Carriage Of Antimicrobial Resistance Genes Research Articles

Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug-resistant Gram-negative bacterial pathogens.

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug-resistant organisms (MDROs) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by transmission of MDROs, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focusing primarily on E. coli as both a widespread commensal and a globally disseminated multi-drug-resistant pathogen. Samples were taken during highly restrictive coronavirus disease 2019 (COVID-19) control measures from May to December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain-level colonisation of patients, focusing on E. coli sequence types (STs), their colonisation dynamics and antimicrobial resistance gene carriage. We found a lack of multi-drug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying bla KPC-2. There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli. Whilst 22 different E. coli STs were identified, only 1 strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result that may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR.

Antimicrobial Resistance Gene Carriage is not Associated with Uremic Toxin Levels in Chronic Kidney Disease Patients

In chronic kidney disease (CKD), uremic toxin molecules build up and risk of infection increase overtime, making the presence of antimicrobial resistant (AMR) bacteria in the gut more problematic with advancing disease. This study assessed if serum levels of uremic toxins and gut carriage of AMR genes are correlated in individuals with CKD. Whole metagenomic sequencing of stool samples and measured levels of 4 uremic toxins from 21 individuals with CKD were obtained. Multivariable, multivariate linear regression was performed with total abundance of AMR genes and abundance of specific drug classes of AMR genes as outcome variables and serum levels of four uremic toxins as predictor variables. Beta diversity differences of AMR genes were visualized using non-metric multidimensional scaling (NMDS) based on robust Aitchison distances and tested using PERMANOVA. There were no significant associations between uremic toxin levels and AMR gene abundance, or with any of the drug classes of AMR genes. There were no significant differences in beta diversity of AMR genes. These null findings suggest that increased uremic toxin levels are not associated with an increase in AMR carriage, and therefore uremia does not appear to be a direct cause of increased resistant bacteria in patients with more severe CKD.

Antibiotic resistance gene dynamics in the commensal infant gut microbiome over the first year of life

Colonization of the infant gut is an important developmental process characterized by high carriage of antimicrobial resistance genes (ARGs) and high abundances of pathobionts. The horizontal transfer of ARGs to pathogenic bacteria represents a major public health concern. However, there is still a paucity of longitudinal studies surveilling ARGs in healthy infant guts at high temporal resolution. Furthermore, we do not yet have a clear view of how temporal variation in ARG carriage relates to the dynamics of specific bacterial populations, as well as community virulence potential. Here, we performed deep shotgun metagenomic sequencing of monthly fecal samples from a cohort of 12 infants, covering the first year of life to interrogate the infant gut microbiome for ARG content. We further relate ARG dynamics to the dynamics of taxa, virulence potential, as well as the potential for ARG mobilization. We identify a core resistome dominated by efflux systems typically associated with Enterobacteriaceae. Overall ARG carriage declined over the first year of life and showed strong contemporaneous correlation with the population dynamics of Proteobacteria. Furthermore, the majority of ARGs could be further mapped to metagenome-assembled genomes (MAGs) classified to this phylum. We were able to assign a large number of ARGs to E. coli by correlating the temporal dynamics of individual genes with species dynamics, and we show that the temporal dynamics of ARGs and virulence factors are highly correlated, suggesting close taxonomic associations between these two gene classes. Finally, we identify ARGs linked with various categories of mobile genetic elements, demonstrating preferential linkage among mobility categories and resistance to different drug classes. While individual variation in ARG carriage is substantial during infancy there is a clear reduction over the first year of life. With few exceptions, ARG abundances closely track the dynamics of pathobionts and community virulence potential. These findings emphasize the potential for development of resistant pathogens in the developing infant gut, and the importance of effective surveillance in order to detect such events.

Breaking the Cycle: A Yeast Mannan-Rich Fraction Beneficially Modulates Egg Quality and the Antimicrobial Resistome Associated with Layer Hen Caecal Microbiomes under Commercial Conditions.

Antibiotics and antibiotic growth promoters have been extensively employed in poultry farming to enhance growth performance, maintain bird health, improve nutrient uptake efficiency, and mitigate enteric diseases at both sub-therapeutic and therapeutic doses. However, the extensive use of antimicrobials in poultry farming has led to the emergence of antimicrobial resistance (AMR) in microbial reservoirs, representing a significant global public health concern. In response, non-antibiotic dietary interventions, such as yeast mannan-rich fraction (MRF), have emerged as a promising alternative to modulate the gut microbiota and combat the AMR crisis. This study investigated whether a yeast mannan-rich fraction containing feed supplement impacted the performance of laying hens, their microbiomes, and the associated carriage of antimicrobial resistance genes under commercial conditions. High-throughput DNA sequencing was utilised to profile the bacterial community and assess changes in the antibiotic resistance genomes detected in the metagenome, the "resistome", in response to MRF supplementation. It was found that supplementation favourably influenced laying hen performance and microbial composition. Notably, there was a compositional shift in the MRF supplemented group associated with a lower relative abundance of pathobionts, e.g., Escherichia, Brachyspira and Trueperella, and their AMR-encoded genes, relative to beneficial microbes. Overall, the findings further demonstrate the ability of prebiotics to improve laying hen performance through changes associated with their microbiome and resistome.

Comprehensive Metagenomic Analysis of Veterinary Probiotics in Broiler Chickens.

Probiotics are widely used in broiler chickens to support the gut microbiome, gut health, and to reduce the amount of antibiotics used. Despite their benefits, there is concern over their ability to carry and spread antimicrobial resistance genes (ARGs), posing a significant public health risk. This study utilized next-generation sequencing to investigate ARGs in probiotics approved for poultry, focusing on their potential to be transferred via mobile genetic elements such as plasmids and phages. We examined the gut microbiome and resistome changes in 60 broiler chickens over their rearing period, correlating these changes with different probiotic treatments. Specific resistance mechanisms against critically important antibiotics were identified, including genes related to fluoroquinolone resistance and peptide antibiotic resistance. We also found genes with significant relevance to public health (aadK, AAC(6')-Ii) and multiple drug-resistance genes (vmlR, ykkC, ykkD, msrC, clbA, eatAv). Only one phage-encoded gene (dfrA43) was detected, with no evidence of plasmid or mobile genetic element transmission. Additionally, metagenomic analysis of fecal samples showed no significant changes corresponding to time or diet across groups. Our findings highlight the potential risks associated with the use of probiotics in poultry, particularly regarding the carriage of ARGs. It is crucial to conduct further research into the molecular genetics of probiotics to develop strategies that mitigate the risk of resistance gene transfer in agriculture, ensuring the safe and effective use of probiotics in animal husbandry.

Prevalence and clinical significance of the genotypic carriage among ESBL phenotype-negative Escherichia coli and Klebsiella pneumoniae clinical isolates in bacteremia: a study in a Malaysian tertiary center.

Antimicrobial resistance (AMR) can lead to fatal consequences. AMR genes carriage by phenotypically susceptible bacteria, such as Extended-Spectrum β-Lactamases (ESBL)s in Enterobacteriaceae, have potential implications for AMR spread and therapeutic outcomes. This phenomenon should be investigated. Positive blood cultures from hospitalized patients in a Malaysian tertiary center between April 2022 and March 2023 were reviewed. A total of 137 clinical isolates of Escherichia coli (E.coli), Klebsiella pneumoniae (K.pneumoniae), and Klebsiella oxytoca were included. The antibiotic susceptibility and ESBL phenotypes were determined by disk diffusion method and the identification of genotypes by multiplex polymerase chain reaction. The clinical characteristics and outcome information were extracted by reviewing patients' medical records to evaluate the clinical significance of the ESBL genotype-positive but phenotype-negative isolates in bacteremia. All 137 isolates were positive for at least one genotype (bla CTX-M, n = 71, 51.8%; bla SHV, n = 87, 63.5%; bla TEM, n = 95, 69.3%; bla OXA-1, n = 38, 27.7%). While bla CTX-M was proportionately higher in the ESBL phenotype-positive isolates than ESBL phenotype-negative isolates (33/37, 89.2% vs 38/100, 38%; p < 0.001), more than half of those harboring bla CTX-M remained susceptible to third-generation cephalosporins (3GC). The sensitivity (Sen) of bla CTX-M for ESBL phenotypes prediction was 89.19% (95% confidence interval [CI], 74.58 - 96.97%); however, specificity (Sp) was low (46.47%; 95% CI 39.75 - 53.32). The patient characteristics were similar among 98 ESBL phenotype-negative cases, except that the non-bla CTX-M carrier group had significantly more renal impairment (0/37 vs 7/61, p = 0.043) and gastrointestinal sources of bacteremia (9/37 vs 27/61, p = 0.047). No differences were observed in infection severity, in-hospital mortality, and length of stay (LOS) between the bla CTX-M and non-bla CTX-M carrier groups. The current study provides insight into the gene carriage in E.coli and Klebsiella species clinical isolates, including bla CTX-M genotypes in antibiotic-susceptible strains from a Malaysian hospital. The ESBL encoding genotypes such as bla CTX-M presented substantially beyond one-third of the ESBL phenotype-negative or 3GC susceptible E.coli and K.pneumoniae isolated from bloodstream infection. Although clinical outcomes were not worsened with bla CTX-M genotype-positive but ESBL phenotype-negative isolates in bacteremia, the potential implications for AMR spread deserve further investigation.

Genomic analysis of Citrobacter from Australian wastewater and silver gulls reveals novel sequence types carrying critically important antibiotic resistance genes

Antimicrobial resistance (AMR) is a major public health concern, and environmental bacteria have been recognized as important reservoirs of antimicrobial resistance genes (ARGs). Citrobacter, a common environmental bacterium and opportunistic pathogen in humans and other animals, has been largely understudied in terms of its diversity and AMR potential. Whole-genome (short-read) sequencing on a total of 77 Citrobacter isolates obtained from Australian silver gull (Chroicocephalus novaehollandiae) (n = 17) and influent wastewater samples (n = 60) was performed, revealing a diverse Citrobacter population, with seven different species and 33 sequence types, 17 of which were novel. From silver gull using non-selective media we isolated a broader range of species with little to no mobilised ARG carriage. Wastewater isolates (selected using Carbapenem- Resistant Enterobacterales (CRE) selective media) carried a heavy burden of ARGs (up to 21 ARGs, conferring resistance to nine classes of antibiotics), with several novel multidrug-resistant (MDR) lineages identified, including C. braakii ST1110, which carried ARGs conferring resistance to eight to nine classes of antibiotics, and C. freundii ST1105, which carried two carbapenemase genes, blaIMP-4 in class 1 integron structure, and blaKPC-2. Additionally, we identified an MDR C. portucalensis isolate carrying blaNDM-1, blaSHV-12, and mcr-9. We identified IncC, IncM2, and IncP6 plasmids as the likely vectors for many of the critically important mobilised ARGs. Phylogenetic analyses were performed to assess any epidemiological linkages between isolation sources, demonstrating low relatedness across sources beyond the ST level. However, these analyses did reveal some closer relationships between strains from disparate wastewater sources despite their collection some 13,000 km apart. These findings support the need for future surveillance of Citrobacter populations in wastewater and wildlife populations to monitor for potential opportunistic human pathogens.

Mobilization of the blaKPC-14 gene among heterogenous plasmids in extensively drug-resistant hypervirulent Klebsiella pneumoniae.

Ceftazidime/avibactam (CZA) is an effective alternative for the treatment of infections caused by KPC-producing carbapenem-resistant Klebsiella pneumoniae (CRKP). However, KPC variants with CZA resistance have been observed in clinical isolates, further limiting the treatment options of clinical use. In this study, we isolated three KPC-14-producing CRKP from two patients in intensive care units without CZA therapy. The antimicrobial susceptibility was determined using the broth microdilution method. Three CRKP were subjected to whole-genome sequencing to analyze the phylogenetic relatedness and the carriage of antimicrobial resistance genes and virulence factors. Long-read sequencing was also performed to obtain the complete sequences of the plasmids. The horizontal transfer of the blaKPC-14 gene was evaluated by conjugation experiments. Three CRKP displayed resistance or reduced susceptibility to ceftazidime/avibactam, colistin, and tigecycline. Single-nucleotide polymorphism (SNP) analysis demonstrated the close phylogenetic distance between these strains. A highly similar IncFII/IncR plasmid encoding blaKPC-14 was shared by three CRKP, with blaKPC-14 located in an NTEKPC-Ib element with the core region of ISKpn27- blaKPC-14-ISKpn6. This structure containing blaKPC-14 was also observed in another tet(A)-carrying plasmid that belonged to an unknown Inc-type in two out of three isolates. The horizontal transferability of these integrated plasmids to Escherichia coli EC600 was confirmed by the cotransmission of tet(A) and blaKPC-14 genes, but the single transfer of blaKPC-14 on the IncFII/IncR plasmid failed. Three CRKP expressed yersiniabactin and carried a hypervirulence plasmid encoding rmpA2 and aerobactin-related genes, and were thus classified as carbapenem-resistant hypervirulent K. pneumoniae (hvKP). In this study, we reported the evolution of a mosaic plasmid encoding the blaKPC-14 gene via mobile elements in extensively drug-resistant hvKP. The blaKPC-14 gene is prone to integrate into other conjugative plasmids via the NTEKPC-Ib element, further facilitating the spread of ceftazidime/avibactam resistance.

Multidrug-resistant E. coli encoding high genetic diversity in carbohydrate metabolism genes displace commensal E. coli from the intestinal tract.

Extra-intestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside of the intestine and are a major causative agent of urinary tract infections. Treatment of these infections is increasingly frustrated by antimicrobial resistance (AMR) diminishing the number of effective therapies available to clinicians. Incidence of multidrug resistance (MDR) is not uniform across the phylogenetic spectrum of E. coli. Instead, AMR is concentrated in select lineages, such as ST131, which are MDR pandemic clones that have spread AMR globally. Using a gnotobiotic mouse model, we demonstrate that an MDR E. coli ST131 is capable of out-competing and displacing non-MDR E. coli from the gut in vivo. This is achieved in the absence of antibiotic treatment mediating a selective advantage. In mice colonised with non-MDR E. coli strains, challenge with MDR E. coli either by oral gavage or co-housing with MDR E. coli colonised mice results in displacement and dominant intestinal colonisation by MDR E. coli ST131. To investigate the genetic basis of this superior gut colonisation ability by MDR E. coli, we assayed the metabolic capabilities of our strains using a Biolog phenotypic microarray revealing altered carbon metabolism. Functional pangenomic analysis of 19,571 E. coli genomes revealed that carriage of AMR genes is associated with increased diversity in carbohydrate metabolism genes. The data presented here demonstrate that independent of antibiotic selective pressures, MDR E. coli display a competitive advantage to colonise the mammalian gut and points to a vital role of metabolism in the evolution and success of MDR lineages of E. coli via carriage and spread.

Antimicrobial Resistance Genes (ARGs), the Gut Microbiome, and Infant Nutrition.

The spread of antimicrobial resistance genes (ARGs) is a major public health crisis, with the ongoing spread of ARGs leading to reduced efficacy of antibiotic treatments. The gut microbiome is a key reservoir for ARGs, and because diet shapes the gut microbiome, diet also has the potential to shape the resistome. This diet-gut microbiome-resistome relationship may also be important in infants and young children. This narrative review examines what is known about the interaction between the infant gut microbiome, the infant resistome, and infant nutrition, including exploring the potential of diet to mitigate infant ARG carriage. While more research is needed, diet has the potential to reduce infant and toddler carriage of ARGs, an important goal as part of maintaining the efficacy of available antibiotics and preserving infant and toddler health.

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

Microbial diversity and antimicrobial resistance in faecal samples from acute medical patients assessed through metagenomic sequencing.

Antimicrobial resistance (AMR) is a threat to global public health. However, unsatisfactory approaches to directly measuring the AMR burden carried by individuals has hampered efforts to assess interventions aimed at reducing selection for AMR. Metagenomics can provide accurate detection and quantification of AMR genes within an individual person's faecal flora (their gut "resistome"). Using this approach, we aimed to test the hypothesis that differences in antimicrobial use across different hospitals in the United Kingdom will result in observable differences in the resistome of individual patients. Three National Health Service acute Hospital Trusts with markedly different antibiotic use and Clostridioides difficile infection rates collected faecal samples from anonymous patients which were discarded after C. difficile testing over a period of 9 to 15 months. Metagenomic DNA was extracted from these samples and sequenced using an Illumina NovaSeq 6000 platform. The resulting sequencing reads were analysed for taxonomic composition and for the presence of AMR genes. Among 683 faecal metagenomes we found huge variation between individuals in terms of taxonomic diversity (Shannon Index range 0.10-3.99) and carriage of AMR genes (Median 1.50 genes/cell/sample overall). We found no statistically significant differences in diversity (median Shannon index 2.16 (IQR 1.71-2.56), 2.15 (IQR 1.62-2.50) and 2.26 (IQR 1.55-2.51)) or carriage of AMR genes (median 1.37 genes/cell/sample (IQR 0.70-3.24), 1.70 (IQR 0.70-4.52) and 1.43 (IQR 0.55-3.71)) at the three trusts respectively. This was also the case across the sample collection period within the trusts. While we have not demonstrated differences over place or time using metagenomic sequencing of faecal discards, other sampling frameworks may be more suitable to determine whether organisational level differences in antibiotic use are associated with individual-level differences in burden of AMR carriage.

Aeromonas spp. from hospital sewage act as a reservoir of genes resistant to last-line antibiotics.

Aeromonas species are opportunistic pathogens distributed widely in the ecosystem. They are known to be capable of acquiring antibiotic resistance genes, including those encoding proteins against last-line antibiotics, such as the tmexCD-toprJ, mcr and carbapenemase genes. We investigated the genomic and phenotypic characteristics of tmexCD-toprJ-positive Aeromonas strains collected from human, animals, and water samples, particularly those from hospital wastewater in China. Samples were collected from living animals, meat, water and human. Aeromonas strains in these samples were isolated in selective media. Antimicrobial resistance profiles of all Aeromonas strains were tested by the broth microdilution method. The presence of tmexCD-toprJ was verified by polymerase chain reaction (PCR). All tmexCD-toprJ-positive (n=36) and selected tmexCD-toprJ-negative (n=18) Aeromonas strains were subjected to whole genome sequencing. Carriage of antimicrobial resistance genes, the genetic environment of tmexCD-toprJ and genetic diversity of tmexCD-toprJ-positive Aeromonas strains were determined by bioinformatics analysis. Phylogenetic tree of the Aeromonas strains was built by using the Harvest Suite. Among the 636 Aeromonas strains isolated from different sources, 36 were positive for tmexCD-toprJ, with the highest prevalence of tmexCD-toprJ being found in fishes (8.8%, 95 CI% 3.6-17.2%), followed by hospital wastewater (6.5%, 95 CI% 4.3-9.3%), river water (2.0%, 0.1-10.9) and duck (1.2%, 95 CI% 3.6-17.2%). All tmexCD-toprJ-positive Aeromonas strains carried multiple antimicrobial resistance genes and exhibited resistance to different classes of antibiotics. Co-existence of tmexCD-toprJ, mcr and blaKPC-2 were identified in 21 strains. The tmexCD-toprJ-positive Aeromonas strains were genetically diverse and found to belong to four different species that could be clustered into three major lineages. The tmexCD-toprJ gene clusters were predominantly located in the chromosome (35/36) of Aeromonas spp., with only one strain carrying the plasmid-borne tmexCD-toprJ cluster. The tmexCD-toprJ genes were associated with seven different types of genetic environments, each of which carried distinct types of mobile elements that may be responsible for mediating transmission of this gene cluster.

Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries.

Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.

Antibiotic resistance genes in the gut microbiota of mothers and linked neonates with or without sepsis from low- and middle-income countries

Early development of the microbiome has been shown to affect general health and physical development of the infant and, although some studies have been undertaken in high-income countries, there are few studies from low- and middle-income countries. As part of the BARNARDS study, we examined the rectal microbiota of 2,931 neonates (term used up to 60 d) with clinical signs of sepsis and of 15,217 mothers screening for blaCTX-M-15, blaNDM, blaKPC and blaOXA-48-like genes, which were detected in 56.1%, 18.5%, 0% and 4.1% of neonates’ rectal swabs and 47.1%, 4.6%, 0% and 1.6% of mothers’ rectal swabs, respectively. Carbapenemase-positive bacteria were identified by MALDI-TOF MS and showed a high diversity of bacterial species (57 distinct species/genera) which exhibited resistance to most of the antibiotics tested. Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae/E. cloacae complex, the most commonly found isolates, were subjected to whole-genome sequencing analysis and revealed close relationships between isolates from different samples, suggesting transmission of bacteria between neonates, and between neonates and mothers. Associations between the carriage of antimicrobial resistance genes (ARGs) and healthcare/environmental factors were identified, and the presence of ARGs was a predictor of neonatal sepsis and adverse birth outcomes.

Close genetic linkage between human and companion animal extraintestinal pathogenic Escherichia coli ST127.

Escherichia coli ST127, a recently emerged global pathogen noted for high virulence gene carriage, is a leading cause of urinary tract and blood stream infections. ST127 is frequently isolated from humans and companion animals; however, it is unclear if they are distinct or related populations of ST127. We performed a phylogenomic analysis of 299 E. coli ST127 of diverse epidemiological origin to characterize their population structure, genetic determinants of virulence, antimicrobial resistance, and repertoire of mobile genetic elements with a focus on plasmids. The core gene phylogeny was divided into 13 clusters, the largest of which (BAP4) contained the majority of human and companion animal origin isolates. This dominant cluster displayed genetic differences to the remainder of the phylogeny, most notably alternative gene alleles encoding important virulence factors including lipid A, flagella, and K capsule. Furthermore, numerous close genetic linkages (<30 SNPs) between human and companion animal isolates were observed within the cluster. Carriage of antimicrobial resistance genes in the collection was limited, but virulence gene carriage was extensive. We found evidence of pUTI89-like virulence plasmid carriage in over a third of isolates, localised to four of the major phylogenetic clusters. Our study supports global scale repetitive transfer of E. coli ST127 lineages between humans and companion animals, particularly within the dominant BAP4 cluster.

Secrets of the Hospital Underbelly: Patterns of Abundance of Antimicrobial Resistance Genes in Hospital Wastewater Vary by Specific Antimicrobial and Bacterial Family.

Background: Hospital wastewater is a major source of antimicrobial resistance (AMR) outflow into the environment. This study uses metagenomics to study how hospital clinical activity impacts antimicrobial resistance genes (ARGs) abundances in hospital wastewater.Methods: Sewage was collected over a 24-h period from multiple wastewater collection points (CPs) representing different specialties within a tertiary hospital site and simultaneously from community sewage works. High throughput shotgun sequencing was performed using Illumina HiSeq4000. ARG abundances were correlated to hospital antimicrobial usage (AMU), data on clinical activity and resistance prevalence in clinical isolates.Results: Microbiota and ARG composition varied between CPs and overall ARG abundance was higher in hospital wastewater than in community influent. ARG and microbiota compositions were correlated (Procrustes analysis, p=0.014). Total antimicrobial usage was not associated with higher ARG abundance in wastewater. However, there was a small positive association between resistance genes and antimicrobial usage matched to ARG phenotype (IRR 1.11, CI 1.06–1.16, p<0.001). Furthermore, analyzing carbapenem and vancomycin resistance separately indicated that counts of ARGs to these antimicrobials were positively associated with their increased usage [carbapenem rate ratio (RR) 1.91, 95% CI 1.01–3.72, p=0.07, and vancomycin RR 10.25, CI 2.32–49.10, p<0.01]. Overall, ARG abundance within hospital wastewater did not reflect resistance patterns in clinical isolates from concurrent hospital inpatients. However, for clinical isolates of the family Enterococcaceae and Staphylococcaceae, there was a positive relationship with wastewater ARG abundance [odds ratio (OR) 1.62, CI 1.33–2.00, p<0.001, and OR 1.65, CI 1.21–2.30, p=0.006 respectively].Conclusion: We found that the relationship between hospital wastewater ARGs and antimicrobial usage or clinical isolate resistance varies by specific antimicrobial and bacterial family studied. One explanation, we consider is that relationships observed from multiple departments within a single hospital site will be detectable only for ARGs against parenteral antimicrobials uniquely used in the hospital setting. Our work highlights that using metagenomics to identify the full range of ARGs in hospital wastewater is a useful surveillance tool to monitor hospital ARG carriage and outflow and guide environmental policy on AMR.

Analysis of Virulence and Antimicrobial Resistance Gene Carriage in Staphylococcus aureus Infections in Equids Using Whole-Genome Sequencing.

ABSTRACTWhile Staphylococcus aureus is associated with significant morbidity and mortality in equids (horses, donkeys, and mules), few studies have performed whole-genome sequencing to fully categorize large collections of equine isolates. Such sequencing allows for a comprehensive analysis of the genetic lineage and relationships of isolates, as well as the virulence genes present in each, which can be important for understanding the epidemiology of strains and their range of infections. Seventy-two clinical Staphylococcus aureus isolates from equids were collected at the Texas A&M University Veterinary Medical Teaching Hospital between 2007 and 2017. Whole-genome sequencing was performed to characterize the isolates according to sequence typing, biofilm association, antimicrobial resistance, and toxin gene carriage. Of the 72 isolates, 19% were methicillin resistant, of which the majority belonged to clonal complex 8. Eighteen distinct sequence types (STs) were represented, with the most common being ST1, ST133, ST8, and ST97. Most isolates had weak or negative overall biofilm production. Toxin and antimicrobial resistance gene carriage was varied; of note, this study revealed that a large proportion of North American equine isolates carry the leucocidin PQ toxin (66% of isolates). One isolate (17-021) carried genes imparting lincosamide and high-level mupirocin resistance, a combination not previously reported in equine-derived S. aureus isolates.IMPORTANCE This is one of the first studies to perform whole-genome sequencing (WGS) of a large collection of Staphylococcus aureus isolates, both methicillin resistant and susceptible, collected from horses. A large proportion of the isolates carry leucocidin PQ (LukPQ), making this one of the first reports of such carriage in the United States. The presence of lincosamide and high-level mupirocin resistance in a methicillin-susceptible S. aureus (MSSA) isolate highlights the importance of MSSA as a reservoir of important antimicrobial resistance genes. As microbial resistance genes on mobile genetic elements can pass between S. aureus strains and livestock-associated strains can be transferred to humans, these findings have important public health implications.

Population structure and uropathogenic potential of extended-spectrum cephalosporin-resistant Escherichia coli from retail chicken meat

BackgroundFood-producing animals and their products are considered a source for human acquisition of antimicrobial resistant (AMR) bacteria, and poultry are suggested to be a reservoir for Escherichia coli resistant to extended-spectrum cephalosporins (ESC), a group of antimicrobials used to treat community-onset urinary tract infections in humans. However, the zoonotic potential of ESC-resistant E. coli from poultry and their role as extraintestinal pathogens, including uropathogens, have been debated. The aim of this study was to characterize ESC-resistant E. coli isolated from domestically produced retail chicken meat regarding their population genetic structure, the presence of virulence-associated geno- and phenotypes as well as their carriage of antimicrobial resistance genes, in order to evaluate their uropathogenic potential.ResultsA collection of 141 ESC-resistant E. coli isolates from retail chicken in the Norwegian monitoring program for antimicrobial resistance in bacteria from food, feed and animals (NORM-VET) in 2012, 2014 and 2016 (n = 141) were whole genome sequenced and analyzed. The 141 isolates, all containing the beta-lactamase encoding gene blaCMY-2, were genetically diverse, grouping into 19 different sequence types (STs), and temporal variations in the distribution of STs were observed. Generally, a limited number of virulence-associated genes were identified in the isolates. Eighteen isolates were selected for further analysis of uropathogen-associated virulence traits including expression of type 1 fimbriae, motility, ability to form biofilm, serum resistance, adhesion- and invasion of eukaryotic cells and colicin production. These isolates demonstrated a high diversity in virulence-associated phenotypes suggesting that the uropathogenicity of ESC-resistant E. coli from chicken meat is correspondingly highly variable. For some isolates, there was a discrepancy between the presence of virulence-associated genes and corresponding expected phenotype, suggesting that mutations or regulatory mechanisms could influence their pathogenic potential.ConclusionOur results indicate that the ESC-resistant E. coli from chicken meat have a low uropathogenic potential to humans, which is important knowledge for improvement of future risk assessments of AMR in the food chains.

Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38–9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.