Advancing Source Tracking: Systematic Review and Source-Specific Genome Database Curation of Fecally Shed Prokaryotes.

Advancements within fecal source tracking (FST) studies are complicated by a lack of knowledge regarding the genetic content and distribution of fecally shed microbial populations. To address this gap, we performed a systematic literature review and curated a large collection of genomes (n = 26,018) representing fecally shed prokaryotic species across broad and narrow source categories commonly implicated in FST studies of recreational waters (i.e., cats, dogs, cows, seagulls, chickens, pigs, birds, ruminants, human feces, and wastewater). We find that across these sources the total number of prokaryotic genomes recovered from materials meeting our initial inclusion criteria varied substantially across fecal sources: from none in seagulls to 9,085 in pigs. We examined genome sequences recovered from these metagenomic and isolation-based studies extensively via comparative genomic approaches to characterize trends across source categories and produce a finalized genome database for each source category which is available online (n = 12,730). On average, 81% of the genomes representing species-level populations occur only within a single source. Using fecal slurries to test the performance of each source database, we report read capture rates that vary with fecal source alpha diversity and database size. We expect this resource to be useful to FST-related objectives, One Health research, and sanitation efforts globally.

More sensitive microbial responses to the interactive effects of warming and altered precipitation in subsoil than topsoil of an alpine grassland ecosystem.

Subsoil is a large organic carbon reservoir, storing more than half of the total soil organic carbon (SOC) globally. Conventionally, subsoil is assumed to not be susceptible to climate change, but recent studies document that climate change could significantly alter subsoil carbon cycling. However, little is known about subsoil microbial responses to the interactive effects of climate warming and altered precipitation. Here, we investigated carbon cycling and associated microbial responses in both subsoil (30-40 cm) and topsoil (0-10 cm) in a Tibetan alpine grassland over 4 years of warming and altered precipitation. Compared to the unchanged topsoil carbon (ß = .55, p = .587), subsoil carbon exhibited a stronger response to the interactive effect of warming and altered precipitation (ß = 2.04, p = .021), that is, warming decreased subsoil carbon content by 28.20% under decreased precipitation while warming increased subsoil carbon content by 18.02% under increased precipitation.Furthermore, 512 metagenome-assembled genomes (MAGs) were recovered, including representatives of phyla with poor genomic representation. Compared to only one changed topsoil MAG, 16 subsoil MAGs were significantly affected by altered precipitation, and 5 subsoil MAGs were significantly affected by the interactive effect of warming and precipitation. More than twice as many populations whose MAG abundances correlated significantly with the variations of carbon content, components and fluxes were observed in the subsoil than topsoil, suggesting their potential contribution in mediating subsoil carbon cycling. Collectively, our findings highlight the more sensitive responses of specific microbial lineages to the interactive effects of warming and altered precipitation in the subsoil than topsoil, and provide key information for predicting subsoil carbon cycling under future climate change scenarios.

New Drinking Water Genome Catalog Identifies a Globally Distributed Bacterial Genus Adapted to Disinfected Drinking Water Systems.

Genome-resolved insights into the structure and function of the drinking water microbiome can advance the effective management of drinking water quality. To enable this, we constructed and curated thousands of metagenome-assembled and isolate genomes from drinking water distribution systems globally to develop a Drinking Water Genome Catalog (DWGC). The current DWGC disproportionately represents disinfected drinking water systems due to a paucity of metagenomes from nondisinfected systems. Using the DWGC, we identify core genera of the drinking water microbiome including a genus (UBA4765) within the order Rhizobiales that is frequently detected and highly abundant in disinfected drinking water systems. We demonstrate that this genus has been widely detected but incorrectly classified in previous amplicon sequencing-based investigations of the drinking water microbiome. Further, we show that a single genome variant (genomovar) within this genus is detected in 75% of drinking water systems included in this study. We propose a name for this uncultured bacterium as "Raskinella chloraquaticus" and describe the genus as "Raskinella" (endorsed by SeqCode). Metabolic annotation and modeling-based predictions indicate that this bacterium is capable of necrotrophic growth, is able to metabolize halogenated compounds, proliferates in a biofilm-based environment, and shows clear indications of disinfection-mediated selection.

Genomic insights into organic acid production and plant growth promotion by different species of phosphate-solubilizing bacteria.

Bacteria can solubilize phosphorus (P) through the secretion of low-molecular-weight organic acids and acidification. However, the genes involved in the production of these organic acids are poorly understood. The objectives of this study were to verify the calcium phosphate solubilization and the production of low-molecular-weight organic acids by diverse genera of phosphate solubilizing bacterial strains (PSBS); to identify the genes related to the synthesis of the organic acids in the genomes of these strains and; to evaluate growth and nutrient accumulation of maize plants inoculated with PSBS and fertilized with Bayóvar rock phosphate. Genomic DNA was extracted for strain identification and annotation of genes related to the organic acids production. A greenhouse experiment was performed with five strains plus 150 mg dm- 3 P2O5 as Bayóvar rock phosphate (BRP) to assess phosphate solubilization contribution to maize growth and nutrition. Paraburkholderia fungorum UFLA 04-21 and Pseudomonas anuradhapurensis UFPI B5-8A solubilized over 60% of Ca phosphate and produced high amounts of citric/maleic and gluconic acids in vitro, respectively. Eleven organic acids were identified in total, although not all strains produced all acids. Besides, enzymes related to the organic acids production were found in all bacterial genomes. Plants inoculated with strains UFPI B5-6 (Enterobacter bugandensis), UFPI B5-8A, and UFLA 03-10 (Paenibacillus peoriae) accumulated more biomass than the plants fertilized with BRP only. Strains UFLA 03-10 and UFPI B5-8A increased the accumulation of most macronutrients, including P. Collectively, the results show that PSBS can increase maize growth and nutrient accumulation based on Bayóvar rock phosphate fertilization.

A natural ANI gap that can define intra-species units of bacteriophages and other viruses.

Despite the importance of intra-species variants of viruses for causing disease and/or disrupting ecosystem functioning, there is no universally applicable standard to define these. A (natural) gap in whole-genome average nucleotide identity (ANI) values around 95% is commonly used to define species, especially for bacteriophages, but whether a similar gap exists within species that can be used to define intra-species units has not been evaluated yet. Whole-genome comparisons among members of 1,016 bacteriophage (Caudoviricetes) species revealed a region of low frequency of ANI values around 99.2%-99.8%, showing threefold or fewer pairs than expected for an even distribution. This second gap is prevalent in viruses infecting various cultured or uncultured hosts from a variety of environments, although a few exceptions to this pattern were also observed (3.7% of total species) and are likely attributed to cultivation biases or other factors. Similar results were observed for a limited set of eukaryotic viruses that are adequately sampled, including SARS-CoV-2, whose ANI-based clusters matched well with the WHO-defined variants of concern, indicating that our findings from bacteriophages might be more broadly applicable and the ANI-based clusters may represent functionally and/or ecologically distinct units. These units appear to be predominantly driven by (high) ecological cohesiveness coupled to either frequent recombination for bacteriophages or selection and clonal evolution for other viruses such as SARS-CoV-2, indicating that fundamentally different underlying mechanisms could lead to similar diversity patterns. Accordingly, we propose the ANI gap approach outlined above for defining viral intra-species units, for which we propose the term genomovars. IMPORTANCE: Viral species are composed of an ensemble of intra-species variants whose individual dynamics may have major implications for human and animal health and/or ecosystem functioning. However, the lack of universally accepted standards to define these intra-species variants has led researchers to use different approaches for this task, creating inconsistent intra-species units across different viral families and confusion in communication. By comparing hundreds of mostly bacteriophage genomes, we show that there is a widely distributed natural gap in whole-genome average nucleotide identity values in most, but not all, of these species that can be used to define intra-species units. Therefore, these results advance the molecular toolbox for tracking viral intra-species units and should facilitate future epidemiological and environmental studies.

GSearch: ultra-fast and scalable genome search by combining K-mer hashing with hierarchical navigable small world graphs.

Genome search and/or classification typically involves finding the best-match database (reference) genomes and has become increasingly challenging due to the growing number of available database genomes and the fact that traditional methods do not scale well with large databases. By combining k-mer hashing-based probabilistic data structures (i.e. ProbMinHash, SuperMinHash, Densified MinHash and SetSketch) to estimate genomic distance, with a graph based nearest neighbor search algorithm (Hierarchical Navigable Small World Graphs, or HNSW), we created a new data structure and developed an associated computer program, GSearch, that is orders of magnitude faster than alternative tools while maintaining high accuracy and low memory usage. For example, GSearch can search 8000 query genomes against all available microbial or viral genomes for their best matches (n = ∼318 000 or ∼3 000 000, respectively) within a few minutes on a personal laptop, using ∼6 GB of memory (2.5 GB via SetSketch). Notably, GSearch has an O(log(N)) time complexity and will scale well with billions of genomes based on a database splitting strategy. Further, GSearch implements a three-step search strategy depending on the degree of novelty of the query genomes to maximize specificity and sensitivity. Therefore, GSearch solves a major bottleneck of microbiome studies that require genome search and/or classification.

Why and how to use the SeqCode.

The SeqCode, formally called the Code of Nomenclature of Prokaryotes Described from Sequence Data, is a new code of nomenclature in which genome sequences are the nomenclatural types for the names of prokaryotic species. While similar to the International Code of Nomenclature of Prokaryotes (ICNP) in structure and rules of priority, it does not require the deposition of type strains in international culture collections. Thus, it allows for the formation of permanent names for uncultured prokaryotes whose nearly complete genome sequences have been obtained directly from environmental DNA as well as other prokaryotes that cannot be deposited in culture collections. Because the diversity of uncultured prokaryotes greatly exceeds that of readily culturable prokaryotes, the SeqCode is the only code suitable for naming the majority of prokaryotic species. The start date of the SeqCode was January 1, 2022, and the online Registry (https://seqco.de/) was created to ensure valid publication of names. The SeqCode recognizes all names validly published under the ICNP before 2022. After that date, names validly published under the SeqCode compete with ICNP names for priority. As a result, species can have only one name, either from the SeqCode or ICNP, enabling effective communication and the creation of unified taxonomies of uncultured and cultured prokaryotes. The SeqCode is administered by the SeqCode Committee, which is comprised of the SeqCode Community and elected administrative components. Anyone with an interest in the systematics of prokaryotes is encouraged to join the SeqCode Community and participate in the development of this resource.

Organic fertilization co-selects genetically linked antibiotic and metal(loid) resistance genes in global soil microbiome.

Antibiotic resistance genes (ARGs) and metal(loid) resistance genes (MRGs) coexist in organic fertilized agroecosystems based on their correlations in abundance, yet evidence for the genetic linkage of ARG-MRGs co-selected by organic fertilization remains elusive. Here, an analysis of 511 global agricultural soil metagenomes reveals that organic fertilization correlates with a threefold increase in the number of diverse types of ARG-MRG-carrying contigs (AMCCs) in the microbiome (63 types) compared to non-organic fertilized soils (22 types). Metatranscriptomic data indicates increased expression of AMCCs under higher arsenic stress, with co-regulation of the ARG-MRG pairs. Organic fertilization heightens the coexistence of ARG-MRG in genomic elements through impacting soil properties and ARG and MRG abundances. Accordingly, a comprehensive global map was constructed to delineate the distribution of coexistent ARG-MRGs with virulence factors and mobile genes in metagenome-assembled genomes from agricultural lands. The map unveils a heightened relative abundance and potential pathogenicity risks (range of 4-6) for the spread of coexistent ARG-MRGs in Central North America, Eastern Europe, Western Asia, and Northeast China compared to other regions, which acquire a risk range of 1-3. Our findings highlight that organic fertilization co-selects genetically linked ARGs and MRGs in the global soil microbiome, and underscore the need to mitigate the spread of these co-resistant genes to safeguard public health.

Sustained bacterial N2O reduction at acidic pH.

Nitrous oxide (N2O) is a climate-active gas with emissions predicted to increase due to agricultural intensification. Microbial reduction of N2O to dinitrogen (N2) is the major consumption process but microbial N2O reduction under acidic conditions is considered negligible, albeit strongly acidic soils harbor nosZ genes encoding N2O reductase. Here, we study a co-culture derived from acidic tropical forest soil that reduces N2O at pH 4.5. The co-culture exhibits bimodal growth with a Serratia sp. fermenting pyruvate followed by hydrogenotrophic N2O reduction by a Desulfosporosinus sp. Integrated omics and physiological characterization revealed interspecies nutritional interactions, with the pyruvate fermenting Serratia sp. supplying amino acids as essential growth factors to the N2O-reducing Desulfosporosinus sp. Thus, we demonstrate growth-linked N2O reduction between pH 4.5 and 6, highlighting microbial N2O reduction potential in acidic soils.

pH selects for distinct N2O-reducing microbiomes in tropical soil microcosms.

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH < 5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02 mM) and high (2 mM) N2O assessed N2O reduction at pH 4.5 and 7.3. All microcosms consumed N2O, with lag times of up to 7 months observed in microcosms with 2 mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. At pH 4.5, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen high-quality metagenome-assembled genomes (MAGs) recovered from the N2O-reducing microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the Clade II type nosZ and lacking nitrite reductase genes (nirS/K). Five of the eight MAGs recovered from pH 4.5 microcosms represent novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH 3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

Cold-chain free nucleic acid preservation using porous super-absorbent polymer (PSAP) beads to facilitate wastewater surveillance.

The instability of viral targets including SARS-CoV-2 in sewage is an important challenge in wastewater monitoring projects. The unrecognized interruptions in the 'cold-chain' transport from the sample collection to RNA quantification in the laboratory may undermine the accurate quantification of the virus. In this study, bovine serum albumin (BSA)-modified porous superabsorbent polymer (PSAP) beads were applied to absorb raw sewage samples as a simple method for viral RNA preservation. The preservation efficiency for SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA were examined during storage for 14 days at 4 °C or room temperature against the control (no beads applied). While a non-significant difference was observed at 4 °C (∼80 % retention for both control and PSAP-treated sewage), the reduction of SARS-CoV-2 RNA concentrations was significantly lower in sewage retrieved from PSAP beads (25-40 % reduction) compared to control (>60 % reduction) at room temperature. On the other hand, the recovery of PMMoV, known for its high persistence in raw sewage, from PSAP beads or controls were consistently above 85 %, regardless of the storage temperature. Our results demonstrate the applicability of PSAP beads to wastewater-based epidemiology (WBE) projects for preservation of SARS-CoV-2 RNA in sewage, especially in remote settings with no refrigeration capabilities.

Potential routes of plastics biotransformation involving novel plastizymes revealed by global multi-omic analysis of plastic associated microbes.

Despite increasing efforts across various disciplines, the fate, transport, and impact of synthetic plastics on the environment and public health remain poorly understood. To better elucidate the microbial ecology of plastic waste and its potential for biotransformation, we conducted a large-scale analysis of all publicly available meta-omic studies investigating plastics (n = 27) in the environment. Notably, we observed low prevalence of known plastic degraders throughout most environments, except for substantial enrichment in riverine systems. This indicates rivers may be a highly promising environment for discovery of novel plastic bioremediation products. Ocean samples associated with degrading plastics showed clear differentiation from non-degrading polymers, showing enrichment of novel putative biodegrading taxa in the degraded samples. Regarding plastisphere pathogenicity, we observed significant enrichment of antimicrobial resistance genes on plastics but not of virulence factors. Additionally, we report a co-occurrence network analysis of 10 + million proteins associated with the plastisphere. This analysis revealed a localized sub-region enriched with known and putative plastizymes-these may be useful for deeper investigation of nature's ability to biodegrade man-made plastics. Finally, the combined data from our meta-analysis was used to construct a publicly available database, the Plastics Meta-omic Database (PMDB)-accessible at plasticmdb.org. These data should aid in the integrated exploration of the microbial plastisphere and facilitate research efforts investigating the fate and bioremediation potential of environmental plastic waste.

Multi-omics analysis of the gut microbiome and metabolites associated with the psychoneurological symptom cluster in children with cancer receiving chemotherapy.

BACKGROUND: Children with cancer receiving chemotherapy commonly report a cluster of psychoneurological symptoms (PNS), including pain, fatigue, anxiety, depression, and cognitive dysfunction. The role of the gut microbiome and its functional metabolites in PNS is rarely studied among children with cancer. This study investigated the associations between the gut microbiome-metabolome pathways and PNS in children with cancer across chemotherapy as compared to healthy children. METHODS: A case-control study was conducted. Cancer cases were recruited from Children's Healthcare of Atlanta and healthy controls were recruited via flyers. Participants reported PNS using the Pediatric Patient-Reported Outcomes Measurement Information System. Data for cases were collected pre-cycle two chemotherapy (T0) and post-chemotherapy (T1), whereas data for healthy controls were collected once. Gut microbiome and its metabolites were measured using fecal specimens. Gut microbiome profiling was performed using 16S rRNA V4 sequencing, and metabolome was performed using an untargeted liquid chromatography-mass spectrometry approach. A multi-omics network integration program analyzed microbiome-metabolome pathways of PNS. RESULTS: Cases (n = 21) and controls (n = 14) had mean ages of 13.2 and 13.1 years. For cases at T0, PNS were significantly associated with microbial genera (e.g., Ruminococcus, Megasphaera, and Prevotella), which were linked with carnitine shuttle (p = 0.0003), fatty acid metabolism (p = 0.001) and activation (p = 0.001), and tryptophan metabolism (p = 0.008). Megasphaera, clustered with aspartate and asparagine metabolism (p = 0.034), carnitine shuttle (p = 0.002), and tryptophan (p = 0.019), was associated with PNS for cases at T1. Gut bacteria with potential probiotic functions, along with fatty acid metabolism, tryptophan, and carnitine shuttle, were more clustered in cancer cases than the control network and this linkage with PNS needs further studies. CONCLUSIONS: Using multi-omics approaches, this study indicated specific microbiome-metabolome pathways linked with PNS in children with cancer across chemotherapy. Due to limitations such as antibiotic use in cancer cases, these findings need to be further confirmed in a larger cohort.

Metagenomic insights into the impact of litter from poultry Concentrated Animal Feeding Operations (CAFOs) to adjacent soil and water microbial communities.

In recent decades, human food consumption has led to an increased demand for animal-based foods, particularly chicken meat production. The state of Georgia, USA is one of the top broiler chicken producers in the United States, where animals are raised in Concentrated Animal Feeding Operations (CAFOs). Without proper management, CAFOs could negatively impact the environment and become a public health risk as a source of water and air pollution and/or by spreading antimicrobial resistance genes. In this study, we used metagenome sequencing to investigate the impact of the application of the CAFO's litter on adjacent soils and downstream creek waters in terms of microbial diversity and antimicrobial resistance profile changes. Our data indicate that while a few microbial groups increased in abundance within a short period of time after litter application, these populations subsequently decreased to levels similar to those found prior to the litter application or to below the detection limit of our metagenome sequencing effort. Microbial taxonomic composition analyses, relative abundance of Metagenome-Assembled Genomes (MAGs) and detection of Antimicrobial Resistance Genes (ARGs) allow us to conclude that this practice of litter application had a negligible effect on the microbiome or resistome profile of these soils and nearby waterways, likely due to its dilution in the field and/or outcompetition by indigenous microbes, revealing a minimal impact of these poultry facilities on the natural microbial communities.

Metagenomic evaluation of the performance of passive Moore swabs for sewage monitoring relative to composite sampling over time resolved deployments.

Moore swabs have re-emerged as a versatile tool in the field of wastewater-based epidemiology during the COVID-19 pandemic and offer unique advantages for monitoring pathogens in sewer systems, especially at the neighborhood-level. However, whether Moore swabs provide comparable results to more commonly used composite samples remains to be rigorously tested including the optimal duration of Moore swab deployment. This study provides new insights into these issues by comparing the results from Moore swab samples to those of paired composite samples collected from the same sewer lines continuously over six to seventy-two hours post-deployment, during low COVID-19 prevalence periods. Our results show that Moore swabs accumulated approximately 10-fold higher PMMoV concentrations (on a basis of mL of Moore swab squeezed filtrate to mL of composite sewage) and showed comparable trends in terms of bacterial species abundance when compared to composite samples. Moore swabs also generally captured higher SARS-CoV-2 N1/N2 RNA concentrations than composite samples. Moore swabs showed comparable trends in terms of abundance dynamics of the sewage microbiome to composite samples and variable signs of saturation over time that were site and/or microbial population-specific. Based on our dual ddRT-PCR and shotgun metagenomic approach, we find that Moore swabs at our sites were optimally deployed for 6 h at a time at two sites.

Judicial Opinion 129.

Opinion 129 addresses the status of Firmicutes corrig. Gibbons and Murray 1978 (Approved Lists 1980). The name has the category 'division' and was included in the Approved Lists of Bacterial Names, although that category had previously been removed from the International Code of Nomenclature of Bacteria (1975 revision onwards). When the category 'phylum' was introduced into the International Code of Nomenclature of Prokaryotes (ICNP) in 2021, equivalence between 'phylum' and 'division' was not stipulated. Since the definition of the taxonomic categories and their relative order is one of the principal tasks of every code of nomenclature, the inclusion of Firmicutes corrig. Gibbons and Murray 1978 in the Approved Lists was an error. The name is either not validly published or illegitimate because its category is not covered by the ICNP. If Firmicutes corrig. Gibbons and Murray 1978 (Approved Lists 1980) was a validly published phylum name, it would be illegitimate because it would contravene Rule 8, which does not permit any deviation from the requirement to derive a phylum name from the name of the type genus. Since Firmicutes corrig. Gibbons and Murray 1978 is also part of a 'misfitting megaclassification' recognized in Opinion 128, the name is rejected, without any pre-emption regarding a hypothetically validly published name Firmicutes at the rank of phylum. Gracilicutes Gibbons and Murray 1978 (Approved Lists 1980) and Anoxyphotobacteriae Gibbons and Murray 1978 (Approved Lists 1980) are also rejected. The validly published phylum names have a variety of advantages over their not validly published counterparts and cannot be replaced with ad hoc names suggested in the literature. To ease the transition, it is recommended to mention the not validly published phylum names which strongly deviate in spelling from their validly published counterparts along with the latter in publications during the next years.

Towards estimating the number of strains that make up a natural bacterial population.

What a strain is and how many strains make up a natural bacterial population remain elusive concepts despite their apparent importance for assessing the role of intra-population diversity in disease emergence or response to environmental perturbations. To advance these concepts, we sequenced 138 randomly selected Salinibacter ruber isolates from two solar salterns and assessed these genomes against companion short-read metagenomes from the same samples. The distribution of genome-aggregate average nucleotide identity (ANI) values among these isolates revealed a bimodal distribution, with four-fold lower occurrence of values between 99.2% and 99.8% relative to ANI >99.8% or <99.2%, revealing a natural "gap" in the sequence space within species. Accordingly, we used this ANI gap to define genomovars and a higher ANI value of >99.99% and shared gene-content >99.0% to define strains. Using these thresholds and extrapolating from how many metagenomic reads each genomovar uniquely recruited, we estimated that -although our 138 isolates represented about 80% of the Sal. ruber population- the total population in one saltern pond is composed of 5,500 to 11,000 genomovars, the great majority of which appear to be rare in-situ. These data also revealed that the most frequently recovered isolate in lab media was often not the most abundant genomovar in-situ, suggesting that cultivation biases are significant, even in cases that cultivation procedures are thought to be robust. The methodology and ANI thresholds outlined here should represent a useful guide for future microdiversity surveys of additional microbial species.

An ANI gap within bacterial species that advances the definitions of intra-species units.

IMPORTANCE: Bacterial strains and clonal complexes are two cornerstone concepts for microbiology that remain loosely defined, which confuses communication and research. Here we identify a natural gap in genome sequence comparisons among isolate genomes of all well-sequenced species that has gone unnoticed so far and could be used to more accurately and precisely define these and related concepts compared to current methods. These findings advance the molecular toolbox for accurately delineating and following the important units of diversity within prokaryotic species and thus should greatly facilitate future epidemiological and micro-diversity studies across clinical and environmental settings.

A user's guide to the bioinformatic analysis of shotgun metagenomic sequence data for bacterial pathogen detection.

Metagenomics, i.e., shotgun sequencing of the total microbial community DNA from a sample, has become a mature technique but its application to pathogen detection in clinical, environmental, and food samples is far from common or standardized. In this review, we summarize ongoing developments in metagenomic sequence analysis that facilitate its wider application to pathogen detection. We examine theoretical frameworks for estimating the limit of detection for a particular level of sequencing effort, current approaches for achieving species and strain analytical resolution, and discuss some relevant modern tools for these tasks. While these recent advances are significant and establish metagenomics as a powerful tool to provide insights not easily attained by culture-based approaches, metagenomics is unlikely to emerge as a widespread, routine monitoring tool in the near future due to its inherently high detection limits, cost, and inability to easily distinguish between viable and non-viable cells. Instead, metagenomics seems best poised for applications involving special circumstances otherwise challenging for culture-based and molecular (e.g., PCR-based) approaches such as the de novo detection of novel pathogens, cases of co-infection by more than one pathogen, and situations where it is important to assess the genomic composition of the pathogenic population(s) and/or its impact on the indigenous microbiome.

Fecal microbiota transplantation promotes reduction of antimicrobial resistance by strain replacement.

Multidrug-resistant organism (MDRO) colonization is a fundamental challenge in antimicrobial resistance. Limited studies have shown that fecal microbiota transplantation (FMT) can reduce MDRO colonization, but its mechanisms are poorly understood. We conducted a randomized, controlled trial of FMT for MDRO decolonization in renal transplant recipients called PREMIX (NCT02922816). Eleven participants were enrolled and randomized 1:1 to FMT or an observation period followed by delayed FMT if stool cultures were MDRO positive at day 36. Participants who were MDRO positive after one FMT were treated with a second FMT. At last visit, eight of nine patients who completed all treatments were MDRO culture negative. FMT-treated participants had longer time to recurrent MDRO infection versus PREMIX-eligible controls who were not treated with FMT. Key taxa (Akkermansia muciniphila, Alistipes putredinis, Phocaeicola dorei, Phascolarctobacterium faecium, Alistipes species, Mesosutterella massiliensis, Barnesiella intestinihominis, and Faecalibacterium prausnitzii) from the single feces donor used in the study that engrafted in recipients and metabolites such as short-chain fatty acids and bile acids in FMT-responding participants uncovered leads for rational microbiome therapeutic and diagnostic development. Metagenomic analyses revealed a previously unobserved mechanism of MDRO eradication by conspecific strain competition in an FMT-treated subset. Susceptible Enterobacterales strains that replaced baseline extended-spectrum ß-lactamase-producing strains were not detectable in donor microbiota manufactured as FMT doses but in one case were detectable in the recipient before FMT. These data suggest that FMT may provide a path to exploit strain competition to reduce MDRO colonization.