A decline in the occurrence of extended-spectrum ß-lactamase-producing Escherichia coli in retail chicken meat in the UK between 2013 and 2018.

AIMS: The aim of this study was to report on the occurrence of antimicrobial resistant (AMR) Escherichia coli from retail chicken meat samples in the UK, with particular focus on AmpC and extended spectrum beta-lactamase (ESBL) production and carbapenem resistance. METHODS AND RESULTS: Methods from EU protocols were used for selective isolation of AmpC-/ESBL-producing E. coli, carbapenem-resistant E. coli and for performing minimum inhibitory concentrations. Additional work not part of EU protocols included viable counts, detection by PCR of blaCTX-M , blaOXA, blaSHV and blaTEM genes in ESBL-phenotype E. coli and screening for mcr plasmid-mediated colistin resistance. From the 313/309 retail chicken meat samples tested in 2016/2018, carbapenem or mcr plasmid-mediated colistin-resistant E. coli were not detected. For 2016/2018 chicken samples, 141/42 (45·0%/13·6%), 90/23 (28·8%/7·4%), 48/16 (15·3%/5·2%) and 3/3 (1·0%/1·0%) were positive for ESBL- and/or AmpC-, ESBL- alone AmpC- alone and AmpC+ESBL-phenotype E. coli respectively. ESBL-producing E. coli were predominantly blaCTX-M-1 . All AmpC and/or ESBL-phenotype E. coli were sensitive to colistin, ertapenem, imipenem, meropenem, temocillin and tigecycline, applying epidemiological cut-off values. CONCLUSIONS: A previous study in 2013/14 showed that 65·4% of retail chicken meat samples tested in the UK were positive for ESBL-producing (mainly CTX-M) E. coli. Since then the proportion of samples positive in the UK has dropped significantly to 7·4% in 2018. SIGNIFICANCE AND IMPACT OF THE STUDY: Significant reductions in antimicrobials used in the UK poultry meat sector between 2012 and 2016 may be linked to significant reductions in AmpC/ESBL-phenotype E. coli in retail chicken between 2013/14 and 2018.

Detection of extended-spectrum ß-lactam, AmpC and carbapenem resistance in Enterobacteriaceae in beef cattle in Great Britain in 2015.

AIMS: This study investigated the occurrence and genetic diversity of Enterobacteriaceae with extended-spectrum ß-lactamase (ESBL)-, AmpC- and carbapenemase-mediated resistance in British beef cattle, and related risk factors. METHODS AND RESULTS: Faecal samples (n = 776) were obtained from farms in England and Wales (n = 20) and Scotland (n = 20) in 2015. Isolates from selective agars were identified by MALDI ToF mass spectrometry. Selected isolates were characterized by multiplex PCR (blaCTX -M, blaOXA , blaSHV and blaTEM genes), whole-genome sequencing (WGS), minimum inhibitory concentrations and pulsed-field gel electrophoresis. None of the faecal samples yielded carbapenem-resistant Escherichia coli. Ten (25%) of the farms tested positive for ESBL-producing CTX-M Enterobacteriaceae, 15 (37·5%) of the farms were positive for AmpC phenotype E. coli and none were positive for carbapenem-resistant E. coli. WGS showed a total of 30 different resistance genes associated with E. coli, Citrobacter and Serratia from ESBL agars, and colocation of resistance genes with blaCTX -M1 . Buying bulls and bringing in fattening cattle from another farm were identified as significant risk factors for positive samples harbouring CTX-M Enterobacteriaceae or AmpC phenotype E. coli respectively. CONCLUSIONS: Beef cattle on a proportion of farms in GB carry ESBL-producing Enterobacteriaceae. Factors, such as operating as a closed herd, may have an important role in reducing introduction and transmission of resistant Enterobacteriaceae. The results indicate management factors may play an important role in impacting ESBL prevalence. In particular, further study would be valuable to understand the impact of maintaining a closed herd on reducing the introduction of resistant Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing the presence of ESBL-producing Enterobacteriaceae in British beef cattle.

Longitudinal study on the occurrence in pigs of colistin-resistant Escherichia coli carrying mcr-1 following the cessation of use of colistin.

AIMS: In 2015, colistin-resistant Escherichia coli and Salmonella with the mcr-1 gene were isolated from a pig farm in Great Britain. Pigs were subsequently monitored over a ~20-month period for the occurrence of mcr-1-mediated colistin resistance and the risk of mcr-1 E. coli entering the food chain was assessed. METHODS AND RESULTS: Pig faeces and slurry were cultured for colistin-resistant E. coli and Salmonella, tested for the mcr-1 gene by PCR and selected isolates were further analysed. Seventy-eight per cent of faecal samples (n = 275) from pigs yielded mcr-1 E. coli after selective culture, but in positive samples only 0·2-1·3% of the total E. coli carried mcr-1. Twenty months after the initial sampling, faecal samples (n = 59) were negative for E. coli carrying mcr-1. CONCLUSIONS: The risk to public health from porcine E. coli carrying mcr-1 was assessed as very low. Twenty months after cessation of colistin use, E. coli carrying mcr-1 was not detected in pig faeces on a farm where it was previously present. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that cessation of colistin use may help over time to reduce or possibly eliminate mcr-1 E. coli on pig farms where it occurs.

Degradation of cefquinome in spiked milk as a model for bioremediation of dairy farm waste milk containing cephalosporin residues.

AIMS: The aims of this work were to develop a model of dairy farm waste milk and to investigate methods for the bioremediation of milk containing cefquinome residues. METHODS AND RESULTS: Unpasteurized milk and UHT milk that had both been spiked with cefquinome at a concentration of 2 µg ml(-1) were used as a model for waste milk containing cephalosporin residues. Adjustment of the spiked UHT milk to pH 10 or treatment with conditioned medium from bacterial growth producing cefotaximase, were the most effective methods for decreasing the cefquinome concentrations within 24 h. A large-scale experiment (10 l of cefquinome-spiked unpasteurized milk) suggested that fermentation for 22 h at 37°C followed by heating at 60°C for 2 h was sufficient to decrease cefquinome concentrations to below the limit of quantification (<125 µg kg(-1) ) and to kill the majority of the enriched bacterial population. CONCLUSIONS: One or a combination of the bioremediation methods described may have potential as a practical treatment for dairy farm waste milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of waste milk to decrease cephalosporin residue concentrations and also to kill bacteria prior to feeding to dairy calves could decrease the risk of selection for ESBL bacteria on dairy farms.

Prevalence of extended-spectrum-ß-lactamase-producing Escherichia coli from pigs at slaughter in the UK in 2013.

OBJECTIVES: To determine the prevalence and types of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli occurring in pigs at slaughter in the UK in 2013. METHODS: Caecal samples from 637 pigs, sampled via a UK-wide monitoring programme in 2013, were enriched overnight in buffered peptone water, before plating to CHROMagar CTX and Oxoid Brilliance ESBL agar. Presumptive ESBL-producing E. coli from both media were tested for ESBL phenotype using MAST ESßL ID discs. Isolates with an ESBL phenotype were examined for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes using a multiplex PCR. All blaCTX-M and blaSHV genes identified by PCR were sequenced. RESULTS: A total of 23.4% (95% CI 19.2-27.6) of pigs were positive for ESBL-producing E. coli; 22% (95% CI 17.8-26.1) of the pigs carried E. coli producing CTX-M enzymes [comprising enzyme types 1 (18.7% of pigs), 3 (0.2%), 14 (0.5%), 15 (1.4%), 27 (0.5%), 32 (0.5%) and 55 (0.3%)] and 2.2% (95% CI 0.8-3.6) of the pigs carried E. coli producing SHV-12. Five pigs carried both CTX-M- and SHV-12-producing E. coli as different isolates. There were no statistically significant differences observed between the two medium types in terms of the proportions of each CTX-M enzyme type isolated. CONCLUSIONS: In this UK study, 23.4% of pigs were found to be positive for ESBL-producing E. coli using selective culture media. The use of two different commercially available ESBL isolation media was found to improve the detection of ESBL-producing E. coli.

Characteristics of ciprofloxacin and cephalosporin resistant Escherichia coli isolated from turkeys in Great Britain.

1. A field study was performed to investigate the presence and characteristics of ciprofloxacin-resistant, extended spectrum ß-lactamase (ESBL) and AmpC Escherichia coli from turkeys in Great Britain. E. coli were isolated from ~9000 boot swab samples from 27 different farms owned by four different companies. Between 1 and 14 visits were made to each farm (mean 3) at between 0 and 15 m intervals (mean ~5 m). 2. CHROMagar ECC with and without ciprofloxacin or cephalosporin antibiotics was used as selective isolation media. Representative isolates with different phenotypes were tested for mutations in gyrA and for: qnrA, B, S, qepA and aac(6')-Ib genes, for ESBL phenotype, the presence of bla genes and plasmid type, and for ampC genes Representative ciprofloxacin-resistant and CTX-M isolates were further tested for serotype and PFGE type. On ciprofloxacin selective media 55% of samples yielded ciprofloxacin resistant E. coli and of those further analysed, most had ciprofloxacin MICs >4 mg/l and mutations in gyrA. 3. For the different companies, the mean number of samples per farm with cefoxitin- or cefotaxime-resistant isolates ranged from 1·0% to 61·9% and 4·7% to 31·7% respectively. Cefotaxime-resistance was most commonly associated with an ESBL phenotype, a CTX-M-1 or CTX-M-14 sequence type and an I1-γ or K plasmid inc type. The mechanism of cefoxitin resistance was not determined for most isolates, but where determined it was bla . 4. PFGE and serotyping showed clonally-related isolates persisting over multiple visits suggesting both more prudent use of antibiotics and improved farm hygiene are needed to address the issue of antimicrobial resistance in isolates from turkeys.

Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales.

OBJECTIVES: To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. METHODS: UK CTX-M-14-producing E. coli (n=70) from cattle (n=33), turkeys (n=9), sheep (n=2) and humans (n=26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of the bla(CTX-M-14) gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence. RESULTS: The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 isolates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxigenic E. coli ('VTEC'); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of the bla(CTX-M-14) gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n=9), turkeys (n=8) and humans (n=4). All pCT-like plasmids were conjugative, and most were IncK (n=21) and had the same local genetic environment flanking the bla(CTX-M-14) gene (n=23). RFLP analysis demonstrated ≥ 75% similarity among most plasmids (n=22). CONCLUSIONS: pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla(CTX-M-14) genes to different E. coli isolates from humans, cattle and turkeys.

Ciprofloxacin resistance in E. coli isolated from turkeys in Great Britain.

Fluoroquinolones are a widely used group of antimicrobials in both human and animal medicine, with ciprofloxacin being a critically important fluoroquinolone for serious human infections. The present study reports on a 1-year survey for the presence of ciprofloxacin-resistant Escherichia coli in turkeys from Great Britain. Boot swabs were taken from 442 turkey flocks comprised of 125 breeding flocks and 317 meat flocks from 337 different farms over a 1-year period (2006 to 2007). CHROMagar ECC containing 1 mg/l ciprofloxacin was used to obtain ciprofloxacin-resistant isolates. Isolates were tested for sensitivity to 16 different antimicrobials. Isolates with ciprofloxacin minimum inhibitory concentrations ≥8 mg/l were tested for mutations in gyrA by polymerase chain reaction and DNA sequencing. Selected isolates were tested by multiplex polymerase chain reaction for qnrA, qnrB and qnrS, qepA and aac(6')-Ib-cr genes. Conjugations were performed to assess the transferability of resistance to quinolones. Ciprofloxacin-resistant E. coli was found in 22.4% of turkey breeding flocks and 60.9% of meat flocks. Two main mutations in gyrA, as well as a range of silent mutations, were identified in resistant isolates. Flocks with transferable resistance genes qnrB, qnrS, and aac(6')-Ib-cr were found at a low flock prevalence of 4.2%, 1.6% and 1.0%, respectively; however, under laboratory conditions only transfer of qnrS genes could be demonstrated. This work has confirmed the occurrence of ciprofloxacin-resistant E. coli strains throughout turkey breeding and meat flocks, with almost one-third of E. coli isolates being resistant to ciprofloxacin. Of those, more than 87% were also resistant to three or more antimicrobial classes.

Prevalence of Escherichia coli carrying extended-spectrum ß-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009.

OBJECTIVES: to determine the prevalence of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli from poultry in Great Britain (GB). METHODS: E. coli was isolated from 388 broiler chicken caecal samples from 22 abattoirs and from boot swabs from 442 turkey flocks over successive 1 year periods. CHROMagar ECC with and without cephalosporin antibiotics was used as isolation medium and the chicken study also used CHROMagar CTX. ESBL phenotype isolates were tested for the presence of bla(CTX-M,) bla(OXA), bla(SHV), bla(TEM) and ampC genes(.) CTX-M isolates were tested for O25 serogroup, replicon, CTX-M sequence, multilocus sequence type (MLST), PFGE type, plasmid transfer and qnrA, qnrB, qnrS, qepA and aac(6')-Ib genes. RESULTS: CTX-M-carrying E. coli were isolated from 54.5% of the broiler abattoirs and from 3.6% of individual broiler caecal samples and were CTX-M sequence types 1 (mainly), 3 and 15 with replicon types I1-γ, A/C and P/F, and I1-γ, respectively. CTX-M-carrying E. coli were isolated from 5.2% of turkey meat production farms and 6.9% of turkey breeder farms and were CTX-M sequence types 1, 14 (mainly), 15 and 55 with mainly replicon types F, FIA, K and I1-γ, respectively. None of the CTX-M isolates was serogroup O25. PFGE/MLST showed the CTX-M isolates to be clonally diverse, although MLST 156 with CTX-M-15 was isolated from both chickens and turkeys and has been previously reported in gulls. CTX-M-negative, ESBL- and bla(TEM)-positive strains were mainly TEM-52C. CONCLUSIONS: poultry-derived CTX-M E. coli in GB are different from major CTX-M sequence types causing disease in humans.

Fecal carriage and shedding density of CTX-M extended-spectrum {beta}-lactamase-producing escherichia coli in cattle, chickens, and pigs: implications for environmental contamination and food production.

The number and proportion of CTX-M positive Escherichia coli organisms were determined in feces from cattle, chickens, and pigs in the United Kingdom to provide a better understanding of the risk of the dissemination of extended-spectrum ß-lactamase (ESBL) bacteria to humans from food animal sources. Samples of bovine (n = 35) and swine (n = 20) feces were collected from farms, and chicken cecal contents (n = 32) were collected from abattoirs. There was wide variation in the number of CTX-M-positive E. coli organisms detected; the median (range) CFU/g were 100 (100 × 10(6) to 1 × 10(6)), 5,350 (100 × 10(6) to 3.1 × 10(6)), and 2,800 (100 × 10(5) to 4.7 × 10(5)) for cattle, chickens, and pigs, respectively. The percentages of E. coli isolates that were CTX-M positive also varied widely; median (range) values were 0.013% (0.001 to 1%) for cattle, 0.0197% (0.00001 to 28.18%) for chickens, and 0.121% (0.0002 to 5.88%) for pigs. The proportion of animals designated high-density shedders (≥1 × 10(4) CFU/g) of CTX-M E. coli was 3/35, 15/32, and 8/20 for cattle, chickens, and pigs, respectively. We postulate that high levels of CTX-M E. coli in feces facilitate the dissemination of bla(CTX-M) genes during the rearing of animals for food, and that the absolute numbers of CTX-M bacteria should be given greater consideration in epidemiological studies when assessing the risks of food-borne transmission.

Evaluation of CHROMagar CTX, a novel medium for isolating CTX-M-ESBL-positive Enterobacteriaceae while inhibiting AmpC-producing strains.

OBJECTIVES: The aim of this study was to evaluate CHROMagar CTX (CHROMagar France), a novel agar for the selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene in the presence of enteric bacteria expressing AmpC enzymes. METHODS: A panel of 150 Gram-negative bacteria (mainly Escherichia coli, Enterobacter, Klebsiella, Pseudomonas and Salmonella) isolated from humans and animals were assembled for the purpose of evaluating CHROMagar CTX and comparing it with CHROMagar ECC with the addition of 1, 2, 4 and 8 mg/L cefotaxime or ceftazidime and with bioMérieux extended-spectrum beta-lactamase (ESBL)-Bx agar. CHROMagar CTX was also assessed for its ability to isolate bla(CTX-M) strains from farm animal faeces (n = 342). RESULTS: The panel contained CTX-M-positive (n = 70) strains (CTX-M types 1, 9, 14 and 15), ESBLs (n = 31) belonging to other families (OXA, PER, SHV, TEM, VEB), strains positive for ampC genes (n = 31), strains that overexpressed ampC (n = 6), non-ESBL/AmpC strains (n = 11) and Klebsiella oxytoca (n = 1). CHROMagar CTX was superior to other agars tested for selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene with 100% sensitivity and 64.2% specificity for CTX-M strains in the panel and 90.1% of the colonies from animal faeces plated on CHROMagar CTX were CTX-M strains. CONCLUSIONS: CHROMagar CTX is a valuable agar in situations where it is important to isolate bla(CTX-M) strains in the presence of AmpC strains. The agar may be particularly useful in veterinary studies, where AmpC-producing commensal E. coli can be encountered reasonably frequently in the enteric flora of some animal species and may also be useful, following further evaluation, for samples from humans.

Evaluation of meat, fruit and vegetables from retail stores in five United Kingdom regions as sources of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli.

We determined the prevalence and types of extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli in raw retail beef, chicken, pork, fruit and vegetables in five UK regions in 2013-14. Raw meat (n=397), and fruit and vegetable samples (n=400) were purchased from retail stores in London, East Anglia, North West England, Scotland and Wales. Samples were tested for the presence of ESBL-producing E. coli by plating enriched samples on CHROMagar CTX and CHROMagar ESBL, for AmpC-type E. coli by plating on "CHROMagar FOX" (CHROMagar ECC+16mg/L cefoxitin), and for carbapenem-resistant E. coli by plating on CHROMagar KPC. Additionally, pre-enrichment counts were performed on the above agars, and on CHROMagar ECC. Isolates of interest were characterised by MALDI-ToF to confirm identification, by PCR for blaCIT,blaCTX-M,blaOXA, blaSHV and blaTEM genes; ESBL or blaCIT genes were sequenced. Only 1.9% and 2.5% of beef and pork samples, respectively were positive for ESBL-producing E. coli after enrichment compared with 65.4% of chicken samples. 85.6% positive samples from chicken meat carried blaCTX-M-1; blaCTX-M-15 was not detected. None of the fruits or vegetables yielded ESBL-producing E. coli and none of the meat, fruit or vegetable samples yielded carbapenem-resistant E. coli. Retail chicken was more frequently a source of ESBL-producing E. coli than were beef, pork, fruit or vegetables. None of the foodstuffs yielded E. coli with CTX-M-15 ESBL, which dominates in human clinical isolates in the UK, and none yielded carbapenem-resistant E. coli.

Differentiation of UK endemic strains of Salmonella enterica serovar Newport from epidemic North American strains by PCR detection of a truncated bapA chromosomal gene.

The aim of this study was to develop a PCR test to detect chromosomal differences between epidemic multidrug resistant (epi-MDR) strains of Salmonella enterica serovar Newport (S. Newport) and non-epi-MDR strains of S. Newport that are endemic to the United Kingdom (UK). Sequence analysis of the biofilm-associated protein A gene (bapA) showed that epi-MDR strains of S. Newport from the United States of America (USA) had a deletion of 309 bp, which was not present in non-epi-MDR strains of S. Newport from the UK. A PCR test was developed using primers designed to target this difference and was applied to a panel of S. Newport isolates comprising of strains from the UK (n=20, non-epi-MDR), from the USA (n=10, epi-MDR) and from Canada (n=7). A second panel of isolates (n=73) was used to assess the test specificity, and these isolates consisted of non-Newport Salmonella serovars (n=25), and other epidemic serovars (n=48). Epi-MDR S. Newport isolates produced a characteristic 505 bp amplicon, whereas non-epi-MDR S. Newport isolates produced an 814 bp amplicon. The bapA PCR has potential to discriminate between these S. Newport strains irrespective of their carrying resistance genes.

Longitudinal study of CTX-M ESBL-producing E. coli strains on a UK dairy farm.

The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm.

Role of the mar locus in virulence of Salmonella enterica serovar Typhimurium DT104 in chickens.

The virulence of a Salmonella enterica serovar Typhimurium DT014 strain in which marA was insertionally inactivated was compared to its isogenic parent in vitro and in vivo. In vitro, the numbers of the marA mutant phagocytosed by porcine lung macrophages were significantly increased, while survival at 24 h inside macrophages and adherence to human gut cells were significantly reduced in comparison with the parent strain. In vivo, the marA inactivated strain, in competition with its parent strain, persisted for a shorter period in chickens, was present in the caeca at significantly lower levels and invaded the deeper organs to a significantly lesser extent. Therapeutic antibiotic treatment of one group of chickens with oxytetracycline favoured the persistence of both the parent strain and, to a lesser extent, the marA inactivated strain; but interestingly, increased tetracycline resistance of Salmonella isolates after treatment of birds with antibiotic was seen only for the parent strain. Further work is needed to elucidate how mar is involved in virulence and if its inactivation can minimise the ability of bacteria to become antibiotic-resistant in vivo.

The multiple antibiotic resistance (mar) locus and its significance.

Chromosomally encoded systems involved in low level resistance of bacteria to different classes of antibiotics (mainly beta-lactams, chloramphenicol, quinolones and tetracycline), disinfectants and in resistance to organic solvents have been the focus of considerable interest in recent years. The multiple antibiotic resistance (mar) locus of Escherichia coli and Salmonella is perhaps the best described system involved in this type of resistance which is induced by MarA, the activator protein encoded by the marRAB locus. The mar -locus is reported to mediate resistance primarily by up-regulating efflux of some antibiotics, disinfectants and organic solvents via the AcrAB-TolC efflux pump and down regulating influx through Outer Membrane Protein F (OmpF). Whilst the level of antibiotic resistance conferred by marRAB is only low level, there are increasing data to suggest that marRAB and related systems are important in clinical antibiotic resistance, possibly as a 'stepping stone' to higher levels of resistance. Other related systems include up-regulation of RobA, SoxS and AcrAB which give rise to a similar resistance phenotype to that conferred by up-regulation of MarA. The aim of this paper is to review the function and significance of the mar -locus and related systems with a particular focus on its implications in veterinary medicine.

Studies on the development and use of a monoclonal sandwich ELISA for the detection of verotoxic Escherichia coli in animal faeces.

A sandwich ELISA using monoclonal antibodies to Escherichia coli verocytotoxins 1 and 2 for capture and detection was developed for detection of verocytotoxin-producing Escherichia coli (VTEC) in animal faeces. For optimal toxin detection, the faeces were cultured in MacConkey broth before subculture on to trypticase soy agar containing mitomycin C. It was possible to detect between 10 and 10(3) VTI-producing and 1 to 10 VT2-producing E coli/g faeces, and overall the sensitivity and specificity of the ELISA for VTEC in faeces were 80.5 per cent and 91.2 per cent, respectively, when compared with a verocell assay. A limited survey of faeces from healthy animals (89 cattle, 16 sheep, 22 pigs, 11 goats and six domestic fowl) and faeces from 144 cattle with enteric disease detected VTEC in 64 per cent, 63 per cent, 5 per cent, 45 per cent, none and 16 per cent of the samples, respectively.

Natural and experimental infection of normal cattle with Escherichia coli O157.

The objective of the study was to determine the effects of inoculating cattle orally with a strain of Escherichia coli O157:H7 (A84/92). However, before they were challenged two of the six calves were found to be infected naturally with a wild-type strain of E coli O157 and two more of them became infected later. The number of daily faeces samples from which the wild-type E coli O157 was isolated ranged from one to 10. After they were inoculated, A84/92 was detected in all the calves' faeces on one to six of the next 14 days, and later from the faeces samples of three calves on two, three, and 11 occasions, the last occasion being between 19 and 51 days after inoculation. Two calves were redosed with A84/92, and the organism was isolated on a further five and 15 occasions, the last being after 20 and 58 days. In three dry cows, A84/92 was isolated from the faeces on three to 11 of the 14 days after they were inoculated. Two of the cows were redosed and from one of them it was isolated on 15 occasions, the last being 44 days after the initial infection; in the other cow no further isolation was made. In three lactating cows, it was detected on three to four of the 14 days after they were inoculated, and similar results were obtained after they were reinoculated. None of the animals showed clinical signs and no lesions were detected in the intestines of the calves. Three calves had a serological response to E coli O157 but, with the exception of one cow which had a slight increase to IgM levels, no serological changes were observed in the adult cattle.

Evaluation of an autogenous vaccine in cattle against Escherichia coli bearing the CTX-M-14 plasmid.

Enteric bacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing the blaCTX-M gene, have been detected in a wide range of food producing animals. However, commercial vaccines for these organisms are not currently available. An autogenous vaccine was prepared from E. coli bearing the blaCTX-M-14 gene and evaluated as a potential control measure to reduce shedding and dissemination of these organisms in cattle. Calves (n=30) received either an autogenous vaccine prepared from E. coli serotype O33 bearing the blaCTX-M-14 gene or a placebo by intramuscular injection on three separate occasions. Two weeks after the final vaccination, all calves were challenged by oral gavage with the O33 CTX-M-14 strain of E. coli (1×10(10) CFU). Faeces, intestinal mucosa and blood samples were taken for enumeration of total and CTX-M-14 E. coli and for assessment of the humoral immune response. The cumulative number of total E. coli excreted at 7 days post-challenge was significantly (p=0.006) lower in the vaccinated group than the placebo group. However, there was no significant difference in the shedding of either CTX-M-14 E. coli or total E. coli between vaccinated and placebo calves throughout the study period. The systemic immune response to E. coli O33 antigen was tested by ELISA and was significantly higher (p<0.001) in vaccinated than placebo calves. However, there was no significant difference in the mucosal immune response. These findings do not support the use of autogenous vaccination for the control of CTX-M-14 E. coli in calves.

Isolation and detection of extended spectrum ß-lactamase (ESBL)-producing enterobacteriaceae from meat using chromogenic agars and isothermal loop-mediated amplification (LAMP) assays.

The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity.