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387400.9994Culture-enriched human gut microbiomes reveal core and accessory resistance genes. BACKGROUND: Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. Unfortunately, these less-abundant bacteria can be overlooked by deep shotgun sequencing. In addition, it is a challenge to associate the presence of resistance genes with their risk of acquisition by pathogens. In this study, we used liquid culture enrichment of stools to assemble the genome of lower-abundance bacteria from fecal samples. We then investigated the gene content recovered from these culture-enriched and culture-independent metagenomes in relation with their taxonomic origin, specifically antibiotic resistance genes. We finally used a pangenome approach to associate resistance genes with the core or accessory genome of Enterobacteriaceae and inferred their propensity to horizontal gene transfer. RESULTS: Using culture-enrichment approaches with stools allowed assembly of 187 bacterial species with an assembly size greater than 1 million nucleotides. Of these, 67 were found only in culture-enriched conditions, and 22 only in culture-independent microbiomes. These assembled metagenomes allowed the evaluation of the gene content of specific subcommunities of the gut microbiome. We observed that differentially distributed metabolic enzymes were associated with specific culture conditions and, for the most part, with specific taxa. Gene content differences between microbiomes, for example, antibiotic resistance, were for the most part not associated with metabolic enzymes, but with other functions. We used a pangenome approach to determine if the resistance genes found in Enterobacteriaceae, specifically E. cloacae or E. coli, were part of the core genome or of the accessory genome of this species. In our healthy volunteer cohort, we found that E. cloacae contigs harbored resistance genes that were part of the core genome of the species, while E. coli had a large accessory resistome proximal to mobile elements. CONCLUSION: Liquid culture of stools contributed to an improved functional and comparative genomics study of less-abundant gut bacteria, specifically those associated with antibiotic resistance. Defining whether a gene is part of the core genome of a species helped in interpreting the genomes recovered from culture-independent or culture-enriched microbiomes.201930953542
466510.9994A comprehensive survey of integron-associated genes present in metagenomes. BACKGROUND: Integrons are genomic elements that mediate horizontal gene transfer by inserting and removing genetic material using site-specific recombination. Integrons are commonly found in bacterial genomes, where they maintain a large and diverse set of genes that plays an important role in adaptation and evolution. Previous studies have started to characterize the wide range of biological functions present in integrons. However, the efforts have so far mainly been limited to genomes from cultivable bacteria and amplicons generated by PCR, thus targeting only a small part of the total integron diversity. Metagenomic data, generated by direct sequencing of environmental and clinical samples, provides a more holistic and unbiased analysis of integron-associated genes. However, the fragmented nature of metagenomic data has previously made such analysis highly challenging. RESULTS: Here, we present a systematic survey of integron-associated genes in metagenomic data. The analysis was based on a newly developed computational method where integron-associated genes were identified by detecting their associated recombination sites. By processing contiguous sequences assembled from more than 10 terabases of metagenomic data, we were able to identify 13,397 unique integron-associated genes. Metagenomes from marine microbial communities had the highest occurrence of integron-associated genes with levels more than 100-fold higher than in the human microbiome. The identified genes had a large functional diversity spanning over several functional classes. Genes associated with defense mechanisms and mobility facilitators were most overrepresented and more than five times as common in integrons compared to other bacterial genes. As many as two thirds of the genes were found to encode proteins of unknown function. Less than 1% of the genes were associated with antibiotic resistance, of which several were novel, previously undescribed, resistance gene variants. CONCLUSIONS: Our results highlight the large functional diversity maintained by integrons present in unculturable bacteria and significantly expands the number of described integron-associated genes.202032689930
965720.9994Machine Learning Leveraging Genomes from Metagenomes Identifies Influential Antibiotic Resistance Genes in the Infant Gut Microbiome. Antibiotic resistance in pathogens is extensively studied, and yet little is known about how antibiotic resistance genes of typical gut bacteria influence microbiome dynamics. Here, we leveraged genomes from metagenomes to investigate how genes of the premature infant gut resistome correspond to the ability of bacteria to survive under certain environmental and clinical conditions. We found that formula feeding impacts the resistome. Random forest models corroborated by statistical tests revealed that the gut resistome of formula-fed infants is enriched in class D beta-lactamase genes. Interestingly, Clostridium difficile strains harboring this gene are at higher abundance in formula-fed infants than C. difficile strains lacking this gene. Organisms with genes for major facilitator superfamily drug efflux pumps have higher replication rates under all conditions, even in the absence of antibiotic therapy. Using a machine learning approach, we identified genes that are predictive of an organism's direction of change in relative abundance after administration of vancomycin and cephalosporin antibiotics. The most accurate results were obtained by reducing annotated genomic data to five principal components classified by boosted decision trees. Among the genes involved in predicting whether an organism increased in relative abundance after treatment are those that encode subclass B2 beta-lactamases and transcriptional regulators of vancomycin resistance. This demonstrates that machine learning applied to genome-resolved metagenomics data can identify key genes for survival after antibiotics treatment and predict how organisms in the gut microbiome will respond to antibiotic administration. IMPORTANCE The process of reconstructing genomes from environmental sequence data (genome-resolved metagenomics) allows unique insight into microbial systems. We apply this technique to investigate how the antibiotic resistance genes of bacteria affect their ability to flourish in the gut under various conditions. Our analysis reveals that strain-level selection in formula-fed infants drives enrichment of beta-lactamase genes in the gut resistome. Using genomes from metagenomes, we built a machine learning model to predict how organisms in the gut microbial community respond to perturbation by antibiotics. This may eventually have clinical applications.201829359195
464430.9994Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes. Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.202235982656
510140.9994Identification of Key Features Pivotal to the Characteristics and Functions of Gut Bacteria Taxa through Machine Learning Methods. BACKGROUND: Gut bacteria critically influence digestion, facilitate the breakdown of complex food substances, aid in essential nutrient synthesis, and contribute to immune system balance. However, current knowledge regarding intestinal bacteria remains insufficient. OBJECTIVE: This study aims to discover essential differences for different intestinal bacteria. METHODS: This study was conducted by investigating a total of 1478 gut bacterial samples comprising 235 Actinobacteria, 447 Bacteroidetes, and 796 Firmicutes, by utilizing sophisticated machine learning algorithms. By building on the dataset provided by Chen et al., we engaged sophisticated machine learning techniques to further investigate and analyze the gut bacterial samples. Each sample in the dataset was described by 993 unique features associated with gut bacteria, including 342 features annotated by the Antibiotic Resistance Genes Database, Comprehensive Antibiotic Research Database, Kyoto Encyclopedia of Genes and Genomes, and Virulence Factors of Pathogenic Bacteria. We employed incremental feature selection methods within a computational framework to identify the optimal features for classification. RESULTS: Eleven feature ranking algorithms selected several key features as pivotal to the characteristics and functions of gut bacteria. These features appear to facilitate the identification of specific gut bacterial species. Additionally, we established quantitative rules for identifying Actinobacteria, Bacteroidetes, and Firmicutes. CONCLUSION: This research underscores the significant potential of machine learning in studying gut microbes and enhances our understanding of the multifaceted roles of gut bacteria.202540671232
964950.9993Bacteria of the order Burkholderiales are original environmental hosts of type II trimethoprim resistance genes (dfrB). It is consensus that clinically relevant antibiotic resistance genes have their origin in environmental bacteria, including the large pool of primarily benign species. Yet, for the vast majority of acquired antibiotic resistance genes, the original environmental host(s) has not been identified to date. Closing this knowledge gap could improve our understanding of how antimicrobial resistance proliferates in the bacterial domain and shed light on the crucial step of initial resistance gene mobilization in particular. Here, we combine information from publicly available long- and short-read environmental metagenomes as well as whole-genome sequences to identify the original environmental hosts of dfrB, a family of genes conferring resistance to trimethoprim. Although this gene family stands in the shadow of the more widespread, structurally different dfrA, it has recently gained attention through the discovery of several new members. Based on the genetic context of dfrB observed in long-read metagenomes, we predicted bacteria of the order Burkholderiales to function as original environmental hosts of the predominant gene variants in both soil and freshwater. The predictions were independently confirmed by whole-genome datasets and statistical correlations between dfrB abundance and taxonomic composition of environmental bacterial communities. Our study suggests that Burkholderiales in general and the family Comamonadaceae in particular represent environmental origins of dfrB genes, some of which now contribute to the acquired resistome of facultative pathogens. We propose that our workflow centered on long-read environmental metagenomes allows for the identification of the original hosts of further clinically relevant antibiotic resistance genes.202439658215
466460.9993Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes. Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70 % amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria. This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.202033125315
464370.9993Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes. Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs (as measured through PERMANOVA, P < 0.001) and that the relative abundance of many individual AMR genes/variants increased over time (as measured with MaAsLin2, P(adj) < 0.05). Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.202336455460
391380.9993Determining the prevalence, identity and possible origin of bacterial pathogens in soil. Soil biomes are vast, exceptionally diverse and crucial to the health of ecosystems and societies. Soils also contain an appreciable, but understudied, diversity of opportunistic human pathogens. With climate change and other forms of environmental degradation potentially increasing exposure risks to soilborne pathogens, it is necessary to gain a better understanding of their ecological drivers. Here we use the Galleria mellonella insect virulence model to selectively isolate pathogenic bacteria from soils in Cornwall (UK). We find a high prevalence of pathogenic soil bacteria with two genera, Providencia and Serratia, being especially common. Providencia alcalifaciens, P. rustigianii, Serratia liquefaciens and S. plymuthica strains were studied in more detail using phenotypic virulence and antibiotic resistance assays and whole-genome sequencing. Both genera displayed low levels of antibiotic resistance and antibiotic resistance gene carriage. However, Serratia isolates were found to carry the recently characterized metallo-β-lactamase blaSPR-1 that, although not conferring high levels of resistance in these strains, poses a potential risk of horizontal transfer to other pathogens where it could be fully functional. The Galleria assay can be a useful approach to uncover the distribution and identity of pathogenic bacteria in the environment, as well as uncover resistance genes with an environmental origin.202032990385
454490.9993Identification of aminoglycoside and β-lactam resistance genes from within an infant gut functional metagenomic library. The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2-512x), increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.201425247417
3882100.9993Clusters of Antibiotic Resistance Genes Enriched Together Stay Together in Swine Agriculture. Antibiotic resistance is a worldwide health risk, but the influence of animal agriculture on the genetic context and enrichment of individual antibiotic resistance alleles remains unclear. Using quantitative PCR followed by amplicon sequencing, we quantified and sequenced 44 genes related to antibiotic resistance, mobile genetic elements, and bacterial phylogeny in microbiomes from U.S. laboratory swine and from swine farms from three Chinese regions. We identified highly abundant resistance clusters: groups of resistance and mobile genetic element alleles that cooccur. For example, the abundance of genes conferring resistance to six classes of antibiotics together with class 1 integrase and the abundance of IS6100-type transposons in three Chinese regions are directly correlated. These resistance cluster genes likely colocalize in microbial genomes in the farms. Resistance cluster alleles were dramatically enriched (up to 1 to 10% as abundant as 16S rRNA) and indicate that multidrug-resistant bacteria are likely the norm rather than an exception in these communities. This enrichment largely occurred independently of phylogenetic composition; thus, resistance clusters are likely present in many bacterial taxa. Furthermore, resistance clusters contain resistance genes that confer resistance to antibiotics independently of their particular use on the farms. Selection for these clusters is likely due to the use of only a subset of the broad range of chemicals to which the clusters confer resistance. The scale of animal agriculture and its wastes, the enrichment and horizontal gene transfer potential of the clusters, and the vicinity of large human populations suggest that managing this resistance reservoir is important for minimizing human risk. IMPORTANCE: Agricultural antibiotic use results in clusters of cooccurring resistance genes that together confer resistance to multiple antibiotics. The use of a single antibiotic could select for an entire suite of resistance genes if they are genetically linked. No links to bacterial membership were observed for these clusters of resistance genes. These findings urge deeper understanding of colocalization of resistance genes and mobile genetic elements in resistance islands and their distribution throughout antibiotic-exposed microbiomes. As governments seek to combat the rise in antibiotic resistance, a balance is sought between ensuring proper animal health and welfare and preserving medically important antibiotics for therapeutic use. Metagenomic and genomic monitoring will be critical to determine if resistance genes can be reduced in animal microbiomes, or if these gene clusters will continue to be coselected by antibiotics not deemed medically important for human health but used for growth promotion or by medically important antibiotics used therapeutically.201627073098
9658110.9993Functional metagenomic libraries generated from anthropogenically impacted environments reveal importance of metabolic genes in biocide and antibiotic resistance. Anthropogenic activities result in the release of antimicrobial resistant bacteria and a cocktail of antimicrobial compounds into the environment that may directly select or indirectly co-select for antimicrobial resistance (AMR). Many studies use metagenome sequencing or qPCR-based approaches to study the environmental resistome but these methods are limited by a priori knowledge. In this study, a functional metagenomic approach was used to explore biocide resistance mechanisms in two contaminated environments and a pristine site, and to identify whether potentially novel genes conferring biocide resistance also conferred resistance or reduced susceptibility to antibiotics. Resistance was predominately mediated through novel mechanisms exclusive of the well-known qac efflux genes. UDP-galactose 4-epimerase (galE) -like genes were identified in both contaminated environments and were shown to confer cross-resistance to biocides and clinically important antibiotics for the first time (to our knowledge), compared to knockout mutants. GalE -like genes were also co-located with transposons, suggesting mobilisation potential. These results show that housekeeping genes may play a significant yet underappreciated role in AMR in environmental microbiomes.202336908773
4666120.9993Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes. Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.201930808697
3865130.9993Assessing Transmission of Antimicrobial-Resistant Escherichia coli in Wild Giraffe Contact Networks. There is growing evidence that anthropogenic sources of antibiotics and antimicrobial-resistant bacteria can spill over into natural ecosystems, raising questions about the role wild animals play in the emergence, maintenance, and dispersal of antibiotic resistance genes. In particular, we lack an understanding of how resistance genes circulate within wild animal populations, including whether specific host characteristics, such as social associations, promote interhost transmission of these genes. In this study, we used social network analysis to explore the forces shaping population-level patterns of resistant Escherichia coli in wild giraffe (Giraffa camelopardalis) and assess the relative importance of social contact for the dissemination of resistant E. coli between giraffe. Of 195 giraffe sampled, only 5.1% harbored E. coli isolates resistant to one or more tested antibiotics. Whole-genome sequencing on a subset of resistant isolates revealed a number of acquired resistance genes with linkages to mobile genetic elements. However, we found no evidence that the spread of resistance genes among giraffe was facilitated by interhost associations. Giraffe with lower social degree were more likely to harbor resistant E. coli, but this relationship was likely driven by a correlation between an individual's social connectedness and age. Indeed, resistant E. coli was most frequently detected in socially isolated neonates, indicating that resistant E. coli may have a selective advantage in the gastrointestinal tracts of neonates compared to other age classes. Taken together, these results suggest that the maintenance of antimicrobial-resistant bacteria in wild populations may, in part, be determined by host traits and microbial competition dynamics within the host.IMPORTANCE Antimicrobial resistance represents a significant threat to human health, food security, and the global economy. To fully understand the evolution and dissemination of resistance genes, a complete picture of antimicrobial resistance in all biological compartments, including natural ecosystems, is required. The environment and wild animals may act as reservoirs for anthropogenically derived resistance genes that could be transferrable to clinically relevant bacteria of humans and domestic animals. Our study investigated the possible transmission mechanisms for antimicrobial-resistant bacteria within a wild animal population and, more broadly, contributes to our understanding of how resistance genes are spread and maintained in natural ecosystems.201930413480
8383140.9993Novel insights into carbohydrate utilisation, antimicrobial resistance, and sporulation potential in Roseburia intestinalis isolates across diverse geographical locations. Roseburia intestinalis is one of the most abundant and important butyrate-producing human gut anaerobic bacteria that plays an important role in maintaining health and is a potential next-generation probiotic. We investigated the pangenome of 16 distinct strains, isolated over several decades, identifying local and time-specific adaptations. More than 50% of the genes in each individual strain were assigned to the core genome, and 77% of the cloud genes were unique to individual strains, revealing the high level of genome conservation. Co-carriage of the same enzymes involved in carbohydrate binding and degradation in all strains highlighted major pathways in carbohydrate utilization and reveal the importance of xylan, starch and mannose as key growth substrates. A single strain had adapted to use rhamnose as a sole growth substrate, the first time this has been reported. The ubiquitous presence of motility and sporulation gene clusters demonstrates the importance of these phenotypes for gut survival and acquisition of this bacterium. More than half the strains contained functional, potentially transferable, tetracycline resistance genes. This study advances our understanding of the importance of R. intestinalis within the gut ecosystem by elucidating conserved metabolic characteristics among different strains, isolated from different locations. This information will help to devise dietary strategies to increase the abundance of this species providing health benefits.202540089923
4357150.9993Comparative genomic analysis of 255 Oenococcus oeni isolates from China: unveiling strain diversity and genotype-phenotype associations of acid resistance. Oenococcus oeni, the only species of lactic acid bacteria capable of fully completing malolactic fermentation under challenging wine conditions, continues to intrigue researchers owing to its remarkable adaptability, particularly in combating acid stress. However, the mechanism underlying its superior adaptation to wine stresses still remains elusive due to the lack of viable genetic manipulation tools for this species. In this study, we conducted genomic sequencing and acid resistance phenotype analysis of 255 O. oeni isolates derived from diverse wine regions across China, aiming to elucidate their strain diversity and genotype-phenotype associations of acid resistance through comparative genomics. A significant correlation between phenotypes and evolutionary relationships was observed. Notably, phylogroup B predominantly consisted of acid-resistant isolates, primarily originating from Shandong and Shaanxi wine regions. Furthermore, we uncovered a noteworthy linkage between prophage genomic islands and acid resistance phenotype. Using genome-wide association studies, we identified key genes correlated with acid resistance, primarily involved in carbohydrates and amino acid metabolism processes. This study offers profound insights into the genetic diversity and genetic basis underlying adaptation mechanisms to acid stress in O. oeni.IMPORTANCEThis study provides valuable insights into the genetic basis of acid resistance in Oenococcus oeni, a key lactic acid bacterium in winemaking. By analyzing 255 isolates from diverse wine regions in China, we identified significant correlations between strain diversity, genomic islands, and acid resistance phenotypes. Our findings reveal that certain prophage-related genomic islands and specific genes are closely linked to acid resistance, offering a deeper understanding of how O. oeni adapts to acidic environments. These discoveries not only advance our knowledge of microbial stress responses but also pave the way for selecting and engineering acid-resistant strains, enhancing malolactic fermentation efficiency and wine quality. This research underscores the importance of genomics in improving winemaking practices and addressing challenges posed by high-acidity wines.202540261018
3866160.9993The evolution of superbugs in space: a genomic perspective on pathogens in the International Space Station environment. Microgravity, pressure, and temperature variations in the International Space Station (ISS) create conditions leading to the emergence of superbugs. Due to technical issues in spacecraft, astronauts are forced to stay in ISS for extended periods; prolonged stay and exposure in stressful ISS environment weakens their immune systems, increasing susceptibility to infections. The presence of hypervirulent and antibiotic-resistant pathogens in space station is a worrisome feature as these might cause serious life-threatening infections in astronauts staying in high stress environments with weakened immune systems. In the present study, we compared antimicrobial resistance genes (ARGs) and virulence factors (VFs) in bacterial genomes from ISS with Earth counterparts. ISS genomes exhibited elevated counts of defense-related genes, particularly in E. ludwigii and E. cancerogenus. Among genes uniquely found in ISS genomes, CRISPR-Cas system components were notably prevalent. Though Earth genomes harbored higher number of ARGs overall, several species from ISS possessed modestly higher ARG counts. VFs profiling showed a slightly lower count in ISS genomes, but P. conspicua, E. ludwigii, and K. pneumoniae from ISS carried exclusive VFs linked to metal ion uptake and secretion systems, suggesting environment-driven functional adaptations. The adaptation of pathogenic bacteria in ISS is alarming and therefore periodic monitoring of bacterial genomic surveillance is important. Our findings shed light on genomic profiles in bacterial strains from both ISS and Earth, enhancing our understanding of the bacterial pathogens' potential impact on drug resistance and pathogenicity in space-missions and the possible threat of spread from ISS.202540854655
3830170.9993Resistance Gene Carriage Predicts Growth of Natural and Clinical Escherichia coli Isolates in the Absence of Antibiotics. Bacterial pathogens that carry antibiotic resistance alleles sometimes pay a cost in the form of impaired growth in antibiotic-free conditions. This cost of resistance is expected to be a key parameter for understanding how resistance spreads and persists in pathogen populations. Analysis of individual resistance alleles from laboratory evolution and natural isolates has shown they are typically costly, but these costs are highly variable and influenced by genetic variation at other loci. It therefore remains unclear how strongly resistance is linked to impaired antibiotic-free growth in bacteria from natural and clinical scenarios, where resistance alleles are likely to coincide with other types of genetic variation. To investigate this, we measured the growth of 92 natural and clinical Escherichia coli isolates across three antibiotic-free environments. We then tested whether variation of antibiotic-free growth among isolates was predicted by their resistance to 10 antibiotics, while accounting for the phylogenetic structure of the data. We found that isolates with similar resistance profiles had similar antibiotic-free growth profiles, but it was not simply that higher average resistance was associated with impaired growth. Next, we used whole-genome sequences to identify antibiotic resistance genes and found that isolates carrying a greater number of resistance gene types grew relatively poorly in antibiotic-free conditions, even when the resistance genes they carried were different. This suggests that the resistance of bacterial pathogens is linked to growth costs in nature, but it is the total genetic burden and multivariate resistance phenotype that predict these costs, rather than individual alleles or mean resistance across antibiotics.IMPORTANCE Managing the spread of antibiotic resistance in bacterial pathogens is a major challenge for global public health. Central to this challenge is understanding whether resistance is linked to impaired bacterial growth in the absence of antibiotics, because this determines whether resistance declines when bacteria are no longer exposed to antibiotics. We studied 92 isolates of the key bacterial pathogen Escherichia coli; these isolates varied in both their antibiotic resistance genes and other parts of the genome. Taking this approach, rather than focusing on individual genetic changes associated with resistance as in much previous work, revealed that growth without antibiotics was linked to the number of specialized resistance genes carried and the combination of antibiotics to which isolates were resistant but was not linked to average antibiotic resistance. This approach provides new insights into the genetic factors driving the long-term persistence of antibiotic-resistant bacteria, which is important for future efforts to predict and manage resistance.201930530714
4380180.9993Comparative genome analysis of ciprofloxacin-resistant Pseudomonas aeruginosa reveals genes within newly identified high variability regions associated with drug resistance development. The alarming rise of ciprofloxacin-resistant Pseudomonas aeruginosa has been reported in several clinical studies. Though the mutation of resistance genes and their role in drug resistance has been researched, the process by which the bacterium acquires high-level resistance is still not well understood. How does the genomic evolution of P. aeruginosa affect resistance development? Could the exposure of antibiotics to the bacteria enrich genomic variants that lead to the development of resistance, and if so, how are these variants distributed through the genome? To answer these questions, we performed 454 pyrosequencing and a whole genome analysis both before and after exposure to ciprofloxacin. The comparative sequence data revealed 93 unique resistance strain variation sites, which included a mutation in the DNA gyrase subunit A gene. We generated variation-distribution maps comparing the wild and resistant types, and isolated 19 candidates from three discrete resistance-associated high variability regions that had available transposon mutants, to perform a ciprofloxacin exposure assay. Of these region candidates with transposon disruptions, 79% (15/19) showed a reduction in the ability to gain high-level resistance, suggesting that genes within these high variability regions might enrich for certain functions associated with resistance development.201323808957
6597190.9993Exploiting a targeted resistome sequencing approach in assessing antimicrobial resistance in retail foods. BACKGROUND: With the escalating risk of antimicrobial resistance (AMR), there are limited analytical options available that can comprehensively assess the burden of AMR carried by clinical/environmental samples. Food can be a potential source of AMR bacteria for humans, but its significance in driving the clinical spread of AMR remains unclear, largely due to the lack of holistic-yet-sensitive tools for surveillance and evaluation. Metagenomics is a culture-independent approach well suited for uncovering genetic determinants of defined microbial traits, such as AMR, present within unknown bacterial communities. Despite its popularity, the conventional approach of non-selectively sequencing a sample's metagenome (namely, shotgun-metagenomics) has several technical drawbacks that lead to uncertainty about its effectiveness for AMR assessment; for instance, the low discovery rate of resistance-associated genes due to their naturally small genomic footprint within the vast metagenome. Here, we describe the development of a targeted resistome sequencing method and demonstrate its application in the characterization of the AMR gene profile of bacteria associated with several retail foods. RESULT: A targeted-metagenomic sequencing workflow using a customized bait-capture system targeting over 4,000 referenced AMR genes and 263 plasmid replicon sequences was validated against both mock and sample-derived bacterial community preparations. Compared to shotgun-metagenomics, the targeted method consistently provided for improved recovery of resistance gene targets with a much-improved target detection efficiency (> 300-fold). Targeted resistome analyses conducted on 36 retail-acquired food samples (fresh sprouts, n = 10; ground meat, n = 26) and their corresponding bacterial enrichment cultures (n = 36) reveals in-depth features regarding the identity and diversity of AMR genes, most of which were otherwise undetected by the whole-metagenome shotgun sequencing method. Furthermore, our findings suggest that foodborne Gammaproteobacteria could be the major reservoir of food-associated AMR genetic determinants, and that the resistome structure of the selected high-risk food commodities are, to a large extent, dictated by microbiome composition. CONCLUSIONS: For metagenomic sequencing-based surveillance of AMR, the target-capture method presented herein represents a more sensitive and efficient approach to evaluate the resistome profile of complex food or environmental samples. This study also further implicates retail foods as carriers of diverse resistance-conferring genes indicating a potential impact on the dissemination of AMR.202336991496