Carriage Of Antibiotic Resistance Genes Research Articles

Whole-genome analysis of a ST45-SCCmec IVa (2B)-t116 methicillin-resistant Staphylococcus aureus strain isolated from the sputum of a 5-year-old child with pneumonia

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 is a major global MRSA lineage with huge strain diversity and a high clinical impact. In Hainan and Guangzhou of China, the ST45-MRSA was mainly associated with t116.MethodsThe MRSA strain SA2107 was isolated from the sputum of a 5-year-old child with pneumonia. The whole genome of SA2107 was sequence using Illumina (Novaseq 6000) and PacBio (Sequel IIe) sequencers, and the sequences were assembled using hybrid assembly. The carriage of antibiotic resistance genes, virulence genes, and mobile genetic elements were identified using bioinformatics tools. The comparative genomic analyses of MRSA strain SA2107 with other MRSA strains worldwide were performed.FindingsThe genome size of ST45-SCCmec IVa (2B)-t116 MRSA strain SA2107 was ~2.9 Mb. Mobile genetic elements analysis of SA2107 revealed two plasmids (30,064-bp pSA2107-1 and 8,033-bp pSA2107-2), three prophages, two integrative and conjugative elements (ICEs), and two insertion sequences (ISs, IS431 and IS1272). The SCCmec IVa (2B) carried by SA2107 contained the class B mec gene complex (IS431-mecA-ΔmecR1-IS1272) and type 2 ccr gene complex (ccrA2 and ccrB2). Besides mecA, another beta-lactam resistance gene blaZ was found to located on six copies of bla complex (blaZ, blaR1, and blaI) on the chromosome of SA2107. Three kinds of virulence factors were detected on the chromosome of SA2107, including genes encoding toxins, exoenzyme, and immune-modulating protein. Notably, the three prophages harbored by the chromosome of SA2107 all carried virulence genes.ConclusionThus far, only three complete genomes available for ST45-SCCmec IVa (2B)-t116 strain from United States, Germany, and Australia, respectively. The strain SA2107 was the first complete genome data (CP104559) from China for ST45-SCCmec IVa (2B)-t116 MRSA.

Exposure to doxycycline increases risk of carrying a broad range of enteric antimicrobial resistance determinants in an elderly cohort

High rates of antibiotic prescription in residential aged care are likely to promote enteric carriage of antibiotic-resistant pathogens and increase the risk of antibiotic treatment failure. Despite their importance, relationships between antibiotic exposures and patterns of enteric resistance carriage in this population remain poorly understood. We conducted a cross-sectional metagenomic cohort analysis of stool samples from residents of five long-term aged-care facilities in South Australia. Taxonomic composition was determined, and enteric carriage of antibiotic resistance genes (ARGs) was identified and quantified against the Comprehensive Antibiotic Resistance Database. Both the detection and abundance of stool taxa and ARGs were related to antibiotic exposures up to 12 months prior. Factors associated with the abundance of ARGs of high clinical concern were identified. Stool samples were provided by 164 participants (median age: 88 years, IQR 81-93; 72% female). Sixty-one percent (n=100) of participants were prescribed antibiotics at least once in the prior 12 months (median prescriptions: 4, range: 1-52), most commonly a penicillin (n=55, 33.5%), cephalosporin (n=53, 32.3%), diaminopyrimidine (trimethoprim) (n=36, 22%), or tetracycline (doxycycline) (n=21, 12.8%). More than 1100 unique ARGs, conferring resistance to 38 antibiotic classes, were identified, including 20 ARGs of high clinical concern. Multivariate logistic regression showed doxycycline exposure to be the greatest risk factor for high ARG abundance (adjusted odds ratio [aOR]=14.8, q<0.001) and a significant contributor to inter-class selection, particularly for ARGs relating to penicillins (aOR=3.1, q=0.0004) and cephalosporins (aOR=3.4, q=0.003). High enteric ARG abundance was associated with the number of separate antibiotic exposures (aOR: 6.4, q<0.001), exposures within the prior 30 days (aOR: 4.6, q=0.008) and prior 30-100 days (aOR: 2.6, q=0.008), high duration of antibiotic exposure (aOR: 7.9, q<0.001), and exposure to 3 or more antibiotic classes (aOR: 7.4, q<0.001). Carriage of one or more ARGs of high clinical concern was identified in 99% of participants (n=162, median: 3, IQR: 2-4), involving 11 ARGs conferring resistance to aminoglycosides, four to beta-lactams, one to glycopeptides, three to fluoroquinolones, and one to oxazolidinones. Carriage of ARGs of high clinical concern was positively associated with exposure to doxycycline (aminoglycoside, fluoroquinolone, and oxazolidinone ARGs) and trimethoprim (fluoroquinolone and beta-lactam ARGs). Analysis of doxycycline impact on microbiota composition suggested that observed resistome changes arose principally through direct ARG selection, rather than through the antibiotic depletion of sensitive bacterial populations. The gut microbiome of aged care residents is a major reservoir of antibiotic resistance. As a critical antibiotic in medical practice, a comprehensive understanding of the impact of doxycycline exposure on the gut resistome is paramount for informed antibiotic use, particularly in an evolving landscape of prophylactic applications. Near-universal asymptomatic carriage of clinically critical resistance determinants is highly concerning and reinforces the urgent need for improved management of antibiotic use in long-term aged care.

Prolonged hospitalization signature and early antibiotic effects on the nasopharyngeal resistome in preterm infants

Respiratory pathogens, commonly colonizing nasopharynx, are among the leading causes of death due to antimicrobial resistance. Yet, antibiotic resistance determinants within nasopharyngeal microbial communities remain poorly understood. In this prospective cohort study, we investigate the nasopharynx resistome development in preterm infants, assess early antibiotic impact on its trajectory, and explore its association with clinical covariates using shotgun metagenomics. Our findings reveal widespread nasopharyngeal carriage of antibiotic resistance genes (ARGs) with resistomes undergoing transient changes, including increased ARG diversity, abundance, and composition alterations due to early antibiotic exposure. ARGs associated with the critical nosocomial pathogen Serratia marcescens persist up to 8–10 months of age, representing a long-lasting hospitalization signature. The nasopharyngeal resistome strongly correlates with microbiome composition, with inter-individual differences and postnatal age explaining most of the variation. Our report on the collateral effects of antibiotics and prolonged hospitalization underscores the urgency of further studies focused on this relatively unexplored reservoir of pathogens and ARGs.

Variability of faecal microbiota and antibiotic resistance genes in flocks of migratory gulls and comparison with the surrounding environment

Gulls commonly rely on human-generated waste as their primary food source, contributing to the spread of antibiotic-resistant bacteria and their resistance genes, both locally and globally. Our understanding of this process remains incomplete, particularly in relation to its potential interaction with surrounding soil and water. We studied the lesser black-backed gull, Larus fuscus, as a model to examine the spatial variation of faecal bacterial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) and its relationship with the surrounding water and soil. We conducted sampling campaigns within a connectivity network of different flocks of gulls moving across functional units (FUs), each of which represents a module of highly interconnected patches of habitats used for roosting and feeding. The FUs vary in habitat use, with some gulls using more polluted sites (notably landfills), while others prefer more natural environments (e.g., wetlands or beaches). Faecal bacterial communities in gulls from flocks that visit and spend more time in landfills exhibited higher richness and diversity. The faecal microbiota showed a high compositional overlap with bacterial communities in soil. The overlap was greater when compared to landfill (11%) than to wetland soils (6%), and much lower when compared to bacterial communities in surrounding water (2% and 1% for landfill and wetland water, respectively). The relative abundance of ARGs and MGEs were similar between FUs, with variations observed only for specific families of ARGs and MGEs. When exploring the faecal carriage of ARGs and MGEs in bird faeces relative to soil and water compartments, gull faeces were enriched in ARGs classified as High-Risk. Our results shed light on the complex dynamics of antibiotic resistance spread in wild bird populations, providing insights into the interactions among gull movement and feeding behavior, habitat characteristics, and the dissemination of antibiotic resistance determinants across environmental reservoirs.

Winged resistance: Storks and gulls increase carriage of antibiotic resistance by shifting from paddy fields to landfills

Waterbirds are vectors for the dissemination of antimicrobial resistance across environments, with some species increasingly reliant on highly anthropized habitats for feeding. However, data on the impact of their feeding habits on the carriage of antibiotic resistance genes (ARGs) are still scarce. To fill this gap, we examined the microbiota (16S rRNA amplicon gene sequencing) and the prevalence of ARG (high-throughput qPCR of 47 genes) in faeces from white storks (Ciconia ciconia) and lesser black-backed gulls (Larus fuscus) feeding in highly (landfill) and less (paddy fields) polluted habitats. Faecal bacterial richness and diversity were higher in gulls feeding upon landfills and showed a greater abundance of potential pathogens, such as Staphylococcus. In contrast, faecal bacterial communities from storks were similar regardless of habitat preferences, maybe due to a less intense habitat use compared to gulls. In addition, birds feeding in the landfill carried a higher burden of ARGs compared to the surrounding soil and surface waters. Network analysis revealed strong correlations between ARGs and potential pathogens, particularly between tetM (resistance to tetracyclines), blaCMY (beta-lactam resistance), sul1 (sulfonamide resistance) and members of the genera Streptococcus, Peptostreptococcus, and Peptoclostridium. Our work demonstrates how transitioning from paddy fields to landfills fosters the carriage of ARGs and potential pathogens in the bird gut, shedding light on the ecological role of these avian vectors in antimicrobial resistance dissemination.

Investigation of the antimicrobial susceptibility patterns of marine cyanobacteria in Bohai Bay: Cyanobacteria may be important hosts of antibiotic resistance genes in marine environment

Marine cyanobacteria, as widely distributed and photosynthetically autotrophic bacteria in the ocean, may contribute to the global dissemination of antibiotic resistance genes (ARGs) and develop a different antimicrobial susceptibility pattern from heterotrophic bacteria and cyanobacteria from freshwater environments. However, studies on antimicrobial susceptibility and the carriage of ARGs in marine cyanobacteria are still very limited. In this study, the antibiotic resistance characteristics of cyanobacteria in nearshore waters were examined through field monitoring and laboratory investigations, which included PCR detection and ARG transformation. The results showed a positive correlation between marine cyanobacteria and some ARGs in the nearshore waters of Bohai Bay. Moreover, most screened cyanobacteria showed high minimum inhibitory concentration (MIC) values for polymyxins, tetracyclines, kanamycin, and sulfonamides, moderate MIC values for streptomycin, chloramphenicol, rifampicin, and norfloxacin, and low MIC values for roxithromycin and cephalosporins. The blaTEM, blaKPC, sul1, sul2, strA, tetA, tetB, tetC, tetM, mdfA, and intI1 genes were detected in the screened marine cyanobacteria. The highest detection rates were observed for blaTEM (93.3 %), sul1 (56.6 %), sul2 (90 %), and strA (73.3 %). The detection rate of tetA (33.3 %) was the highest among the tetracycline resistance genes, and mdfA, a multidrug-resistant pump gene with resistance to tetracycline, also showed a high detection level (23.3 %). Overall, most of the screened marine cyanobacteria were found to tolerate multiple antibiotics in seawater, and the condition of the ARGs carriage was serious. Furthermore, the screened marine Synechocystis sp. C12-2 demonstrated the ability to accept ARGs on the RP4 plasmid through natural transformation and showed reduced sensitivity to ampicillin, suggesting the possibility that some marine cyanobacteria could acquire ARGs from the environment through horizontal gene transfer. Thus, marine cyanobacteria may play an important role in the propagation of marine ARGs.

Carriage of antibiotic resistance genes to treatments for chlamydial disease in koalas (Phascolarctos cinereus): A comparison of occurrence before and during catastrophic wildfires

Growing reports of diverse antibiotic resistance genes in wildlife species around the world symbolises the extent of this global One Health issue. The health of wildlife is threatened by antimicrobial resistance in situations where wildlife species develop disease and require antibiotics. Chlamydial disease is a key threat for koalas in Australia, with infected koalas frequently entering wildlife hospitals and requiring antibiotic therapy, typically with chloramphenicol or doxycycline. This study investigated the occurrence and diversity of target chloramphenicol and doxycycline resistance genes (cat and tet respectively) in koala urogenital and faecal microbiomes. DNA was extracted from 394 urogenital swabs and 91 faecal swabs collected from koalas in mainland Australia and on Kangaroo Island (KI) located 14 km off the mainland, before (n = 145) and during (n = 340) the 2019–2020 wildfires. PCR screening and DNA sequencing determined 9.9% of samples (95%CI: 7.5% to 12.9%) carried cat and/or tet genes, with the highest frequency in fire-affected KI koalas (16.8%) and the lowest in wild KI koalas sampled prior to fires (6.5%). The diversity of cat and tet was greater in fire-affected koalas (seven variants detected), compared to pre-fire koalas (two variants detected). Fire-affected koalas in care that received antibiotics had a significantly higher proportion (p < 0.05) of cat and/or tet genes (37.5%) compared to koalas that did not receive antibiotics (9.8%). Of the cat and/or tet positive mainland koalas, 50.0% were Chlamydia-positive by qPCR test. Chloramphenicol and doxycycline resistance genes in koala microbiomes may contribute to negative treatment outcomes for koalas receiving anti-chlamydial antibiotics. Thus a secondary outcome of wildfires is increased risk of acquisition of cat and tet genes in fire-affected koalas that enter care, potentially exacerbating the already significant threat of chlamydial disease on Australia's koalas. This study highlights the importance of considering impacts to wildlife health within the One Health approach to AMR and identifies a need for greater understanding of AMR ecology in wildlife.

Metagenomic profiles of planktonic bacteria and resistome along a salinity gradient in the Pearl River Estuary, South China

Estuarine ecosystems undergo pronounced and intricate changes due to the mixing of freshwater and saltwater. Additionally, urbanization and population growth in estuarine regions result in shifts in the planktonic bacterial community and the accumulation of antibiotic resistance genes (ARGs). The dynamic changes in bacterial communities, environmental factors, and carriage of ARGs from freshwater to seawater, as well as the complex interrelationships among these factors, have yet to be fully elucidated. Here, we conducted a comprehensive study based on metagenomic sequencing and full-length 16S rRNA sequencing, covering the entire Pearl River Estuary (PRE) in Guangdong, China. The abundance and distribution of the bacterial community, ARGs, mobile genetic elements (MGEs), and bacterial virulence factors (VFs) were analyzed on a site-by-site basis through sampling along the salinity gradient in PRE, from upstream to downstream. The structure of the planktonic bacterial community undergoes continuous changes in response to variations in estuarine salinity, with the phyla Proteobacteria and Cyanobacteria being dominant bacterial throughout the entire region. The diversity and abundance of ARGs and MGEs gradually decreased with the direction of water flow. A large number of ARGs were carried by potentially pathogenic bacteria, especially in Alpha-proteobacteria and Beta-proteobacteria. Multi-drug resistance genes have the highest abundance and subtypes in PRE. In addition, ARGs are more linked to some MGEs than to specific bacterial taxa and disseminate mainly by HGT and not by vertical transfer in the bacterial communities. Various environmental factors, such as salinity and nutrient concentrations, have a significantly impact on the community structure and distribution of bacteria. In conclusion, our results represent a valuable resource for further investigating the intricate interplay between environmental factors and anthropogenic disturbances on bacterial community dynamics. Moreover, they contribute to a better understanding of the relative impact of these factors on the dissemination of ARGs.

Factors associated with plasmid antibiotic resistance gene carriage revealed using large-scale multivariable analysis

Plasmids are major vectors of bacterial antibiotic resistance, but understanding of factors associated with plasmid antibiotic resistance gene (ARG) carriage is limited. We curated > 14,000 publicly available plasmid genomes and associated metadata. Duplicate and replicate plasmids were excluded; where possible, sample metadata was validated externally (BacDive database). Using Generalised Additive Models (GAMs) we assessed the influence of 12 biotic/abiotic factors (e.g. plasmid genetic factors, isolation source, collection date) on ARG carriage, modelled as a binary outcome. Separate GAMs were built for 10 major ARG types. Multivariable analysis indicated that plasmid ARG carriage patterns across time (collection years), isolation sources (human/livestock) and host bacterial taxa were consistent with antibiotic selection pressure as a driver of plasmid-mediated antibiotic resistance. Only 0.42% livestock plasmids carried carbapenem resistance (compared with 12% human plasmids); conversely, tetracycline resistance was enriched in livestock vs human plasmids, reflecting known prescribing practices. Interpreting results using a timeline of ARG type acquisition (determined by literature review) yielded additional novel insights. More recently acquired ARG types (e.g. colistin and carbapenem) showed increases in plasmid carriage during the date range analysed (1994–2019), potentially reflecting recent onset of selection pressure; they also co-occurred less commonly with ARGs of other types, and virulence genes. Overall, this suggests that following acquisition, plasmid ARGs tend to accumulate under antibiotic selection pressure and co-associate with other adaptive genes (other ARG types, virulence genes), potentially re-enforcing plasmid ARG carriage through co-selection.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

885. Integrated Blood Culture and Molecular Diagnostic Device Reduces Time-to-Result to a Quarter of Traditional Methods

Abstract Background Prolonged antibiotic exposure in infants with negative blood cultures is associated with adverse outcomes, disruption of the developing microbiome, and increased carriage of antibiotic resistance genes. However, early administration of antibiotics significantly reduces risk of infant morbidity and mortality caused by infections, and therefore faster culture-to-identification is highly desirable. We combined blood culture (BC) and PCR-based pathogen identification into a single digital microfluidic (DMF) device to speed up BC results. Methods Our DMF BC+PCR device has a custom-built BC bottle incubator, which is connected to a disposable cartridge that performs sample preparation and PCR. Standard BC bottles were inoculated with K. pneumoniae, then incubated in either the DMF BC device or a traditional BC device (BD BACTEC 9050). Samples were drawn from the DMF BC bottle at 3, 4 and 5 hours and loaded in the cartridge for automated sample processing and PCR. Time-to-positivity for the DMF BC+PCR device was determined using cycle threshold and compared with the traditional method, which uses fluorescence from BC bottle followed by PCR on a separate device. Results Bacterial presence could be detected as quickly as 3 hours post inoculation (Ct values of 34, 31 and 28 at 3, 4, and 5 hrs) with five replicates of K. pneumoniae samples on the DMF BC+PCR device (growing 10x every hr), compared to &amp;gt; 12 hrs for the traditional method (10.5 hr culture and 2 hr PCR identification). Additionally, for 5 common pathogens that cause neonatal sepsis (K. pneumoniae, E. coli, S. aureus, S. agalactiae, or S. epidermidis), we also demonstrated that the DMF BC bottle incubator and the traditional BC device show similar growth curves using optical methods to determine growth. Conclusion The DMF BC system yields bacterial growth rates equivalent to traditional methods, but can drastically reduce time to pathogen identification, from 12 hrs to 3 hrs by leveraging PCR sensitivity to determine growth rather than fluorescence in the BC bottles. To our knowledge, this is the first demonstration of using an integrated microfluidic PCR platform to speed up culture and identification. Further work is ongoing to include antibiotic susceptibility testing and expand the panel of pathogens detected on the same cartridge. Disclosures Laura Huning, PhD, Baebies, Inc.: Stocks/Bonds Devika Varma, PhD, Baebies, Inc.: Stocks/Bonds Abbey Jackson, PhD, Baebies, Inc.: Stocks/Bonds Peter Bohlen, n/a, Baebies, Inc.: Stocks/Bonds Rainer Ng, PhD, Baebies, Inc.: Stocks/Bonds Vamsee Pamula, PhD, Baebies, Inc.: Stocks/Bonds.

Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.

This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.

High prevalence of antibiotic resistance in pathogenic foodborne bacteria isolated from bovine milk

This study aimed to investigate the prevalence of foodborne pathogenic bacteria in bovine milk, their antibiogram phenotype, and the carriage of antibiotic resistance genes. Raw bovine milk samples (n = 100) were randomly collected from different suppliers in the northwest of Iran. Antibiotic-resistant patterns and the presence of antibiotic resistance genes were evaluated in the isolates. Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella spp. were isolated from 78%, 47%, 25%, and 21% of samples, respectively. All isolates showed high rates of resistance to amoxicillin, penicillin, and cefalexin. The blaTEM and blaSHV genes were detected in 50.0% and 6.4% of E. coli isolates, respectively. Also, 28.5% and 19.0% of Salmonella isolates were positive for blaTEM and blaSHV. The frequency of mecA and blaZ in S. aureus isolates was 20.0% and 12.0%, respectively. The high prevalence of bovine milk contamination with antimicrobial-resistant species in this study necessitates precise control on antibiotic prescription in veterinary medicine.

INfrastructure for a PHAge REference Database: Identification of Large-Scale Biases in the Current Collection of Cultured Phage Genomes.

Background: With advances in sequencing technology and decreasing costs, the number of phage genomes that have been sequenced has increased markedly in the past decade. Materials and Methods: We developed an automated retrieval and analysis system for phage genomes (https://github.com/RyanCook94/inphared) to produce the INfrastructure for a PHAge REference Database (INPHARED) of phage genomes and associated metadata. Results: As of January 2021, 14,244 complete phage genomes have been sequenced. The INPHARED data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated on only 30 bacterial genera. There is further bias, with significantly more lytic phage genomes (∼70%) than temperate (∼30%) within our database. Collectively, this results in ∼54% of temperate phage genomes originating from just three host genera. With much debate on the carriage of antibiotic resistance genes and their potential safety in phage therapy, we searched for putative antibiotic resistance genes. Frequency of antibiotic resistance gene carriage was found to be higher in temperate phages than in lytic phages and again varied with host. Conclusions: Given the bias of currently sequenced phage genomes, we suggest to fully understand phage diversity, efforts should be made to isolate and sequence a larger number of phages, in particular temperate phages, from a greater diversity of hosts.

Fecal Putative Uropathogen Abundance and Antibiotic Resistance Gene Carriage in Women With Refractory Recurrent Urinary Tract Infection Treated With Fecal Microbiota Transplantation.

The aims of this study were to describe the fecal relative abundance of potentially uropathogenic bacteria and to analyze antibiotic resistance genes before and after fecal microbiota transplantation in women with recurrent urinary tract infection (UTI). Shotgun sequencing was performed on fecal samples from 3 donors and 4 women with recurrent UTI who underwent transplantation. Recipient samples were sequenced at baseline and at 4 time points through 6 months postintervention. Relative fecal uropathogen abundance was analyzed by species and participant using descriptive statistics. Antibiotic resistance gene abundance was assigned, normalized, and compared between donors and recipients at baseline and postintervention using an abundance bar plot, nonmetric multidimensional scaling, and pairwise permutational multivariate analysis of variance. The median (range) relative abundance of Escherichia coli in all fecal samples from women with recurrent UTI was 0% (0%-5.10%); Enterococcus faecalis, 0% (0%-0.20%); Enterococcus faecium, 0% (0%-1.90%); Klebsiella pneumoniae, 0% (0%-0.10%); and Pseudomonas aeruginosa, 0% (0%-0.10%). Gut microbes carried genes conferring resistance to antibiotics used for UTI. No significant difference was seen in antibiotic resistance gene carriage after transplantation compared with baseline (P=0.22, R2=0.08 at 3 months). Antibiotic gene composition and abundance were significantly associated with the individual from whom the sample came (P=0.004, R2=0.78 at 3 months). Exploratory analysis of gut microbiomes in women with recurrent UTI identifies no or low relative putative uropathogen abundance for all species examined. Antibiotic resistance gene carriage persisted after fecal microbiota transplantation, although conclusions are limited by small sample size.

Profiling the antibiotic resistome in soils between pristine and human-affected sites on the Tibetan Plateau

With increasing pressure from anthropogenic activity in pristine environments, the comprehensive profiling of antibiotic resistance genes (ARGs) is essential to evaluate the potential risks from human-induced antibiotic resistance in these under-studied places. Here, we characterized the microbial resistome in relatively pristine soil samples collected from four distinct habitats on the Tibetan Plateau, using a Smart chip based high-throughput qPCR approach. We compared these to soils from the same habitats that had been subjected to various anthropogenic activities, including residential sewage discharge, animal farming, atmospheric deposition, and tourism activity. Compared to pristine samples, an average of 23.7% more ARGs were detected in the human-affected soils, and the ARGs enriched in these soils mainly encoded resistances to aminoglycoside and beta-lactam. Of the four habitats studied, soils subjected to animal farming showed the highest risks of ARG enrichment and dissemination. As shown, the number of ARGs enriched (a total of 42), their fold changes (17.6 fold on average), and the co-occurrence complexity between ARGs and mobile genetic elements were all the highest in fecal-polluted soils. As well as antibiotics themselves, heavy metals also influenced ARG distributional patterns in Tibetan environments. However, compared to urban areas, the Tibetan Plateau had a low potential for ARG selection and exhibited low carriage of ARGs by mobile genetic elements, even in environments impacted by humans, suggesting that these ARGs have a limited capacity to disseminate. The present study examined the effects of multiple anthropogenic activities on the soil resistomes in relatively pristine environments.

High fecal carriage of blaCTX-M, blaCMY-2, and plasmid-mediated quinolone resistance genes among healthy Korean people in a metagenomic analysis

To characterize the carriage of antibiotic resistance genes (ARGs) in the gut microbiome of healthy individuals. Fecal carriage of ARGs was investigated in 61 healthy individuals aged 30 to 59 years through whole metagenome sequencing of the gut microbiome and a targeted metagenomic approach. The number of ARGs in the gut microbiome was counted and normalized per million predicted genes (GPM). In the Korean population, the resistome ranged from 49.7 to 292.5 GPM (median 89.7). Based on the abundance of ARGs, the subjects were categorised into high (> 120 GPM), middle (60‒120 GPM), and low (< 60 GPM) ARG groups. Individuals in the high ARG group tended to visit hospitals more often (P = 0.065), particularly for upper respiratory tract infections (P = 0.066), and carried more blaCTX-M (P = 0.008). The targeted metagenome approach for bla and plasmid-mediated quinolone resistance (PMQR) genes revealed a high fecal carriage rate; 23% or 13.1% of the subjects carried blaCTX-M or blaCMY-2, respectively. Regarding PMQR genes, 59% of the subjects carried PMQR, and 83% of them harboured 2‒4 PMQR genes (qnrB 44.3%, qnrS 47.5% etc.). The presence of blaCTX-M correlated with ARG abundance in the gut resistome, whereas PMQR genes were irrelevant to other ARGs (P = 0.176). Fecal carriage of blaCTX-M and PMQR genes was broad and multiplexed among healthy individuals.

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Microbiota restoration reduces antibiotic-resistant bacteria gut colonization in patients with recurrent Clostridioides difficile infection from the open-label PUNCH CD study

BackgroundOnce antibiotic-resistant bacteria become established within the gut microbiota, they can cause infections in the host and be transmitted to other people and the environment. Currently, there are no effective modalities for decreasing or preventing colonization by antibiotic-resistant bacteria. Intestinal microbiota restoration can prevent Clostridioides difficile infection (CDI) recurrences. Another potential application of microbiota restoration is suppression of non-C. difficile multidrug-resistant bacteria and overall decrease in the abundance of antibiotic resistance genes (the resistome) within the gut microbiota. This study characterizes the effects of RBX2660, a microbiota-based investigational therapeutic, on the composition and abundance of the gut microbiota and resistome, as well as multidrug-resistant organism carriage, after delivery to patients suffering from recurrent CDI.MethodsAn open-label, multi-center clinical trial in 11 centers in the USA for the safety and efficacy of RBX2660 on recurrent CDI was conducted. Fecal specimens from 29 of these subjects with recurrent CDI who received either one (N = 16) or two doses of RBX2660 (N = 13) were analyzed secondarily. Stool samples were collected prior to and at intervals up to 6 months post-therapy and analyzed in three ways: (1) 16S rRNA gene sequencing for microbiota taxonomic composition, (2) whole metagenome shotgun sequencing for functional pathways and antibiotic resistome content, and (3) selective and differential bacterial culturing followed by isolate genome sequencing to longitudinally track multidrug-resistant organisms.ResultsSuccessful prevention of CDI recurrence with RBX2660 correlated with taxonomic convergence of patient microbiota to the donor microbiota as measured by weighted UniFrac distance. RBX2660 dramatically reduced the abundance of antibiotic-resistant Enterobacteriaceae in the 2 months after administration. Fecal antibiotic resistance gene carriage decreased in direct relationship to the degree to which donor microbiota engrafted.ConclusionsMicrobiota-based therapeutics reduce resistance gene abundance and resistant organisms in the recipient gut microbiome. This approach could potentially reduce the risk of infections caused by resistant organisms within the patient and the transfer of resistance genes or pathogens to others.Trial registrationClinicalTrials.gov, NCT01925417; registered on August 19, 2013.

Gut Microbiota Modulation and Fecal Transplantation: An Overview on Innovative Strategies for Hepatic Encephalopathy Treatment.

Simple SummaryTreatment of advanced liver disease and its complications continue to be a challenge due to the complexity of this illness. In recent years, the gut microbiome has been recognized to play a beneficial role in our health. Studies have shown that overgrowth of harmful organisms in the gut can contribute to worsened outcomes in liver disease. Fecal microbiota transplant (FMT) is an approved and effective treatment in other gastrointestinal conditions. FMT involves the administration of a solution of a fecal suspension from a healthy donor into the intestinal tract of a recipient. This has led researchers to attempt this treatment in liver disease. There have now been small clinical trials showing that FMT is safe and could be effective in improving outcomes in advanced liver disease. There remain several questions to be answered before FMT is implanted in clinical practice, including the best route to administer this treatment, how many doses are needed to achieve a therapeutic response, and how long we need to wait between treatments. In this review paper, we explore the role of the gut microbiome in the human body with emphasis on the gastrointestinal system, how it changes in liver disease, and how we can improve it with fecal microbiota transplant.Hepatic encephalopathy (HE) is a major complication of cirrhosis, which is associated with gut microbial composition and functional alterations. Current treatments largely focus on gut microbiota using lactulose, rifaximin and other agents. However, despite these treatments, patients with HE have a high rate of readmission, morbidity and cognitive impairment. Fecal microbiota transplant (FMT) involves introduction of a donor microbiota into a recipient and is currently mainly used for recurrent C. difficile infection (rCDI). The role of FMT in cirrhosis and HE is evolving. There have been two randomized clinical trials (RCT) and several case reports/series in cirrhosis. Both RCTs were safety-focused phase 1 trials. One involved pre-FMT antibiotics and FMT enema versus standard of care, while the other involved 15 FMT capsules versus placebo without pre-FMT antibiotics. There was evidence of safety in both trials and the FMT group demonstrated reduction in hospitalizations compared to the non-FMT group. Changes in microbial function centered around short-chain fatty acids, bile acids and brain function showed improvement in the FMT groups. Long-term follow-up demonstrated continued safety and reduction in the antibiotic-resistance gene carriage. However, larger trials of FMT in HE are needed that can refine the dose, duration and route of FMT administration.