree with a near-adder model where the added size has a dependence on the single-cell growth rate. Our findings define relevant constraints that any theoretical description should reproduce, and will help future studies aiming to falsify some of the competing models of the cell cycle existing today in the literature.

Aerobic methane-oxidizing bacteria (MOB) have the ability to thrive on methane, a potent greenhouse gas with critical climatic implications. They use methane as their sole source of carbon and energy. They phylogenetically mainly group as Alpha- and Gammaproteobacteria, as well as Verrucomicrobia. The metalloenzyme methane-monooxygenase (MMO) catalyses the initial oxidation step, the conversion of methane to methanol. The membrane-bound, particulate MMO (pMMO) is widespread among MOB, however, its atomic structure and active site composition remains a matter of debate. Nevertheless, it is generally agreed that pMMO is a copper-dependent enzyme.
Freshwater lakes are significant contributors to global natural methane emissions. However, MOB inhabiting their water column substantially reduce methane fluxes from the sediments to the atmosphere. MOB are commonly highly enriched at oxic-anoxic transition zones, but increasing evidence suggests that methane oxidation by MOB is also occurring at depths depleted in oxygen. [...]

Methan ist ein starkes Treibhausgas mit kritischen klimatischen Auswirkungen. Aerobe Methan oxidierende Bakterien (MOB) haben die Fähigkeit, Methan als einzige Kohlenstoff- und Energiequelle zu nutzen. Phylogenetisch werden sie hauptsächlich in Alpha- und Gammaproteobakterien sowie Verrucomicrobia eingeteilt. Das Enzym Methan-Monooxygenase (MMO) katalysiert den ersten Oxidationsschritt, die Umsetzung von Methan zu Methanol. Die membrangebundene, partikuläre MMO (pMMO) ist unter MOB weit verbreitet, jedoch sind seine chemische Struktur und die Zusammensetzung seines aktiven Zentrums weiterhin umstritten. Nichtsdestotrotz besteht ein allgemeiner Konsens, dass pMMO ein kupferabhängiges Enzym ist.
Süsswasserseen tragen wesentlich zu den globalen natürlichen Methanemissionen bei. Die in ihrer Wassersäule lebenden MOB konsumieren eine erhebliche Menge an Methan, welches aus den Sedimenten austritt, und andernfalls in die Atmosphäre gelangen würde. MOB findet man normalerweise stark angereichert in oxisch-anoxischen Übergangszonen, es gibt jedoch zunehmend Hinweise, dass Methanoxidation durch MOB auch in sauerstoffarmen Tiefen stattfindet. [...]

To optimize removal of organic micropollutants from the water cycle, understanding the processes during activated sludge treatment is essential. In this study, we hypothesize that aliphatic amines, which are highly abundant among organic micropollutants, are partly removed from the water phase in activated sludge through ion trapping in protozoa. In ion trapping, which has been extensively investigated in medical research, the neutral species of amine-containing compounds diffuse through the cell membrane and further into acidic vesicles present in eukaryotic cells such as protozoa. There they become trapped because diffusion of the positively charged species formed in the acidic vesicles is strongly hindered. We tested our hypothesis with two experiments. First, we studied the distribution of the fluorescent amine acridine orange in activated sludge by confocal fluorescence imaging. We observed intense fluorescence in distinct compartments of the protozoa, but not in the bacterial biomass. Second, we investigated the distribution of 12 amine-containing and eight control micropollutants in both regular activated sludge and sludge where the protozoa had been inactivated. In contrast to most control compounds, the amine-containing micropollutants displayed a distinctly different behavior in the noninhibited sludge compared to the inhibited one: (i) more removal from the liquid phase; (ii) deviation from first-order kinetics for the removal from the liquid phase; and (iii) higher amounts in the solid phase. These results provide strong evidence that ion trapping in protozoa occurs and that it is an important removal mechanism for amine-containing micropollutants in batch experiments with activated sludge that has so far gone unnoticed. We expect that our findings will trigger further investigations on the importance of this process in full-scale wastewater treatment systems, including its relevance for accumulation of ammonium.

LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P_1δ, P_2δ) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T_1δ, T_2δ). Enzymatic transformations of δ-HBCD, α₁-PBCDol, one of the transformation products, and α₂-PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters K_M, v_max, k_cat/K_M indicated that δ-HBCD is the best LinB substrate followed by α₂- and α₁-PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P_1δ, identical to α₂-PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An S_N2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α₂-PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with α₂-PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T_1δ. In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at Å-resolution and predictions of their stereochemistry.

As more countries engage in water reuse, either intended or de facto, there is an urgent need to more comprehensively evaluate resulting environmental and public health concerns. While antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are increasingly coming under the spotlight, as emerging contaminants, existing water reuse regulations and guidelines do not adequately address these concerns. This perspectives paper seeks to frame the various challenges that need to be resolved to identify meaningful and realistic target types and levels of antibiotic resistance benchmarks for water reuse. First, there is the need for standardized and agreed-upon methodologies to identify and quantify ARB and ARGs. Second, even if methodologies are available, identifying which ARB and ARGs to monitor that would best relate to the occurrence of disease burden remains unknown. Third, a framework tailored to assessing the risks associated with ARB and ARGs during reuse is urgently needed. Fourth, similar to protecting drinking water sources, strategies to prevent dissemination of ARB and ARGs via wastewater treatment and reuse are required to ensure that appropriate barriers are emplaced. Finally, current wastewater treatment technologies could benefit from modification or retrofit to more effectively remove ARB and ARGs while also producing a high quality product for water and resource recovery. This perspectives paper highlights the need to consider ARB and ARGs when evaluating the overall safety aspects of water reuse and ways by which this may be accomplished.

Noch immer sind die Quellen und Übertragungswege von E. coli-Kontaminationen nicht ganz verstanden. Um Antworten auf offene Fragen zu finden, wurden im Rahmen eines Forschungsprojekts 142 Haushalte in der simbabwischen Hauptstadt Harare auf das Vorhandensein von E. coli untersucht. Zudem wurden Haushaltsmerkmale gesammelt. Dadurch erhoffte sich das Forschungsteam, signifikante Zusammenhänge aufzuzeigen.

Infectious disease transmission is frequently mediated by the environment, where people's movements through and interactions with the environment dictate risks of infection and/or illness. Capturing these interactions, and quantifying their importance, offers important insights into effective interventions. In this study, we capture high time-resolution activity data for twenty-five Vietnamese farmers during collection and land application of human excreta for agriculture. Although human excreta use improves productivity, the use increases risks of enteric infections for both farmers and end users. In our study, the activity data are integrated with environmental microbial sampling data into a stochastic-mechanistic simulation of E. coli contamination on hands and E. coli ingested. Results from the study include frequent and variable contact rates for farmers' hands (from 34 to 1344 objects contacted per hour per hand), including highly variable hand-to-mouth contact rates (from 0 to 9 contacts per hour per hand). The frequency of hand-to-mouth contacts was substantially lower than the widely-used frequency previously reported for U.S. Office Workers. Environmental microbial contamination data highlighted ubiquitous E. coli contamination in the environment, including excreta, hands, toilet pit, handheld tools, soils, surfaces, and water. Results from the simulation suggest dynamic changes in E. coli contamination on hands, and wide variation in hand contamination and E. coli ingested amongst the farmers studied. Sensitivity analysis suggests that E. coli contamination on hands and ingested doses are most influenced by contamination of handheld tools, excreta, and the toilet pit as well as by frequency of hand-to-mouth contacts. The study findings are especially relevant given the context: no farmers reported adequate storage time of human excreta, and personal protective mask availability did not prevent hand-to-mouth contacts. Integrating high time-resolution activity data into exposure assessments highlights variation in exposures amongst farmers, and offers greater insight into effective interventions and their potential impacts.

Quantitative data on human-environment interactions are needed to fully understand infectious disease transmission processes and conduct accurate risk assessments. Interaction events occur during an individual’s movement through, and contact with, the environment, and can be quantified using diverse methodologies. Methods that utilize videography, coupled with specialized software, can provide a permanent record of events, collect detailed interactions in high resolution, be reviewed for accuracy, capture events difficult to observe in real-time, and gather multiple concurrent phenomena. In the accompanying video, the use of specialized software to capture human-environment interactions for human exposure and disease transmission is highlighted. Use of videography, combined with specialized software, allows for the collection of accurate quantitative representations of human-environment interactions in high resolution. Two specialized programs include the Virtual Timing Device for the Personal Computer, which collects sequential microlevel activity time series of contact events and interactions, and LiveTrak, which is optimized to facilitate annotation of events in real-time. Opportunities to annotate behaviors at high resolution using these tools are promising, permitting detailed records that can be summarized to gain information on infectious disease transmission and incorporated into more complex models of human exposure and risk.

We have shown for the first time that a natural protein (human insulin) can be acylated at the N-terminus with a β-amino acid (H-β³hAla-), in a process catalyzed by the β-peptidyl aminopeptidase 3-2W4-BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases.

Microbial communities often exhibit incredible taxonomic diversity, raising questions regarding the mechanisms enabling species coexistence and the role of this diversity in community functioning. On the one hand, many coexisting but taxonomically distinct microorganisms can encode the same energy-yielding metabolic functions, and this functional redundancy contrasts with the expectation that species should occupy distinct metabolic niches. On the other hand, the identity of taxa encoding each function can vary substantially across space or time with little effect on the function, and this taxonomic variability is frequently thought to result from ecological drift between equivalent organisms. Here, we synthesize the powerful paradigm emerging from these two patterns, connecting the roles of function, functional redundancy and taxonomy in microbial systems. We conclude that both patterns are unlikely to be the result of ecological drift, but are inevitable emergent properties of open microbial systems resulting mainly from biotic interactions and environmental and spatial processes.

While antibiotics are a mainstay of contemporary medicine, many basic questions about how they inactivate and kill bacteria are still unresolved. The main goal of this thesis was to address some of these questions. Specifically, we aimed at expanding our understanding of two central themes that are difficult to explore in traditional microbiological approaches. The first theme is about how antibiotics affect the basic components of bacterial growth: cell division and death. We exposed bacterial populations to various concentrations of antibiotics and analyzed how the population growth patterns that we observed emerged from the interplay between cell division and cell death. The second theme is nongenetic factors that contribute to differences in the response and survival to antibiotics, either between individual cells or between environments with plenty or limited resources. We used Escherichia coli and ribosome-binding antibiotics as our model systems and employed microfluidics and time-lapse microscopy to investigate the effects of antibiotics at the level of individual cells. [...]

Antibiotika spielen eine zentrale Rolle in der zeitgenössischen Medizin. Nichtsdestotrotz verstehen wir aber nicht im Detail, wie Bakterien durch Antibiotika inaktiviert und abgetötet werden. Das zentrale Ziel dieser Dissertation war es, an diesen Wissenslücken zu arbeiten. Konkret haben wir uns auf zwei Aspekte fokussiert, die mit traditionellen Methoden aus der Mikrobiologie schwierig anzugehen sind. Der erste Aspekt ist, wie Antibiotika die grundlegenden Komponenten des bakteriellen Wachstums – Zellteilung und Zelltod – beeinflussen. Wir haben Bakterien verschiedenen Konzentrationen von Antibiotika ausgesetzt und haben untersucht, wie das beobachtete Populationswachstum zu Stande kommt durch ein Wechselspiel von Zellteilung und – tod. Der zweite Aspekt ist, dass nichtgenetische Faktoren einen erheblichen Effekt haben können darauf, wie Bakterien Antibiotika-Exposition überleben, und Unterschiede im Überleben zwischen individuellen Zellen innerhalb von klonalen Population oder zwischen verschiedenen Wachstumsbedingungen verursachen können. Wir haben das Bakterium Escherichia coli als Modellsystem verwendet, und mit Antibiotika gearbeitet, die an das bakterielle Ribosom binden. Um die Effekte von Antibiotika auf Einzelzell-Ebene zu untersuchen, haben wir Experimente in Mikrofluidik-Kammer und mit Zeitraffer-Mikroskopen durchgeführt. [...]

Polymeric materials are widely used in drinking water distribution systems. These materials could release organic carbon that supports bacterial growth. To date, the available migration assays for polymeric materials have not included the potential influence of chlorination on organic carbon migration behavior. Hence, we established a migration and growth potential protocol specifically for analysis of carbon migration from materials in contact with chlorinated drinking water. Four different materials were tested, including ethylene propylene dienemethylene (EPDM), poly-ethylene (PEX b and PEX c) and poly-butylene (PB). Chlorine consumption rates decreased gradually over time for EPDM, PEXc and PB. In contrast, no free chlorine was detected for PEXb at any time during the 7 migration cycles. Total organic carbon (TOC) and assimilable organic carbon (AOC) was evaluated in both chlorinated and non-chlorinated migrations. TOC concentrations for EPDM and PEXb in chlorinated migrations were significantly higher than non-chlorinated migrations. The AOC results showed pronounced differences among tested materials. AOC concentrations from chlorinated migration waters of EPDM and PB were higher compared to non-chlorinated migrations, whereas the opposite trend was observed for PEXb and PEXc. There was also a considerable difference between tested materials with regards to bacterial growth potential. The results revealed that the materials exposed to chlorine-influenced migration still exhibited a strong biofilm formation potential. The overall results suggested that the choice in material would make a considerable difference in chlorine consumption and carbon migration behavior in drinking water distribution systems.

Coastal oceans receive large amounts of anthropogenic fixed nitrogen (N), most of which is denitrified in the sediment before reaching the open ocean. Sandy sediments, which are common in coastal regions, seem to play an important role in catalysing this N-loss. Permeable sediments are characterized by advective porewater transport, which supplies high fluxes of organic matter into the sediment, but also leads to fluctuations in oxygen and nitrate concentrations. Little is known about how the denitrifying communities in these sediments are adapted to such fluctuations. Our combined results indicate that denitrification in eutrophied sandy sediments from the world's largest tidal flat system, the Wadden Sea, is carried out by different groups of microorganisms. This segregation leads to the formation of N₂O which is advectively transported to the overlying waters and thereby emitted to the atmosphere. At the same time, the production of N₂O within the sediment supports a subset of Flavobacteriia which appear to be specialized on N₂O reduction. If the mechanisms shown here are active in other coastal zones, then denitrification in eutrophied sandy sediments may substantially contribute to current marine N₂O emissions.

A cell can divide only upon completion of chromosome segregation; otherwise, its daughters would lose genetic material. However, we do not know whether the partitioning of chromosomes is the key event for the decision to divide. We show how key trends in single-cell data reject the classic idea of replication-segregation as the rate-limiting process for cell division. Instead, the data agree with a model where two concurrent processes (setting replication initiation and interdivision time) set cell division on competing time scales. During each cell cycle, division is set by the slowest process (an "AND" gate). The concept of transitions between cell cycle stages as decisional processes integrating multiple inputs instead of cascading from orchestrated steps can affect the way we think of the cell cycle in general.

Understanding the classic problem of how single E. coli cells coordinate cell division with genome replication would open the way to addressing cell-cycle progression at the single-cell level. Recent studies produced new data, but the contrast in their conclusions and proposed mechanisms makes the emerging picture fragmented and unclear. Here, we re-evaluate available data and models, including generalizations based on the same assumptions. We show that although they provide useful insights, none of the proposed models captures all correlation patterns observed in data. We conclude that the assumption that replication is the bottleneck process for cell division is too restrictive. Instead, we propose that two concurrent cycles responsible for division and initiation of DNA replication set the time of cell division. This framework allows us to select a nearly constant added size per origin between subsequent initiations as the most likely mechanism setting initiation of replication.

Soils in household environments in low- and middle-income countries may play an important role in the persistence, proliferation, and transmission of Escherichia coli. Our goal was to investigate the risk factors for detection, survival, and growth of E. coli in soils collected from household plots. E. coli was enumerated in soil and fecal samples from humans, chickens, and cattle from 52 households in rural Bangladesh. Associations between E. coli concentrations in soil, household-level risk factors, and soil physicochemical characteristics were investigated. Susceptibility to 16 antibiotics and the presence of intestinal pathotypes were evaluated for 175 E. coli isolates. The growth and survival of E. coli in microcosms using soil collected from the households were also assessed. E. coli was isolated from 44.2% of the soil samples, with an average of 1.95 log₁₀ CFU/g dry soil. Soil moisture and clay content were associated with E. coli concentrations in soil, whereas no household-level risk factor was significantly correlated. Antibiotic resistance and pathogenicity were common among E. coli isolates, with 42.3% resistant to at least one antibiotic, 12.6% multidrug resistant (≥3 classes), and 10% potentially pathogenic. Soil microcosms demonstrate growth and/or survival of E. coli, including an enteropathogenic extended-spectrum beta-lactamase (ESBL)-producing isolate, in some, but not all, of the household soils tested. In rural Bangladesh, defined soil physicochemical characteristics appear more influential for E. coli detection in soils than household-level risk factors. Soils may act as reservoirs in the transmission of antibiotic-resistant and potentially pathogenic E. coli and therefore may impact the effectiveness of water, sanitation, and hygiene interventions.

Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli, which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households (N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environment within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli—and enteric pathogens generally—to identify effective interventions.

Changes in bacterial concentration and composition in drinking water during distribution are often attributed to biological (in)stability. Here we assessed temporal biological stability in a full-scale distribution network (DN) supplied with different types of source water: treated and chlorinated surface water and chlorinated groundwater produced at three water treatment plants (WTP). Monitoring was performed weekly during 12 months in two locations in the DN. Flow cytometric total and intact cell concentration (ICC) measurements showed considerable seasonal fluctuations, which were different for two locations. ICC varied between 0.1–3.75 × 10⁵ cells mL⁻¹ and 0.69–4.37 × 10⁵ cells mL⁻¹ at two locations respectively, with ICC increases attributed to temperature-dependent bacterial growth during distribution. Chlorinated water from the different WTP was further analysed with a modified growth potential method, identifying primary and secondary growth limiting compounds. It was observed that bacterial growth in the surface water sample after chlorination was primarily inhibited by phosphorus limitation and secondly by organic carbon limitation, while carbon was limiting in the chlorinated groundwater samples. However, the ratio of available nutrients changed during distribution, and together with disinfection residual decay, this resulted in higher bacterial growth potential detected in the DN than at the WTP. In this study, bacterial growth was found to be higher (i) at higher water temperatures, (ii) in samples with lower chlorine residuals and (iii) in samples with less nutrient (carbon, phosphorus, nitrogen, iron) limitation, while this was significantly different between the samples of different origin. Thus drinking water microbiological quality and biological stability could change during different seasons, and the extent of these changes depends on water temperature, the water source and treatment. Furthermore, differences in primary growth limiting nutrients in different water sources could contribute to biological instability in the network, where mixing occurs.

Biofilme in Duschschläuchen sind ein kritischer Punkt in Hausinstallationen und deshalb zunehmend Gegenstand von Forschungsprojekten. Ausmass und Zusammensetzung der Biofilme werden massgeblich von der Wahl des Schlauchmaterials beeinflusst. Dies ist insbesondere deshalb wichtig, da sich Bakterien aus dem Biofilm in die Wasserphase ablösen (v. a. während der Stagnation). Somit nimmt die Materialwahl direkten Einfluss auf die asserqualität und letztendlich auf den Konsumenten.

Bath toys pose an interesting link between flexible plastic materials, potable water, external microbial and nutrient contamination, and potentially vulnerable end-users. Here, we characterized biofilm communities inside 19 bath toys used under real conditions. In addition, some determinants for biofilm formation were assessed, using six identical bath toys under controlled conditions with either clean water prior to bathing or dirty water after bathing. All examined bath toys revealed notable biofilms on their inner surface, with average total bacterial numbers of 5.5 × 10⁶ cells/cm² (clean water controls), 9.5 × 10⁶ cells/cm² (real bath toys), and 7.3 × 10⁷ cells/cm² (dirty water controls). Bacterial community compositions were diverse, showing many rare taxa in real bath toys and rather distinct communities in control bath toys, with a noticeable difference between clean and dirty water control biofilms. Fungi were identified in 58% of all real bath toys and in all dirty water control toys. Based on the comparison of clean water and dirty water control bath toys, we argue that bath toy biofilms are influenced by (1) the organic carbon leaching from the flexible plastic material, (2) the chemical and biological tap water quality, (3) additional nutrients from care products and human body fluids in the bath water, as well as, (4) additional bacteria from dirt and/or the end-users’ microbiome. The present study gives a detailed characterization of bath toy biofilms and a better understanding of determinants for biofilm formation and development in systems comprising plastic materials in contact with potable water.

Microorganisms inhabiting filtration media of a drinking water treatment plant can be beneficial, because they metabolize biodegradable organic matter from source waters and those formed during disinfection processes, leading to the production of biologically stable drinking water. However, which microbial consortia colonize filters and what metabolic capacity they possess remain to be investigated. To gain insights into these issues, we performed metagenome sequencing and analysis of microbial communities in three different filters of a full-scale drinking water treatment plant (DWTP). Filter communities were sampled from a rapid sand filter (RSF), granular activated carbon filter (GAC), and slow sand filter (SSF), and from the Schmutzdecke (SCM, a biologically active scum layer accumulated on top of SSF), respectively. Analysis of community phylogenetic structure revealed that the filter bacterial communities significantly differed from those in the source water and final effluent communities, respectively. Network analysis identified a filter-specific colonization pattern of bacterial groups. Bradyrhizobiaceae were abundant in GAC, whereas Nitrospira were enriched in the sand-associated filters (RSF, SCM, and SSF). The GAC community was enriched with functions associated with aromatics degradation, many of which were encoded by Rhizobiales (~30% of the total GAC community). Predicting minimum generation time (MGT) of prokaryotic communities suggested that the GAC community potentially select fast-growers (<15 h of MGT) among the four filter communities, consistent with the highest dissolved organic matter removal rate by GAC. Our findings provide new insights into the community phylogenetic structure, colonization pattern, and metabolic capacity that potentially contributes to organic matter removal achieved in the biofiltration stages of the full-scale DWTP.

Biological richness is positively associated with the rates of some metabolic processes performed by microbial communities. It remains unclear, however, whether these positive associations are a general feature of the metabolic processes performed by microbial communities or whether they are specific to certain types of metabolic processes. For example, it was hypothesized that the strength of any particular positive association depends on how many different genotypes within a microbial community perform the metabolic process of interest (i.e. the 'rarity hypothesis'). We tested the generality of these positive associations by measuring the taxonomic richness, functional gene richness and rate constants for 71 different metabolic processes across 30 independent microbial communities. We found that both taxonomic and functional gene richness do indeed tend to positively associate with the rates of metabolic processes. In addition, we found that positive associations occur across a wide range of different environmental conditions. Counter to the 'rarity hypothesis', however, we did not detect a relationship between the strengths of the positive associations and the rarity of each metabolic process. Together, our data provide empirical evidence that positive associations with biological richness may indeed be a general feature of the metabolic processes performed by microbial communities.

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin.

Deux détecteurs en ligne (cytomètre de flux et analyseur d’adénosine triphosphate) ont été testés à grande échelle dans une usine de traitement d’eau potable. Des variations quotidiennes dans la concentration et la viabilité bactériennes ont été relevées et mises en relation avec les conditions d’opération de l’usine. La combinaison des deux technologies d’analyse microbiologique a fourni des informations complémentaires entre elles, ainsi que de nouvelles perspectives pour l’analyse en ligne de l’eau potable.

Shower hoses offer an excellent bacterial growth environment in close proximity to a critical end-user exposure route within building drinking water plumbing. However, the health risks associated with and processes underlying the development of biofilms in shower hoses are poorly studied. In a global survey, biofilms from 78 shower hoses from 11 countries were characterized in terms of cell concentration (4.1 × 10⁴–5.8 × 10⁸ cells/cm²), metal accumulation (including iron, lead, and copper), and microbiome composition (including presence of potential opportunistic pathogens). In countries using disinfectant, biofilms had on average lower cell concentrations and diversity. Metal accumulation (up to 5 μg-Fe/cm², 75 ng-Pb/cm², and 460 ng-Cu/cm²) seemed to be partially responsible for discoloration in biofilms, and likely originated from other pipes upstream in the building. While some genera that may contain potential opportunistic pathogens (Legionella, detected in 21/78 shower hoses) were positively correlated with biofilm cell concentration, others (Mycobacterium, Pseudomonas) had surprisingly non-existent or negative correlations with biofilm cell concentrations. In a controlled study, 15 identical shower hoses were installed for the same time period in the same country, and both stagnant and flowing water samples were collected. Ecological theory of dispersal and selection helped to explain microbiome composition and diversity of different sample types. Shower hose age was related to metal accumulation but not biofilm cell concentration, while frequency of use appeared to influence biofilm cell concentration. This study shows that shower hose biofilms are clearly a critical element of building drinking water plumbing, and a potential target for building drinking water plumbing monitoring.

Here we used flow cytometry (FCM) and filtration paired with amplicon sequencing to determine the abundance and composition of small low nucleic acid (LNA)-content bacteria in a variety of freshwater ecosystems. We found that FCM clusters associated with LNA-content bacteria were ubiquitous across several ecosystems, varying from 50 to 90% of aquatic bacteria. Using filter-size separation, we separated small LNA-content bacteria (passing 0.4 µm filter) from large bacteria (captured on 0.4 µm filter) and characterized communities with 16S amplicon sequencing. Small and large bacteria each represented different sub-communities within the ecosystems’ community. Moreover, we were able to identify individual operational taxonomical units (OTUs) that appeared exclusively with small bacteria (434 OTUs) or exclusively with large bacteria (441 OTUs). Surprisingly, these exclusive OTUs clustered at the phylum level, with many OTUs appearing exclusively with small bacteria identified as candidate phyla (i.e. lacking cultured representatives) and symbionts. We propose that LNA-content bacteria observed with FCM encompass several previously characterized categories of bacteria (ultramicrobacteria, ultra-small bacteria, candidate phyla radiation) that share many traits including small size and metabolic dependencies on other microorganisms.

Detecting disturbances in microbial communities is an important aspect of managing natural and engineered microbial communities. Here, we implemented a custom-built continuous staining device in combination with real-time flow cytometry (RT-FCM) data acquisition, which, combined with advanced FCM fingerprinting methods, presents a powerful new approach to track and quantify disturbances in aquatic microbial communities. Through this new approach we were able to resolve various natural community and single-species microbial contaminations in a flow-through drinking water reactor. Next to conventional FCM metrics, we applied metrics from a recently developed fingerprinting technique in order to gain additional insight into the microbial dynamics during these contamination events. Importantly, we found that multiple community FCM metrics based on different statistical approaches were required to fully characterize all contaminations. Furthermore we found that for accurate cell concentration measurements and accurate inference from the FCM metrics (coefficient of variation ≤ 5%), at least 1000 cells should be measured, which makes the achievable temporal resolution a function of the prevalent bacterial concentration in the system-of-interest. The integrated RT-FCM acquisition and analysis approach presented herein provides a considerable improvement in the temporal resolution by which microbial disturbances can be observed and simultaneously provides a multi-faceted toolset to characterize such disturbances.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Pinoresinol is a dimer of two β-β′ linked coniferyl alcohol molecules. It is both a plant defence molecule synthesized from the shikimic acid pathway and a representative of several β-β linked dimers produced during the microbial degradation of lignin in dead plant material. Until now little has been known about the bacterial catabolism of such dimers. Here we report the isolation of an efficient (+)-pinoresinol mineralizing Pseudomonas sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding ketoalcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate as above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both the routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by the strain SG-MS2.

Whether mutations in bacteria exhibit a noticeable delay before expressing their corresponding mutant phenotype was discussed intensively in the 1940s to 1950s, but the discussion eventually waned for lack of supportive evidence and perceived incompatibility with observed mutant distributions in fluctuation tests. Phenotypic delay in bacteria is widely assumed to be negligible, despite the lack of direct evidence. Here, we revisited the question using recombineering to introduce antibiotic resistance mutations into E. coli at defined time points and then tracking expression of the corresponding mutant phenotype over time. Contrary to previous assumptions, we found a substantial median phenotypic delay of three to four generations. We provided evidence that the primary source of this delay is multifork replication causing cells to be effectively polyploid, whereby wild-type gene copies transiently mask the phenotype of recessive mutant gene copies in the same cell. Using modeling and simulation methods, we explored the consequences of effective polyploidy for mutation rate estimation by fluctuation tests and sequencing-based methods. For recessive mutations, despite the substantial phenotypic delay, the per-copy or per-genome mutation rate is accurately estimated. However, the per-cell rate cannot be estimated by existing methods. Finally, with a mathematical model, we showed that effective polyploidy increases the frequency of costly recessive mutations in the standing genetic variation (SGV), and thus their potential contribution to evolutionary adaptation, while drastically reducing the chance that de novo recessive mutations can rescue populations facing a harsh environmental change such as antibiotic treatment. Overall, we have identified phenotypic delay and effective polyploidy as previously overlooked but essential components in bacterial evolvability, including antibiotic resistance evolution.

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9 kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested.

Rapid quantification of absolute microbial cell abundances is important for a comprehensive interpretation of microbiome surveys and crucial to support theoretical modelling and the design of engineered systems. In this paper, we propose a protocol specifically optimised for the quantification of microbial abundances in water biofilters using flow cytometry (FCM). We optimised cell detachment from sand biofilter particles for FCM quantification through the evaluation of five chemical dispersants (NaCl, Triton-X100, CaCl₂, sodium pyrophosphate (PP), Tween 80 combined with PP), different mechanical pre-treatments (low and high energy sonication and shaking) and two fixation methods (glutaraldehyde and ethanol). The developed protocol was cross-compared using other established and commonly employed methods for biomass quantification in water filter samples (adenosine triphosphate (ATP) quantification, real-time quantitative PCR (qPCR) and volatile solids (VS)). The highest microbial count was obtained by detaching the biofilm from biofilter grains and dispersing clusters into singles cells using Tween 80 and sodium pyrophosphate combined with four steps of high energy sonication (27W, for 80 s each step); glutaraldehyde was shown to be the best fixative solution. The developed protocol was reliable and highly reproducible and produced results that are comparable to data from alternative quantification methods. Indeed, high correlations were found with trends obtained through ATP and qPCR (ρ = 0.98 and ρ = 0.91) measurements. The VS content was confirmed as an inaccurate method to express biomass in sand samples since it correlated poorly with all the other three methods (ρ = 0.005 with FCM, 0.002 with ATP and 0.177 with qPCR). FCM and ATP showed the strongest agreement between absolute counts with a slope of the correlation equal to 0.7, while qPCR seemed to overestimate cell counts by a factor of ten. The rapidity and reproducibility of the method developed make its application ideal for routine quantification of microbial cell abundances on sand from water biofilters and thus useful in revealing the ecological patterns and quantifying the metabolic kinetics involved in such systems.

Persisters are a subpopulation of bacteria that are not killed by antibiotics even though they lack genetic resistance. Here we provide evidence that persisters can manifest as small colony variants (SCVs) in clinical infections. We analyze growth kinetics of Staphylococcus aureus sampled from in vivo conditions and in vitro stress conditions that mimic growth in host compartments. We report that SCVs arise as a result of a long lag time, and that this phenotype emerges de novo during the growth phase in various stress conditions including abscesses and acidic media. We further observe that long lag time correlates with antibiotic usage. These observations suggest that treatment strategies should be carefully tailored to address bacterial persisters in clinics.

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N₂ and CO₂ fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H₂S (i.e. electron donor) limited chemostats show that varying levels of NH₄⁺ limitation induce heterogeneity in N₂ fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H₂S) populations indicates that electron donor limitation drives heterogeneity in N₂ and CO₂ fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.

Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO₂ and microbial biomass. The approach is based on ¹³C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate-co-terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.

Gene expression levels in clonal bacterial groups have been found to be spatially correlated. These correlations can partly be explained by the shared lineage history of nearby cells, although they could also arise from local cell-cell interactions. Here, we present a quantitative framework that allows us to disentangle the contributions of lineage history, long-range spatial gradients, and local cell-cell interactions to spatial correlations in gene expression. We study pathways involved in toxin production, SOS stress response, and metabolism in Escherichia coli microcolonies and find for all pathways that shared lineage history is the main cause of spatial correlations in gene expression levels. However, long-range spatial gradients and local cell-cell interactions also contributed to spatial correlations in SOS response, amino acid biosynthesis, and overall metabolic activity. Together, our data show that the phenotype of a cell is influenced by its lineage history and population context, raising the question of whether bacteria can arrange their activities in space to perform functions they cannot achieve alone.

Even in the absence of genetic or environmental differences, cells differ from each other in their molecular make-up. The consequences of these phenotypic differences are often not well understood. New work by Waite et al (2016) directly links variation in the molecular composition of individual bacterial cells to their population-level performance.

Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells' close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies-or bet-hedging-as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood.We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria.

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies.

Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells μl^–1 to less than 30 cells μl^–1 within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice.

Recent theory predicts that the fitness of pioneer populations can decline when species expand their range, due to high rates of genetic drift on wave fronts making selection less efficient at purging deleterious variants. To test these predictions, we studied the fate of mutator bacteria expanding their range for 1650 generations on agar plates. In agreement with theory, we find that growth abilities of strains with a high mutation rate (HMR lines) decreased significantly over time, unlike strains with a lower mutation rate (LMR lines) that present three to four times fewer mutations. Estimation of the distribution of fitness effect under a spatially explicit model reveals a mean negative effect for new mutations (–0.38%), but it suggests that both advantageous and deleterious mutations have accumulated during the experiment. Furthermore, the fitness of HMR lines measured in different environments has decreased relative to the ancestor strain, whereas that of LMR lines remained unchanged. Contrastingly, strains with a HMR evolving in a well-mixed environment accumulated less mutations than agar-evolved strains and showed an increased fitness relative to the ancestor. Our results suggest that spatially expanding species are affected by deleterious mutations, leading to a drastic impairment of their evolutionary potential.

Der vorliegende Bericht fasst die Ergebnisse des Forschungsprojektes "Biologische Nachbehandlung von kommunalem Abwasser nach Ozonung", kurz "ReTREAT", zusammen. Das Projekt wurde durch das Bundesamtes für Umwelt (BAFU) im Rahmen der Umwelttechnologieförderung (UTF) finanziert. Zudem wurde das Projekt durch hohe Eigen- und Cash-Leistungen der Industriepartner finanziell und technisch unterstützt.
Ziel des Projektes war es Untersuchungen zur Leistungsfähigkeit und Effizienz verschiedener Verfahren der biologischen Nachbehandlung zur Reduktion der ökotoxikologischen Wirkung und zur Reduktion von Transformationsprodukten von ozonten, kommunalem Abwassers durchzuführen.

Marine microbes are ubiquitous and play a fundamental role in shaping the oceans as we know them, through key processes such as photosynthesis and biogeochemical cycles. Their monitoring requires sampling at the correct time and space, since they show high turnover rates and patchy distributions. We have used two on-line flow cytometry systems to monitor marine microbes in the coastal area of the Gulf of Naples, as a proof-of-concept that this technology can represent a useful tool for real-time alert systems of water biological quality. Bacterial and picophytoplankton concentrations are proposed as general indicators of water quality, the first step necessary to assess the effects of changes in environmental conditions, to be followed by in-depth analyses of community composition for presence of pathogens or other indicators. During the survey we could detect hotspots of high bacterial concentrations located in areas of discharges of untreated urban wastes or subject to riverine inputs. In general, bacterial abundances were inversely correlated to salinity, suggesting that they use organic matter of terrestrial origin for their growth. The technological approach used represents a useful and fast method to monitor coastal areas also in the context of the European Directive 2008/56/EC, aimed at assessing and improving European marine waters quality.

Bacteriophage transfer (lysogenic conversion) promotes bacterial virulence evolution. There is limited understanding of the factors that determine lysogenic conversion dynamics within infected hosts. A murine Salmonella Typhimurium (S.Tm) diarrhea model was used to study the transfer of SopEΦ, a prophage from S.Tm SL1344, to S.Tm ATCC14028S. Gut inflammation and enteric disease triggered >55% lysogenic conversion of ATCC14028S within 3 days. Without inflammation, SopEΦ transfer was reduced by up to 10⁵-fold. This was because inflammation (e.g., reactive oxygen species, reactive nitrogen species, hypochlorite) triggers the bacterial SOS response, boosts expression of the phage antirepressor Tum, and thereby promotes free phage production and subsequent transfer. Mucosal vaccination prevented a dense intestinal S.Tm population from inducing inflammation and consequently abolished SopEΦ transfer. Vaccination may be a general strategy for blocking pathogen evolution that requires disease-driven transfer of temperate bacteriophages.

Flussnahe Trinkwasserfassungen sind oft gekennzeichnet durch variierende Anteile an jungem Flusswasserinfiltrat mit kurzen Aufenthaltszeiten und regionalem älterem Grundwasser. Durch die Überlagerung von natürlichen Randbedingungen und dem Pumpbetrieb in einer Trinkwasserfassung entsteht somit eine hohe Variabilität der Rohwasserqualität in den Trinkwasserfassungen.

Background: Consistent domestic hand hygiene can reduce diarrhea-related morbidity and mortality and the spread of other communicable diseases. However, it remains uncertain which technique of handwashing is most effective and practicable during everyday life. The goal of this study is to determine how the handwashing technique, as performed in the daily life by the participants of this case study in Harare, Zimbabwe, influences microbial handwashing effectiveness.
Methods: Handwashing technique of 173 primary caregivers was observed in their homes and hand rinse samples were collected before and after handwashing. Samples were analyzed for Escherichia coli and total coliform concentrations. Generalized linear models were used to predict fecal hand contamination after washing from observed handwashing technique.
Results: Cleaning under fingernails, scrubbing the fingertips, using soap, and drying hands through rubbing on clothes or a clean towel statistically significantly reduced E coli contamination of hands after washing. Tap use, scrubbing fingertips, and rubbing hands on clothes to dry them statistically significantly reduced total coliform contamination.
Conclusions: Recommendations for effective and practicable domestic handwashing in Harare, Zimbabwe, should include performing specific handscrubbing steps (ie, cleaning under the fingernails and rubbing the fingertips), and soap and tap use. This calls for further research to develop behavior change interventions that explicitly promote effective handwashing technique at critical times.

Successive range expansions occur within all domains of life, where one population expands first (primary expansion) and one or more secondary populations then follow (secondary expansion). In general, genetic drift reduces diversity during range expansion. However, it is not clear whether the same effect applies during successive range expansion, mainly because the secondary population must expand into space occupied by the primary population. Here we used an experimental microbial model system to show that, in contrast to primary range expansion, successive range expansion promotes local population diversity. Because of mechanical constraints imposed by the presence of the primary population, the secondary population forms fractal-like dendritic structures. This divides the advancing secondary population into many small sub-populations and promotes intermixing between the primary and secondary populations. We further developed a mathematical model to simulate the formation of dendritic structures in the secondary population during succession. By introducing mutations in the primary or dendritic secondary populations, we found that mutations are more likely to accumulate in the dendritic secondary populations. Our results thus show that successive range expansion can promote intermixing over the short term and increase genetic diversity over the long term. Our results therefore have potentially important implications for predicting the ecological processes and evolutionary trajectories of microbial communities.

Modifications to transcriptional regulators play a major role in adaptation. Here, we compared the effects of multiple beneficial mutations within and between Escherichia coli rpoB, the gene encoding the RNA polymerase β subunit, and rho, which encodes a transcriptional terminator. These two genes have harbored adaptive mutations in numerous E. coli evolution experiments but particularly in our previous large-scale thermal stress experiment, where the two genes characterized alternative adaptive pathways. To compare the effects of beneficial mutations, we engineered four advantageous mutations into each of the two genes and measured their effects on fitness, growth, gene expression and transcriptional termination at 42.2 °C. Among the eight mutations, two rho mutations had no detectable effect on relative fitness, suggesting they were beneficial only in the context of epistatic interactions. The remaining six mutations had an average relative fitness benefit of ≈20%. The rpoB mutations affected the expression of ≈1,700 genes; rho mutations affected the expression of fewer genes but most (83%) were a subset of those altered by rpoB mutants. Across the eight mutants, relative fitness correlated with the degree to which a mutation restored gene expression back to the unstressed, 37.0 °C state. The beneficial mutations in the two genes did not have identical effects on fitness, growth or gene expression, but they caused parallel phenotypic effects on gene expression and genome-wide transcriptional termination.

Bacterial evolution has resulted in the appearance of several Sphingomonadacea strains that gained the ability to metabolize hexachlorocyclohexanes (HCHs). HCHs have been widely used as pesticides but were banned under the Stockholm Convention on persistent organic pollutants (POPs) in 2009. Here we present evidence for bacterial transformation reactions of hexabromocyclododecanes (HBCDs), which are structurally related to HCHs. HBCDs were used as flame retardants. They are now also considered as POPs and their production and use is restricted since 2013. Racemic α-, β-, and γ-HBCDs and their mixture were exposed to Sphingobium chinhatense IP26 in resting cell assays in parallel to β-HCH. All HBCD stereoisomers were converted with (−)β-HBCD being the best and both α-HBCD enantiomers the poorest substrates. HBCD conversion rates were 27–430 times slower than that of β-HCH. Three generations of hydroxylated transformation products were observed, 7 pentabromocyclododecanol isomers (PeBCD-ols), 11 tetrabromocyclododecadiols (TeBCD-diols) and 3 tribromocyclododecatriols (TrBCD-triols). The conversion of (+)α-, (−)β- and (−)γ-HBCD was faster than those of their enantiomers. Therefore the respective enantiomeric excess increased to 3 ± 1%, 36 ± 1% and 6 ± 2% during 48 h of bacterial exposure. PeBCD-ols appeared first, followed by TeBCD-diols and TrBCD-triols indicating stepwise hydrolytic dehalogenation reactions. In conclusion, severe HCH pollution at geographically distinct dumpsites triggered bacterial evolution to express enzymes transforming such compounds. We used S. chinhatense IP26 bacteria to transform structurally related HBCDs, also regulated under the Stockholm Convention. Such bacteria might be useful for bioremediation but the toxicity of the numerous transformation products observed must be assessed in advance.

Die Herausforderung zum Erhalt der Trinkwasserqualität im Gebäude nimmt zu. Ein bekanntes Risiko ist die Einnistung krankheitserregender Mikroorganismen in Gebäudeinstallationen. Trotzdem wird der Trinkwasserqualität im Gebäude oft zu wenig Aufmerksamkeit entgegengebracht. Im Rahmen eines dreijährigen KTI-Projekts wurden vier Teilbereiche mit dem Ziel bearbeitet, den Wissensstand auszubauen und dadurch geeignete Präventionsstrategien abzuleiten, die in die zukünftige Entwicklung des Bereiches der Trinkwasserqualität in Gebäuden einfliessen sollen.

Background: The production of toxic metabolites has shaped the spatial and temporal arrangement of metabolic processes within microbial cells. While diverse solutions to mitigate metabolite toxicity have evolved, less is known about how evolution itself is affected by metabolite toxicity. We hypothesized that the pace of molecular evolution should increase as metabolite toxicity increases. At least two mechanisms could cause this. First, metabolite toxicity could increase the mutation rate. Second, metabolite toxicity could increase the number of available mutations with large beneficial effects that selection could act upon (e.g., mutations that provide tolerance to toxicity), which consequently would increase the rate at which those mutations increase in frequency.

Results: We tested this hypothesis by experimentally evolving the bacterium Pseudomonas stutzeri under denitrifying conditions. The metabolite nitrite accumulates during denitrification and has pH-dependent toxic effects, which allowed us to evolve P. stutzeri at different magnitudes of nitrite toxicity. We demonstrate that increased nitrite toxicity results in an increased pace of molecular evolution. We further demonstrate that this increase is generally due to an increased number of available mutations with large beneficial effects and not to an increased mutation rate.

Conclusions: Our results demonstrate that the production of toxic metabolites can have important impacts on the evolutionary processes of microbial cells. Given the ubiquity of toxic metabolites, they could also have implications for understanding the evolutionary histories of biological organisms.

Background: While mutualistic interactions between different genotypes are pervasive in nature, their evolutionary origin is not clear. The dilemma is that, for mutualistic interactions to emerge and persist, an investment into the partner genotype must pay off: individuals of a first genotype that invest resources to promote the growth of a second genotype must receive a benefit that is not equally accessible to individuals that do not invest. One way for exclusive benefits to emerge is through spatial structure (i.e., physical barriers to the movement of individuals and resources).
Results: Here we propose that organisms can evolve their own spatial structure based on physical attachment between individuals, and we hypothesize that attachment evolves when spatial proximity to members of another species is advantageous. We tested this hypothesis using experimental evolution with combinations of E. coli strains that depend on each other to grow. We found that attachment between cells repeatedly evolved within 8 weeks of evolution and observed that many different types of mutations potentially contributed to increased attachment.
Conclusions: We postulate a general principle by which passive beneficial interactions between organisms select for attachment, and attachment then provides spatial structure that could be conducive for the evolution of active mutualistic interactions.

Background. The ability to form a cellular memory and use it for cellular decision-making could help bacteria to cope with recurrent stress conditions. We analyzed whether bacteria would form a cellular memory specifically if past events are predictive of future conditions. We worked with the asymmetrically dividing bacterium Caulobacter crescentus where past events are expected to only be informative for one of the two cells emerging from division, the sessile cell that remains in the same microenvironment and does not migrate.
Results. Time-resolved analysis of individual cells revealed that past exposure to low levels of antibiotics increases tolerance to future exposure for the sessile but not for the motile cell. Using computer simulations, we found that such an asymmetry in cellular memory could be an evolutionary response to situations where the two cells emerging from division will experience different future conditions.
Conclusions. Our results raise the question whether bacteria can evolve the ability to form and use cellular memory conditionally in situations where it is beneficial.

Improved micropollutant (MP) biotransformation during biological wastewater treatment has been associated with high ammonia oxidation activities, suggesting co-metabolic biotransformation by ammonia oxidizing bacteria as an underlying mechanism. The goal of this study was to clarify the contribution of ammonia oxidizing bacteria to increased MP degradation in nitrifying activated sludge (NAS) communities using a series of inhibition experiments. To this end, we treated a NAS community with two different ammonia oxidation inhibitors, namely octyne (OCT), a mechanistic inhibitor that covalently binds to ammonia monooxygenases, and allylthiourea (ATU), a copper chelator that depletes copper ions from the active center of ammonia monooxygenases. We investigated the biotransformation of 79 structurally different MPs by the inhibitor-treated and untreated sludge communities. Fifty-five compounds exhibited over 20% removal in the untreated control after a 46 h-incubation. Of these, 31 compounds were significantly inhibited by either ATU and/or OCT. For 17 of the 31 MPs, the inhibition by ATU at 46 h was substantially higher than by OCT despite the full inhibition of ammonia oxidation by both inhibitors. This was particularly the case for almost all thioether and phenylurea compounds tested, suggesting that in nitrifying activated sludge communities, ATU does not exclusively act as an inhibitor of bacterial ammonia oxidation. Rather, ATU also inhibited enzymes contributing to MP biotransformation but not to bulk ammonia oxidation. Thus, inhibition studies with ATU tend to overestimate the contribution of ammonia-oxidizing bacteria to MP biotransformation in nitrifying activated sludge communities. Biolog tests revealed only minor effects of ATU on the heterotrophic respiration of common organic substrates by the sludge community, suggesting that ATU did not affect enzymes that were essential in energy conservation and central metabolism of heterotrophs. By comparing ATU- and OCT-treated samples, as well as before and after ammonia oxidation was recovered in OCT-treated samples, we were able to demonstrate that ammonia-oxidizing bacteria were highly involved in the biotransformation of four compounds: asulam, clomazone, monuron and trimethoprim.

Chemotaxis of the bacterium Escherichia coli is well understood in shallow chemical gradients, but its swimming behavior remains difficult to interpret in steep gradients. By focusing on single-cell trajectories from simulations, we investigated the dependence of the chemotactic drift velocity on attractant concentration in an exponential gradient. Whereas maxima of the average drift velocity can be interpreted within analytical linear-response theory of chemotaxis in shallow gradients, limits in drift due to steep gradients and finite number of receptor-methylation sites for adaptation go beyond perturbation theory. For instance, we found a surprising pinning of the cells to the concentration in the gradient at which cells run out of methylation sites. To validate the positions of maximal drift, we recorded single-cell trajectories in carefully designed chemical gradients using microfluidics.

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium. The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium. We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.

Vaccine-induced high-avidity IgA can protect against bacterial enteropathogens by directly neutralizing virulence factors or by poorly defined mechanisms that physically impede bacterial interactions with the gut tissues (‘immune exclusion’)^1–3. IgAmediated cross-linking clumps bacteria in the gut lumen and is critical for protection against infection by non-typhoidal Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium). However, classical agglutination, which was thought to drive this process, is efficient only at high pathogen densities (≥10⁸ non-motile bacteria per gram). In typical infections, much lower densities^4,5 (10⁰–10⁷ colony-forming units per gram) of rapidly dividing bacteria are present in the gut lumen. Here we show that a different physical process drives formation of clumps in vivo: IgA-mediated cross-linking enchains daughter cells, preventing their separation after division, and clumping is therefore dependent on growth. Enchained growth is effective at all realistic pathogen densities, and accelerates pathogen clearance from the gut lumen. Furthermore, IgA enchains plasmid-donor and -recipient clones into separate clumps, impeding conjugative plasmid transfer in vivo. Enchained growth is therefore a mechanism by which IgA can disarm and clear potentially invasive species from the intestinal lumen without requiring high pathogen densities, inflammation or bacterial killing. Furthermore, our results reveal an untapped potential for oral vaccines in combating the spread of antimicrobial resistance.

The Blue Diversion AUTARKY Toilet is a urine-diverting toilet with on-site treatment. The toilet is being developed to provide a safe and affordable sanitation technology for people who lack access to sewer-based sanitation. Water used for personal hygiene, hand washing, and flushing to rinse urine- and feces-collection bowls is treated, stored, and recycled for reuse to reduce reliance on external water supplies. The system provides an opportunity to investigate hygiene of water for reuse following treatment. Treatment in the toilet includes a Biologically Activated Membrane Bioreactor (BAMBi) followed by a secondary treatment technology. To identify effective secondary treatment, three options, including granular activated carbon (GAC) only, GAC+chlorine (sodium hypochlorite), and GAC+electrolysis are considered based on the bacterial inactivation and growth inhibition efficiency. Four different hygiene-relevant bacteria are tested: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Salmonella typhimurium. Our evaluation demonstrates that—despite treatment of water with the BAMBi—E. coli, P. aeruginosa, and S. typhimurium have the potential to grow during storage in the absence of microbial competition. Including the indigenous microbial community influences bacterial growth in different ways: E. coli growth decreases but P. aeruginosa growth increases relative to no competition. The addition of the secondary treatment options considerably improves water quality. A column of GAC after the BAMBi reduces E. coli growth potential by 2 log₁₀, likely due to the reduction of carbon sources. Additional treatments including chlorination and electrolysis provide further safety margins, with more than 5 log₁₀ inactivation of E. coli. However, reactivation and/or regrowth of E. coli and P. aeruginosa occurs under in the absence of residual disinfectant. Treatment including the BAMBi, GAC, and electrolysis appear to be promising technologies to control bacterial growth during storage in water intended for reuse.

While we have good understanding of bacterial metabolism at the population level, we know little about the metabolic behavior of individual cells: do single cells in clonal populations sometimes specialize on different metabolic pathways? Such metabolic specialization could be driven by stochastic gene expression and could provide individual cells with growth benefits of specialization. We measured the degree of phenotypic specialization in two parallel metabolic pathways, the assimilation of glucose and arabinose. We grew Escherichia coli in chemostats, and used isotope-labeled sugars in combination with nanometer-scale secondary ion mass spectrometry and mathematical modeling to quantify sugar assimilation at the single-cell level. We found large variation in metabolic activities between single cells, both in absolute assimilation and in the degree to which individual cells specialize in the assimilation of different sugars. Analysis of transcriptional reporters indicated that this variation was at least partially based on cell-to-cell variation in gene expression. Metabolic differences between cells in clonal populations could potentially reduce metabolic incompatibilities between different pathways, and increase the rate at which parallel reactions can be performed.

We have studied the dynamics of water quality in three karst springs taking advantage of new technological developments that enable high-resolution measurements of bacterial load (total cell concentration: TCC) as well as online measurements of abiotic parameters. We developed a novel data analysis approach, using self-organizing maps and non-linear projection methods, to approximate the TCC dynamics using the multivariate data sets of abiotic parameter time-series, thus providing a method that could be implemented in an online water quality management system for water suppliers. The (TCC) data, obtained over several months, provided a good basis to study the microbiological dynamics in detail. Alongside the TCC measurements, online abiotic parameter time-series, including spring discharge, turbidity, spectral absorption coefficient at 254 nm (SAC254) and electrical conductivity, were obtained. High-density sampling over an extended period of time, i.e. every 45 min for 3 months, allowed a detailed analysis of the dynamics in karst spring water quality. Substantial increases in both the TCC and the abiotic parameters followed precipitation events in the catchment area. Differences between the parameter fluctuations were only apparent when analyzed at a high temporal scale. Spring discharge was always the first to react to precipitation events in the catchment area. Lag times between the onset of precipitation and a change in discharge varied between 0.2 and 6.7 h, depending on the spring and event. TCC mostly reacted second or approximately concurrent with turbidity and SAC254, whereby the fastest observed reaction in the TCC time series occurred after 2.3 h. The methodological approach described here enables a better understanding of bacterial dynamics in karst springs, which can be used to estimate risks and management options to avoid contamination of the drinking water.

Enzymatic oxygenations are among the most important biodegradation and detoxification reactions of organic pollutants. In the environment, however, such natural attenuation processes are extremely difficult to monitor. Changes of stable isotope ratios of aromatic pollutants at natural isotopic abundances serve as proxies for isotope effects associated with oxygenation reactions. Such isotope fractionations offer new avenues for revealing the pathway and extent of pollutant transformation and provide new insights into the mechanisms of catalysis by Rieske non-heme ferrous iron oxygenases. Based on compound-specific C, H, N, and O isotope analysis, we present a comprehensive methodology with which isotope effects can be derived from the isotope fractionation measured in substrates, the cosubstrate O₂, and organic oxygenation products. We use dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase as illustrative examples to introduce different mathematical procedures for deriving apparent substrate and product isotope effects. We present two experimental approaches to control reactant and product turnover for isotope fractionation analysis in experimental systems containing purified enzymes, E. coli clones, and pure strains of environmental microorganisms. Finally, we present instrumental procedures and sample treatment instructions for analysis of C, H, and N isotope analysis in organic compounds and O isotope analysis in aqueous O₂ by gas and liquid chromatography coupled to isotope ratio mass spectrometry.

Understanding virus transfer between liquid and skin is necessary to estimate transmission during water-related activities. Here, we modeled virus transfer from liquid-to-skin and skin-to-liquid. We performed human subject studies using three bacteriophages as pathogenic virus surrogates: nonenveloped MS2 and Qβ and enveloped Φ6. Our study shows that transfer from liquid-to-skin is describable by a single model based on (1) virus concentration and (2) volume of liquid remaining on skin. Contact times (0.1–30 min), and virus species had little-to-no influence on virus transfer. Likewise, liquid conditions (pH 6–9, ionic strength 10–550 mM) had no influence on transfer as shown for MS2. The model accounts for both, virus adsorbed onto the skin, and virus in the liquid retained on skin. In comparison, virus transfer from skin-to-liquid was influenced by the wetness of the skin and by liquid type (water, saliva). 90 ± 19% of the virus inoculated on the skin are transferred to the water when the skin remains wet compared to 30 ± 17% when the skin is dry. The transfer from skin-to-liquid was 41% higher when the recipient liquid was water as compared with saliva. This study quantifies virus transfer between liquid and skin and guides risk assessments of water-related activities.

Since different wavelengths of light impact different cellular targets, microorganisms exposed to natural sunlight experience a combination of multiple stressors. In order to better understand the effects of sunlight on microorganisms we, therefore, need to understand how different wavelength act alone and in combination. Here, we describe a synergistic effect between UVA and UVB irradiation on viability of Escherichia coli bacteria. To investigate the basis of this synergistic effect we analysed mutant strains that were obtained through selection for increased resistance to combined UVA and UVB. By identifying and reconstructing genetic changes in the resistant strains we provide evidence that UVA-absorbing thiouridine residues in tRNAs are the key to the synergistic effect. Our study provides insights into how naturally occurring combinations of stressors can interact, and points to new ways for controlling microbial populations.

Background: Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 µg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing.

Results: Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 °C in the presence of PEX (1.58 x 10⁵ gene copies/mL). At 53 °C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 °C and 20% at 53 °C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log₁₀ lower) at temperatures ≤ 41 °C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤ 45 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing.

Conclusions: Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers, but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors.

Drinking water, even when distributed safely, deteriorates in quality after entering the home. The microbiological quality of this building plumbing water is of particular interest because opportunistic pathogens (e.g. Legionella pneumophila) are known to proliferate there. Beyond the potential threat to health, microbial growth results in customer complaints about taste, odor, color, turbidity, slime formation, and filter clogging in building plumbing.
While water distributors strive to deliver a product that remains stable in the home, they have minimal influence on the design, maintenance, and operation of building plumbing systems. At the property line, legal responsibility for the water shifts away from the water distributor to the building owner. On top of this, monitoring water stability in the home requires invasive sampling and has potential privacy implications. All of these factors contribute to a poor understanding of the microbiology of water in building plumbing.
Many factors give building plumbing a unique growth environment. Pipe materials are varied and sometimes unregulated. In the building multiple materials are found within inches of each other. With high surface area to volume ratios, and more frequent replacement (e.g. with bathroom remodels), these materials have a considerably higher impact in buildings than in the distribution network. Temperature is high (e.g. hot and mixed water distribution) compared to the distribution network, creating favorable growth conditions. Building distalends are also only flowing intermittently (i.e. long stagnation times), allowing for biofilm and water phase interactions. While all of these factors are known to contribute to growth, their impact on bacterial concentration and composition is not completely understood. Moreover, each building is different with respect to design, operation, and location, complicating synthesis of various building plumbing studies.
The research presented here primarily addresses three factors in building plumbing, namely material quality, stagnation, and temperature. These three factors come together at shower hoses, which have (1) flexible polymeric materials that leach large amounts of biodegradable carbon, (2) long stagnation intervals (i.e. once daily showers), and (3) temperatures ranging only from warm to room temperature (i.e. neither hot nor cold). Shower hoses were thus often used as a model system throughout this thesis, for studies of both biofilm development and stagnation dependent water dynamics. As this research was supported by highly quantitative culture-independent methods (i.e., flow cytometry and qPCR), some critical aspects of these methods were also further investigated.
Throughout this work, building plumbing is approached as an ecological system governed by simple ecological principles, primarily that of dispersal (i.e., movement of bacteria) and selection (i.e., favorable growth conditions). This approach allowed for a cohesive understanding of building plumbing behavior in both large data sets from around the world and a series of highly controlled systems. For example, the quality of polymeric material was selective, but the effect could be surpassed by dispersal from the distribution network, and the selective factors ‘upstream’, like chlorination or temperature. During stagnation, spatial dispersal along the continuum of water delivery was insignificant compared to local dispersal from the biofilm to the water phase, which affected both concentration and microbiome composition. Ultimately, controlling bacterial growth in buildings will take advantage of these principles, and will furthermore embrace the inevitable growth in building plumbing pipes and water. Altogether, this research presents valuable steps towards a better understanding and better management of the microbiology within building plumbing.

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

Recent recognition that ecological and evolutionary processes can operate on similar timescales has led to a rapid increase in theoretical and empirical studies on eco-evolutionary dynamics. Progress in the fields of evolutionary biology, genomics and ecology is greatly enhancing our understanding of rapid adaptive processes, the predictability of adaptation and the genetics of ecologically important traits. However, progress in these fields has proceeded largely independently of one another. In an attempt to better integrate these fields, the centre for Adaptation to a Changing Environment' organized a conference entitled The genomic basis of eco-evolutionary change' and brought together experts in ecological genomics and eco-evolutionary dynamics. In this review, we use the work of the invited speakers to summarize eco-evolutionary dynamics and discuss how they are relevant for understanding and predicting responses to contemporary environmental change. Then, we show how recent advances in genomics are contributing to our understanding of eco-evolutionary dynamics. Finally, we highlight the gaps in our understanding of eco-evolutionary dynamics and recommend future avenues of research in eco-evolutionary dynamics.

A central question in microbial ecology is whether microbial interactions are predominantly cooperative or competitive. The secretion of siderophores, microbial iron chelators, is a model system for cooperative interactions. However, siderophores have also been shown to mediate competition by sequestering available iron and making it unavailable to competitors. The details of how siderophores mediate competition are not well understood, especially considering the complex distribution of iron phases in the environment. One pertinent question is whether sequestering iron through siderophores can indeed be effective in natural conditions; many natural environments are characterized by large pools of precipitated iron, and it is conceivable that any soluble iron that is sequestered by siderophores is replenished by the dissolution of these precipitated iron sources. Our goal here was to address this issue, and investigate the magnitude and mechanism of siderophore-mediated competition in the presence of precipitated iron. We combined experimental work with thermodynamic modeling, using Pseudomonas aeruginosa as a model system and ferrihydrite precipitates as the iron source with low solubility. Our experiments show that competitive growth inhibition by the siderophore pyochelin is indeed efficient, and that inhibition of a competitor can even have a stronger growth-promoting effect than solubilization of precipitated iron. Based on the results of our thermodynamic models we conclude that the observed inhibition of a competitor is effective because sequestered iron is only very slowly replenished by the dissolution of precipitated iron. Our research highlights the importance of competitive benefits mediated by siderophores, and underlines that the dynamics of siderophore production and uptake in environmental communities could be a signature of competitive, not just cooperative, dynamics.

Bacterial resistance against antibiotic substances is a growing problem worldwide. Antibiotic resistance increases the risk of prolonged treatment, treatment failure and mortality. The prevalence and distribution of antibiotic resistance varies around the world and there is currently neither an organized global data collection nor a methodological standard. This leads to gaps of surveillance, especially in developing countries. In this study, we developed a multiplex-ligation dependent probe amplification (MLPA) assay for the detection of antibiotic resistance genes in large numbers of samples with a focus on b-lactamase genes. Thirty distinct DNA probe pairs were designed to target 1114 different b-lactamases genes and were successfully tested with artificial DNA templates. Additionally, twenty out of thirty probe pairs were effectively evaluated on 26 Gram-negative control strains carrying 29 b-lactamase genes. This MLPA assay is a promising approach for the cost effective detection of antibiotic resistance genes in large numbers of samples to contribute to the global surveillance of antibiotic resistance gene epidemiology.

LinA-type1 and LinA-type2 are two well characterized variants of the enzyme 'hexachlorocyclohexane (HCH)-dehydrochlorinase'. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinAtype1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.

Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

Combined partial nitritation–anammox (PN/A) systems are increasingly being employed for sustainable removal of nitrogen from wastewater, but process instabilities present ongoing challenges for practitioners. The goal of this study was to elucidate differences in process stability between PN/A process variations employing two distinct aggregate types: biofilm [in moving bed biofilm reactors (MBBRs)] and suspended growth biomass. Triplicate reactors for each process variation were studied under baseline conditions and in response to a series of transient perturbations. MBBRs displayed elevated NH₄⁺ removal rates relative to those of suspended growth counterparts over six months of unperturbed baseline operation but also exhibited significantly greater variability in performance. Transient perturbations led to strikingly divergent yet reproducible behavior in biofilm versus suspended growth systems. A temperature perturbation prompted a sharp reduction in NH₄⁺ removal rates with no accumulation of NO₂^– and rapid recovery in MBBRs, compared to a similar reduction in NH₄⁺ removal rates but a high level of accumulation of NO₂^– in suspended growth reactors. Pulse additions of a nitrification inhibitor (allylthiourea) prompted only moderate declines in performance in suspended growth reactors compared to sharp decreases in NH₄⁺ removal rates in MBBRs. Quantitative fluorescence in situ hybridization demonstrated a significant enrichment of anammox in MBBRs compared to suspended growth reactors, and conversely a proportionally higher AOB abundance in suspended growth reactors. Overall, MBBRs displayed significantly increased susceptibility to transient perturbations employed in this study compared to that of suspended growth counterparts (stability parameter), including significantly longer recovery times (resilience). No significant difference in the maximal impact of perturbations (resistance) was apparent. Taken together, our results suggest that aggregate architecture (biofilm vs suspended growth) in PN/A processes exerts an unexpectedly strong influence on process stability.

Biodegradable polyesters have a large potential to replace persistent polymers in numerous applications and to thereby reduce the accumulation of plastics in the environment. Ester hydrolysis by extracellular carboxylesterases is considered the rate-limiting step in polyester biodegradation. In this work, we systematically investigated the effects of polyester and carboxylesterase structure on the hydrolysis of nanometer-thin polyester films using a quartz-crystal microbalance with dissipation monitoring. Hydrolyzability increased with increasing polyester-chain flexibility as evidenced from differences in the hydrolysis rates and extents of aliphatic polyesters varying in the length of their dicarboxylic acid unit and of poly(butylene adipate-co-terephthalate) (PBAT) polyesters varying in their terephthalate-to-adipate ratio by Rhizopus oryzae lipase and Fusarium solani cutinase. Nanoscale nonuniformities in the PBAT films affected enzymatic hydrolysis and were likely caused by domains with elevated terephthalate contents that impaired enzymatic hydrolysis. Yet, the cutinase completely hydrolyzed all PBAT films, including films with a terephthalate-to-adipate molar ratio of one, under environmentally relevant conditions (pH 6, 20 °C). A comparative analysis of the hydrolysis of two model polyesters by eight different carboxylesterases revealed increasing hydrolysis with increasing accessibility of the enzyme active site. Therefore, this work highlights the importance of both polyester and carboxylesterase structure to enzymatic polyester hydrolysis.

Biodegradable polyesters have the potential to replace nondegradable, persistent polymers in numerous applications and thereby alleviate plastic accumulation in the environment. Herein, we present an analytical approach to study enzymatic hydrolysis of polyesters, the key step in their overall biodegradation process. The approach is based on embedding fluorescein dilaurate (FDL), a fluorogenic ester substrate, into the polyester matrix and on monitoring the enzymatic cohydrolysis of FDL to fluorescein during enzymatic hydrolysis of the polyester. We validated the approach against established techniques using FDL-containing poly(butylene adipate) films and Fusarium solani cutinase (FsC). Implemented on a microplate reader platform, the FDL-based approach enabled sensitive and high-throughput analysis of the enzymatic hydrolysis of eight aliphatic polyesters by two fungal esterases (FsC and Rhizopus oryzae lipase) at different temperatures. While hydrolysis rates for both enzymes increased with decreasing differences between the polyester melting temperatures and the experimental temperatures, this trend was more pronounced for the lipase than the cutinase. These trends in rates could be ascribed to a combination of temperature-dependent polyester chain flexibility and accessibility of the enzyme active site. The work highlights the capability of the FDL-based approach to be utilized in both screening and mechanistic studies of enzymatic polyester hydrolysis.

Biology is marked by a hierarchical organization: all life consists of cells; in some cases, these cells assemble into groups, such as endosymbionts or multicellular organisms; in turn, multicellular organisms sometimes assemble into yet other groups, such as primate societies or ant colonies. The construction of new organizational layers results from hierarchical evolutionary transitions, in which biological units (e.g., cells) form groups that evolve into new units of biological organization (e.g., multicellular organisms). Despite considerable advances, there is no bottom-up, dynamical account of how, starting from the solitary ancestor, the first groups originate and subsequently evolve the organizing principles that qualify them as new units. Guided by six central questions, we propose an integrative bottom-up approach for studying the dynamics underlying hierarchical evolutionary transitions, which builds on and synthesizes existing knowledge. This approach highlights the crucial role of the ecology and development of the solitary ancestor in the emergence and subsequent evolution of groups, and it stresses the paramount importance of the life cycle: only by evaluating groups in the context of their life cycle can we unravel the evolutionary trajectory of hierarchical transitions. These insights also provide a starting point for understanding the types of subsequent organizational complexity. The central research questions outlined here naturally link existing research programs on biological construction (e.g., on cooperation, multilevel selection, self-organization, and development) and thereby help integrate knowledge stemming from diverse fields of biology.

Individuality and collective behavior are two terms that the general public might not readily associate with bacteria, yet both concepts have become central in recent studies of bacterial populations. Bacteria in clonal populations share the same genetic information, yet they can differ substantially in their phenotype. This is partly due to cells adapting their phenotype to local environmental differences. However, the stochastic nature of gene expression can lead to phenotypic heterogeneity even in homogenous environments, a concept that is known as bacterial individuality. Furthermore, bacteria do not live in isolation, but rather interact with other members of their population. By exchanging molecules through the environment cells can affect each other’s activities. These interactions can have important consequences: they allow cells to achieve functionality at the level of the group that is not available to cells in isolation. Interactions can thus give rise to emergent collective behavior.

Both phenotypic differentiation and intercellular interactions can take place in well mixed systems (as will be shown in Chapter 4), however both are much more pronounced in spatially structured environments. The uptake and release of molecules by cells living in these structured environments can quickly generate gradients in environmental conditions that promote phenotypic differentiation (as will be shown in Chapter 3). Furthermore, the sharing of a micro-environment can facilitate intercellular interactions (as will be shown in Chapter 2).

The central theme addressed in these thesis is the question of how the behavior of a cell is affected by the presence of other cells in the population. Specifically, I will address the following three questions: i) how can clonal population arrange their activities in space? ii) how can interactions between cells give rise to collective behavior? and iii) what are the functional consequences of these two processes?

In Chapter 2 I asked the question if and how cells can arrange their activities in space. To address this question we used fluorescent reporters and time-lapse microscopy to follow the growth and gene expression dynamics in Escherichia coli microcolonies. We found that the gene expression dynamics of neighboring cells were strongly correlated for genes involved in stress response and metabolism. Using a newly developed statistical approach we showed that these spatial correlations had three main causes: i) lineage history dependence; ii) emergent gradients in environmental conditions; and iii) local intercellular interactions. Taken together, our data showed that expression patterns in bacterial colonies were highly non-random.

In Chapter 3 I studied how emergent gradients in nutrients can cause phenotypic differentiation. We investigated the consequences of this differentiation for the ability of the population to respond to changes in the environment. We grew E. coli cells in two-dimensional growth chambers in microfluidic devices under glucose limiting conditions. We found evidence that the population diversified in metabolically distinct subpopulations that engaged in a cross-feeding interaction. Furthermore, we showed that the different subpopulations varied strongly in their response to a change in nutrient conditions. Taken together, our data showed that by modifying their environment cells could generate a highly structured and phenotypically diverse population.

In Chapter 4 I studied how interactions between cells can affect their ability to survive periods of antibiotic exposure. We studied E. coli populations that produce colicin, a toxic protein (bacteriocin) that is thought to play an important role in inter-strain warfare. Here we showed that colicins can also lead to tolerance to antibiotic exposure. Importantly, we found that cells only became tolerant when they could take up colicin produced by clonemates; intracellularly produced colicin did not have any measurable effect. The tolerance to antibiotics is thus an emergent property of the population.

In all three chapters we observed that interactions between cells led to emergent behavior at the population level. Emergent behavior can have important consequences for bacterial populations: it can create functionality at the level of the group that is not available to cells living in isolation. Chapter 5 addresses the question of how such emergent behavior could have evolved.

To survive antibiotics, bacteria use two different strategies: counteracting antibiotic effects by expression of resistance genes or evading their effects e.g. by persisting inside host cells. Since bacterial adhesins provide access to the shielded, intracellular niche and the adhesin type 1 fimbriae increases bacterial survival chances inside macrophages, we asked if fimbriae also influenced survival by antibiotic evasion. Combined gentamicin survival assays, flow cytometry, single cell microscopy and kinetic modeling of dose response curves showed that type 1 fimbriae increased the adhesion and internalization by macrophages. This was caused by strongly decreased off-rates and affected the number of intracellular bacteria but not the macrophage viability and morphology. Fimbriae thus promote antibiotic evasion which is particularly relevant in the context of chronic infections.

Within 2 months, two water sources in a karst area in Switzerland were sampled 9 times each, and analyzed by real-time PCR for 6 EHEC O-types, Shiga-like-toxin (stx1 and stx2) and intimin (eae) genes. With the exception of O111, 5 O-types were recorded regularly and at high frequencies (O26: 33.3 %; O157: 33.3 %; O104: 66.6 %; O103: 72.2 %; O145: 94.4 %). Genes for Shiga-like-toxins and intimin were almost omnipresent (stx1: 77.8 %; stx2: 83.3 %; eae: 77.8 %). Strain isolation was undertaken for O-groups 26, 103, 104, 145 and 157. Sample selection for strain isolation was based on Cq-values for the O-groups and stx1, stx2 and eae. From selected samples, frozen enrichment cultures were cultivated on EHLY-agar and 50 typical colonies screened for the O-type and genes encoding for stx1, stx2 and eae. With this approach, only one virulent EHEC-strain could be isolated (Escherichia coli O103, stx1 +; stx2 -; eae +). We carried out one extensive testing with 800 colonies of O-group O145, and no virulent strain was isolated. Our findings showed that PCR-results are not sufficient to formulate epidemiological conclusions and that the isolation of strains is necessary. However, as the detection procedure of EHEC in foods is cumbersome and expensive, the appropriateness of such an approach in official food control is a matter of debate.

Bacteria are an integral part of natural and engineered aquatic ecosystems. They contribute to many ecosystem processes (e.g., food webs, nutrient cycling) but are also used to remove nutrients in drinking water treatment (e.g., in biological filtration). However, bacteria can also have negative impacts on drinking water quality and infrastructure (e.g., biologically induced corrosion, aesthetic problems) and even pose a health risk.
All aquatic ecosystems experience perturbations at relatively short time scales from seconds to days and thus dynamics in bacterial concentrations are to be expected on similar time scales. Knowledge on such short-term microbial dynamics is important for a better understanding of natural and engineered ecosystem functioning. In drinking water production, knowledge on fluctuations in raw water quality as well as during treatment, storage, and distribution is crucial for appropriate design and operation of these systems. Furthermore, many basic laboratory experiments would strongly benefit from highly resolved, direct detection of bacterial concentrations. [...]

Bakterien sind ein fester Bestandteil von natürlichen und technischen Ökosystemen. Sie wirken bei vielen Prozessen in Ökosystemen mit (z.B. Nahrungsnetze, Nährstoffkreisläufe), werden aber auch für die Reduktion von Nährstoffen in der Trinkwasseraufbereitung eingesetzt (z.B. biologische Filtration). Allerdings können Bakterien auch negative Auswirkungen auf die Trinkwasserqualität und -infrastruktur haben (z.B. biologisch induzierte Korrosion, ästhetische Probleme) und sogar eine Gesundheitsgefährdung darstellen.
Alle aquatischen Ökosysteme erfahren Perturbationen auf relativ kurzen Zeitskalen von Sekunden bis Tagen und folglich ist zu erwarten, dass auch Dynamiken in der Bakterienkonzentration auf diesen Zeitskalen auftreten. Kenntnisse über solche kurzzeitige mikrobiologische Dynamiken sind wichtig für ein besseres Verständnis des Funktionierens von natürlichen und technischen Ökosystemen. In der Trinkwasserproduktion ist Wissen über Fluktuationen im Rohwasser aber auch in der Aufbereitung, Speicherung und Verteilung essentiell für die angemessene Planung und den Betrieb solcher Systeme. Des Weiteren würden auch viele grundlegende Laborexperimente stark von hochaufgelösten, direkten Messungen von Bakterienkonzentrationen profitieren. [...]

Detailed measurements of physical, chemical and biological dynamics in groundwater are key to understanding the important processes in place and their influence on water quality – particularly when used for drinking water. Measuring temporal bacterial dynamics at high frequency is challenging due to the limitations in automation of sampling and detection of the conventional, cultivation-based microbial methods. In this study, fully automated online flow cytometry was applied in a groundwater system for the first time in order to monitor microbial dynamics in a groundwater extraction well. Measurements of bacterial concentrations every 15 minutes during 14 days revealed both aperiodic and periodic dynamics that could not be detected previously, resulting in total cell concentration (TCC) fluctuations between 120 and 280 cells μL⁻¹. The aperiodic dynamic was linked to river water contamination following precipitation events, while the (diurnal) periodic dynamic was attributed to changes in hydrological conditions as a consequence of intermittent groundwater extraction. Based on the high number of measurements, the two patterns could be disentangled and quantified separately. This study i) increases the understanding of system performance, ii) helps to optimize monitoring strategies, and iii) opens the possibility for more sophisticated (quantitative) microbial risk assessment of drinking water treatment systems.

Der Einsatz der Online-Durchflusszytometrie eröffnet neue spannende Einblicke in mikrobiologische Dynamiken auf kurzen Zeitskalen in natürlichen sowie technischen Wassersystemen. In Trinkwassersystemen treten solche Dynamiken sowohl im Rohwasser als auch am Wasserhahn auf. Das mit solchen neuen Methoden generierte Wissen über kurzzeitige Schwankungen der mikrobiologischen Wasserqualität eröffnet eine neue Dimension im gezielten Management von Wasserressourcen.

Short-term fluctuations in bacterial concentrations in drinking water systems, occurring on time scales of hours-to-weeks, are essentially unexplored due to a lack of microbial monitoring tools that allow high frequency measurements. Here, we applied fully automated online flow cytometry to measure the total cell concentrations (TCC) in both raw water (karstic groundwater) and treated water (flocculation – ultrafiltration (UF) – ozonation – granular active carbon (GAC) filtration) during a period of 70 days at high temporal resolution (n > 4000 for both water types). We detected and characterized in considerable detail aperiodic fluctuations in the raw water following regional precipitation, with TCC increasing up to 50-fold from a dry weather baseline of approximately 120 cells μl⁻¹ to an event peak of > 5000 cells μl⁻¹. Moreover, we observed the buffering of the treatment plant against these fluctuations, but in addition we recorded a completely unexpected periodic fluctuation of TCC in the treated water after GAC filtration. We concluded that the latter was the result of fluctuating water abstraction from the treatment plant reservoir by two connected water utilities, which resulted in variations in water throughput in the plant. This in turn influenced bacterial detachment and dilution in the GAC filter. This study provides strong evidence of multiple different microbial dynamics occurring in a drinking water treatment system. Given numerous possible sources of natural and operational fluctuations in raw water and drinking water treatment plants, such microbial fluctuations should be expected in many systems. The high-frequency monitoring approach presented herein can improve the understanding and eventual mitigation of such fluctuations.

In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported.

Hintergrund: Die Häufigkeit antibiotikaresistenter Keime nimmt weltweit seit Jahren zu. Infektionen mit solchen Keimen sind schwieriger, manchmal sogar gar nicht mehr mit Antibiotika behandelbar, was erhebliche medizinische Probleme und zusätzliche Kosten für das Gesundheitswesen mit sich bringt. Die Bakterien in der Umwelt sind ein natürliches Reservoir für Resistenzgene. Aber antibiotikaresistente Bakterien gelangen zum Beispiel auch mit menschlichen oder tierischen Ausscheidungen in die Umwelt. Mit dem Abwasser oder mit dem Oberflächenabfluss gelangen so zusätzlich resistente Organismen in die Gewässer. Die Verbreitung von Resistenzen in der Umwelt wird zunehmend mit Sorge beobachtet. Zur Abschätzung des Risikos solcher Emissionen von resistenten Keimen ist es wichtig die Exposition der Population abzuschätzen. Ein möglicher Weg für die Aufnahme von Resistenten Bakterien durch den Menschen stellt das Trinkwasser dar. Für den SVGW war deshalb die Frage relevant in wie weit resistente Bakterien im Trinkwasser von Schweizer Trinkwasserversorgern vorhanden sind und ob für die Qualität des Trinkwassers in dieser Hinsicht Handlungsbedarf besteht. Das durchgeführte Projekt sollte einen ersten Überblick über das Vorkommen von antibiotikaresistenten Bakterien im Trinkwasser und die Dynamik während der Trinkwasseraufbereitung geben.
Vorgehen: Durchgeführt wurde eine Untersuchung von acht Trinkwasserproduzenten der Schweiz. An jedem Standort wurden Proben von Rohwasser, aufbereitetem Wasser, und Netzwasser auf das Vorkommen von Bakterien mit Resistenzen gegen verschiedene Kombinationen von Antibiotika und auf die Häufigkeit verschiedener Resistenzgene untersucht.
Ergebnisse: Resistente Bakterien waren erwartungsgemäss im Rohwasser häufiger als in aufbereitetem Trinkwasser. Die Belastung war lokal sehr unterschiedlich, Seewasser wies dabei tendenziell höhere und zeitlich variablere Werte auf als Grund- oder Infiltrationswasser. Die Trinkwasseraufbereitung führte in den meisten Fällen zu einer deutlichen Reduktion der Zahl der kultivierbaren resistenten Bakterien. Resistenzen gegen Antibiotika der neueren Generation (Norfloxacin und Ceftazidim) wurden im aufbereiteten Wasser praktisch gar nicht gefunden. Allerdings nahm die Zahl resistenter Bakterien an mehreren Standorten nach der Netzpassage wieder zu. Auch deuten die Ergebnisse für bestimmte Standorte auf eine erhöhte relative Abundanz resistenter Bakterien nach der Aufbereitung hin, also auf eine mögliche Selektion von Resistenzen durch die Aufbereitung. Resistenzgene konnten an allen Standorten nur teilweise nachgewiesen werden, was ebenso wie die geringe Zahl der kultivierbaren antibiotikaresistenten Bakterien die insgesamt geringe Belastung der untersuchten Wasserproben belegt. Die Ergebnisse liefern daher keine Hinweise darauf, dass Trinkwasser in der Schweiz eine bedeutende Rolle für die Exposition der Bevölkerung gegenüber resistenten Bakterien bzw. genetischen Resistenzfaktoren spielen könnte. Allerdings zeigen die Ergebnisse auch, dass es in einigen Trinkwasserbetrieben noch Verbesserungspotential bei der Elimination von resistenten Bakterien oder bei der mikrobiologischen Stabilisierung des Trinkwassers für den Netztransport gibt.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

Assemblages of microbial genotypes growing together can display surprisingly complex and unexpected dynamics and result in community-level functions and behaviors that are not readily expected from analyzing each genotype in isolation. This complexity has, at least in part, inspired a discipline of synthetic microbial ecology. Synthetic microbial ecology focuses on designing, building and analyzing the dynamic behavior of ‘ecological circuits’ (i.e. a set of interacting microbial genotypes) and understanding how community-level properties emerge as a consequence of those interactions. In this review, we discuss typical objectives of synthetic microbial ecology and the main advantages and rationales of using synthetic microbial assemblages. We then summarize recent findings of current synthetic microbial ecology investigations. In particular, we focus on the causes and consequences of the interplay between different microbial genotypes and illustrate how simple interactions can create complex dynamics and promote unexpected community-level properties. We finally propose that distinguishing between active and passive interactions and accounting for the pervasiveness of competition can improve existing frameworks for designing and predicting the dynamics of microbial assemblages.

Environmental conditions unsuitable for microbial growth are the rule rather than the exception in most habitats. In response to this, microorganisms have developed various strategies to withstand environmental conditions that limit active growth. Endospore-forming Firmicutes (EFF) deploy a myriad of survival strategies in order to resist adverse conditions. Like many bacterial groups, they can form biofilms and detect nutrient scarcity through chemotaxis. Moreover, within this paraphyletic group of Firmicutes, ecophysiological optima are diverse. Nonetheless, a response to adversity that delimits this group is the formation of wet-heat resistant spores. These strategies are energetically demanding and therefore might affect the biological success of EFF. Therefore, we hypothesize that abundance and diversity of EFF should be maximized in those environments in which the benefits of these survival strategies offsets the energetic cost. In order to address this hypothesis, geothermal and mineral springs and drillings were selected because in these environments of steep physicochemical gradients, diversified survival strategies may become a successful strategy.We collected 71 samples from geothermal and mineral environments characterized by none (null), single or multiple limiting environmental factors (temperature, pH, UV radiation, and specific mineral composition). To measure success, we quantified EFF gene copy numbers (GCN; spo0A gene) in relation to total bacterial GCN (16S rRNA gene), as well as the contribution of EFF to community composition. The quantification showed that relative GCN for EFF reached up to 20% at sites characterized by multiple limiting environmental factors, whereas it corresponded to less than 1% at sites with one or no limiting environmental factor. Pyrosequencing of the 16S rRNA gene supports a higher contribution of EFF at sites with multiple limiting factors. Community composition suggested a combination of phylotypes for which active growth could be expected, and phylotypes that are most likely in the state of endospores, in all the sites. In summary, our results suggest that diversified survival strategies, including sporulation and metabolic adaptations, explain the biological success of EFF in geothermal and natural springs, and that multiple extreme environmental factors favor the prevalence of EFF.

Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz^® solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high.

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and β-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of β- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of β- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.

The main removal process for polar organic micropollutants during activated sludge treatment is biotransformation, which often leads to the formation of stable transformation products (TPs). Because the analysis of TPs is challenging, the use of pathway prediction systems can help by generating a list of suspected TPs. To complete and refine pathway prediction, comprehensive biotransformation studies for compounds exhibiting pertinent functional groups under environmentally relevant conditions are needed. Because many polar organic micropollutants present in wastewater contain one or several amine functional groups, we systematically explored amine biotransformation by conducting experiments with 19 compounds that contained 25 structurally diverse primary, secondary, and tertiary amine moieties. The identification of 144 TP candidates and the structure elucidation of 101 of these resulted in a comprehensive view on initial amine biotransformation reactions. The reactions with the highest relevance were N-oxidation, N-dealkylation, N-acetylation, and N-succinylation. Whereas many of the observed reactions were similar to those known for the mammalian metabolism of amine-containing xenobiotics, some N-acylation reactions were not previously described. In general, different reactions at the amine functional group occurred in parallel. Finally, recommendations on how these findings can be implemented to improve microbial pathway prediction of amine-containing micropollutants are given.

Phosphate remobilization from digested sewage sludge containing iron phosphate was scaled-up in a microbial fuel cell (MFC). A 3 litre triple chambered MFC was constructed. This reactor was operated as a microbial fuel cell and later as a microbial electrolysis cell to accelerate cathodic phosphate remobilization. Applying an additional voltage and exceeding native MFC power accelerated chemical base formation and the related phosphate remobilization rate. The electrolysis approach was extended using a platinum-RVC cathode. The pH rose to 12.6 and phosphate was recovered by 67% in 26 h. This was significantly faster than using microbial fuel cell conditions. Shrinking core modelling particle fluid kinetics showed that the reaction resistance has to move inside the sewage sludge particle for considerable rate enhancement. Remobilized phosphate was subsequently precipitated as struvite and inductively coupled plasma mass spectrometry indicated low levels of cadmium, lead, and other metals as required by law for recycling fertilizers.

Cooperation and diversity abound in nature despite cooperators risking exploitation from defectors and superior competitors displacing weaker ones. Understanding the persistence of cooperation and diversity is therefore a major problem for evolutionary ecology, especially in the context of wellmixed populations, where the potential for exploitation and displacement is greatest. Here, we demonstrate that a 'loner effect', described by economic game theorists, can maintain cooperation and diversity in real-world biological settings. We use mathematical models of public-good-producing bacteria to show that the presence of a loner strain, which produces an independent but relatively inefficient good, can lead to rock–paper–scissor dynamics, whereby cooperators outcompete loners, defectors outcompete cooperators and loners outcompete defectors. These model predictions are supported by our observations of evolutionary dynamics in well-mixed experimental communities of the bacterium Pseudomonas aeruginosa.We find that the coexistence of cooperators and defectors that produce and exploit, respectively, the iron-scavenging siderophore pyoverdine, is stabilized by the presence of lonerswith an independent iron-uptake mechanism. Our results establish the loner effect as a simple and general driver of cooperation and diversity in environments that would otherwise favour defection and the erosion of diversity.

Every year, more than half a million children die due to diarrheal diseases. Recent studies have identified the most important etiologies of diarrheal disease are enterotoxigenic and enteropathogenic E. coli, Shigella spp., rotavirus, norovirus and Cryptosporidium spp. These etiologies are unsurprisingly characterized by a combination of high shedding, high infectivity, and transmissibility through multiple environmental reservoirs. The relative importance of the transmission routes is likely site-specific. So the impact of interventions, which typically target only one or two environmental reservoirs, is likely also site-specific. The factors influencing the transmission routes most important for diarrheal disease are complex, including – at a minimum – etiology of endemic disease; and water, sanitation, and hygiene infrastructure and practices. The site-specific nature – and complexity of transmission – helps explain the observed variation in impacts of water, sanitation, and hygiene interventions. It may also render efforts to estimate or quantify global means for interventions' impacts irrelevant. The theme of this Perspective is that greater reductions in diarrheal disease transmission in LMICs can be achieved by designing interventions to interrupt the most important environmental transmission pathways. Intervention choice should be informed by site-specific conditions, most notably: diarrheal etiology and existing water, sanitation, and hygiene infrastructure and practices. The theme is discussed through the lens of the characteristics of the most important diarrheal diseases (shedding, infectivity, growth, and persistence) and the general characteristics of environmental reservoirs (exposure pathways and fecal contamination). The discussion highlights when interventions – and combinations of interventions – will be most effective at reducing diarrheal disease burden.

There is a growing interest in controlling—promoting or avoiding—the invasion of microbial communities by new community members. Resource availability and community structure have been reported as determinants of invasion success. However, most invasion studies do not adhere to a coherent and consistent terminology nor always include rigorous interpretations of the processes behind invasion. Therefore, we suggest that a consistent set of definitions and a rigorous conceptual framework are needed. We define invasion in a microbial community as the establishment of an alien microbial type in a resident community and argue how simple criteria to define aliens, residents, and alien establishment can be applied for a wide variety of communities. In addition, we suggest an adoption of the community ecology framework advanced by Vellend (2010) to clarify potential determinants of invasion. This framework identifies four fundamental processes that control community dynamics: dispersal, selection, drift and diversification. While selection has received ample attention in microbial community invasion research, the three other processes are often overlooked. Here, we elaborate on the relevance of all four processes and conclude that invasion experiments should be designed to elucidate the role of dispersal, drift and diversification, in order to obtain a complete picture of invasion as a community process.

Kunststoffe, die in Kontakt mit Trinkwasser kommen, müssen hohen Anforderungen genügen. Unter anderem sollte durch deren Migrationseigenschaften weder der chemische noch der mikrobiologische Zustand eines Trinkwassers zu stark beeinflusst werden. Im Rahmen eines Projekts der Kommission für Technologie und Innovation (KTI) wurde die Aussagekraft unterschiedlicher Testverfahren zur Ermittlung des Verkeimungspotenzials von Kunststoffen verglichen.

Die Erstbefüllung und Inbetriebnahme einer neuen Trinkwasserinstallation stellt hohe Anforderungen an den Planer und Installateur. Gerade die Mikrobiologie ist ein oft vernachlässigter Mitspieler, der bei mangelnder Sorgfalt schnell zum Spielverderber wird. Denn haben sich krankheitserregende Mikroorganismen im Leitungsnetz erst mal eingenistet, lassen sie sich meist nur durch kostenintensive Massnahmen entfernen. Das mikrobiologische Kontaminationsrisiko kann jedoch bereits mit einfachen Mitteln und ohne grösseren Aufwand reduziert werden.

DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface.

Inactivation kinetics of autochthonous bacteria during ozonation of wastewater effluents were investigated using cultivation-independent flow cytometry (FCM) with total cell count (TCC) and intact cell count (ICC) and intracellular adenosine triphosphate (ATP) analysis. The principles of the methods including ozone inactivation kinetics were demonstrated with laboratory-cultured Escherichia coli spiked into filtered and sterilized wastewater effluent. Both intracellular ATP and ICC decreased with increasing ozone doses, with ICC being the more conservative parameter. The log-inactivation levels (−log(N/N₀) of E. coli reached the method detection limits for FCM (∼3) and ATP (∼1.7) at specific ozone doses of ≥0.5 gO₃/gDOC. During ozonation of four real wastewater effluents, the log-inactivation of autochthonous bacteria with FCM ICC was 0.3–1.0 for 0.25 gO₃/gDOC and increased to 1.1–2.1 for 0.5 gO₃/gDOC, but remained at a similar level of 1.5–2.8 for a further increase of the specific ozone doses to 1.0 and 1.5 gO₃/gDOC. The FCM data also showed that autochthonous bacteria were composed of communities with high and low ozone reactivity. The inactivation levels measured with intracellular ATP were reasonably correlated to ICC (r² = 0.8). Overall, FCM and ATP measurements were demonstrated to be useful tools to monitor the inactivation of autochthonous bacteria during ozonation of municipal wastewater effluents.

Different microbial cell types typically specialize at performing different metabolic processes. A canonical example is substrate cross-feeding, where one cell type consumes a primary substrate into an intermediate and another cell type consumes the intermediate. While substrate cross-feeding is widely observed, its consequences on ecosystem processes is often unclear. How does substrate cross-feeding affect the rate or extent of substrate consumption? We hypothesized that substrate cross-feeding eliminates competition between different enzymes and reduces the accumulation of growth-inhibiting intermediates, thus accelerating substrate consumption. We tested this hypothesis using isogenic mutants of the bacterium Pseudomonas stutzeri that either completely consume nitrate to dinitrogen gas or cross-feed the intermediate nitrite. We demonstrate that nitrite cross-feeding eliminates inter-enzyme competition and, in turn, reduces nitrite accumulation. We further demonstrate that nitrite cross-feeding accelerates substrate consumption, but only when nitrite has growth-inhibiting effects. Knowledge about inter-enzyme competition and the inhibitory effects of intermediates could therefore be important for deciding how to best segregate different metabolic processes into different microbial cell types to optimize a desired biotransformation.

Filamentous organisms represent an example where incomplete separation after cell division underlies the development of multicellular formations. With a view to understanding the evolution of more complex multicellular structures, we explore the transition of multicellular growth from one to two dimensions. We develop a computational model to simulate multicellular development in populations where cells exhibit density-dependent division and death rates. In both the one- and two-dimensional contexts, multicellular formations go through a developmental cycle of growth and subsequent decay. However, the model shows that a transition to a higher dimension increases the size of multicellular formations and facilitates the maintenance of large cell clusters for significantly longer periods of time. We further show that the turnover rate for cell division and death scales with the number of iterations required to reach the stationary multicellular size at equilibrium. Although size and life cycles of multicellular organisms are affected by other environmental and genetic factors, the model presented here evaluates the extent to which the transition of multicellular growth from one to two dimensions contributes to the maintenance of multicellular structures during development.

Most bacteria live in ever-changing environments where periods of stress are common. One fundamental question is whether individual bacterial cells have an increased tolerance to stress if they recently have been exposed to lower levels of the same stressor. To address this question, we worked with the bacterium Caulobacter crescentus and asked whether exposure to a moderate concentration of sodium chloride would affect survival during later exposure to a higher concentration. We found that the effects measured at the population level depended in a surprising and complex way on the time interval between the two exposure events: The effect of the first exposure on survival of the second exposure was positive for some time intervals but negative for others. We hypothesized that the complex pattern of history dependence at the population level was a consequence of the responses of individual cells to sodium chloride that we observed: (i) exposure to moderate concentrations of sodium chloride caused delays in cell division and led to cell-cycle synchronization, and (ii) whether a bacterium would survive subsequent exposure to higher concentrations was dependent on the cell-cycle state. Using computational modeling, we demonstrated that indeed the combination of these two effects could explain the complex patterns of history dependence observed at the population level. Our insight into how the behavior of single cells scales up to processes at the population level provides a perspective on how organisms operate in dynamic environments with fluctuating stress exposure.

The biotransformation of some micropollutants has previously been observed to be positively associated with ammonia oxidation activities and the transcript abundance of the archaeal ammonia monooxygenase gene (amoA) in nitrifying activated sludge. Given the increasing interest in and potential importance of ammonia-oxidizing archaea (AOA), we investigated the capabilities of an AOA pure culture, Nitrososphaera gargensis, to biotransform ten micropollutants belonging to three structurally similar groups (i.e., phenylureas, tertiary amides, and tertiary amines). N. gargensis was able to biotransform two of the tertiary amines, mianserin (MIA) and ranitidine (RAN), exhibiting similar compound specificity as two ammonia-oxidizing bacteria (AOB) strains that were tested for comparison. The same MIA and RAN biotransformation reactions were carried out by both the AOA and AOB strains. The major transformation product (TP) of MIA, α-oxo MIA was likely formed via a two-step oxidation reaction. The first hydroxylation step is typically catalyzed by monooxygenases. Three RAN TP candidates were identified from nontarget analysis. Their tentative structures and possible biotransformation pathways were proposed. The biotransformation of MIA and RAN only occurred when ammonia oxidation was active, suggesting cometabolic transformations. Consistently, a comparative proteomic analysis revealed no significant differential expression of any protein-encoding gene in N. gargensis grown on ammonium with MIA or RAN compared with standard cultivation on ammonium only. Taken together, this study provides first important insights regarding the roles played by AOA in micropollutant biotransformation.

Following the success of the inaugural games, the Microbial Olympics return with a new series of events and microbial competitors. The games may have moved to a new hosting venue, but the dedication to training, fitness, competition (and yes, education and humour) lives on.

The increasing use of flow cytometry (FCM) for analyses of environmental samples has resulted in a large variety of staining protocols with varying results and limited comparability. Viability assessment with FCM is in this context of particular interest because incorrect staining could severely affect the outcome/interpretation of the results. Here we propose a pipeline for the development and optimization of staining protocols for environmental samples, demonstrated with the common viability dye combination of SYBR Green I (SG) and propidium iodide (PI). Optimization steps included the assessment of dye solvents, determination of suitable PI concentration, and determining the optimal staining temperature and staining time. We demonstrated that dimethyl sulfoxide (DMSO) could impair membrane integrity, when used for SGPI stock solution preparation, and TRIS buffer was chosen as an alternative. Moreover we selected 6 μM as optimal PI final concentration: less than 3 μM resulted in incomplete staining of damaged cells, while concentrations higher that 12 μM resulted in false PI-positive staining of intact cells. Low temperatures (25 °C) resulted in a slow reaction and did not enable the staining of all bacteria, while high temperatures (44 °C) caused damage to cells and false PI-positive results. Hence, 35 °C was selected as optimal staining temperature. We further showed that a minimum of 15 min were necessary to obtain stable staining results. Moreover, we showed that addition of EDTA resulted in 1–39% more PI-positive results compared to an EDTA-free sample, and argue that insufficient evidence currently exist in favor of adding EDTA to all samples in general. Altogether, the data clearly shows the need to be careful, precise and reproducible when staining cells for flow cytometric analyses, and the need to assess and optimize staining protocols with both viable and non-viable bacteria.

Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0–22.4 mg-Cl₂ L⁻¹; 5 min exposure) were applied to an Escherichiacoli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L⁻¹. The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl₂ L⁻¹; 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h⁻¹) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h⁻¹) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

Freshwater lakes represent a major natural source of methane to the atmosphere, a potent greenhouse gas with critical climatic implications. Particularly in stratified lakes methane can potentially build up to high concentrations in the anoxic hypolimnion and be emitted by a variety of flux mechanisms. Microbial methane oxidation, which is principally mediated by anaerobic archaea and aerobic proteobacteria, is the only process impeding methane emissions from lacustrine waters. The majority of occurring methane oxidation in lakes has been ascribed to aerobic methanotrophs, which are preferably located at oxic/anoxic interfaces in both sediments and water columns. However, there is increasing evidence that methane is also oxidized in anoxic hypolimnia of lakes, where oxygen is not available as an oxidizing agent. It is speculated that electron acceptors such as nitrate, nitrite and iron- or manganese oxides could be of relevance there. Though the microorganisms mediating these processes remain elusive, recent laboratory studies revealed novel methanotrophs and pathways for methane oxidation, which could also be essential in lakes. [...]

Methan ist ein starkes Treibhausgas und hoch relevant für Klimafragen. Wichtige Quellen des natürlichen Ausstosses von Methan in die Atmosphäre sind Süsswasserseen. Besonders in geschichteten Seen können hohe Konzentrationen von Methan im anoxischen Hypolimnion akkumuliert und auf verschiedenste Weise emittiert werden. Einzig durch mikrobielle Methanoxidation, welche prinzipiell durch anaerobe Archeaen und aerobe Proteobakterien durchgeführt wird, kann der Ausstoss von Methan aus Seen unterbunden werden. Der Hauptteil der mikrobiellen Methanoxidation wird aeroben methanotrophen Bakterien zugeschrieben, welche sich vorzugsweise in oxisch/anoxischen Grenzschichten von Sedimenten oder Wassersäulen aufhalten. Zunehmend gibt es jedoch Hinweise, dass Methan auch im anoxischen Hypolimnion oxidiert wird, also ohne Verfügbarkeit von Sauerstoff als Oxidationsmittel. Stattdessen könnten unter diesen Bedingungen alternative Elektronenakzeptoren, wie Nitrat, Nitrit und Eisen- oder Manganoxide eine Rolle spielen. Bislang konnte nicht geklärt werden, welche Mikroorganismen diese Prozesse steuern, jedoch wurden kürzlich in Laborstudien neue methanotrophe Mikroorganismen entdeckt, welche ebenfalls eine wichtige Rolle in Seen spielen könnten. [...]

Rationale: Oxygen isotope fractionation of molecular O₂ is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism.
Methods: We developed a simple method to measure the δ¹⁸O values of dissolved O₂ in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N₂ headspace, the dissolved O₂ partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph.
Results: In aqueous samples of 10 mL and in diluted air samples, we quantified the δ¹⁸O values at O₂ concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O₂ and N₂ with a molecular sieve column made it possible to use N₂ as the headspace gas for the extraction of dissolved O₂ from water. We were therefore able to apply a rigorous δ¹⁸O blank correction for the quantification of ¹⁸O/¹⁶O ratios in 20 nmol of injected O₂.
Conclusions: The successful quantification of ¹⁸O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O₂ illustrates how the proposed method can be applied for studying enzymatic O₂ activation mechanisms in a variety of (bio)chemical processes.

Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent ¹³C- and ²H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O₂ activation and aromatic C oxygenation. The contribution of enzymatic O₂ activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O₂ activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

Carbon nanotubes (CNTs) have numerous exciting potential applications and some that have reached commercialization. As such, quantitative measurements of CNTs in key environmental matrices (water, soil, sediment, and biological tissues) are needed to address concerns about their potential environmental and human health risks and to inform application development. However, standard methods for CNT quantification are not yet available. We systematically and critically review each component of the current methods for CNT quantification including CNT extraction approaches, potential biases, limits of detection, and potential for standardization. This review reveals that many of the techniques with the lowest detection limits require uncommon equipment or expertise, and thus, they are not frequently accessible. Additionally, changes to the CNTs (e.g., agglomeration) after environmental release and matrix effects can cause biases for many of the techniques, and biasing factors vary among the techniques. Five case studies are provided to illustrate how to use this information to inform responses to real-world scenarios such as monitoring potential CNT discharge into a river or ecotoxicity testing by a testing laboratory. Overall, substantial progress has been made in improving CNT quantification during the past ten years, but additional work is needed for standardization, development of extraction techniques from complex matrices, and multimethod comparisons of standard samples to reveal the comparability of techniques.

A systematic approach is presented for the assessment of (i) bacterial growth-controlling factors in drinking water and (ii) the impact of distribution conditions on the extent of bacterial growth in full-scale distribution systems. The approach combines (i) quantification of changes in autochthonous bacterial cell concentrations in full-scale distribution systems with (ii) laboratory-scale batch bacterial growth potential tests of drinking water samples under defined conditions. The growth potential tests were done by direct incubation of water samples, without modification of the original bacterial flora, and with flow cytometric quantification of bacterial growth. This method was shown to be reproducible (ca. 4% relative standard deviation) and sensitive (detection of bacterial growth down to 5 µg L⁻¹ of added assimilable organic carbon). The principle of step-wise assessment of bacterial growth-controlling factors was demonstrated on bottled water, shown to be primarily carbon limited at 133 (±18) × 10³ cells mL⁻¹ and secondarily limited by inorganic nutrients at 5,500 (±1,700) × 10³ cells mL⁻¹. Analysis of the effluent of a Dutch full-scale drinking water treatment plant showed (1) bacterial growth inhibition as a result of end-point chlorination, (2) organic carbon limitation at 192 (±72) × 10³ cells mL⁻¹ and (3) inorganic nutrient limitation at 375 (±31) × 10³ cells mL⁻¹. Significantly lower net bacterial growth was measured in the corresponding full-scale distribution system (176 (±25) × 10³ cells mL⁻¹) than in the laboratory-scale growth potential test of the same water (294 (±35) × 10³ cells mL⁻¹), highlighting the influence of distribution on bacterial growth. The systematic approach described herein provides quantitative information on the effect of drinking water properties and distribution system conditions on biological stability, which can assist water utilities in decision-making on treatment or distribution system improvements to better control bacterial growth during water distribution.

Biological stability of drinking water refers to the concept of providing consumers with drinking water of same microbial quality at the tap as produced at the water treatment facility. However, uncontrolled growth of bacteria can occur during distribution in water mains and premise plumbing, and can lead to hygienic (e.g., development of opportunistic pathogens), aesthetic (e.g., deterioration of taste, odor, color) or operational (e.g., fouling or biocorrosion of pipes) problems. Drinking water contains diverse microorganisms competing for limited available nutrients for growth. Bacterial growth and interactions are regulated by factors, such as (i) type and concentration of available organic and inorganic nutrients, (ii) type and concentration of residual disinfectant, (iii) presence of predators, such as protozoa and invertebrates, (iv) environmental conditions, such as water temperature, and (v) spatial location of microorganisms (bulk water, sediment, or biofilm). Water treatment and distribution conditions in water mains and premise plumbing affect each of these factors and shape bacterial community characteristics (abundance, composition, viability) in distribution systems. Improved understanding of bacterial interactions in distribution systems and of environmental conditions impact is needed for better control of bacterial communities during drinking water production and distribution. This article reviews (i) existing knowledge on biological stability controlling factors and (ii) how these factors are affected by drinking water production and distribution conditions. In addition, (iii) the concept of biological stability is discussed in light of experience with well-established and new analytical methods, enabling high throughput analysis and in-depth characterization of bacterial communities in drinking water. We discussed, how knowledge gained from novel techniques will improve design and monitoring of water treatment and distribution systems in order to maintain good drinking water microbial quality up to consumer's tap. A new definition and methodological approach for biological stability is proposed.

Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 10³ cells mL^-1 in winter time up to 455 × 10³ cells mL^-1 in summer time) and in bacterial ATP concentrations (<1–3.6 ng L^-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 10³ cells mL^-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously undocumented seasonal dynamics in the distribution network. Moreover, high-resolution FCM data enabled prediction of bacterial cell concentrations at specific water temperatures and time of year. The study highlights the need to systematically assess temporal fluctuations in parallel to spatial dynamics for individual drinking water distribution systems.

Flexible polymeric pipe materials are commonly used as shower hoses or connections to faucets in the last meters of building plumbing, but these tend to leach high concentrations of carbon that encourage bacterial growth. Here we compared the microbiological impact of six such materials, with both a short-term material comparison test and a daily shower simulator operating for eight months. The materials ranked differently in the migration potential and biomass formation potential assays of the comparison test, but overall these results correlated (R² > 0.77) with long-term biofilm development in the shower simulator. The biofilm concentration after eight months ranged from 2 × 10⁶ cells per cm² on the control material (PE-Xc) to 2 × 10⁸ cells per cm² on a typical shower hose (PVC-P). However, the differences in four of the six materials were much less pronounced after eight months than in the early months. The communities were characterized with 16S rRNA amplicon sequencing clustered with both material and time (R² = 0.31; R² = 0.25), and correlated strongly with the biofilm concentration (R² > 0.74). A universal core consisting of 7 genera accounted for 44% of all sequences, and accounted for more of young and high-biomass biofilms. Genera containing opportunistic pathogens were more common in low-biomass pipes. We conclude that choice of materials is not only critical for determining biofilm concentration, but also community composition. Our results show that a seemingly small choice in plumbing material in the final meters of distribution can make a considerable difference in the building plumbing 'exposome'.

Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity¹ in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future^2-9. It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N₂-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH₄⁺ limitation shapes phenotypic heterogeneity in N₂ fixation. In turn, the N₂ fixation rate of single cells during NH₄⁺ limitation correlates positively with their growth rate after a shift to NH₄⁺ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges—nutrient limitation and fluctuations—that many microorganisms face.

Human-induced environmental changes are causing major shifts in ecosystems around the globe. To support environmental management, scientific research has to infer both general trends and context dependency in these shifts at global and local scales. Combining replicated real-world experiments, which take advantage of implemented mitigation measures or other forms of human impact, with research-led experimental manipulations can provide powerful scientific tools for inferring causal drivers of ecological change and the generality of their effects. Additionally, combining these two approaches can facilitate communication with stakeholders involved in implementing management strategies. We demonstrate such an integrative approach using the case study EcoImpact, which aims at empirically unravelling the impacts of wastewater-born micropollutants on aquatic ecosystems.

The importance of microbial communities (MCs) cannot be overstated. MCs underpin the biogeochemical cycles of the earth's soil, oceans and the atmosphere, and perform ecosystem functions that impact plants, animals and humans. Yet our ability to predict and manage the function of these highly complex, dynamically changing communities is limited. Building predictive models that link MC composition to function is a key emerging challenge in microbial ecology. Here, we argue that addressing this challenge requires close coordination of experimental data collection and method development with mathematical model building. We discuss specific examples where model-experiment integration has already resulted in important insights into MC function and structure. We also highlight key research questions that still demand better integration of experiments and models. We argue that such integration is needed to achieve significant progress in our understanding of MC dynamics and function, and we make specific practical suggestions as to how this could be achieved.

Cleavage of ester bonds by extracellular microbial hydrolases is considered a key step during the breakdown of biodegradable polyester materials in natural and engineered systems. Here we present a novel analytical approach for simultaneous detection of changes in the masses and rigidities of polyester thin films during enzymatic hydrolysis using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). In experiments with poly(butylene succinate) (PBS) and the lipase of Rhizopus oryzae (RoL), we detected complete hydrolysis of PBS thin films at pH 5 and 40 °C that proceeded through soft and water-rich film intermediates. Increasing the temperature from 20 to 40 °C resulted in a larger increase of the enzymatic hydrolysis rate of PBS than of nonpolymeric dibutyl adipate. This finding was ascribed to elevated accessibility of ester bonds to the catalytic site of RoL due to increasing polyester chain mobility. When the pH of the solution was changed from 5 to 7, initial hydrolysis rates were little affected, while a softer film intermediate that lead to incomplete film hydrolysis was formed. Hydrolysis dynamics of PBS, poly(butylene adipate), poly(lactic acid), and poly(ethylene terephthalate) in assays with RoL showed distinct differences that we attribute to differences in the polyester structure.

Die Bevölkerung wird via Medien regelmässig über verkeimte Wasserinstallationen und deren Gefahren informiert. Dies führt dazu, dass Planer ind Installateure vermehrt über mögliche hygienische Präventivmassnamen Auskunft geben müssen. Weiter werden Planer durch neu definierte Grenzwerte in Normen und Labels zu neuen Konzepten gezwungen. Es gilt, ernauerbare Energien oder Abwärme aus thermischen Prozessen zu nutzen. Dabei kommt es oft zum Widerspruch zwischen energetisch optimaler und hygienisch einwandfreier Warmwasserversorgung.