Biology subjects

Avison, M. B.

Publications and source records attributed to Avison, M. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Local Temperature and Humidity are Associated with Proportion of Antimicrobial-Resistant Escherichia coli isolates in Farm Environments: Considerations for On-Farm Surveillance

Evidence suggests that increased local temperatures are associated with higher prevalence of antimicrobial resistance (AMR) in environmental bacteria. This study investigates the association between local climate and the proportion of antimicrobial-resistant Escherichia coli isolated from 2,766 farm environment samples from 53 English dairy farms. To do this, a non-linear Bayesian model that specifically accounts for decreased test sensitivity at low E. coli abundance was developed and used to estimate the proportion of isolates resistant to four antimicrobials (amoxicillin, cephalexin, streptomycin and tetracycline) from colony count data. Mean 7-day temperature and relative humidity at the farm location was modelled using a generalised additive model formulation. A higher proportion of E. coli isolates were resistant to cephalexin and streptomycin in samples collected from adult cow collecting yards, than heifer housing sheds. In contrast, a greater proportion of E. coli isolates from heifer housing sheds were resistant to amoxicillin and tetracycline. Evidence that local temperature is associated with an increase in the proportion of E. coli isolates resistant to streptomycin (20{degrees}C increase associated with a 5.0-fold increase; 95% CI: 1.03-33.0) and tetracycline (2.6-fold increase; 90% CI: 1.1-5.2) was observed. Additionally, relative humidity was associated with an increase in the proportion of isolates resistant to amoxicillin streptomycin and tetracycline. The influence of weather on the proportion of antimicrobial-resistant E. coli varied between samples collected from adult animals in collecting yards and heifers in housing sheds. These findings highlight the importance of considering weather conditions, sample characterises and seasonality when designing on-farm AMR surveillance systems. ImportanceUnderstanding how environmental conditions are associated with variability in AMR prevalence is critical for developing robust livestock AMR surveillance and anticipating the potential effects of climate change. The non-linear Bayesian modelling approach developed here adjusts for E. coli abundance associated variability in test sensitivity, enabling the influence of risk factors associated with the proportion of antimicrobial-resistant E. coli within samples to be more accurately estimated. Applying this approach to 2,766 faecal samples from 53 dairy farms in Southwest England indicated that the proportion of antimicrobial-resistant E. coli generally increased under warmer and wetter conditions. These findings suggest that environmental conditions can influence the prevalence of AMR E. coli in dairy farm environments and demonstrate the importance of accounting for weather related variability in livestock AMR surveillance. Adjusting for these associations in livestock AMR surveillance could improve the accuracy of modelling AMR trends and strengthen the assessment of climate-associated AMR risks.

microbiology↗

bioRxiv2025

Genomic Analyses of Antibiotic-Resistant Escherichia coli From Extensive Beef Cattle and Sheep Farms Identifies Inter-Species and Farm-Farm Sharing as Clonal Dissemination Pathways

SynopsisO_ST_ABSBackgroundC_ST_ABSGlobally, there is a large gap in our understanding of the prevalence, ecology and transmission dynamics of antibiotic resistance (ABR) in extensively reared ruminants, despite these animals contributing to the food chain and frequently sharing land with humans. MethodsFive hundred and seventy one visits to 33 Welsh beef cattle and/or sheep farms resulted in 1874 samples being collected at faecally contaminated sites from April 2022 to March 2023 (ADGC1) and September 2023 to December 2024 (ADGC2). Samples were tested for resistant Escherichia coli using amoxicillin, streptomycin, spectinomycin, cefotaxime and ciprofloxacin. WGS used Illumina technology. Clonal relationships were determined following core-genome alignment. ResultsA significant reduction in positivity for spectinomycin-resistant E. coli in sheep samples from ADGC1 to ADGC2 was observed, coincident with market withdrawal of a spectinomycin-containing preparation widely used in sheep. Reductions were seen in 19/22 sheep flocks with nine seeing a >50% reduction. Resistance to other tested antibiotics was unchanged. Phenotypic analysis and WGS for 713 E. coli showed that resistance to antibacterials important for human medicine was rare and genetically diverse. We identified 77 E. coli clones (<100 SNP cutoff) circulating among study farms with mixed farms contributing most; clones were also shared between animal species on mixed farms. ConclusionsFor extensively reared ruminants, ABR-reducing efforts can have significant impacts on antibiotic resistance on farms. Focusing these efforts onto farms contributing to the most animal movement and mixing events may generate the greatest reductions in overall on-farm ABR prevalence at regional and national levels.

microbiology↗

bioRxiv2024

The Zoonotic Potential of Poultry-Associated Nitrofurantoin-Resistant Escherichia coli

Escherichia coli is the most common cause of urinary tract infection (UTI) in humans. The nitrofuran-class antibacterial drug nitrofurantoin is a frequent UTI therapy, with resistance rarely observed. Here we show that nitrofurantoin resistant (NFT-R) E. coli are sometimes excreted by dogs fed a raw meat diet in the city of Bristol, United Kingdom, and that NFT-R and pre-resistant (one mutation away from NFT-R) E. coli can be found contaminating chicken meat sold for human consumption and chicken-based raw dog food in the same city. Using whole genome sequencing, we identified multiple NFT-R or pre-resistant E. coli clones spanning several phylogroups. These clones were dominated by isolates from poultry farms and poultry meat in Europe, Canada, the United States and Japan, and we identified instances where closely related NFT-R and pre-resistant isolates have colonised humans and caused UTIs. The origins of these poultry-associated NFT-R and pre-resistant E. coli clones are uncertain, but nitrofuran-class antibacterials (particularly furazolidone, furaltadone, and nitrofurazone) were used in poultry production during the 1970s and 80s, though this practice has been banned since the 1990s. It is possible, therefore, that this caused an initial selective pressure for the emergence of NFT-R and pre-resistant E. coli clones on poultry farms. Our findings have potentially important implications for domestic hygiene, particularly among people receiving nitrofurantoin therapy.

microbiology↗

bioRxiv2024

E. coli phylogeny drives co-amoxiclav resistance through variable expression of blaTEM-1

Co-amoxiclav resistance in E. coli is a clinically important phenotype associated with increased mortality. The class A beta-lactamase blaTEM-1 is often carried by co- amoxiclav-resistant pathogens, but exhibits high phenotypic heterogeneity, making genotype-phenotype predictions challenging. We present a curated dataset of n=377 E. coli isolates representing all 8 known phylogroups, where the only acquired beta- lactamase is blaTEM-1. For all isolates, we generate hybrid assemblies and co-amoxiclav MICs, and for a subset (n=67/377), blaTEM-1 qPCR expression data. First, we test whether certain E. coli lineages are intrinsically better or worse at expressing blaTEM-1, for example, due to lineage differences in regulatory systems, which are challenging to directly quantify. Using genotypic features of the isolates (blaTEM-1 promoter variants and copy number), we develop a hierarchical Bayesian model for blaTEM-1 expression that controls for phylogeny. We establish that blaTEM-1 expression intrinsically varies across the phylogeny, with some lineages (e.g. phylogroups B1 and C, ST12) better at expression than others (e.g. phylogroups E and F, ST372). Next, we test whether phylogenetic variation in expression influences the resistance of the isolates. With a second model, we use genotypic features (blaTEM-1 promoter variants, copy number, duplications; ampC promoter variants; efflux pump AcrF presence) to predict isolate MIC, again controlling for phylogeny. Lastly, we use a third model to demonstrate that the phylogenetic influence on blaTEM-1 expression causally drives the variation in co- amoxiclav MIC. This underscores the importance of incorporating phylogeny into genotype-phenotype predictions, and the study of resistance more generally.

microbiology↗

bioRxiv2024

Antibiotic Resistant Escherichia coli in Uncooked Meat Purchased from Large Chain Grocery Stores and in Raw Dog Food Purchased From Pet Stores in the Same City.

ObjectivesThere is strong evidence linking raw meat or raw dog food (RDF) feeding with dogs shedding antibiotic-resistant (ABR) Escherichia coli. As well as potential risks of ingesting ABR E. coli when handling/consuming meat, therefore, people who raw-feed pets may face additional risks. However, phylogenetic evidence closely linking E. coli found on raw meat/RDF with those causing opportunistic human infections is lacking, partly due to non-contemporaneous sampling. Here, we monitored ABR E. coli in meat/RDF in Bristol, UK in 2022/23, and compared E. coli isolates with E. coli causing human urinary tract and bloodstream infections in Bristol in 2023. MethodsWe monitored ABR E. coli positivity in raw chicken, beef, pork and lamb meat from 15 large-chain stores and chicken-based RDF from 15 pet stores and selected 200 ABR E. coli for Illumina WGS. E. coli causing 1182 human infections were sequenced, irrespective of resistance phenotype. ResultsChicken/RDF were most likely to carry amoxicillin-, spectinomycin-, streptomycin-, cefotaxime-, and ciprofloxacin-resistant E. coli. Phylogroup A/B1 and B2 E. coli dominated meat/RDF and human isolates, respectively. Resistance to antibiotics used predominantly in farmed animals dominated meat/RDF isolates; resistance to antibiotics extensively used in humans dominated human isolates. Nineteen meat/RDF-infection E. coli pairs differed by <50 SNPs across the core-genome, with four pairs (three ST69 and one ST117) differing by <20 SNPs and carrying identical resistance genes, suggestive of recent sharing. ConclusionsUsing contemporaneous, geographically focussed sampling, we have confirmed close phylogenetic relationships between meat/RDF and human infection-causing ABR E. coli. Despite representing a small number of infections, these findings support the consideration of tighter microbiological standards for RDF given that it is explicitly sold to be fed uncooked. Improved risk communication to pet owners and the inclusion of RDF within integrated ABR surveillance is warranted.

microbiology↗

bioRxiv2024

Improving Nitrofurantoin Resistance Prediction in Escherichia coli from Whole Genome Sequence by Integrating NfsA/B Enzyme Assays

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB making it extremely difficult to predict nitrofurantoin resistance from whole genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here we report a WGS survey of 200 E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterised individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and assays of NfsA/B enzyme activity in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, A112T and A122T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D and A188V in NfsA and G66D, M75I, V93A and A174E in NfsB, are functionally silent in this context. We identified that 9/166 (5.4%) of nitrofurantoin susceptible isolates were "pre-resistant", defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme activity assay and proteomics we demonstrated that 9/34 (26.5%) of nitrofurantoin resistant isolates carried functionally wild-type nfsB or nfsB/nfsA. In these cases, enzyme activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis, but is still insufficient to allow its reliable prediction from WGS data.

microbiology↗

bioRxiv2024

Convergence and global epidemiology of Klebsiella pneumoniae plasmids harbouring the iuc3 virulence locus.

BackgroundKlebsiella pneumoniae (Kp) is an important pathogen of humans and animals, and recent reports of convergent strains that carry both virulence and antimicrobial resistance genes (ARGs) have raised serious public health concern. The plasmid-borne iuc locus, encoding the siderophore aerobactin, is a key virulence factor in this species. The variant iuc3 is associated with porcine and human clinical isolates and is carried by mostly uncharacterised IncF plasmids. MethodsWe used a combination of short-read and long-read sequencing to characterise IncFIB(K)/IncFII iuc3-carrying plasmids harboured by 79 Kp isolates and one K. oxytoca isolate recovered as part of two large One-Health studies in Italy (SpARK) and Thailand (OH-DART). Adding data from public repositories gave a combined dataset of 517 iuc3 isolates, and the plasmids were analysed using both clustering and phylogenetic methods. FindingsWe note seven large, convergent, plasmids from Thailand that have emerged through the hybridisation of co-circulating plasmids harbouring iuc3 and antimicrobial resistance genes (ARGs) encoding extended-spectrum beta-lactamases (ESBLs). We were also able to identify putative parental plasmids which were mostly associated with two neighbouring meat markets, as were the hybrid plasmids. Clustering and global phylogenetic analyses resolved an iuc3 plasmid sub-group circulating throughout Asia, with occasional examples in Europe and elsewhere. This variant carries multiple ARGs and is commonly harboured by clinical isolates, thus warranting targeted plasmid surveillance. InterpretationOur study reveals that plasmid hybridisation leading to the convergence of resistance and virulence traits may be very common, even in non-clinical ( One-Health) settings. Population-scale plasmid genomics makes it possible to identify putative parental plasmids, which will help to identify plasmid types that are most likely to hybridise, and what the selective consequences may be for the plasmid and host. A distinct iuc3 plasmid sub-variant is associated with clinical isolates in Asia which requires close monitoring. Research In ContextMultiple reports of convergent clones of Klebsiella pneumoniae that combine both hypervirulence and multidrug resistance (MDR-hvKp) have been published recently; a PubMed search in November 2023 using the key words convergence Klebsiella pneumoniae returned 143 papers, 99 of which were published from 2020 onwards. Our study demonstrates that the hybridisation of plasmids carrying AMR and virulence genes is a frequent, ongoing, process in natural populations. The subsequent transfer of plasmids conferring both traits is thus likely to be a key driver behind the spread of convergent strains. Our study also provides an exemplar of how hybrid assemblies can facilitate large-scale global genomic plasmid epidemiology. Evidence before the studyAlthough multiple recent reports highlight the emergence and spread of convergent Kp strains, the confluence of resistance and virulence genes within the same plasmid has not been studied at a population level, and putative parental plasmids are rarely identified. Moreover, there have been few high-resolution genomic epidemiology studies on closely related plasmids using both long and short-read data on a global scale. Added valueWe more than double the number of complete sequences available for plasmids harbouring iuc3 from 58 to 139 and provide evidence on the host lineages most likely to harbour these plasmids (e.g., ST35), and epidemiological source (e.g., pig, wild animal, human). Our comparative analysis of phylogenetic and clustering approaches will help to inform future plasmid epidemiological studies. ImplicationsThe hybridisation of plasmids harbouring virulence and resistance genes occurs frequently in natural populations, even within One-Health settings. However, the selective drivers (if any) and evolutionary consequences of this phenomenon are unclear. There is clear utility in generating closed plasmid genomes on a population scale, and targeted plasmid surveillance on a clinical sub-variant of iuc3 plasmids is warranted.

genomics↗

bioRxiv2023

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is already globally disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6)-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from, three hospitals serving a population of 1.5 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, transposon copy number amplification is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

microbiology↗

bioRxiv2023

Hound: A novel tool for automated mapping of genotype to phenotype in bacterial genomes assembled de novo

Increasing evidence suggests that microbial species have a strong within species genetic heterogeneity. This can be problematic for the analysis of prokaryote genomes, which commonly relies on a reference genome to guide the assembly process. Any difference between reference and sample genomes can introduce errors in the detection of small insertions, deletions, structural variations and even point mutations. This phenomenon jeopardises the genomic surveillance of antibiotic-resistant bacteria, with predictions of resistance varying between laboratories. Here we present Hound, an analysis pipeline that integrates publicly available tools to locally assemble prokaryote genomes de novo, detect genes by similarity using the proteins they encode as query, and report the mutations found. Three features are exclusive to Hound: it reports relative gene copy number, retrieves sequences upstream the start codon to detect mutations in promoter regions--which allow gene expression signals to be integrated--and, importantly, can merge contigs based on a user-given query sequence to reconstruct genes that are fragmented by the assembler. To demonstrate Hound, we screened through 5,032 bacterial whole-genome sequences isolated from farmed animals and human infections, using the amino acid sequence encoded by blaTEM-1, to predict resistance to amoxicillin/clavulanate which is driven by over-expression of this gene. We believe this tool can facilitate the analysis of prokaryote species that currently lack a reference genome, and can be scaled up to build automated systems for antibiotic susceptibility prediction.

genomics↗

bioRxiv2023

Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms

Control measures are being introduced globally to reduce the prevalence of antibiotic resistant (ABR) bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in key opportunistic human pathogens such as Escherichia coli on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin resistance (3GC-R) in E. coli. 3GC-R isolates were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from faecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal transmission of 3GC-R mechanisms. Despite this, mechanisms such as CTX-M-15 and CTX-M-2 were detected more often in dairy farms, while CTX-M-8 and CMY-2, and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more commonly detected in pig farms. This suggests different selective pressures of antibiotic use in these two animal types, particularly the balance of fourth-versus third-generation cephalosporin use, and of amoxicillin/clavulanate and florfenicol use. We identified the {beta}-lactamase gene blaROB in 3GC-R E. coli, which has previously only been reported in the family Pasteurellaceae, including farmed animal pathogens. blaROB was found alongside a novel florfenicol resistance gene - ydhC - also mobilised from a pig pathogen as part of a new plasmid-mediated composite transposon, which is already widely disseminated. These data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate zoonotic transmission of resistant bacteria in this region. ImportanceLittle is known about the ecology of critically important antibiotic resistance among opportunistic human pathogens (e.g. Escherichia coli) on South American farms. By studying 70 farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species (pigs or dairy cattle) and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli were highly phylogenetically variable and we identified the de novo mobilisation of the resistance gene blaROB, alongside a novel florfenicol resistance gene, from pig pathogens into E. coli on a mobile genetic element that was widespread in the study region. Overall, this shows the importance of surveying poorly studied regions for critically important antibiotic resistance which might impact human health.

microbiology↗

bioRxiv2022

Similarities and differences in molecular epidemiology of third-generation cephalosporin-resistant Escherichia coli carried by dogs living in urban and nearby rural settings and associated behavioural risk factors

SynopsisO_ST_ABSObjectivesC_ST_ABSOur aims were to compare faecal third-generation cephalosporin-resistant (3GC-R) Escherichia coli isolates from dogs living in a city and in a rural area ~30 km away; to compare isolates from dogs, cattle, and humans in these regions; to determine risk factors associated with 3GC-R E. coli carriage in these two cohorts of dogs. Methods600 dogs were included, with faecal samples processed to recover 3GC-R E. coli using 2 mg/L cefotaxime. WGS was by Illumina; risk factor analyses were multivariable linear regression using the results of an owner-completed survey. Results3GC-R E. coli were excreted by 20/303 rural and 31/297 urban dogs. Dog/human sharing was evident for the dominant canine 3GC-R sequence type, ST963(blaCMY-2). Cattle/dog sharing was evident for CTX-M-14 and CTX-M-32-producing E. coli from rural dogs, including sharing of plasmid pMOO-32, which is common on cattle farms in the area. Feeding raw meat was associated with carrying 3GC-R E. coli in rural dogs, but not in urban dogs, where swimming in rivers was a weak risk factor. ConclusionsGiven clear zoonotic potential for resistant canine E. coli, our work suggests interventions that may reduce this threat. In rural dogs, carriage of 3GC-R E. coli, particularly CTX-M producers, was phylogenetically associated with interaction with local cattle and epidemiologically associated with feeding raw meat. In urban dogs, sources of 3GC-R E. coli appear to be more varied and include environments such as rivers.

microbiology↗

bioRxiv2022

Identification and characterisation of Klebsiella pneumoniae and Pseudomonas aeruginosa clinical isolates with atypical β-lactam susceptibility profiles using Orbitrap liquid chromatography-tandem mass spectrometry

There is significant interest in the possibility of predicting antibacterial drug susceptibility directly though the analysis of bacterial DNA or protein. We report the use of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii transformants to define baseline predictive rules for the {beta}- lactam susceptibility profiles of {beta}-lactamase positive clinical isolates. We then deployed a robust and reproducible shotgun proteomics methodology to identify {beta}-lactamase positivity and predict {beta}-lactam susceptibility by reference to our baseline predictive rules both in cultured bacteria and in extracts of culture-positive blood. Proteomics and whole genome sequencing then allowed us to characterise K. pneumoniae and P. aeruginosa isolates that differed from the expected {beta}-lactam susceptibility profile, iteratively expanding our predictive rules. Proteomics added considerable value over and above the information generated by whole genome sequencing, allowing for gene expression, not just gene presence to be considered. Specifically, in K. pneumoniae, we identified key differences between acrR and ramR regulatory mutations and compared the effects of OmpK36 Aspartate-Threonine or Glycine-Aspartate dipeptide porin insertions on susceptibility to cefepime and carbapenems. In P. aeruginosa, we identified differences in the gene expression effects of mexR versus nalC mutations and related these to differences in {beta}-lactam MICs against isolates hyper-producing AmpC {beta}-lactamase and or producing a metallo-{beta}-lactamase.

microbiology↗

bioRxiv2021

Klebsiella pneumoniae mutants resistant to ceftazidime/avibactam plus aztreonam, imipenem/relebactam, meropenem/vaborbactam and cefepime/taniborbactam.

Using modified Klebsiella pneumoniae clinical isolates, we show that ramR plus ompK36 mutation together with production of the V239G variant KPC-3 confirs resistance to ceftazidime/avibactam plus aztreonam, imipenem/relebactam and meropenem/vaborbactam, but not cefepime/taniborbactam. This is because the V239G variant does not generate collateral {beta}-lactam susceptibility as do many other KPC-3 variants associated with ceftazidime/avibactam resistance. Additional mutation of ompK35 and carriage of a plasmid expressing the OXA-48-like carbapenemase OXA-232 was required to confer cefepime/taniborbactam resistance.

microbiology↗

bioRxiv2021

Factors Influencing the Detection of Antibacterial Resistant Escherichia coli in Faecal Samples from Individual Cattle

AimsTo investigate whether on-farm antibacterial usage (ABU), environmental antibacterial resistant (ABR) Escherichia coli prevalence, and sampling and sample handling methodologies are associated with ABR E. coli positivity in individual faecal samples from dairy heifers. Methods and ResultsThree hundred and sixty-four heifers from 37 farms were sampled via rectal or faecal pat sampling. Samples were stored at -80{degrees}C for variable periods before microbiological analysis. Data analysis was through a multilevel, multivariable logistic regression approach. Individual rectal samples had increased odds of positivity for amoxicillin, cefalexin and tetracycline-resistant E. coli. Sample storage for 6-12 month was associated with decreased odds of finding amoxicillin and tetracycline-resistant E. coli. On-farm ABU had little influence, and environmental ABR E. coli prevalence had no significant influence on on the odds of sample-level positivity for ABR E. coli. ConclusionsSampling methodology and sample handling have a greater association than on-farm factors with the detection of ABR E. coli in individual faecal samples from dairy heifers. Significance and Impact of the StudySampling and storage methodologies should be considered carefully at the point of designing ABR surveillance studies in livestock and their environments and, where possible, standardised between and within future studies.

microbiology↗

bioRxiv2021

OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired β-Lactamases.

Cefalexin is a widely used 1st generation cephalosporin, and resistance in Escherichia coli is caused by Extended-Spectrum (e.g. CTX-M) and AmpC {beta}-lactamase production and therefore frequently coincides with 3rd generation cephalosporin resistance. However, we have recently identified large numbers of E. coli isolates from human infections, and from cattle, where cefalexin resistance is not {beta}-lactamase mediated. Here we show, by studying laboratory selected mutants, clinical isolates, and isolates from cattle, that OmpF porin disruption or downregulation is a major cause of cefalexin resistance in E. coli. Importantly, we identify multiple regulatory mutations that cause OmpF downregulation. In addition to mutation of ompR, already known to downregulate OmpF and OmpC porin production, we find that rseA mutation, which strongly activates the Sigma E regulon, greatly increasing DegP production, which degrades OmpF, OmpC and OmpA porins. Furthermore, we reveal that mutations affecting lipopolysaccharide structure, exemplified by the loss of GmhB, essential for lipopolysaccharide heptosylation, also modestly activate DegP production, resulting in OmpF degradation. Remarkably, given the critical importance attached to such systems for normal E. coli physiology, we find evidence for DegP-mediated OmpF downregulation, gmhB and rseA loss of function mutation in E. coli isolates derived from human infections. Finally, we show that these regulatory mutations enhance the ability of group 1 CTX-M {beta}-lactamase to confer reduced carbapenem susceptibility, particularly those mutations that cause OmpC in addition to OmpF downregulation.

microbiology↗

bioRxiv2021

Fluoroquinolone-Resistant Escherichia coli Originating on Dairy Farms are an Infrequent Cause of Bacteriuria in Humans Living in the Same Geographical Region

SynopsisO_ST_ABSBackgroundC_ST_ABSOur primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms were a significant cause of bacteriuria in humans living in the same 50 x 50 km geographical region located in South West England. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms. MethodsFQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularisation approach. ResultsOf 489 FQ-R human isolates, 255 were also 3rd generation cephalosporin-resistant (3GC-R), with strong genetic linkage between aac(6)Ib-cr and blaCTX-M-15. We identified possible farm-to-human sharing for pairs of ST744 and ST162 isolates, but core genome SNP distances (71 and 63, respectively) were smaller in pairs of ST744 and ST162 isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli while total dry cow therapy use showed a negative association. ConclusionsThis work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population, however, this appears greater than observed for 3GC-R E. coli when studied in parallel. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli, and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.

microbiology↗

bioRxiv2021

Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding and that E. coli from these dogs are shared with humans and cause opportunistic infections.

We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 sixteen-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of E. coli resistant to fluoroquinolones, tetracycline, amoxicillin, and streptomycin, but not to cefalexin or cefotaxime. Whole genome sequencing of 30 fluoroquinolone-resistant (FQ-R), 22 cefotaxime-resistant (CTX-R) and seven dual FQ-R/CTX-R E. coli isolates showed a wide range of sequence types (STs), an approximately 50:50 split of CTX-M:AmpC-mediated CTX-R, and almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 x 50 km region with those isolated from human urinary tract and bloodstream infections (isolated in parallel in the same region) identified a clone of ST963 E. coli carrying chromosomal blaCMY.2 in two puppies and causing two urinary tract infections and one bloodstream infection. Furthermore, an ST744 FQ-R clone was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at sixteen weeks of age and that bacteria carried by dogs are shared with humans.

microbiology↗

bioRxiv2020

Trade-Offs Between Antibacterial Resistance and Fitness Cost in the Production of Metallo-β-Lactamase by Enteric Bacteria Manifest as Sporadic Emergence of Carbapenem Resistance in a Clinical Setting.

Meropenem is a clinically important antibacterial reserved for treatment of multi-resistant infections. In meropenem-resistant bacteria of the family Enterobacteriales, NDM-1 is considerably more common than IMP-1, despite both metallo-{beta}-lactamases (MBLs) hydrolysing meropenem with almost identical kinetics. We show that blaNDM-1 consistently confers meropenem resistance in wild-type Enterobacteriales, but blaIMP-1 does not. The reason is higher blaNDM-1 expression because of its stronger promoter. However, the cost of meropenem resistance is reduced fitness of blaNDM-1 positive Enterobacteriales because of amino acid starvation. In parallel, from a clinical case, we identified multiple Enterobacter spp. isolates carrying a plasmid-encoded blaNDM-1 having a modified promoter region. This modification lowered MBL production to a level associated with zero fitness cost but, consequently, the isolates were not meropenem resistant. However, we identified a Klebsiella pneumoniae isolate from this same clinical case carrying the same blaNDM-1 plasmid. This isolate was meropenem resistant despite low-level NDM-1 production because of a ramR mutation, reducing envelope permeability. Overall, therefore, we show how the resistance/fitness trade-off for MBL carriage can be resolved. The result is sporadic emergence of meropenem resistance in a clinical setting.

microbiology↗