Occurrence and characterization of rmtB-harbouring Salmonella and Escherichia coli isolates from a pig farm in the UK.

OBJECTIVES: To characterize and elucidate the spread of amikacin-resistant Enterobacteriaceae isolates from environmental samples on a pig farm in the UK, following the previous identification of index Salmonella isolates harbouring the rmtB gene, a 16S rRNA methylase. METHODS: Environmental samples were collected during two visits to a pig farm in the UK. Isolates were recovered using selective media (amikacin 128 mg/L) followed by real-time PCR and WGS to analyse rmtB-carrying Salmonella and Escherichia coli isolates. RESULTS: Salmonella and E. coli isolates harbouring the rmtB gene were detected at both farm visits. All Salmonella isolates were found to be monophasic S. enterica serovar Typhimurium variant Copenhagen of ST34. rmtB-harbouring E. coli isolates were found to be one of three STs: ST4089, ST1684 and ST34. Long-read sequencing identified the rmtB gene to be chromosomally located in Salmonella isolates and on IncFII-type plasmids in E. coli isolates. The results showed the rmtB gene to be flanked by IS26 elements and several resistance genes. CONCLUSIONS: We report on the occurrence of rmtB-harbouring Enterobacteriaceae on a pig farm in the UK. rmtB confers resistance to multiple aminoglycosides and this work highlights the need for surveillance to assess dissemination and risk.

Genomic surveillance of extended-spectrum cephalosporin-resistant Escherichia coli isolated from poultry in the UK from 2016 to 2020.

Introduction: Surveillance is vital for monitoring the increasing risk of antimicrobial resistance (AMR) in bacteria leading to failures in humans and animals to treat infections. In a One Health context, AMR bacteria from livestock and food can transfer through the food chain to humans, and vice versa, which can be characterized in detail through genomics. We investigated the critical aspects of AMR and the dynamics of AMR in poultry in the UK. Methods: In this study, we performed whole genome sequencing for genomic characterization of 761 extended-spectrum cephalosporinases (ESCs) harboring Escherichia coli isolated from poultry caeca and meat through EU harmonized monitoring of AMR in zoonotic and commensal bacteria from 2016 and 2018 and UK national monitoring in 2020. Results: The most common ESC in 2016 and 2018 was blaCTX-M-1; however, 2020 had a greater diversity of ESCs with blaCTX-M-55 dominant in chickens and blaCTX-M-15 more prevalent in turkeys. Co-resistance to sulphonamides, tetracycline, and trimethoprim was widespread, and there were several positive correlations between the sequence types (STs) and ESC genes. We identified certain AMR genotypes and STs that were frequent each year but not as successful in subsequent years, e.g., ST350 harboring blaCTX-M-1, sul2, and tetA-v4.Phylogenetic comparison of isolates prevalent in our panel with global ones from the same STs available in public databases showed that isolates from the UK generally clustered together, suggesting greater within-country than between-country transmission. Discussion: We conclude that future genomic surveillance of indicator organisms will be invaluable as it will enable detailed comparisons of AMR between and within neighboring countries, potentially identifying the most successful sequence types, plasmids, or emerging threats.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Use of genomics to explore AMR persistence in an outdoor pig farm with low antimicrobial usage.

Food animals may be reservoirs of antimicrobial resistance (AMR) passing through the food chain, but little is known about AMR prevalence in bacteria when selective pressure from antimicrobials is low or absent. We monitored antimicrobial-resistant Escherichia coli over 1 year in a UK outdoor pig farm with low antimicrobial usage (AMU) compared to conventional pig farms in the United Kingdom. Short and selected long-read whole-genome sequencing (WGS) was performed to identify AMR genes, phylogeny and mobile elements in 385 E. coli isolates purified mainly from pig and some seagull faeces. Generally, low levels of antimicrobial-resistant E. coli were present, probably due to low AMU. Those present were likely to be multi-drug resistant (MDR) and belonging to particular Sequence Types (STs) such as ST744, ST88 or ST44, with shared clones (<14 Single Nucleotide Polymorphisms (SNPs) apart) isolated from different time points indicating epidemiological linkage within pigs of different ages, and between pig and the wild bird faeces. Although importance of horizontal transmission of AMR is well established, there was limited evidence of plasmid-mediated dissemination between different STs. Non-conjugable MDR plasmids or large AMR gene-bearing transposons were stably integrated within the chromosome and remained associated with particular STs/clones over the time period sampled. Heavy metal resistance genes were also detected within some genetic elements. This study highlights that although low levels of antimicrobial-resistant E. coli correlates with low AMU, a basal level of MDR E. coli can still persist on farm potentially due to transmission and recycling of particular clones within different pig groups. Environmental factors such as wild birds and heavy metal contaminants may also play important roles in the recycling and dissemination, and hence enabling persistence of MDR E. coli. All such factors need to be considered as any rise in AMU on low usage farms, could in future, result in a significant increase in their AMR burden.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

The importance of using whole genome sequencing and extended spectrum beta-lactamase selective media when monitoring antimicrobial resistance.

To tackle the problem of antimicrobial resistance (AMR) surveillance programmes are in place within Europe applying phenotypic methods, but there are plans for implementing whole genome sequencing (WGS). We tested the benefits of WGS using Escherichia coli collected from pig surveillance performed between 2013 to 2017. WGS was performed on 498 E. coli producing ESBL and AmpC enzymes, recovered from pig caeca on MacConkey + cefotaxime (McC + CTX) agar, as recommended by the European Commission, or ESBL agar, used additionally by United Kingdom. Our results indicated WGS was extremely useful for monitoring trends for specific ESBL genes, as well as a plethora of AMR genotypes, helping to establish their prevalence and co-linkage to certain plasmids. Recovery of isolates with multi-drug resistance (MDR) genotypes was lower from McC + CTX than ESBL agar. The most widespread ESBL genes belonged to the blaCTX-M family. blaCTX-M-1 dominated all years, and was common in two highly stable IncI1 MDR plasmids harbouring (blaCTX-M-1,sul2, tetA) or (blaCTX-M-1, aadA5, sul2, dfrA17), in isolates which were phylogenetically dissimilar, suggesting plasmid transmission. Therefore, WGS provided a wealth of data on prevalence of AMR genotypes and plasmid persistence absent from phenotypic data and, also, demonstrated the importance of culture media for detecting ESBL E. coli.

Streptococcus caledonicus sp. nov., isolated from sheep.

Five strains of an unidentified Gram-positive, catalase-negative, chain-forming coccus-shaped organism recovered from sheep in Scotland were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria, the strains were tentatively identified as streptococci but they did not appear to correspond to any recognised species of the genus. Comparative 16S rRNA gene sequencing showed the strains were highly related to each other and confirmed their placement in the genus Streptococcus, with a maximum nucleotide identity of around 97 % to extant species. Best matches were with Streptococcus hillyeri followed by Streptococcus porci. Average nucleotide identity and in silico DNA-DNA hybridization values determined from whole-genome sequence were also consistent with the group representing a novel species. Best matches, again seen to S. hillyeri, followed by S. porci and S. plurextorum, were below accepted cut-off values for species delineation. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from sheep be assigned to a new species of the genus Streptococcus as Streptococcus caledonicus sp. nov. The type strain of Streptococcus caledonicus is S784/96/1T=CCUG 73951T=NCTC 14363T.

Extended-spectrum ß-lactamase-producing Escherichia coli in human-derived and foodchain-derived samples from England, Wales, and Scotland: an epidemiological surveillance and typing study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli isolates (ESBL-E coli) cause more than 5000 cases of bacteraemias annually in the UK. The contribution of the food chain to these infections is debated. We aimed to identify the most important reservoirs of ESBL-E coli that colonise and infect humans to identify strategic intervention points. METHODS: Sampling for ESBL-E coli was done between Aug 1, 2013, and Dec 15, 2014. We used selective media to seek ESBL-E coli in routinely submitted samples from human faeces, and prospectively collected samples from sewage, farm slurry, and retail foodstuffs in London, East Anglia, northwest England, Scotland, and Wales. We sequenced recovered isolates and compared these isolates with 293 bloodstream and 83 veterinary surveillance ESBL-E coli isolates from the same regions. FINDINGS: 2157 (11%) of 20 243 human faeces samples contained ESBL-E coli, including 678 (17%) of 3995 in London. ESBL-E coli also were frequent in sewage and retail chicken (104 [65%] of 159 meat samples), but were rare in other meats and absent from plant-based foods (0 of 400 fruit and vegetable samples). Sequence type (ST) 131 dominated among ESBL-E coli from human blood (188 [64%] of 293 isolates), faeces (128 [36%] of 360), and sewage (14 [22%] of 65) with STs 38 and 648 also widespread; CTX-M-15 was the predominant ESBL in these lineages (319 [77%] of 416). By contrast, STs 602, 23, and 117-mostly with CTX-M-1 ESBL-dominated among food and veterinary isolates (68 [31%] of 218), with only two ST131 organisms recovered. ST10 occurred in both animals and humans, being frequent in surveillance bovines (11 [22%] of 51 cattle) and representing 15 (4%) of 360 human faecal isolates (but only three [1%] of 293 from bacteraemias); however, both human and animal ST10 isolates were diverse in serotype. INTERPRETATION: Most human bacteraemias with ESBL-E coli in the UK involve internationally prevalent human-associated STs, particularly ST131; non-human reservoirs made little contribution to invasive human disease. Any interventions that seek to target food or livestock can affect the numbers of human infections caused by ESBL-E coli; prevention of the spread of resistant lineages among humans is more vital. FUNDING: NIHR Policy Research.

Molecular epidemiology of isolates with multiple mcr plasmids from a pig farm in Great Britain: the effects of colistin withdrawal in the short and long term.

Background: The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans. Objectives: To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5 month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ∼20 months. Methods: mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined. Results: E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5 months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ∼20 months after colistin cessation. Conclusions: We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.

mcr-1 and mcr-2 variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015.

Objectives: To determine the occurrence of mcr-1 and mcr-2 genes in Gram-negative bacteria isolated from healthy pigs in Great Britain. Methods: Gram-negative bacteria (n = 657) isolated from pigs between 2014 and 2015 were examined by WGS. Results: Variants of mcr-1 and mcr-2 were identified in Moraxella spp. isolated from pooled caecal contents of healthy pigs at slaughter collected from six farms in Great Britain. Other bacteria, including Escherichia coli from the same farms, were not detected harbouring mcr-1 or mcr-2. A Moraxella porci-like isolate, MSG13-C03, harboured MCR-1.10 with 98.7% identity to MCR-1, and a Moraxella pluranimalium-like isolate, MSG47-C17, harboured an MCR-2.2 variant with 87.9% identity to MCR-2, from E. coli; the isolates had colistin MICs of 1-2 mg/L. No intact insertion elements were identified in either MSG13-C03 or MSG47-C17, although MSG13-C03 harboured the conserved nucleotides abutting the ISApl1 composite transposon found in E. coli plasmids and the intervening ∼2.6 kb fragment showed 97% identity. Six Moraxella osloensis isolates were positive for phosphoethanolamine transferase (EptA). They shared 62%-64.5% identity to MCR-1 and MCR-2, with colistin MICs from 2 to 4 mg/L. Phylogenetic analysis indicated that MCR and EptA have evolved from a common ancestor. In addition to mcr, the ß-lactamase gene, blaBRO-1, was found in both isolates, whilst the tetracycline resistance gene, tetL, was found in MSG47-C17. Conclusions: Our results add further evidence for the mobilization of the mcr-pap2 unit from Moraxella via composite transposons leading to its global dissemination. The presence of mcr-pap2 from recent Moraxella isolates indicates they may comprise a reservoir for mcr.

Risk for the development of Antimicrobial Resistance (AMR) due to feeding of calves with milk containing residues of antibiotics.

EFSA was requested to: 1) assess the risk for the development of antimicrobial resistance (AMR) due to feeding on farm of calves with colostrum potentially containing residues of antibiotics; 2) assess the risk for the development of AMR due to feeding on farm of calves with milk of cows treated during lactation with an antibiotic and milked during the withdrawal period, and 3) propose possible options to mitigate the risk for the development of AMR derived from such practices. Treatment of dairy cows during the dry period and during lactation is common in the EU Member States. Penicillins, alone or in combination with aminoglycosides, and cephalosporins are most commonly used. Residue levels of antimicrobials decrease with the length of the dry period. When the interval from the start of the drying-off treatment until calving is as long as or longer than the minimum specified in the Summary of Product Characteristics of the antimicrobial, faecal shedding of antimicrobial-resistant bacteria will not increase when calves are fed colostrum from treated cows. Milk from cows receiving antimicrobial treatment during lactation contains substantial residues during the treatment and withdrawal period. Consumption of such milk will lead to increased faecal shedding of antimicrobial-resistant bacteria by calves. A range of possible options exist for restricting the feeding of such milk to calves, which could be targeting the highest priority critically important antimicrobials. ß-Lactamases can reduce the concentration of ß-lactams which are the most frequently used antimicrobials in milking cows. Options to mitigate the presence of resistant bacteria in raw milk or colostrum are mainly based on thermal inactivation.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015.

Objectives: To determine the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Methods: Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT-PCR. mcr-1 -harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Results: Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%-1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1 -positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1 -plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1 -plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%-99%. Conclusions: mcr-1- harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1 -positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

In vitro investigations into the use of antimicrobials in combination to maintain efficacy of fluoroquinolones in poultry.

The aim of this study was to determine if apramycin, colistin or lincomycin-spectinomycin, in combination with enrofloxacin, was able prevent the emergence of mutants with reduced susceptibility to fluoroquinolone antibiotics in vitro. MICs were determined for enrofloxacin alone and in combination for panels of Campylobacter (n=37), Escherichia coli (n=52) and Salmonella (n=52) isolates. MIC results suggested that apramycin, colistin and lincomycin-spectinomycin worked in an additive/indifferent way when each was combined with enrofloxacin. Apramycin was considered the most promising antibiotic for combination-therapy in conjunction with enrofloxacin, and further evaluations (MBCs, MPCs and time-kill-curves) were performed for this combination for selected isolates. Results suggest combination-therapy of enrofloxacin with apramycin increases the efficacy, as well as decreasing the emergence and survival of bacteria with mutational resistance to fluoroquinolone antibiotics. Such combination-therapy, minimising the development of mutational resistance, may have relevance for Campylobacter, E. coli and Salmonella infections in poultry.

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain.

OBJECTIVES: The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS: Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS: Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured ß-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS: Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory.

Matrix-Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) Mass Spectrometry with Bruker MALDI Biotyper software was evaluated as a method for identifying veterinary bacteria. For 620 isolates (~100 bacterial species), identification by MALDI-ToF and non-16S rDNA methods (mainly phenotypic/biochemical) agreed to species-level (95.3%) and to species/genus-level (100%), but in the absence of 16S rDNA as a gold standard. For a further panel of 107 anaerobes and 234 aerobes (~100 bacteria species) using 16S rDNA results as the gold standard, MALDI-ToF/biochemical tests showed 97.8/96.6% species-level and 99.6/93.5% genus-level agreement for aerobes and 95.3/93.6% species-level and 100/95.3% genus-level agreement for anaerobes compared to the gold standard. Where results were obtained from direct spots, direct spots overlaid with formic acid and extracts, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-ToF. MALDI-ToF was shown to be a rapid and reliable method to identify veterinary bacteria.

Detection of antibiotic residues and association of cefquinome residues with the occurrence of Extended-Spectrum ß-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011.

Waste milk samples from 103 farms in England and Wales were examined for the presence of ß-lactam antibiotics and ESBL-producing Enterobacteriaceae. Approximately 10 months after the initial sampling, further waste milk, environmental and faecal samples from farms shown to be positive for CTX-M Escherichia coli were investigated further. Isolates with an ESBL phenotype were tested by PCR for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes. Isolates positive for blaCTX-M were sequenced to determine CTX-M type. Representative isolates were further examined by PFGE, plasmid replicon typing and serotyping. Of particular interest, 21.4% of waste milk samples contained residues of the cephalosporin cefquinome, which was significantly associated with CTX-M bacteria. Such bacteria occurred in 5.8% of the waste milk samples (including 3.9% CTX-M E. coli). CTX-M types identified were 1, 14, 14b and 15, but none of the E. coli were serotype O25, the serotype of the human pandemic strain.

Quantification and characterization of mucosa-associated and intracellular Escherichia coli in inflammatory bowel disease.

BACKGROUND: Mucosa-associated Escherichia coli are abundant in inflammatory bowel disease (IBD), but whether these bacteria gain intracellular access within the mucosa is uncertain. If E. coli does gain intracellular access, the contribution of bacterial pathogenicity to this requires further elucidation. This study aimed to quantify and characterize mucosa-associated and intracellular E. coli in patients with IBD and in healthy control subjects (HC). METHODS: Mucosal biopsies from 30 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC), and 14 HC were cultured with or without gentamicin protection to recover intracellular or mucosa-associated E. coli, respectively. Overall, 40 strains (CD: n = 24, UC: n = 9, and HC: n = 7) were characterized by phylogenetic typing, adhesion and invasion assays, detection of virulence factors, antimicrobial resistance genes, and proteomic analysis. RESULTS: Mucosa-associated E. coli were more abundant in CD and UC than in HC (2750 versus 1350 versus 230 median colony-forming units per biopsy; P = 0.01). Intracellular E. coli were more prevalent in CD (90%) than in UC (47%) or HC mucosal biopsies (0%) (P < 0.001). Of 24 CD strains, 2 were adherent and invasive, but there were no unifying pathogenicity determinants that could distinguish most CD strains from UC or HC strains, or intracellular isolates from mucosa-associated isolates. CONCLUSIONS: Intracellular E. coli are more common in CD than in UC and not identified in HC. Most intracellular E. coli did not have characterizing pathogenic features, suggesting a significant role for defects in mucosal immunity or barrier dysfunction in their ability to gain intracellular access.

Molecular and phenotypic characterisation of extended spectrum ß-lactamase CTX-M Escherichia coli from farm animals in Great Britain.

The aim of this study was to characterise CTX-M Escherichia coli isolates from cattle, chickens and turkeys in Great Britain with respect to CTX-M sequence type, replicon type, ability to transfer plasmids, and for the presence of antibiotic resistance, fitness and virulence genes as determined by micro-arrays. The main CTX-M enzymes identified in E. coli from cattle, chicken and turkeys were 14 and 15, 1 and 15, and 1 and 14 respectively. Most isolates from different animal species transferred their plasmids with similar frequencies. The plasmid replicon type I1-λ was most common and seen in 23%, 95% and 50% of the isolates tested from cattle, chickens and turkeys respectively, whilst types F, FIA, FIB and K were common to isolates from cattle and turkeys only. Thirty-eight different antibiotic resistance genes were detected by micro-array including aad genes, blaCTX-M, blaTEM, cat genes dfrA, floR, strA, strB, sul, sul2 tetA and tetB. Thirty-nine different fitness and virulence genes were also detected by-micro-array, including espP, ireA, lpfA, mchF, prfB and tsh. Fisher exact test and hierarchical clustering of the antibiotic resistance and virulence gene results showed some genes were more commonly associated with isolates from chickens or cattle. This study provides a baseline of the characteristics of CTX-M E. coli isolates from animals in Great Britain and suggests that chicken and cattle CTX-M E. coli represent different populations.