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Cholera is a diarrhoeal disease caused by Vibrio cholerae . It remains a major public health challenge in the endemic region around the Bay of Bengal. Over decadal time scales, one lineage typically dominates the others and spreads in global pandemic waves. However, it remains unclear to what extent diverse lineages co-circulate during a single outbreak season. Defining the pool of diversity during finer time scales is important because the selective pressures that impact V. cholerae - namely antibiotics and phages - are dynamic on these time scales. To study the nationwide diversity of V. cholerae , we long-read sequenced 273 V. cholerae genomes from seven hospitals over one year (2018) in Bangladesh. Four major V. cholerae lineages were identified: known lineages BD-1, BD-2a, and BD-2b, and a novel lineage that we call BD-3. In 2022, BD-1 caused a large cholera outbreak in Dhaka, apparently outcompeting BD-2 lineages. We show that, in 2018, BD-1 was already dominant in the five northern regions, including Dhaka, consistent with an origin from India in the north. By contrast, we observed a higher diversity of lineages in the two southern regions near the coast. The four lineages differed in pangenome content, including integrative and conjugative elements (ICEs) and genes involved in resistance to bacteriophages and antibiotics. Notably, BD-2a lacked an ICE and is predicted to be more sensitive to phages and antibiotics, but nevertheless persisted throughout the year-long sampling period. Genes associated with antibiotic resistance in V. cholerae from Bangladesh in 2006 were entirely absent from all lineages in 2018-19, suggesting shifting costs and benefits of encoding these genes. Together, our results highlight the dynamic nature of the V. cholerae pangenome and the geographic structure of its lineage diversity.

The COVID-19 pandemic led to a large global effort to sequence SARS-CoV-2 genomes from patient samples to track viral evolution and inform the public health response. Millions of SARS-CoV-2 genome sequences have been deposited in global public repositories. The Canadian COVID-19 Genomics Network (CanCOGeN - VirusSeq), a consortium tasked with coordinating expanded sequencing of SARS-CoV-2 genomes across Canada early in the pandemic, created the Canadian VirusSeq Data Portal, with associated data pipelines and procedures, to support these efforts. The goal of VirusSeq was to allow open access to Canadian SARS-CoV-2 genomic sequences and enhanced, standardized contextual data that were unavailable in other repositories and that meet FAIR standards (Findable, Accessible, Interoperable and Reusable). In addition, the portal data submission pipeline contains data quality checking procedures and appropriate acknowledgement of data generators that encourages collaboration. From inception to execution, the portal was developed with a conscientious focus on strong data governance principles and practices. Extensive efforts ensured a commitment to Canadian privacy laws, data security standards, and organizational processes. This portal has been coupled with other resources, such as Viral AI, and was further leveraged by the Coronavirus Variants Rapid Response Network (CoVaRR-Net) to produce a suite of continually updated analytical tools and notebooks. Here we highlight this portal (https://virusseq-dataportal.ca/), including its contextual data not available elsewhere, and the Duotang (https://covarr-net.github.io/duotang/duotang.html), a web platform that presents key genomic epidemiology and modelling analyses on circulating and emerging SARS-CoV-2 variants in Canada. Duotang presents dynamic changes in variant composition of SARS-CoV-2 in Canada and by province, estimates variant growth, and displays complementary interactive visualizations, with a text overview of the current situation. The VirusSeq Data Portal and Duotang resources, alongside additional analyses and resources computed from the portal (COVID-MVP, CoVizu), are all open source and freely available. Together, they provide an updated picture of SARS-CoV-2 evolution to spur scientific discussions, inform public discourse, and support communication with and within public health authorities. They also serve as a framework for other jurisdictions interested in open, collaborative sequence data sharing and analyses.

During the COVID-19 pandemic, the monitoring of SARS-CoV-2 RNA in wastewater was used to track the evolution and emergence of variant lineages and gauge infection levels in the community, informing appropriate public health responses without relying solely on clinical testing. As more sublineages were discovered, it increased the difficulty in identifying distinct variants in a mixed population sample, particularly those without a known lineage. Here, we compare the sequencing technology from Illumina and from Oxford Nanopore Technologies, in order to determine their efficacy at detecting variants of differing abundance, using 248 wastewater samples from various Quebec and Ontario cities. Our study used two analytical approaches to identify the main variants in the samples: the presence of signature and marker mutations and the co-occurrence of signature mutations within the same amplicon. We observed that each sequencing method detected certain variants at different frequencies as each method preferentially detects mutations of distinct variants. Illumina sequencing detected more mutations with a predominant lineage that is in low abundance across the population or unknown for that time period, while Nanopore sequencing had a higher detection rate of mutations that are predominantly found in the high abundance B.1.1.7 (Alpha) lineage as well as a higher sequencing rate of co-occurring mutations in the same amplicon. We present a workflow that integrates short-read and long-read sequencing to improve the detection of SARS-CoV-2 variant lineages in mixed population samples, such as wastewater.

A spatial-genomic analysis reveals that bird species living closer to humans have higher diversity of the pathogen Campylobacter and its antimicrobial resistance genes. This suggests that urbanization could promote pathogen transmission among wild animals and, potentially, humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract., Importance: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution., Competing Interests: The authors declare no conflict of interest.

Wastewater-based surveillance (WBS) is an important epidemiological and public health tool for tracking pathogens across the scale of a building, neighbourhood, city, or region. WBS gained widespread adoption globally during the SARS-CoV-2 pandemic for estimating community infection levels by qPCR. Sequencing pathogen genes or genomes from wastewater adds information about pathogen genetic diversity, which can be used to identify viral lineages (including variants of concern) that are circulating in a local population. Capturing the genetic diversity by WBS sequencing is not trivial, as wastewater samples often contain a diverse mixture of viral lineages with real mutations and sequencing errors, which must be deconvoluted computationally from short sequencing reads. In this study we assess nine different computational tools that have recently been developed to address this challenge. We simulated 100 wastewater sequence samples consisting of SARS-CoV-2 BA.1, BA.2, and Delta lineages, in various mixtures, as well as a Delta-Omicron recombinant and a synthetic 'novel' lineage. Most tools performed well in identifying the true lineages present and estimating their relative abundances and were generally robust to variation in sequencing depth and read length. While many tools identified lineages present down to 1 % frequency, results were more reliable above a 5 % threshold. The presence of an unknown synthetic lineage, which represents an unclassified SARS-CoV-2 lineage, increases the error in relative abundance estimates of other lineages, but the magnitude of this effect was small for most tools. The tools also varied in how they labelled novel synthetic lineages and recombinants. While our simulated dataset represents just one of many possible use cases for these methods, we hope it helps users understand potential sources of error or bias in wastewater sequencing analysis and to appreciate the commonalities and differences across methods.

Despite an increasingly detailed picture of the molecular mechanisms of bacteriophage (phage)-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. In this work, we report a year-long, nationwide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative polymerase chain reaction while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of antiphage defenses, predation was "effective," with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of antiphage defenses, predation was "ineffective," with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.

Cyanobacteria are known to produce diverse secondary metabolites that are toxic to aquatic ecosystems and human health. However, data about the cyanotoxins occurrence and cyanobacterial diversity in Pakistan's drinking water reservoirs is scarce. In this study, we first investigated the presence of microcystin, saxitoxin, and anatoxin in 12 water bodies using an enzyme-linked immunosorbent assay (ELISA). The observed cyanotoxin values for the risk quotient (RQ) determined by ELISA indicated a potential risk for aquatic life and human health. Based on this result, we made a more in-depth investigation with a subset of water bodies (served as major public water sources) to analyze the cyanotoxins dynamics and identify potential producers. We therefore quantified the distribution of 17 cyanotoxins, including 12 microcystin congeners using a high-performance liquid chromatography-high-resolution tandem mass spectrometry/mass spectrometry (HPLC-HRMS/MS). Our results revealed for the first time the co-occurrence of multiple cyanotoxins and the presence of cylindrospermopsin in an artificial reservoir (Rawal Lake) and a semi-saline lake (Kallar Kahar). We also quantified several microcystin congeners in a river (Panjnad) with MC-LR and MC-RR being the most prevalent and abundant. To identify potential cyanotoxin producers, the composition of the cyanobacterial community was characterized by shotgun metagenomics sequencing. Despite the noticeable presence of cyanotoxins, Cyanobacteria were not abundant. Synechococcus was the most abundant cyanobacterial genus found followed by a small amount of Anabaena, Cyanobium, Microcystis, and Dolichospermum. Moreover, when we looked at the cyanotoxins genes coverage, we never found a complete microcystin mcy operon. To our knowledge, this is the first snapshot sampling of water bodies in Pakistan. Our results would not only help to understand the geographical spread of cyanotoxin in Pakistan but would also help to improve cyanotoxin risk assessment strategies by screening a variety of cyanobacterial toxins and confirming that cyanotoxin quantification is not necessarily related to producer abundance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Colonization with multidrug-resistant Escherichia coli strains causes a substantial health burden in hospitalized patients. We performed a longitudinal genomics study to investigate the colonization of resistant E. coli strains in critically ill patients and to identify evolutionary changes and strain replacement events within patients. Patients were admitted to the intensive care unit and hematology wards at a major hospital in Lebanon. Perianal swabs were collected from participants on admission and during hospitalization, which were screened for extended-spectrum beta-lactamases and carbapenem-resistant Enterobacterales. We performed whole-genome sequencing and analysis on E. coli strains isolated from patients at multiple time points. The E. coli isolates were genetically diverse, with 11 sequence types (STs) identified among 22 isolates sequenced. Five patients were colonized by E. coli sequence type 131 (ST131)-encoding CTX-M-27, an emerging clone not previously observed in clinical samples from Lebanon. Among the eight patients whose resident E. coli strains were tracked over time, five harbored the same E. coli strain with relatively few mutations over the 5 to 10 days of hospitalization. The other three patients were colonized by different E. coli strains over time. Our study provides evidence of strain diversity within patients during their hospitalization. While strains varied in their antimicrobial resistance profiles, the number of resistance genes did not increase over time. We also show that ST131-encoding CTX-M-27, which appears to be emerging as a globally important multidrug-resistant E. coli strain, is also prevalent among critical care patients and deserves further monitoring.IMPORTANCEUnderstanding the evolution of bacteria over time in hospitalized patients is of utmost significance in the field of infectious diseases. While numerous studies have surveyed genetic diversity and resistance mechanisms in nosocomial infections, time series of within-patient dynamics are rare, and high-income countries are over-represented, leaving low- and middle-income countries understudied. Our study aims to bridge these research gaps by conducting a longitudinal survey of critically ill patients in Lebanon. This allowed us to track Escherichia coli evolution and strain replacements within individual patients over extended periods. Through whole-genome sequencing, we found extensive strain diversity, including the first evidence of the emerging E. coli sequence type 131 clone encoding the CTX-M-27 beta-lactamase in a clinical sample from Lebanon, as well as likely strain replacement events during hospitalization., Competing Interests: The authors declare no conflict of interest.

A long-standing question is to what degree genetic drift and selection drive the divergence in rare accessory gene content between closely related bacteria. Rare genes, including singletons, make up a large proportion of pangenomes (all genes in a set of genomes), but it remains unclear how many such genes are adaptive, deleterious or neutral to their host genome. Estimates of species' effective population sizes (N e ) are positively associated with pangenome size and fluidity, which has independently been interpreted as evidence for both neutral and adaptive pangenome models. We hypothesized that pseudogenes, used as a neutral reference, could be used to distinguish these models. We find that most functional categories are depleted for rare pseudogenes when a genome encodes only a single intact copy of a gene family. In contrast, transposons are enriched in pseudogenes, suggesting they are mostly neutral or deleterious to the host genome. Thus, even if individual rare accessory genes vary in their effects on host fitness, we can confidently reject a model of entirely neutral or deleterious rare genes. We also define the ratio of singleton intact genes to singleton pseudogenes (s i /s p ) within a pangenome, compare this measure across 668 prokaryotic species and detect a signal consistent with the adaptive value of many rare accessory genes. Taken together, our work demonstrates that comparing with pseudogenes can improve inferences of the evolutionary forces driving pangenome variation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objectives: Many cisgender women affected by homelessness and substance use desire pregnancy and parenthood. Provider discomfort with patient-centered counseling about reproductive choices and supporting reproductive decisions of these women poses barriers to reproductive healthcare access., Methods: We used participatory research methods to develop a half-day workshop for San Francisco-based medical and social service providers to improve reproductive counseling of women experiencing homelessness and/or who use substances. Guided by a stakeholder group comprising cisgender women with lived experience and providers, goals of the workshop included increasing provider empathy, advancing patient-centered reproductive health communication, and eliminating extraneous questions in care settings that perpetuate stigma. We used pre/post surveys to evaluate acceptability and effects of the workshop on participants' attitudes and confidence in providing reproductive health counseling. We repeated surveys one month post-event to investigate lasting effects., Results: Forty-two San Francisco-based medical and social service providers participated in the workshop. Compared to pre-test, post-test scores indicated reduced biases about: childbearing among unhoused women (p < 0.01), parenting intentions of pregnant women using substances (p = 0.03), and women not using contraception while using substances (p < 0.01). Participants also expressed increased confidence in how and when to discuss reproductive aspirations (p < 0.01) with clients. At one month, 90% of respondents reported the workshop was somewhat or very beneficial to their work, and 65% reported increased awareness of personal biases when working with this patient population., Conclusions for Practice: A half-day workshop increased provider empathy and improved provider confidence in reproductive health counseling of women affected by homelessness and substance use., (© 2023. The Author(s).)

Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts., Competing Interests: Volker Stadler is the Chief Executive Officer of PEPperPRINT. All other authors declare no competing interests., (© 2023 The Authors.)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a generalist virus, infecting and evolving in numerous mammals, including captive and companion animals, free-ranging wildlife, and humans. Transmission among non-human species poses a risk for the establishment of SARS-CoV-2 reservoirs, makes eradication difficult, and provides the virus with opportunities for new evolutionary trajectories, including the selection of adaptive mutations and the emergence of new variant lineages. Here, we use publicly available viral genome sequences and phylogenetic analysis to systematically investigate the transmission of SARS-CoV-2 between human and non-human species and to identify mutations associated with each species. We found the highest frequency of animal-to-human transmission from mink, compared with lower transmission from other sampled species (cat, dog, and deer). Although inferred transmission events could be limited by sampling biases, our results provide a useful baseline for further studies. Using genome-wide association studies, no single nucleotide variants (SNVs) were significantly associated with cats and dogs, potentially due to small sample sizes. However, we identified three SNVs statistically associated with mink and 26 with deer. Of these SNVs, ~⅔ were plausibly introduced into these animal species from local human populations, while the remaining ~⅓ were more likely derived in animal populations and are thus top candidates for experimental studies of species-specific adaptation. Together, our results highlight the importance of studying animal-associated SARS-CoV-2 mutations to assess their potential impact on human and animal health., Competing Interests: SN, PC, CM, RP, SK, BP, SS, BS No competing interests declared, (© 2023, Naderi et al.)

The severity and progression of lung disease are highly variable across individuals with cystic fibrosis (CF) and are imperfectly predicted by mutations in the human gene CFTR, lung microbiome variation or other clinical factors. The opportunistic pathogen Pseudomonas aeruginosa ( Pa ) dominates airway infections in most CF adults. Here we hypothesized that within-host genetic variation of Pa populations would be associated with lung disease severity. To quantify Pa genetic variation within CF sputum samples, we used deep amplicon sequencing (AmpliSeq) of 209 Pa genes previously associated with pathogenesis or adaptation to the CF lung. We trained machine learning models using Pa single nucleotide variants (SNVs), microbiome diversity data and clinical factors to classify lung disease severity at the time of sputum sampling, and to predict lung function decline after 5 years in a cohort of 54 adult CF patients with chronic Pa infection. Models using Pa SNVs alone classified lung disease severity with good sensitivity and specificity (area under the receiver operating characteristic curve: AUROC=0.87). Models were less predictive of lung function decline after 5 years (AUROC=0.74) but still significantly better than random. The addition of clinical data, but not sputum microbiome diversity data, yielded only modest improvements in classifying baseline lung function (AUROC=0.92) and predicting lung function decline (AUROC=0.79), suggesting that Pa AmpliSeq data account for most of the predictive value. Our work provides a proof of principle that Pa genetic variation in sputum tracks lung disease severity, moderately predicts lung function decline and could serve as a disease biomarker among CF patients with chronic Pa infections.

How the ecological process of community assembly interacts with intra-species diversity and evolutionary change is a longstanding question. Two contrasting hypotheses have been proposed: Diversity Begets Diversity (DBD), in which taxa tend to become more diverse in already diverse communities, and Ecological Controls (EC), in which higher community diversity impedes diversification. Previously, using 16S rRNA gene amplicon data across a range of microbiomes, we showed a generally positive relationship between taxa diversity and community diversity at higher taxonomic levels, consistent with the predictions of DBD (Madi et al., 2020). However, this positive 'diversity slope' plateaus at high levels of community diversity. Here we show that this general pattern holds at much finer genetic resolution, by analyzing intra-species strain and nucleotide variation in static and temporally sampled metagenomes from the human gut microbiome. Consistent with DBD, both intra-species polymorphism and strain number were positively correlated with community Shannon diversity. Shannon diversity is also predictive of increases in polymorphism over time scales up to ~4-6 months, after which the diversity slope flattens and becomes negative - consistent with DBD eventually giving way to EC. Finally, we show that higher community diversity predicts gene loss at a future time point. This observation is broadly consistent with the Black Queen Hypothesis, which posits that genes with functions provided by the community are less likely to be retained in a focal species' genome. Together, our results show that a mixture of DBD, EC, and Black Queen may operate simultaneously in the human gut microbiome, adding to a growing body of evidence that these eco-evolutionary processes are key drivers of biodiversity and ecosystem function., Competing Interests: NM, DC, RW, BS, NG No competing interests declared, (© 2023, Madi et al.)

Motivation: Microbiome datasets with taxa linked to the functions (e.g. genes) they encode are becoming more common as metagenomics sequencing approaches improve. However, these data are challenging to analyze due to their complexity. Summary metrics, such as the alpha and beta diversity of taxa contributing to each function (i.e. contributional diversity), represent one approach to investigate these data, but currently there are no straightforward methods for doing so., Results: We addressed this gap by developing FuncDiv, which efficiently performs these computations. Contributional diversity metrics can provide novel insights that would be impossible to identify without jointly considering taxa and functions., Availability and Implementation: FuncDiv is distributed under a GNU Affero General Public License v3.0 and is available at https://github.com/gavinmdouglas/FuncDiv., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Women experiencing homelessness with substance use disorders face unique and intersecting barriers to realizing their reproductive goals., Objective: This study explored the reproductive aspirations of this population, as well as the barriers to accessing reproductive services from the perspectives of affected individuals, and the healthcare providers who serve them., Design: This mixed-methods study included surveys and interviews with women experiencing homelessness with substance use disorders and healthcare providers., Methods: We conducted surveys and semi-structured interviews with women recruited from opiate treatment programs and homeless encampments in San Francisco, California in 2018. We also conducted interviews and focus groups with healthcare providers in reproductive health and substance use treatment settings. Interviews were recorded, transcribed, and coded. Descriptive statistics of survey results were performed., Results: Twenty-eight women completed surveys, 96% of whom reported current substance use. Ten women participated in interviews. One-third (9/28) reported desiring pregnancy in the next year; over half (16/28) reported they would be somewhat or very happy to learn they were pregnant. A majority used no contraception at last intercourse (14/28). Twenty-six healthcare providers participated in interviews (n = 15) and focus groups (n = 2). Patients and providers identified similar barriers to care access, including discrimination, logistical and financial challenges, and delayed pregnancy awareness. While providers proposed solutions focused on overcoming logistical challenges, patients emphasized the importance of transforming the healthcare environment to treat patients affected by substance use and homelessness with dignity and respect., Conclusion: Women experiencing homelessness with substance use disorders face intersecting and compounding barriers to accessing reproductive health services. For patients, the impact of stigma and bias on treatment experiences are particularly salient, in contrast to logistical barriers emphasized by providers. Improving access will require structural and individual-level solutions to address stigma and create person-centered, trauma-informed, and respectful care environments.

The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria are exposed to anaerobic conditions within the gastrointestinal tract, yet little is known about how oxygen exposure influences AMR. The facultative anaerobe Vibrio cholerae was chosen as a model to address this knowledge gap. We obtained V. cholerae isolates from 66 cholera patients, sequenced their genomes, and grew them under anaerobic and aerobic conditions with and without three clinically relevant antibiotics (ciprofloxacin, azithromycin, doxycycline). For ciprofloxacin and azithromycin, the minimum inhibitory concentration (MIC) increased under anaerobic conditions compared to aerobic conditions. Using standard resistance breakpoints, the odds of classifying isolates as resistant increased over 10 times for ciprofloxacin and 100 times for azithromycin under anaerobic conditions compared to aerobic conditions. For doxycycline, nearly all isolates were sensitive under both conditions. Using genome-wide association studies, we found associations between genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations. These AMR phenotypes were more heritable, and the AMR-associated genetic elements were more often discovered, under anaerobic conditions. These AMR-associated genetic elements are promising targets for future mechanistic research. Our findings provide a rationale to determine whether increased MICs under anaerobic conditions are associated with therapeutic failures and/or microbial escape in cholera patients. If so, there may be a need to determine new AMR breakpoints for anaerobic conditions.

Health-related concerns about cyanobacteria-laden sludge of drinking water treatment plants (DWTPs) have been raised in the past few years. Microscopic taxonomy, shotgun metagenomic sequencing, and microcystin (MC) measurement were applied to study the fate of cyanobacteria and cyanotoxins after controlled sludge storage (stagnation) in the dark in a full-scale drinking water treatment plant within 7 to 38 days. For four out of eight dates, cyanobacterial cell growth was observed by total taxonomic cell counts during sludge stagnation. The highest observed cell growth was 96% after 16 days of stagnation. Cell growth was dominated by potential MC producers such as Microcystis , Aphanocapsa , Chroococcus , and Dolichospermum . Shotgun metagenomic sequencing unveiled that stagnation stress shifts the cyanobacterial communities from the stress-sensitive Nostocales (e.g., Dolichospermum ) order towards less compromised orders and potential MC producers such as Chroococcales (e.g., Microcystis ) and Synechococcales (e.g., Synechococcus ). The relative increase of cyanotoxin producers presents a health challenge when the supernatant of the stored sludge is recycled to the head of the DWTP or discharged into the source. These findings emphasize the importance of a strategy to manage cyanobacteria-laden sludge and suggest practical approaches should be adopted to control health/environmental impacts of cyanobacteria and cyanotoxins in sludge., Competing Interests: The authors declare no conflict of interest.

The excessive proliferation of cyanobacteria in surface waters is a widespread problem worldwide, leading to the contamination of drinking water sources. Short- and long-term solutions for managing cyanobacterial blooms are needed for drinking water supplies. The goal of this research was to investigate the cyanobacteria community composition using shotgun metagenomics in a short term, in situ mesocosm experiment of two lakes following their coagulation with ferric sulfate (Fe 2 (SO 4 ) 3 ) as an option for source water treatment. Among the nutrient paramenters, dissolved nitrogen was related to Microcystis in both Missisquoi Bay and Petit Lac St. François, while the presence of Synechococcus was related to total nitrogen, dissolved nitrogen, dissolved organic carbon, and dissolved phosphorus. Results from the shotgun metagenomic sequencing showed that Dolichospermum and Microcystis were the dominant genera in all of the mesocosms in the beginning of the sampling period in Missisquoi Bay and Petit Lac St. François, respectively. Potentially toxigenic genera such as Microcystis were correlated with intracellular microcystin concentrations. A principal component analysis showed that there was a change of the cyanobacterial composition at the genus level in the mesocosms after two days, which varied across the studied sites and sampling time. The cyanobacterial community richness and diversity did not change significantly after its coagulation by Fe 2 (SO 4 ) 3 in all of the mesocosms at either site. The use of Fe 2 (SO 4 ) 3 for an onsite source water treatment should consider its impact on cyanobacterial community structure and the reduction of toxin concentrations.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria. IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Accurately identifying the species present in an ecosystem is vital to lake managers and successful bioassessment programs. This is particularly important when monitoring cyanobacteria, as numerous taxa produce toxins and can have major negative impacts on aquatic ecosystems. Increasingly, DNA-based techniques such as metabarcoding are being used for measuring aquatic biodiversity, as they could accelerate processing time, decrease costs and reduce some of the biases associated with traditional light microscopy. Despite the continuing use of traditional microscopy and the growing use of DNA metabarcoding to identify cyanobacteria assemblages, methodological comparisons between the two approaches have rarely been reported from a wide suite of lake types. Here, we compare planktonic cyanobacteria assemblages generated by inverted light microscopy and DNA metabarcoding from a 379-lake dataset spanning a longitudinal and trophic gradient. We found moderate levels of congruence between methods at the broadest taxonomic levels (i.e., Order, RV=0.40, p < 0.0001). This comparison revealed distinct cyanobacteria communities from lakes of different trophic states, with Microcystis, Aphanizomenon and Dolichospermum dominating with both methods in eutrophic and hypereutrophic sites. This finding supports the use of either method when monitoring eutrophication in lake surface waters. The biggest difference between the two methods was the detection of picocyanobacteria, which are typically underestimated by light microscopy. This reveals that the communities generated by each method currently are complementary as opposed to identical and promotes a combined-method strategy when monitoring a range of trophic systems. For example, microscopy can provide measures of cyanobacteria biomass, which are critical data in managing lakes. Going forward, we believe that molecular genetic methods will be increasingly adopted as reference databases are routinely updated with more representative sequences and will improve as cyanobacteria taxonomy is resolved with the increase in available genetic information., (Copyright © 2022. Published by Elsevier B.V.)

Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium is structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20°C or 30°C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100 km; between forests) and small (<1.2 km; within forests) geographical scales, among host tree species, and was dynamic over seasons. By measuring the growth of 79 isolates during different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together, our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This work paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus. IMPORTANCE Methylobacterium is a bacterial group tied to plants. Despite the ubiquity of methylobacteria and the importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and -independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial (within 1.2 km) scales. Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains under laboratory conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasting growth strategies in response to environmental variations.

The genome of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has been sequenced at an unprecedented scale leading to a tremendous amount of viral genome sequencing data. To assist in tracing infection pathways and design preventive strategies, a deep understanding of the viral genetic diversity landscape is needed. We present here a set of genomic surveillance tools from population genetics which can be used to better understand the evolution of this virus in humans. To illustrate the utility of this toolbox, we detail an in depth analysis of the genetic diversity of SARS-CoV-2 in first year of the COVID-19 pandemic. We analyzed 329,854 high-quality consensus sequences published in the GISAID database during the pre-vaccination phase. We demonstrate that, compared to standard phylogenetic approaches, haplotype networks can be computed efficiently on much larger datasets. This approach enables real-time lineage identification, a clear description of the relationship between variants of concern, and efficient detection of recurrent mutations. Furthermore, time series change of Tajima's D by haplotype provides a powerful metric of lineage expansion. Finally, principal component analysis (PCA) highlights key steps in variant emergence and facilitates the visualization of genomic variation in the context of SARS-CoV-2 diversity. The computational framework presented here is simple to implement and insightful for real-time genomic surveillance of SARS-CoV-2 and could be applied to any pathogen that threatens the health of populations of humans and other organisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mostefai, Gamache, N'Guessan, Pelletier, Huang, Murall, Pesaranghader, Gaonac'h-Lovejoy, Hamelin, Poujol, Grenier, Smith, Caron, Craig, Wolf, Krishnaswamy, Shapiro and Hussin.)

Understanding the evolutionary forces shaping prokaryotic pangenome structure is a major goal of microbial evolution research. Recent work has highlighted that a substantial proportion of accessory genes appear to confer niche-specific adaptations. This work has primarily focused on selection acting at the level of individual cells. Herein, we discuss a lower level of selection that also contributes to pangenome variation: genic selection. This refers to cases where genetic elements, rather than individual cells, are the entities under selection. The clearest examples of this form of selection are selfish mobile genetic elements, which are those that have either a neutral or a deleterious effect on host fitness. We review the major classes of these and other mobile elements and discuss the characteristic features of such elements that could be under genic selection. We also discuss how genetic elements that are beneficial to hosts can also be under genic selection, a scenario that may be more prevalent but not widely appreciated, because disentangling the effects of selection at different levels (i.e., organisms vs. genes) is challenging. Nonetheless, an appreciation for the potential action and implications of genic selection is important to better understand the evolution of prokaryotic pangenomes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Québec was the Canadian province most impacted by COVID-19, with 401,462 cases as of September 24th, 2021, and 11,347 deaths due mostly to a very severe first pandemic wave. In April 2020, we assembled the Coronavirus Sequencing in Québec (CoVSeQ) consortium to sequence SARS-CoV-2 genomes in Québec to track viral introduction events and transmission within the province., Methods: Using genomic epidemiology, we investigated the arrival of SARS-CoV-2 to Québec. We report 2921 high-quality SARS-CoV-2 genomes in the context of > 12,000 publicly available genomes sampled globally over the first pandemic wave (up to June 1st, 2020). By combining phylogenetic and phylodynamic analyses with epidemiological data, we quantify the number of introduction events into Québec, identify their origins, and characterize the spatiotemporal spread of the virus., Results: Conservatively, we estimated approximately 600 independent introduction events, the majority of which happened from spring break until 2 weeks after the Canadian border closed for non-essential travel. Subsequent mass repatriations did not generate large transmission lineages (> 50 sequenced cases), likely due to mandatory quarantine measures in place at the time. Consistent with common spring break and "snowbird" destinations, most of the introductions were inferred to have originated from Europe via the Americas. Once introduced into Québec, viral lineage sizes were overdispersed, with a few lineages giving rise to most infections. Consistent with founder effects, the earliest lineages to arrive tended to spread most successfully. Fewer than 100 viral introductions arrived during spring break, of which 7-12 led to the largest transmission lineages of the first wave (accounting for 52-75% of all sequenced infections). These successful transmission lineages dispersed widely across the province. Transmission lineage size was greatly reduced after March 11th, when a quarantine order for returning travellers was enacted. While this suggests the effectiveness of early public health measures, the biggest transmission lineages had already been ignited prior to this order., Conclusions: Combined, our results reinforce how, in the absence of tight travel restrictions or quarantine measures, fewer than 100 viral introductions in a week can ensure the establishment of extended transmission chains., (© 2021. The Author(s).)

Anthropogenic environmental change is causing habitat deterioration at unprecedented rates in freshwater ecosystems. Despite increasing more rapidly than many other agents of global change, synthetic chemical pollution-including agrochemicals such as pesticides-has received relatively little attention in freshwater community and ecosystem ecology. Determining the combined effects of multiple agrochemicals on complex biological systems remains a major challenge, requiring a cross-field integration of ecology and ecotoxicology. Using a large-scale array of experimental ponds, we investigated the response of zooplankton community properties (biomass, composition, and diversity metrics) to the individual and joint presence of three globally widespread agrochemicals: the herbicide glyphosate, the neonicotinoid insecticide imidacloprid, and nutrient fertilizers. We tracked temporal variation in zooplankton biomass and community structure along single and combined pesticide gradients (each spanning eight levels), under low (mesotrophic) and high (eutrophic) nutrient-enriched conditions, and quantified (1) response threshold concentrations, (2) agrochemical interactions, and (3) community resistance and recovery. We found that the biomass of major zooplankton groups differed in their sensitivity to pesticides: ≥0.3 mg/L glyphosate elicited long-lasting declines in rotifer communities, both pesticides impaired copepods (≥3 µg/L imidacloprid and ≥5.5 mg/L glyphosate), whereas some cladocerans were highly tolerant to pesticide contamination. Strong interactive effects of pesticides were only recorded in ponds treated with the combination of the highest doses. Overall, glyphosate was the most influential driver of aggregate community properties of zooplankton, with biomass and community structure responding rapidly but recovering unequally over time. Total community biomass showed little resistance when first exposed to glyphosate, but rapidly recovered and even increased with glyphosate concentration over time; in contrast, taxon richness decreased in more contaminated ponds but failed to recover. Our results indicate that the biomass of tolerant taxa compensated for the loss of sensitive species after the first exposure, conferring greater community resistance upon a subsequent contamination event; a case of pollution-induced community tolerance in freshwater animals. These findings suggest that zooplankton biomass may be more resilient to agrochemical pollution than community structure; yet all community properties measured in this study were affected at glyphosate concentrations below common water quality guidelines in North America., (© 2021 by the Ecological Society of America.)

Agricultural pollution with fertilizers and pesticides is a common disturbance to freshwater biodiversity. Bacterioplankton communities are at the base of aquatic food webs, but their responses to these potentially interacting stressors are rarely explored. To test the extent of resistance and resilience in bacterioplankton communities faced with agricultural stressors, we exposed freshwater mesocosms to single and combined gradients of two commonly used pesticides: the herbicide glyphosate (0-15 mg/L) and the neonicotinoid insecticide imidacloprid (0-60 μg/L), in high or low nutrient backgrounds. Over the 43-day experiment, we tracked variation in bacterial density with flow cytometry, carbon substrate use with Biolog EcoPlates, and taxonomic diversity and composition with environmental 16S rRNA gene amplicon sequencing. We show that only glyphosate (at the highest dose, 15 mg/L), but not imidacloprid, nutrients, or their interactions measurably changed community structure, favouring members of the Proteobacteria including the genus Agrobacterium. However, no change in carbon substrate use was detected throughout, suggesting functional redundancy despite taxonomic changes. We further show that communities are resilient at broad, but not fine taxonomic levels: 24 days after glyphosate application the precise amplicon sequence variants do not return, and tend to be replaced by phylogenetically close taxa. We conclude that high doses of glyphosate - but still within commonly acceptable regulatory guidelines - alter freshwater bacterioplankton by favouring a subset of higher taxonomic units (i.e., genus to phylum) that transiently thrive in the presence of glyphosate. Longer-term impacts of glyphosate at finer taxonomic resolution merit further investigation., (© 2021 John Wiley & Sons Ltd.)

Background: Cyanobacteria from the genus Microcystis can form large mucilaginous colonies with attached heterotrophic bacteria-their microbiome. However, the nature of the relationship between Microcystis and its microbiome remains unclear. Is it a long-term, evolutionarily stable association? Which partners benefit? Here we report the genomic diversity of 109 individual Microcystis colonies-including cyanobacteria and associated bacterial genomes-isolated in situ and without culture from Lake Champlain, Canada and Pampulha Reservoir, Brazil., Results: We identified 14 distinct Microcystis genotypes from Canada, of which only two have been previously reported, and four genotypes specific to Brazil. Microcystis genetic diversity was much greater between than within colonies, consistent with colony growth by clonal expansion rather than aggregation of Microcystis cells. We also identified 72 bacterial species in the microbiome. Each Microcystis genotype had a distinct microbiome composition, and more closely related genotypes had more similar microbiomes. This pattern of phylosymbiosis could be explained by co-phylogeny in only two out of the nine most prevalent associated bacterial genera, Roseomonas and Rhodobacter. These phylogenetically associated genera could enrich the metabolic repertoire of Microcystis, for example by encoding the biosynthesis of complementary carotenoid molecules. In contrast, other colony-associated bacteria showed weaker signals of co-phylogeny, but stronger evidence of horizontal gene transfer with Microcystis. These observations suggest that acquired genes are more likely to be retained in both partners (Microcystis and members of its microbiome) when they are loosely associated, whereas one gene copy is sufficient when the association is physically tight and evolutionarily long-lasting., Conclusions: We have introduced a method for culture-free isolation of single colonies from nature followed by metagenomic sequencing, which could be applied to other types of microbes. Together, our results expand the known genetic diversity of both Microcystis and its microbiome in natural settings, and support their long-term, specific, and potentially beneficial associations. Video Abstract., (© 2021. The Author(s).)

Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of Thalassospira isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of Thalassospira , in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years. IMPORTANCE The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of Thalassospira that have accumulated mutations in their genomes over millions of years.

Vibrio cholerae can cause a range of symptoms, from severe diarrhea to asymptomatic infection. Previous studies using whole-genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous mutations in V. cholerae DNA repair genes in 5 out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome and in 1 of 3 patients with isolate genomes sequenced. Second, these mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, nonmutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCE Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

Pangenomes-the cumulative set of genes encoded by a population or species-arise from the interplay of horizontal gene transfer, drift, and selection. The balance of these forces in shaping pangenomes has been debated, and studies to date focused on ancient evolutionary time scales have suggested that pangenomes generally confer niche adaptation to their bacterial hosts. To shed light on pangenome evolution on shorter evolutionary time scales, we inferred the selective pressures acting on mobile genes within individual human microbiomes from 176 Fiji islanders. We mapped metagenomic sequence reads to a set of known mobile genes to identify single nucleotide variants (SNVs) and calculated population genetic metrics to infer deviations from a neutral evolutionary model. We found that mobile gene sequence evolution varied more by gene family than by human social attributes, such as household or village. Patterns of mobile gene sequence evolution could be qualitatively recapitulated with a simple evolutionary simulation without the need to invoke the adaptive value of mobile genes to either bacterial or human hosts. These results stand in contrast with the apparent adaptive value of pangenomes over longer evolutionary time scales. In general, the most highly mobile genes (i.e., those present in more distinct bacterial host genomes) tend to have higher metagenomic read coverage and an excess of low-frequency SNVs, consistent with their rapid spread across multiple bacterial species in the gut. However, a subset of mobile genes-including those involved in defense mechanisms and secondary metabolism-showed a contrasting signature of intermediate-frequency SNVs, indicating species-specific selective pressures or negative frequency-dependent selection on these genes. Together, our evolutionary models and population genetic data show that gene-specific selective pressures predominate over human or bacterial host-specific pressures during the relatively short time scales of a human lifetime., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Objective: To characterize SARS-CoV-2 transmission following a COVID-19 outbreak in an emergency childcare centre (ECCC) in April 2020 in Quebec, Canada., Methods: The study population consisted of all the children and employees who attended the ECCC as well as household contacts of the confirmed COVID-19 cases. Of the 120 individuals in the study, five cases were confirmed by epidemiological link and 25 were identified as COVID-19 by RT-PCR among which 19 were analyzed by viral whole genome sequencing. Descriptive epidemiology, social network visualization, and phylogenetic analysis were used to characterize viral transmission., Results: Phylogenetic analysis identified two separate introductions of distinct lineages of SARS-CoV-2 and estimated an average effective reproductive number of R e = 1.9 (range 0.9-4.9) with a mean doubling time of 3.2 days (range 2.1-5.2). The first and most prevalent lineage was introduced by two asymptomatic children who were likely infected by their parent, a confirmed COVID-19 case working in a long-term care centre. Among infected household adults, attack rates were significantly higher in mothers than in fathers (risk ratio = 4.5; 95% CI 1.1-18.7). The extent of transmission makes it one of the largest documented outbreaks in a daycare in Canada., Conclusion: The analyses carried out showed the probable origin and direction of the transmission of the infection (adult-child, child-adult, and child-child), thus highlighting how asymptomatic children can efficiently transmit SARS-CoV-2.

Industrialization has impacted the human gut ecosystem, resulting in altered microbiome composition and diversity. Whether bacterial genomes may also adapt to the industrialization of their host populations remains largely unexplored. Here, we investigate the extent to which the rates and targets of horizontal gene transfer (HGT) vary across thousands of bacterial strains from 15 human populations spanning a range of industrialization. We show that HGTs have accumulated in the microbiome over recent host generations and that HGT occurs at high frequency within individuals. Comparison across human populations reveals that industrialized lifestyles are associated with higher HGT rates and that the functions of HGTs are related to the level of host industrialization. Our results suggest that gut bacteria continuously acquire new functionality based on host lifestyle and that high rates of HGT may be a recent development in human history linked to industrialization., Competing Interests: Declaration of interests R.J.X. is a consultant to Novartis and Nestle. E.J.A. is a co-founder and shareholder of Finch Therapeutics, a company that specializes in microbiome-targeted therapeutics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Susceptibility to Vibrio cholerae infection is affected by blood group, age, and preexisting immunity, but these factors only partially explain who becomes infected. A recent study used 16S ribosomal RNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution., Methods: To achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera., Results: Using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism were also correlated with protection., Conclusion: Our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Conventional processes (coagulation, flocculation, sedimentation, and filtration) are widely used in drinking water treatment plants and are considered a good treatment strategy to eliminate cyanobacterial cells and cell-bound cyanotoxins. The diversity of cyanobacteria was investigated using taxonomic cell counts and shotgun metagenomics over two seasons in a drinking water treatment plant before, during, and after the bloom. Changes in the community structure over time at the phylum, genus, and species levels were monitored in samples retrieved from raw water (RW), sludge in the holding tank (ST), and sludge supernatant (SST). Aphanothece clathrata brevis, Microcystis aeruginosa, Dolichospermum spiroides , and Chroococcus minimus were predominant species detected in RW by taxonomic cell counts. Shotgun metagenomics revealed that Proteobacteria was the predominant phylum in RW before and after the cyanobacterial bloom. Taxonomic cell counts and shotgun metagenomic showed that the Dolichospermum bloom occurred inside the plant. Cyanobacteria and Bacteroidetes were the major bacterial phyla during the bloom. Shotgun metagenomics also showed that Synechococcus , Microcystis , and Dolichospermum were the predominant detected cyanobacterial genera in the samples. Conventional treatment removed more than 92% of cyanobacterial cells but led to cell accumulation in the sludge up to 31 times more than in the RW influx. Coagulation/sedimentation selectively removed more than 96% of Microcystis and Dolichospermum . Cyanobacterial community in the sludge varied from raw water to sludge during sludge storage (1-13 days). This variation was due to the selective removal of coagulation/sedimentation as well as the accumulation of captured cells over the period of storage time. However, the prediction of the cyanobacterial community composition in the SST remained a challenge. Among nutrient parameters, orthophosphate availability was related to community profile in RW samples, whereas communities in ST were influenced by total nitrogen, Kjeldahl nitrogen (N- Kjeldahl), total and particulate phosphorous, and total organic carbon (TOC). No trend was observed on the impact of nutrients on SST communities. This study profiled new health-related, environmental, and technical challenges for the production of drinking water due to the complex fate of cyanobacteria in cyanobacteria-laden sludge and supernatant., Competing Interests: The authors declare no conflict of interest.