Véronique Métayer

Co-occurrence of extended spectrum β lactamase and MCR-1 encoding genes on plasmids

Findings reported by Yi-Yun Liu and colleagues identified the plasmidborne gene mcr-1 encoding resistance to colistin with a high prevalence in Escherichia coli isolates from animals, foodstuff , and human beings in China. The same gene was then reported in Europe (Denmark) among extendedspectrum β lactamase (ESBL) and AmpC-producing E coli isolates from chicken meat and human infections, but at a very low prevalence. We screened ESBL-positive E coli isolates collected in France for colistin resistance. Isolates were collected between 2005 and mid-2014 from faeces of diarrhoeic veal calves at farms, as part of a survey in the context of the French antimicrobial resistance Resapath surveillance network for animal pathogens. We screened these isolates for colistin resistance using disk diff usion and minimum inhibitory concentration determination by broth microdilution. We analysed plasmids bearing the mcr-1 gene by conjugation, S1-pulsed-fi eld gel electrophoresis, PCRbased replicon typing, and Southern blot. We analysed clonal relationship of all isolates by enterobacterial repetitive intergenic consensus PCR and pulsedfi eld gel electrophoresis. Of 517 ESBL-producing E coli isolates collected, 106 (21%) were mcr-1 positive. Notably, the oldest mcr-1positive E coli isolate had been collected in 2005. The 106 mcr-1-positive E coli isolates originated from diff erent individuals located in 94 widely distant farms, and they were clonally unrelated. Sequencing of the whole mcr-1 gene in 75 mcr-1-positive isolates revealed a 100% identity compared with the original sequence. Co-occurrence of the mcr-1 and ESBL genes was identifi ed in a subset of seven isolates, with mcr-1 and blaCTX-M-1 being found on a large and conjugative IncHI2type plasmid together with genes conferring resistance to sulfonamides and tetracyclines, two antibiotics widely used in veterinary medicine. These findings demonstrate a colocation of the mcr-1 gene along with an ESBL gene on a single plasmid, and additional studies are needed to clarify the diversity of the plasmid backbones spreading these two genes within our collection. Noticeably, the prevalence of the mcr-1 gene among ESBL producers in veal calves was much higher than that found in ESBL-positive E coli isolates in human beings and chicken meat reported in Denmark. This diff erence may refl ect a major spread of the mcr-1 gene in European live animals. We showed that the dissemination of mcr-1, at least in France, had already occurred more than a decade ago, with one E coli isolate collected in 2005 identifi ed as mcr-1 positive. Altogether, available data reveal the occurrence of mcr-1 among diff erent animals and human contexts over time. Worryingly, we show that selection pressure with broadspectrum cephalosporins may select for colistin resistance and vice-versa, further highlighting the likelihood of a pandemic spread of mcr-1. Of note, the substantial use of tetracyclines and sulfonamides in animals might also substantially contribute to the dissemination of mcr-1 plasmids. In a one-health perspective, and considering the renewed importance of colistin in human medicine, our data and those from others underscore the urgent need to limit the spread of mcr-1-positive plasmids by reconsidering the massive use of colistin in veterinary medicine worldwide.

Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France.

Extended-spectrum beta-lactamases (ESBLs) have become widespread enzymes in food-producing and companion animals worldwide. However, in cattle mastitis, a major cause of economic loss in the dairy industry, ESBL-producers were rarely described. In this study, from a collection of 1427 Escherichia coli and Klebsiella pneumoniae isolates causing clinical mastitis in France, we report 0.4% (6/1427) of the isolates carrying an ESBL gene. These six isolates were genetically unrelated and recovered over a 3-year period of time. The bla(CTX-M-14) gene was found in 4/6 isolates, and was predominantly located on F2:A-:B- IncFII plasmids. The bla(CTX-M-1) IncI1/ST3, which is widespread in various animal species in France, was found as well. Interestingly, among the five E. coli isolates, the ST23 and ST58 clones were found twice, together with the ST10 clone, all of which were previously found as ESBL-carriers in humans. Despite the very limited number of ESBL-producers recovered, this study shows a surprisingly low molecular diversity of the strains causing mastitis in France with respect to ESBL genes, plasmids and clones. Further work is needed to understand the major driving forces of the ESBL epidemiology in animals, including for different infections within the same animal species.

Phylogenetic Grouping and Virulence Potential of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains in Cattle

ABSTRACT In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx 1 and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla CTX-M genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.

Veterinary hospital-acquired infections in pets with a ciprofloxacin-resistant CTX-M-15-producing Klebsiella pneumoniae ST15 clone

Sir, CTX-M-15 is an extended-spectrum b-lactamase (ESBL) extensively reported in humans, and in particular in Escherichia coli clones of sequence types (STs) ST131 and ST405. The blaCTX-M-15 gene has also become prevalent in Klebsiella pneumoniae, causing outbreaks in hospitals worldwide. – 4 In animals, a highly prevalent ESBL gene is the blaCTX-M-1 gene, with the blaCTX-M-15 gene also being found in E. coli isolated from food-producing animals and pets. From 2008 to 2010, 24 K. pneumoniae isolates were recovered from urine samples of unrelated dogs (n1⁄418) and cats (n1⁄46), 17 of which were collected in the same referral veterinary hospital specialized for surgery in the near suburbs of Paris, France. The other seven isolates were from pets attending surrounding regular veterinary clinics. Susceptibility testing to 32 antimicrobials was performed by agar diffusion and interpreted according to approved clinical breakpoints (http://www.sfm-microbiologie. fr). All isolates were resistant to ceftiofur, cefotaxime, ceftazidime and aztreonam, resistant or of intermediate susceptibility to cefepime, and susceptible to cefoxitin and imipenem. ESBL production was systematically confirmed by a positive synergy test between ceftazidime and clavulanate. All isolates were resistant to ciprofloxacin, streptomycin, sulphonamides and trimethoprim, and 19 isolates were also resistant to kanamycin, gentamicin, tobramycin and netilmicin; only 2 isolates were resistant to tetracycline. Patterns of PFGE performed on XbaIdigested genomic DNA were indistinguishable or differed by no more than two bands (data not shown), strongly suggesting that the same clone was responsible for all infections. Furthermore, all isolates belonged to ST15. The blaCTX-M, blaTEM, blaSHV and blaOXA b-lactamase genes and the aac(6′)-Ib-cr, qnrA, qnrB and qnrS genes were determined by PCR and sequenced when detected (Beckman Coulter, London, UK). The blaCTX-M-15 gene, preceded by the ISEcp1 element, and the blaOXA-1, blaTEM-1 and aac(6′)-Ib-cr genes were identified in all 24 K. pneumoniae isolates. None of the qnr genes was detected, including the qnrS1 gene that has been reported in association with the blaCTX-M-15 and aac(6′)-Ib-cr genes. 6 rep typing revealed the IncR and IncF (but not IncFIIk and IncX) replicons in all isolates, as determined using the PCR-based replicon typing scheme. Considering the clonality of the strains, only a randomly chosen subset of them (9/24) was further investigated. The transformation of plasmid DNA into E. coli K12 was performed with selection on cefotaxime-supplemented plates. All transformants displayed the ESBL phenotype along with most co-resistances (Table 1). They also harboured all b-lactam genes detected in the donor, together with the aac(6′)-Ib-cr gene. In the nine donors, S1-PFGE plasmid profiling demonstrated two plasmids, one of 120 kb and the other ranging from 40 to 70 kb, depending on the isolate. In the nine

High Prevalence of blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2 Plasmids in Healthy Urban Dogs in France

ABSTRACT In the community, close contacts between humans and dogs may promote the transfer of extended-spectrum beta-lactamase/plasmidic AmpC cephalosporinase (ESBL/pAmpC) genes. Large-scale prevalence studies on ESBL/pAmpC carriage in dogs are rare, and data on ESBL/pAmpC plasmids are even more limited. Here, a considerable rate of 18.5% ESBL/pAmpC carriers was found among 368 unrelated healthy dogs in Paris, France. This prevalence is much higher than the one found in healthy humans in the same city (6%) but close to that recently reported in dogs in China (24.5%). All isolates were identified as Escherichia coli, except one Salmonella enterica and one Klebsiella pneumoniae isolate. The sequence type 131 (ST131) clone was rare (2/73 isolates). Interestingly, two plasmids (blaCTX-M-1/IncI1/ST3 and blaCMY-2/IncI1/ST2) were unexpectedly highly predominant, raising the question of their successful spread. Considering that CTX-M-1 was recently found to be equally as abundant as CTX-M-15 in healthy Parisian subjects, the question of dogs being a CTX-M-1 reservoir for humans is open. Such a high prevalence of the blaCMY-2/IncI1/ST2 plasmid may result from the use of cephalexin in veterinary medicine, as previously demonstrated experimentally. In all, our study points out healthy urban dogs as a potential source of ESBL/pAmpC genes that can further disseminate to the human community.

Comparative prevalence and characterization of ESBL-producing Enterobacteriaceae in dominant versus subdominant enteric flora in veal calves at slaughterhouse, France.

Food-producing animals have become a growing reservoir of Extended-Spectrum Beta-Lactamase (ESBL)-producing bacteria. In cattle, veal calves are exposed to high amounts of antibiotics but ESBL prevalence data are still limited compared to other food sectors such as poultry production. Based on the investigation of 491 veal calves from different slaughtering batches at 12 abattoirs, this study shows a prevalence of 29.4% of ESBL producers in the faecal flora of veal calves in France in 2012. A variety of blaCTX-M genes was found, reflecting possible diverse pathways of dissemination in cattle. Another major conclusion is the comparison of the ESBL prevalence in the dominant versus sub-dominant Escherichia coli population of the same calves (1% and 29.4%, respectively). Also, the ESBL E. coli clones in the sub-dominant flora mostly differed from the non-ESBL dominant E. coli clones of the same calves. Of note, the distribution of blaCTX-M genes and E. coli phylogroups were similar to the ones previously found in ESBL E. coli clones from diseased calves. The hypothesis that ESBL genes may distribute more abundantly in certain backgrounds of E. coli was also discussed. In all, as recently reported in the Netherlands, these results strongly suggest a recent increase in the prevalence of ESBL carriage in French veal calves, which should be considered one of the major ESBL reservoirs in food animals.

Increasing Trends in mcr-1 Prevalence among Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolates from French Calves despite Decreasing Exposure to Colistin

Since the first description of the plasmid-mediated colistin resistance gene (mcr-1), over 30 follow-up reports have proved the worldwide geographical distribution of this gene (1, 2). The overall picture indicates a very low prevalence in animals, human beings, and retail food, with two exceptions, the first in China, where mcr-1 carriage was observed in 21% and 15% of the animals and raw meat samples, and the second in France, where 21% of the extended-spectrum-lactamase (ESBL)-producing Escherichia coli isolates from calves were mcr-1 positive (1, 3). Accepted manuscript posted online 8 August 2016

High Prevalence of the Animal-Associated blaCTX-M-1 IncI1/ST3 Plasmid in Human Escherichia coli Isolates

In humans and animals, extended-spectrum beta-lactamases (ESBLs) are widespread enzymes conferring resistance to broad-spectrum cephalosporins. Consumption of ESBL-contaminated foodstuffs or contacts with ESBL-colonized/infected animals enhance the risk of human spread of ESBL genes from nonhuman

mecC-positive MRSA in horses

French Agency for Food, Environmental and Occupational Health Safety (Anses), Unité Antibiorésistance et Virulence Bactériennes, Lyon, France; National Reference Centre for Staphylococci, Laboratoire de Bactériologie, Hospices Civils de Lyon, Lyon, France; International Centre for Infectiology Research, INSERM U1111, CNRS UMR5308, Université Lyon 1, ENS de Lyon, Lyon, France; Labéo Frank Duncombe, Saint-Contest, France

Assessment of Adhesins as an Indicator of Pathovar-Associated Virulence Factors in Bovine Escherichia coli

ABSTRACT The CS31A, F17, and F5 adhesins are usually targeted by serology-based methods to detect pathogenic Escherichia coli associated with calf enteritis. However, the virulence traits of the selected isolates are still poorly described. Here, from a set of 349 diarrheagenic E. coli isolates from cattle, we demonstrated a 70.8% concordance rate (Cohens kappa, 0.599) between serology- and PCR-based approaches for the detection of adhesins under field conditions. A 79% to 82.4% correspondence between the two methods was found for fimbrial adhesins, whereas major discrepancies (33%) were observed for CS31A-type antigens. Various F17A variants were found, such as F17Ac (20K) (50%), F17Aa (FY) (18.9%), F17Ab (8.1%), and F17Ad (111K) (5.4%), including a high proportion (17.6%) of new F17A internal combinations (F17Aab, F17Aac, and F17Abc) or untypeable variants. In addition, the highest proportion of pathovar-associated virulence factor (VF) genes was observed among E. coli isolates that produced F5/F41 adhesins. A specific link between the heat-stable toxins related to the enterotoxigenic E. coli (ETEC) pathovar and adhesins was identified. STa was significantly linked to F5/F41 and EAST1 to CS31A adhesins (P < 0.001), respectively, whereas NTEC was associated with F17 adhesin (P = 0.001). Clustering between phylogroups according to the adhesin types was also observed. Also, few Shiga toxin-producing E. coli (STEC) or enteropathogenic E. coli (EPEC) pathovars were identified. Finally, no statistically significant difference was observed in the occurrence of extended-spectrum beta lactamase (ESBL) production according to the adhesins expressed by the isolates (P = 0.09). Altogether, this study gives new insights into the relationship between adhesins, VF, and antimicrobial resistance in calf enteritis and supports the need for further standardization of methodologies for such approaches.