Antimicrobial Resistance Gene Acquisition and Depletion Following Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection. - Related Documents




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332601.0000Antimicrobial Resistance Gene Acquisition and Depletion Following Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection. Fecal microbiota transplantation (FMT) may be a novel approach to eliminate multidrug-resistant bacteria from the gut and to prevent future infections. Using whole metagenome sequencing data from 8 FMT donor-recipient pairs, we identified 37 and 95 antimicrobial resistance genes that were acquired by or removed from FMT recipients, respectively.201829020222
332510.9998Long-term beneficial effect of faecal microbiota transplantation on colonisation of multidrug-resistant bacteria and resistome abundance in patients with recurrent Clostridioides difficile infection. BACKGROUND: Multidrug-resistant (MDR) bacteria are a growing global threat, especially in healthcare facilities. Faecal microbiota transplantation (FMT) is an effective prevention strategy for recurrences of Clostridioides difficile infections and can also be useful for other microbiota-related diseases. METHODS: We study the effect of FMT in patients with multiple recurrent C. difficile infections on colonisation with MDR bacteria and antibiotic resistance genes (ARG) on the short (3 weeks) and long term (1-3 years), combining culture methods and faecal metagenomics. RESULTS: Based on MDR culture (n = 87 patients), we notice a decrease of 11.5% in the colonisation rate of MDR bacteria after FMT (20/87 before FMT = 23%, 10/87 3 weeks after FMT). Metagenomic sequencing of patient stool samples (n = 63) shows a reduction in relative abundances of ARGs in faeces, while the number of different resistance genes in patients remained higher compared to stools of their corresponding healthy donors (n = 11). Furthermore, plasmid predictions in metagenomic data indicate that patients harboured increased levels of resistance plasmids, which appear unaffected by FMT. In the long term (n = 22 patients), the recipients' resistomes are still donor-like, suggesting the effect of FMT may last for years. CONCLUSIONS: Taken together, we hypothesise that FMT restores the gut microbiota to a composition that is closer to the composition of healthy donors, and potential pathogens are either lost or decreased to very low abundances. This process, however, does not end in the days following FMT. It may take months for the gut microbiome to re-establish a balanced state. Even though a reservoir of resistance genes remains, a notable part of which on plasmids, FMT decreases the total load of resistance genes.202438419010
991920.9997An In Vitro Chicken Gut Model Demonstrates Transfer of a Multidrug Resistance Plasmid from Salmonella to Commensal Escherichia coli. The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase bla(CTX-M1) We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies.IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.201728720731
392230.9997Mobile Antimicrobial Resistance Genes in Probiotics. Even though people worldwide tend to consume probiotic products for their beneficial health effects on a daily basis, recently, concerns were outlined regarding the uptake and potential intestinal colonisation of the bacteria that they carry. These bacteria are capable of executing horizontal gene transfer (HGT) which facilitates the movement of various genes, including antimicrobial resistance genes (ARGs), among the donor and recipient bacterial populations. Within our study, 47 shotgun sequencing datasets deriving from various probiotic samples (isolated strains and metagenomes) were bioinformatically analysed. We detected more than 70 ARGs, out of which rpoB mutants conferring resistance to rifampicin, tet(W/N/W) and potentially extended-spectrum beta-lactamase (ESBL) coding TEM-116 were the most common. Numerous ARGs were associated with integrated mobile genetic elements, plasmids or phages promoting the HGT. Our findings raise clinical and public health concerns as the consumption of probiotic products may lead to the transfer of ARGs to human gut bacteria.202134827225
462340.9997Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes. Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the sequences in the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2,000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probe set on DNA libraries generated from multidrug-resistant bacteria to selectively capture resistance genes reproducibly produced higher numbers of reads on target at a greater length of coverage than shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables a sensitive means of gene detection in diverse environments where genes encoding antibiotic resistance represent less than 0.1% of the metagenome.201931611361
371550.9997Deposition of resistant bacteria and resistome through FMT in germ-free piglets. Faecal microbiota transplantation (FMT) has received considerable attention in recent years due to its remarkable efficacy in restoring a normal gut microbiome. Here, we established the groups of post-FMT recipient piglets using germ-free piglets during early life to characterize the colonization of gut microbiota composition and the enrichment of resistance gene acquisition. By metagenomic analysis, we identified 115 bacterial phyla and 2111 bacterial genera that were acquired by the FMT recipients. We found that early-life microbial colonization and the spread of resistomes in recipient piglets were age dependent. A total of 425, 425 and 358 AR genes primarily belonging to 114, 114 and 102 different types were detected in the donors, post-FMT recipients in the FMT-3D group and post-FMT recipients in the FMT-15D group respectively. Genes that encoded tetracycline, macrolide and chloramphenicol resistance proteins were the most dominant AR genes, and the results corresponded with the exposure of antibiotic consumption at farm. Bacteroides, Escherichia, Clostridium, Parabacteroides, Treponema, Lactobacillus and Enterococcus were significantly correlated with the distribution of AR genes. More importantly, the relative abundance of AR genes was positively correlated with the levels of mobile genetic elements. Our results indicate that early-life microbial colonization can persistently shape the gut microbiota and antibiotic resistome.202133894059
510660.9997Metagenomic diagnostics for the simultaneous detection of multiple pathogens in human stool specimens from Côte d'Ivoire: a proof-of-concept study. BACKGROUND: The intestinal microbiome is a complex community and its role in influencing human health is poorly understood. While conventional microbiology commonly attributes digestive disorders to a single microorganism, a metagenomic approach can detect multiple pathogens simultaneously and might elucidate the role of microbial communities in the pathogenesis of intestinal diseases. We present a proof-of-concept that a shotgun metagenomic approach provides useful information on the diverse composition of intestinal pathogens and antimicrobial resistance profiles in human stool samples. METHODS: In October 2012, we obtained stool specimens from patients with persistent diarrhea in south Côte d'Ivoire. Four stool samples were purposefully selected and subjected to microscopy, multiplex polymerase chain reaction (PCR), and a metagenomic approach. For the latter, we employed the National Center for Biotechnology Information nucleotide database and screened for 36 pathogenic organisms (bacteria, helminths, intestinal protozoa, and viruses) that may cause digestive disorders. We further characterized the bacterial population and the prevailing resistance patterns by comparing our metagenomic datasets with a genome-specific marker database and with a comprehensive antibiotic resistance database. RESULTS: In the four patients, the metagenomic approach identified between eight and 11 pathogen classes that potentially cause digestive disorders. For bacterial pathogens, the diagnostic agreement between multiplex PCR and metagenomics was high; yet, metagenomics diagnosed several bacteria not detected by multiplex PCR. In contrast, some of the helminth and intestinal protozoa infections detected by microscopy were missed by metagenomics. The antimicrobial resistance analysis revealed the presence of genes conferring resistance to several commonly used antibiotics. CONCLUSIONS: A metagenomic approach provides detailed information on the presence and diversity of pathogenic organisms in human stool samples. Metagenomic studies allow for in-depth molecular characterization such as the antimicrobial resistance status, which may be useful to develop setting-specific treatment algorithms. While metagenomic approaches remain challenging, the benefits of gaining new insights into intestinal microbial communities call for a broader application in epidemiologic studies. TRIAL REGISTRATION: ISRCTN86951400.201626391184
465470.9997Early Bacterial Colonization and Antibiotic Resistance Gene Acquisition in Newborns. Several studies have recently identified the main factors contributing to the bacterial colonization of newborns and the dynamics of the infant microbiome development. However, most of these studies address large time periods of weeks or months after birth, thereby missing on important aspects of the early microbiome maturation, such as the acquisition of antibiotic resistance determinants during postpartum hospitalization. The pioneer bacterial colonization and the extent of its associated antibiotic resistance gene (ARG) dissemination during this early phase of life are largely unknown. Studies addressing resistant bacteria or ARGs in neonates often focus only on the presence of particular bacteria or genes from a specific group of antibiotics. In the present study, we investigated the gut-, the oral-, and the skin-microbiota of neonates within the first 72 h after birth using 16S rDNA sequencing approaches. In addition, we screened the neonates and their mothers for the presence of 20 different ARGs by directed TaqMan qPCR assays. The taxonomic analysis of the newborn samples revealed an important shift of the microbiota during the first 72 h after birth, showing a clear site-specific colonization pattern in this very early time frame. Moreover, we report a substantial acquisition of ARGs during postpartum hospitalization, with a very high incidence of macrolide resistance determinants and mecA detection across different body sites of the newborns. This study highlights the importance of antibiotic resistance determinant dissemination in neonates during hospitalization, and the need to investigate the implication of the mothers and the hospital environment as potential sources of ARGs.202032754449
464080.9997Genome analysis of probiotic bacteria for antibiotic resistance genes. To date, probiotic bacteria are used in the diet and have various clinical applications. There are reports of antibiotic resistance genes in these bacteria that can transfer to other commensal and pathogenic bacteria. The aim of this study was to use whole-genome sequence analysis to identify antibiotic resistance genes in a group of bacterial with probiotic properties. Also, this study followed existing issues about the importance and presence of antibiotic resistance genes in these bacteria and the dangers that may affect human health in the future. In the current study, a collection of 126 complete probiotic bacterial genomes was analyzed for antibiotic resistance genes. The results of the current study showed that there are various resistance genes in these bacteria that some of them are transferable to other bacteria. The tet(W) tetracycline resistance gene was more than other antibiotic resistance genes in these bacteria and this gene was found in Bifidobacterium and Lactobacillus. In our study, the most numbers of antibiotic resistance genes were transferred with mobile genetic elements. We propose that probiotic companies before the use of a micro-organism as a probiotic, perform an antibiotic susceptibility testing for a large number of antibiotics. Also, they perform analysis of complete genome sequence for prediction of antibiotic resistance genes.202234989942
460190.9996CRISPR tracking reveals global spreading of antimicrobial resistance genes by Staphylococcus of canine origin. The close contact between pets and their owners is a potential source for microorganisms and genetic material exchange. Staphylococcus species considered as harmless inhabitants of animals' and humans' microbiota can act as reservoirs of antimicrobial resistance genes to more virulent species, thereby increasing their potential to resist drug therapy. This process could be inhibited by the antiplasmid immunity conferred by CRISPR systems. On the other hand, CRISPR spacer sequences can be explored as molecular clocks to track the history of genetic invasion suffered by a bacterial strain. To understand better the role of domestic dogs in human health as an antimicrobial resistance genes source, we analyzed 129 genomes of Staphylococcus strains of canine origin for the presence of CRISPR systems. Only 8% of the strains were positive for CRISPR, which is consistent with Staphylococcus role as gene reservoirs. The plasmidial origin or some spacers confirms the unsuccessful attempt of plasmid exchange in strains carrying CRISPRs. Some of these systems are within a staphylococcal cassette chromosome mec (SCCmec), sharing 98% of identity between their harboring strains. These CRISPRs' spacers reveal that this SCCmec was transferred between canine S. pseudintermedius strains, then to S. schleiferi and to Staphylococcus strains isolated from human beings. Our findings shows genetic evidence for the global spreading of pathogenic bacteria and the antimicrobial resistance genes carried by them and reinforce that, in the age of antimicrobial resistance, it is imperative that drug therapies consider the integrated nature of the relationship between pets and humans.201931030846
9918100.9996Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR. The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.202134282723
4322110.9996Multi-Drug Resistance in Bacterial Genomes-A Comprehensive Bioinformatic Analysis. Antimicrobial resistance is presently one of the greatest threats to public health. The excessive and indiscriminate use of antibiotics imposes a continuous selective pressure that triggers the emergence of multi-drug resistance. We performed a large-scale analysis of closed bacterial genomes to identify multi-drug resistance considering the ResFinder antimicrobial classes. We found that more than 95% of the genomes harbor genes associated with resistance to disinfectants, glycopeptides, macrolides, and tetracyclines. On average, each genome encodes resistance to more than nine different classes of antimicrobial drugs. We found higher-than-expected co-occurrences of resistance genes in both plasmids and chromosomes for several classes of antibiotic resistance, including classes categorized as critical according to the World Health Organization (WHO). As a result of antibiotic-resistant priority pathogens, higher-than-expected co-occurrences appear in plasmids, increasing the potential for resistance dissemination. For the first time, co-occurrences of antibiotic resistance have been investigated for priority pathogens as defined by the WHO. For critically important pathogens, co-occurrences appear in plasmids, not in chromosomes, suggesting that the resistances may be epidemic and probably recent. These results hint at the need for new approaches to treating infections caused by critically important bacteria.202337511196
3910120.9996Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages. Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.202438935220
4562130.9996The Dynamics of the Antimicrobial Resistance Mobilome of Salmonella enterica and Related Enteric Bacteria. The foodborne pathogen Salmonella enterica is considered a global public health risk. Salmonella enterica isolates can develop resistance to several antimicrobial drugs due to the rapid spread of antimicrobial resistance (AMR) genes, thus increasing the impact on hospitalization and treatment costs, as well as the healthcare system. Mobile genetic elements (MGEs) play key roles in the dissemination of AMR genes in S. enterica isolates. Multiple phenotypic and molecular techniques have been utilized to better understand the biology and epidemiology of plasmids including DNA sequence analyses, whole genome sequencing (WGS), incompatibility typing, and conjugation studies of plasmids from S. enterica and related species. Focusing on the dynamics of AMR genes is critical for identification and verification of emerging multidrug resistance. The aim of this review is to highlight the updated knowledge of AMR genes in the mobilome of Salmonella and related enteric bacteria. The mobilome is a term defined as all MGEs, including plasmids, transposons, insertion sequences (ISs), gene cassettes, integrons, and resistance islands, that contribute to the potential spread of genes in an organism, including S. enterica isolates and related species, which are the focus of this review.202235432284
3923140.9996Antimicrobial resistance genes in raw milk for human consumption. The increasing prevalence of antimicrobial resistance (AMR) is a significant threat to global health. More and more multi-drug-resistant bacterial strains cause life-threatening infections and the death of thousands of people each year. Beyond disease control animals are often given antibiotics for growth promotion or increased feed efficiency, which further increase the chance of the development of multi-resistant strains. After the consumption of unprocessed animal products, these strains may meet the human bacteriota. Among the foodborne and the human populations, antimicrobial resistance genes (ARGs) may be shared by horizontal gene transfer. This study aims to test the presence of antimicrobial resistance genes in milk metagenome, investigate their genetic position and their linkage to mobile genetic elements. We have analyzed raw milk samples from public markets sold for human consumption. The milk samples contained genetic material from various bacterial species and the in-depth analysis uncovered the presence of several antimicrobial resistance genes. The samples contained complete ARGs influencing the effectiveness of acridine dye, cephalosporin, cephamycin, fluoroquinolone, penam, peptide antibiotics and tetracycline. One of the ARGs, PC1 beta-lactamase may also be a mobile element that facilitates the transfer of resistance genes to other bacteria, e.g. to the ones living in the human gut.202032366826
4559150.9996Systematic detection of horizontal gene transfer across genera among multidrug-resistant bacteria in a single hospital. Multidrug-resistant bacteria pose a serious health threat, especially in hospitals. Horizontal gene transfer (HGT) of mobile genetic elements (MGEs) facilitates the spread of antibiotic resistance, virulence, and environmental persistence genes between nosocomial pathogens. We screened the genomes of 2173 bacterial isolates from healthcare-associated infections from a single hospital over 18 months, and identified identical nucleotide regions in bacteria belonging to distinct genera. To further resolve these shared sequences, we performed long-read sequencing on a subset of isolates and generated highly contiguous genomes. We then tracked the appearance of ten different plasmids in all 2173 genomes, and found evidence of plasmid transfer independent from bacterial transmission. Finally, we identified two instances of likely plasmid transfer within individual patients, including one plasmid that likely transferred to a second patient. This work expands our understanding of HGT in healthcare settings, and can inform efforts to limit the spread of drug-resistant pathogens in hospitals.202032285801
4178160.9996Efficacy and food safety considerations of poultry competitive exclusion products. Competitive exclusion (CE) products are anaerobic cultures of bacteria that are applied to poultry hatchlings to establish a protective enteric microbiota that excludes intestinal colonization by human food-borne pathogens. For safety of the poultry flock and human consumers, the identities of bacteria in CE products need to be known. A CE product is a culture of intestinal contents from adult chickens. It may be microbiologically defined by analysis of bacteria isolated from the culture, but many bacteria are hard to reliably isolate, identify, and characterize with conventional techniques. Sequence analysis of 16S ribosomal RNA (rRNA) genes may be more reliable than conventional techniques to identify CE bacteria. Bacteria in CE products may contain antimicrobial drug resistance and virulence mechanisms that could be transferred to the enteric bacteria of the food animal and to the human consumer. Detection methods for specific antimicrobial drug resistance and virulence genes and the integrase genes of conjugative transposons, mostly utilizing PCR technology, are being developed that can be applied to assess these risks in CE bacteria. With improvements in efficacy, bacterial identification, and detection and control of the possible risks of gene transfer, CE product technology can be made a more effective food safety tool.200617039457
3149170.9996Effect of a probiotic and an antibiotic on the mobilome of the porcine microbiota. Introduction: To consider the growing health issues caused by antibiotic resistance from a "one health" perspective, the contribution of meat production needs to be addressed. While antibiotic resistance is naturally present in microbial communities, the treatment of farm animals with antibiotics causes an increase in antibiotic resistance genes (ARG) in the gut microbiome. Pigs are among the most prevalent animals in agriculture; therefore, reducing the prevalence of antibiotic-resistant bacteria in the pig gut microbiome could reduce the spread of antibiotic resistance. Probiotics are often studied as a way to modulate the microbiome and are, therefore, an interesting way to potentially decrease antibiotic resistance. Methods: To assess the efficacy of a probiotic to reduce the prevalence of ARGs in the pig microbiome, six pigs received either treatment with antibiotics (tylvalosin), probiotics (Pediococcus acidilactici MA18/5M; Biopower(®) PA), or a combination of both. Their faeces and ileal digesta were collected and DNA was extracted for whole genome shotgun sequencing. The reads were compared with taxonomy and ARG databases to identify the taxa and resistance genes in the samples. Results: The results showed that the ARG profiles in the faeces of the antibiotic and combination treatments were similar, and both were different from the profiles of the probiotic treatment (p < 0.05). The effects of the treatments were different in the digesta and faeces. Many macrolide resistance genes were detected in a higher proportion in the microbiome of the pigs treated with antibiotics or the combination of probiotics and antibiotics. Resistance-carrying conjugative plasmids and horizontal transfer genes were also amplified in faeces samples for the antibiotic and combined treatments. There was no effect of treatment on the short chain fatty acid content in the digesta or the faeces. Conclusion: There is no positive effect of adding probiotics to an antibiotic treatment when these treatments are administered simultaneously.202438606356
3874180.9996Culture-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
9912190.9996Comprehensive Genomic Investigation of Coevolution of mcr genes in Escherichia coli Strains via Nanopore Sequencing. Horizontal gene transfer facilitates the spread of antibiotic resistance genes, which constitutes a global challenge. However, the evolutionary trajectory of the mobile colistin resistome in bacteria is largely unknown. To investigate the coevolution and fitness cost of the colistin resistance genes in wild strains, different assays to uncover the genomic dynamics of mcr-1 and mcr-3 in bacterial populations are utilized. Escherichia coli strains harboring both mcr-1 and mcr-3.1/3.5 are isolated and mcr genes are associated with diverse mobile elements. Under exposure to colistin, the mcr-1-bearing resistome is stably inherited during bacterial replication, but mcr-3 is prone to be eliminated in populations of certain strains. In the absence of colistin, the persistence rates of the mcr-1 and mcr-3-bearing subclones varies depending on the genomic background. The decay of the mcr-bearing bacterial populations can be mediated by the elimination of mcr-containing segments, large genomic deletions, and plasmid loss. Mobile elements, including plasmids and transposons, are double-edged swords in the evolution of the resistome. The findings support the idea that antibiotic overuse accounts for global spread of multidrug-resistant (MDR) bacteria. Therefore, stringent regulation of antibiotic prescription for humans and animals should be performed systematically to alleviate the threat of MDR bacteria.202133728052