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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC.

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospiraborgpetersenii, and Leptospirakirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK.

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.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence.

OBJECTIVES: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. METHODS: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. RESULTS: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. CONCLUSIONS: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation.

AIMS: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). METHODS: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. RESULTS: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. CONCLUSIONS: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli.

AIMS: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [indicative of a multiple antibiotic resistance (MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. METHODS AND RESULTS: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli (10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive (n=5) and -resistant (n=5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli (n=389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations (MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers (P < or = 0.023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. CONCLUSIONS: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.