Veterinary Microbiology 162 (2013) 793799

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Characterization of extended-spectrum beta-lactamase (ESBL)-carrying plasmids and clones of Enterobacteriaceae causing cattle mastitis in France
ronique Me tayer a, Emilie Gay b, Jean-Yves Madec a, Marisa Haenni a,* Saa Dahmen a, Ve
a b

curite Sanitaire (Anses), Unite Antibiore sistance et Virulence Bacte riennes, Lyon, France Agence Nationale de Se curite Sanitaire (Anses), Unite Epide miologie, Lyon, France Agence Nationale de Se

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 August 2012 Received in revised form 8 October 2012 Accepted 10 October 2012 Keywords: ESBL Cattle Mastitis Plasmids

Extended-spectrum beta-lactamases (ESBLs) have become widespread enzymes in foodproducing 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 blaCTXM-14 gene was found in 4/6 isolates, and was predominantly located on F2:A-:B- IncFII plasmids. The blaCTX-M-1 IncI1/ST3, which is widespread in various animal species in France, was found as well. Interestingly, among the ve 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. 2012 Elsevier B.V. All rights reserved.

1. Introduction In animals, extended-spectrum beta-lactamases (ESBLs) have become widespread enzymes conferring high levels of resistance to last generations of beta-lactams in Enterobacteriaceae, including ceftiofur and cefquinome, which are cephalosporins widely used for food-producing animals. Similarly to what happened in human medicine, this situation is especially due to the epidemiological success of certain ESBL genes, in particular the blaCTX-M genes. Even though strains can spread clonally, recent data strongly suggest that plasmids play a key role in the horizontal transfer of ESBL genes, and the molecular

* Corresponding author. Tel.: +33 4 78696830; fax: +33 4 78619145. E-mail address: marisa.haenni@anses.fr (M. Haenni). 0378-1135/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.10.015

characterization of ESBL-carrying plasmids contributes to our understanding of the ESBL epidemiology (Dahmen et al., in press; Madec et al., 2012). In cattle, Escherichia coli, and to a lesser extent Klebsiella pneumoniae, are frequently responsible for clinical mastitis, a disease causing major economic loss in the dairy industry worldwide. In a previous study, we found that these strains were susceptible to almost all antibiotics used in veterinary medicine despite recurrent treatments at farms (Botrel et al., 2010). Moreover, contrary to other diseases in cattle, such as diarrhea in veal calves, resistances to last-generation cephalosporins were rarely reported (Geser et al., 2012; Locatelli et al., 2010). However, ESBL-producing Enterobacteriaceae in raw milk may be a risk for public health, even in developed countries such as France where raw cheese is commonly consumed. It also raises the question of the origin of

794

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799

those ESBL-producing isolates, as we recently reported one of the most prevalent Staphylococcus aureus clones infecting humans in France causing mastitis in cattle (Haenni et al., 2011). The goal of this study was to estimate the prevalence of ESBL-producing Enterobacteriaceae in cattle mastitis in France and to thoroughly characterize the collected strains, in order to infer possible routes of transmission of these resistances between humans and animals. 2. Materials and methods 2.1. Bacterial sampling, identication and susceptibility testing From 2009 to 2011, antimicrobial susceptibility data of 1745 E. coli and 137 K. pneumoniae isolates from clinical cattle mastitis were collected through the Resapath network, which ensures the surveillance of antimicrobial resistance in diseased animals in France (www.resapath.anses.fr). Susceptibility testing was performed in all peripheral laboratories by the disc diffusion method according to the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM) (www.sfm-microbiologie.fr). In particular, ceftiofur and cefquinome were tested as third- and fourth-generation cephalosporins for 1342/1745 E. coli and 85/137 K. pneumoniae isolates. Bacteria were classied as susceptible, intermediate or resistant according to the approved clinical breakpoints. E. coli ATCC 25922 was used as the quality control. All isolates that were intermediate or resistant to ceftiofur (<21 mm) and/or to cefquinome (<22 mm) according to the guidelines of the CA-SFM were sent to the Anses laboratory in Lyon, where they were conrmed as E. coli or K. pneumoniae by standard procedures. Antimicrobial susceptibility to 32 antibiotics of veterinary and human interest was tested on those strains by the disc diffusion method as described above. The antibiotics tested were: amoxicillin, piperacillin, ticarcillin, amoxicillinclavulanic acid, piperacillintazobactam, ticarcillinclavulanic acid, cefalotine, cefuroxime, cefotaxime, ceftiofur, ceftazidime, cefoxitin, cefepime, cefquinome, aztreonam, ertapenem, tetracycline, kanamycin, tobramycin, gentamicin, amikacin, apramicin, netilmicin, streptomycin, orfenicol, chloramphenicol, colistin, sulphonamides, trimethoprim, nalidixic acid, ooxacin and enrooxacin. ESBL production was conrmed by the double-disc synergy test according to the guidelines of the CA-SFM. Minimum Inhibitory Concentrations to relevant antibiotics were determined by E-test1. 2.2. Beta-lactamase genes identication The blaTEM, blaSHV and blaOXA genes were screened by PCR (Dierikx et al., 2010; Hasman et al., 2005; Lavollay et al., 2006). The blaCTX-M genes were detected using a CTXM group-specic multiplex PCR (Shibata et al., 2006). The whole coding sequences of the identied blaCTX-M and blaTEM genes were then amplied and fully sequenced

(Beckman Coulter, London, UK) (Eckert et al., 2004; Hasman et al., 2005; Literacka et al., 2009). 2.3. Transferability of the blaESBL genes and plasmid characterization Plasmids were transferred by conjugation in E. coli rifampicin-resistant K-12 J53 recipient strains. All plasmids were rep-typed in both donor and recipient strains using the PCR-based replicon typing (PBRT) scheme (Carattoli et al., 2005), and the sizes of plasmids were determined on S1-PFGE gels. Southern blot hybridizations were performed with the blaCTX-M, IncI1 or IncF probes. Depending on the plasmid types (IncI1 or IncF), plasmid subtypes were determined using the plasmid Multi-Locus Sequence Typing (pMLST) or the replicon sequence typing (RST) scheme (Garcia-Fernandez et al., 2008; Villa et al., 2010). Restriction fragment length polymorphism (RFLP) was carried out on EcoRI- and PstI-digested plasmid DNA from transconjugants. 2.4. Phylogenetic grouping Phylogenetic grouping of the E. coli isolates was performed using the triplex PCR for chuA, yjaA and tspE4C2 genes as described previously (Clermont et al., 2000). 2.5. Typing of the bacteria E. coli were characterized by PFGE and further typed by MLST according to the online method (mlst.ucc.i.e./mlst/ dbs/Ecoli/). The K. pneumoniae isolate was typed by MLST (www.pasteur.fr/recherche/genopole/PF8/mlst/).

3. Results Antimicrobial susceptibility data of the 1745 E. coli and 137 K. pneumoniae were analyzed, and resistant phenotypes were overall rare, except for amoxicillin and tetracycline (Table S1, Supplemental data). Among the 1342/1745 E. coli and 85/137 K. pneumoniae isolates for which ceftiofur and cefquinome were tested, a total of 5 E. coli (5/1342, 0.3%) and 1 K. pneumoniae isolates (1/85, 1.2%) were conrmed for ESBL production by the synergy test, with MIC values of cefotaxime ranging from 12 to 16 mg/ml and of ceftazidime from 0.38 to 1 mg/ml (Table 1). No discrepancy was observed between results obtained for ceftiofur and cefquinome. Five out of the six isolates presented additional resistances to several non betalactams. PCR and sequencing revealed the presence of two blaCTX-M-1 genes with an upstream-located ISEcp1 element, and four blaCTX-M-14 genes. None of the isolates carried a blaSHV or blaOXA gene, whereas the blaTEM-1 gene was detected in two CTX-M-14- and one CTX-M-1-producing isolates (Table 1). The blaESBL-carrying plasmids were transferred by conjugation for all isolates, together with a single replicon and other resistance genes (Table 2). Southern blot analysis of S1-PFGE gels demonstrated that blaCTX-M-1 genes were carried on IncI1 plasmids while

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799 Table 1 Antimicrobial resistance and molecular characterization of ESBL-producing isolates causing cattle mastitis. Isolate Species Year of isolation CM (mg/ml) CTX 26971 24067 26497 26561 26916 27116 K. pneumoniae E. coli E. E. E. E. coli coli coli coli 2011 2009 2010 2010 2011 2011 12 12 16 16 16 16 CAZ 0.38 0.5 1 0.5 1 0.5 CTX-M-14 CTX-M-14 CTX-M-1 CTX-M-14 CTX-M-1 CTX-M-14 + + + None STR, KAN, GEN, APR, TOB, NET, TET, TMP, SUL, ENR, NAL, OFX STR, KAN, TET, TMP, SUL STR, KAN, CHL, TET, SUL, NAL, ENR, OFX STR, KAN, TET, TMP, SUL, NAL, ENR, OFX None F FIB/F/BO I1/N/FIB/F FIA/FIB/F I1/Y/FIB/F I1/F 45 23 58 10 23 58 / A B1 A A B1 ESBL TEM Additional resistancesa Replicon types MLST

795

Phylogenetic group

a APR, apramycin; CHL, chloramphenicol; ENR, enrooxacin GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; NET, netilmicin; STR, streptomycin; TET, tetracyclines; TMP, trimethoprim; TOB, tobramycin; OFX, ooxacin and SUL, sulphonamides.

Table 2 Antimicrobial resistance and molecular characterization of transconjugants (TC) from ESBL-producing isolates causing cattle mastitis. TC number TC-26971 TC-24067 TC-26497 TC-26561 TC-26916 TC-27116 Additional resistancesa None STR, KAN, GEN, APR, TOB, NET, TET, TMP, SUL STR, TMP, SUL STR STR, KAN, TET, TMP, SUL None Replicon types F F I1 F I1 F FABb formula F2:A-:BF18:A-: B F2:A-: B F2:A-: BpMLST ST3 ST87 Plasmid size (kb) 70 130 120 60 100 85

a APR, apramycin; GEN, gentamicin; KAN, kanamycin; NET, netilmicin; STR, streptomycin; TET, tetracyclines; TMP, trimethoprim; TOB, tobramycin and SUL, sulphonamides. b FAB corresponds to FII:FIA:FIB.

blaCTX-M-14 genes were located on IncF ones. Plasmid sizes ranged from 60 to 130 kb (Table 2). blaCTX-M-1 IncI1 plasmids belonged to the ST3 (allelic prole 2/1/4/1/2) or to the closely related ST87 (8/1/4/1/2) sub-types. RFLP proles after digestion with the PstI (which cuts into the blaCTX-M-1 and blaCTX-M-14 coding sequences) or EcoRI (which does not cut into the blaCTX-M-1 and blaCTX-M-14 coding sequences) enzymes were similar for the two plasmids but non-identical (Fig. 1; lane 6 and lane 5, respectively). Southern blot hybridization with a CTX-M probe showed similar proles with PstI but different ones with EcoRI. On the contrary, the IncI1/ST3 prole was identical or highly similar to other IncI1/ST3 plasmids isolated from horses, companion animals and a goat in France, but largely different from the IncI1/ST70 used here for comparison purposes (Fig. 1; lanes 8 and 9) (Dahmen et al., in press). Three blaCTX-M-14 IncF plasmids displayed the F2:A-:Bformula, whereas the last one was subtyped as F18:A-:B-. RFLP analysis using PstI or EcoRI proved that F2:A-:B- and F18:A-:B- plasmids were clearly different, while the three F2:A-:B- plasmids displayed similar though non-identical proles (Fig. 1; lanes 14). Southern blot hybridization of plasmid DNA with a blaCTX-M gene probe showed identical proles for two IncF/F2:A-:B- plasmids (Fig. 1; lanes 2 and 3), with one fragment at 3.5 kb with PstI and one at 5 kb with EcoRI respectively, while the third one presented clearly different proles (Fig. 1; lane 4). All E. coli isolates originated from different regions of France and PFGE analyses showed that they were not clonally related (data not shown). Nevertheless, two isolates belonged to the ST23 clonal group (harboring the blaCTX-M-1 IncI1/ST87 and the blaCTX-M-14 F18:A-:Bplasmids, respectively), and two others were typed as ST58

(harboring the blaCTX-M-1 IncI1/ST3 and one blaCTX-M-14 F2:A-:B- plasmids, respectively). The remaining E. coli belonged to the ST10 clonal group, while the K. pneumoniae belonged to the ST45 one. 4. Discussion This study reports on one of the largest screening of ESBL-producing Enterobacteriaceae causing cattle mastitis published so far. We showed that resistant phenotypes were overall rare and that the estimated prevalence of ESBL-producers was very low (6/1427, 0.4%), even though ceftiofur and cefquinome, which are widely tested in veterinary medicine, might not be the best indicator cephalosporins for the detection of ESBL-producing bacteria (Aarestrup et al., 2010). This prevalence is far lower than the one observed in other diseases in cattle, such as diarrhea in veal calves (www.resapath.anses.fr). This may result from the nature of the antibiotics used to treat cattle mastitis, which are generally local and highly dosed. In addition, the mammary gland, which is usually sterile, does surely not constitute a favorable ecological niche for genetic exchanges and selection of resistance. These results also indicate that milk is not a major risk with regard to the transfer of ESBL genes from cattle to humans and/or to the environment. The six ESBL-producers described in this study were clonally unrelated, originated from different areas and were sampled over a 3-year period of time. Despite this epidemiological heterogeneity, the molecular diversity was surprisingly low with respect to ESBL genes, plasmids types and sub-types and clonal groups. Even though the blaCTX-M-14 and blaCTX-M-15 genes have been already identied in cattle (Liebana et al., 2006;

796

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799

Fig. 1. (A) Restriction fragment length polymorphism (RFLP) analysis of transconjugant (TC)-E. coli plasmid DNA digested with PstI, and Southern blot hybridization with a CTX-M probe. (B) RFLP with EcoRI and Southern blot hybridization with a CTX-M probe M: molecular size (Smart Ladder, Eurogentec), lane 1: TC-24067, lane 2: TC-26971, lane 3: TC-26561, lane 4: TC-27116, lane 5: TC-26916, lane 6: TC-26497, lane 7: TC-21364 (IncI 1/ST3 isolated from a dog (Dahmen et al., in press)), lane 8: TC-26557 (IncI1/ST3 isolated from a horse (Dahmen et al., in press)), lane 9:TC-24431 (IncI1/ST70 (Dahmen et al., in press)).

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799

797

Meunier et al., 2006; Stokes et al., 2012; Teale et al., 2005; Zheng et al., 2012), the blaCTX-M-1 gene was the most prevalent ESBL gene reported in this animal species so far (Dolejska et al., 2011; Hartmann et al., 2012; Madec et al., 2008; Mollenkopf et al., 2012; Valat et al., 2012). Therefore, the nding of 4/6 isolates producing CTX-M-14 was unexpected. Nevertheless, most results published in cattle relate to fecal isolates. It would thus be interesting to investigate whether blaCTX-M-carrying plasmids may be selected differently depending on the types of infections in a particular animal species. The nature of the blaCTX-M-carrying plasmids and bacterial clonal groups reported here is also noticeable. The two blaCTX-M-1 IncI1 plasmids were of very close subtypes (ST3 and ST87) and harbored similar RFLP proles. Interestingly, the blaCTX-M-1-carrying IncI1/ST3 plasmid was recently reported as one of the most abundant blaCTXM-1-carrying plasmid in various animal species in France, including in cattle (Cloeckaert et al., 2010; Dahmen et al., in press; Madec et al., 2011). The hypothesis of a possible animal origin of this plasmid is thus strengthened by this study. This blaCTX-M-1 IncI1/ST3 plasmid was detected in a ST58 E. coli isolate, which has been described in humans, but also very recently in cattle (Wu et al., 2012). Until now, cattle ESBL-producing E. coli isolated in France mostly belonged to STs that were not described in humans (Madec et al., 2012). The present results, in addition to others (Wu et al., 2012), highlight the difculty to accurately determine the animal or human origin of the E. coli strains infecting cattle. Similarly, the IncF plasmids reported here were mostly of the F2:A-:B- replicon sequence type (ST). F2:A-:Bplasmids, such as pEK516 and pC15-1a, have been widely identied as major vehicles of the blaCTX-M-15 gene in humans (Boyd et al., 2004; Woodford et al., 2009) but were also reported in animals, including cattle and pets (Deng et al., 2011; Hou et al., 2012). Notably here, F2:A-:Bplasmids were found to carry the blaCTX-M-14 gene only, and were found in both E. coli and K. pneumoniae. The spread of blaCTX-M-14 was reported to be conferred predominantly by pHK01-like IncFII plasmids in E. coli from human and animal sources in Hong-Kong (including F2:A-:B- plasmids) (Hou et al., 2012). On the contrary, the blaCTX-M-14 gene was carried on IncK plasmids in humans, cattle and turkeys E. coli isolates in England and Wales (Stokes et al., 2012). In France, in humans, the blaCTX-M-14 gene was et al., 2009), mostly described on IncFII plasmids (Marcade so that our ndings in cattle extent the description of possible shared reservoirs of blaCTX-M-14-carrying plasmids between food-producing animals and humans in various countries. blaCTX-M-carrying IncFII plasmids were often associated with human E. coli isolates, as exemplied by the O25ST131 clone carrying a blaCTX-M-15 on an IncFII plasmid. Here, none of the IncFII plasmids were carried by this widely disseminated clone. However, they were harbored by ST10, ST23 and ST58 E. coli isolates, which were all identied in human infections, including as ESBL-producers (Oteo et al., 2009). However, the ST10 clone was also found in cattle (Wu et al., 2012), and both the ST10 and ST58 clones were found in poultry and human isolates in a

context of a probable cross-transmission of ESBL E. coli between Dutch patients, chicken meat and poultry in the Netherlands (Leverstein-van Hall et al., 2011). E. coli isolates of the ST23 complex were often described as harboring extended-spectrum AmpC cephalosporinases (ESAC) in humans (Cremet et al., 2010), but were also recently identied in cattle (Wu et al., 2012). Finally, the only ESBL-producing K. pneumoniae reported from bovine clinical mastitis carried the blaCTX-M-1 gene but the clonal group of the bacteria was not determined (Locatelli et al., 2010). Of note, MLST data on ESBL-producing K. pneumoniae clones from animals are still scarce (Haenni et al., 2012). Besides, the ST45 clone of K. pneumoniae reported here was found in humans (Novais et al., 2012), including in a human blood infection in France (Brisse et al., 2009; Diancourt et al., 2005). In all, the low diversity of ESBL plasmids and clones identied here highly contrasts with the limited number of ESBL isolates recovered. Whether ESBL determinants and clones have an animal or human origin is still an open question, which is probably more difcult to solve in Enterobacteriaceae compared to staphylococci, thanks to the huge diversity of combinations between clones, plasmids and ESBL genes. It has been reported that the blaCTX-M-15 gene together with the O25-ST131 E. coli clone have spread in humans (Nicolas-Chanoine et al., 2008), while others, such as the blaCTX-M-1 IncI1/ST3 plasmid, has largely disseminated in animals independently of the bacterial host (Cloeckaert et al., 2010; Dahmen et al., in press). The data presented here now highlight a more complex picture, where the human origin of the infecting clones and of some plasmids, such as F2:A-:B-, is plausible. This hypothesis would be also reinforced by the known close contacts between farmers and animals during the milking process. On the other hand, blaCTX-M-1 IncI1 plasmids are much more prevalent in animals than in humans, and blaCTX-M-15 F2:A-:B- plasmids were recently reported in cattle (Madec et al., 2012). Further work is therefore needed to better document the distribution of plasmids and clones of Enterobacteriaceae in humans and animals, and in particular in non-ESBL-producers. Acknowledgements This work was supported by the French Food Safety Agency (Anses). We thank Nathalie Jarrige for her help analyzing the data from the Resapath database. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2012.10.015. References
Aarestrup, F.M., Hasman, H., Veldman, K., Mevius, D., 2010. Evaluation of eight different cephalosporins for detection of cephalosporin resistance in Salmonella enterica and Escherichia coli. Microb. Drug Resist. 16, 253261.

798

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799 Hou, J., Huang, X., Deng, Y., He, L., Yang, T., Zeng, Z., Chen, Z., Liu, J.H., 2012. Dissemination of the fosfomycin resistance gene fosA3 with CTX-M beta-lactamase genes and rmtB carried on IncFII plasmids among Escherichia coli isolates from pets in China. Antimicrob. Agents Chemother. 56, 21352138. Lavollay, M., Mamlouk, K., Frank, T., Akpabie, A., Burghoffer, B., Ben Redjeb, S., Bercion, R., Gautier, V., Arlet, G., 2006. Clonal dissemination of a CTX-M-15 beta-lactamase-producing Escherichia coli strain in the Paris area, Tunis, and Bangui. Antimicrob. Agents Chemother. 50, 24332438. Leverstein-van Hall, M.A., Dierikx, C.M., Cohen Stuart, J., Voets, G.M., van den Munckhof, M.P., van Essen-Zandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N., Scharinga, J., Bonten, M.J., Mevius, D.J., 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. Microbiol. Infect. 17, 873880. Liebana, E., Batchelor, M., Hopkins, K.L., Clifton-Hadley, F.A., Teale, C.J., Foster, A., Barker, L., Threlfall, E.J., Davies, R.H., 2006. Longitudinal farm study of extended-spectrum beta-lactamase-mediated resistance. J. Clin. Microbiol. 44, 16301634. Literacka, E., Bedenic, B., Baraniak, A., Fiett, J., Tonkic, M., Jajic-Bencic, I., Gniadkowski, M., 2009. blaCTX-M genes in Escherichia coli strains from Croatian hospitals are located in new (blaCTX-M-3a) and widely spread (blaCTX-M-3a and blaCTX-M-15) genetic structures. Antimicrob. Agents Chemother. 53, 16301635. Locatelli, C., Scaccabarozzi, L., Pisoni, G., Moroni, P., 2010. CTX-M1 ESBLproducing Klebsiella pneumoniae subsp. pneumoniae isolated from cases of bovine mastitis. J. Clin. Microbiol. 48, 38223823. Madec, J.Y., Doublet, B., Ponsin, C., Cloeckaert, A., Haenni, M., 2011. Extended-spectrum beta-lactamase blaCTX-M-1 gene carried on an IncI1 plasmid in multidrug-resistant Salmonella enterica serovar Typhimurium DT104 in cattle in France. J. Antimicrob. Chemother. 66, 942944. Madec, J.Y., Lazizzera, C., Chatre, P., Meunier, D., Martin, S., Lepage, G., Menard, M.F., Lebreton, P., Rambaud, T., 2008. Prevalence of fecal carriage of acquired expanded-spectrum cephalosporin resistance in Enterobacteriaceae strains from cattle in France. J. Clin. Microbiol. 46, 15661567. Madec, J.Y., Poirel, L., Saras, E., Gourguechon, A., Girlich, D., Nordmann, P., Haenni, M., 2012. Non-ST131 Escherichia coli from cattle harbouring human-like blaCTX-M-15-carrying plasmids. J. Antimicrob. Chemother. 67, 578581. , G., Deschamps, C., Boyd, A., Gautier, V., Picard, B., Branger, C., Marcade Denamur, E., Arlet, G., 2009. Replicon typing of plasmids in Escherichia coli producing extended-spectrum beta-lactamases. J. Antimicrob. Chemother. 63, 6771. Meunier, D., Jouy, E., Lazizzera, C., Kobisch, M., Madec, J.Y., 2006. CTX-M1- and CTX-M-15-type beta-lactamases in clinical Escherichia coli isolates recovered from food-producing animals in France. Int. J. Antimicrob. Agents 28, 402407. Mollenkopf, D.F., Weeman, M.F., Daniels, J.B., Abley, M.J., Mathews, J.L., Gebreyes, W.A., Wittum, T.E., 2012. Variable within- and betweenherd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle. Appl. Environ. Microbiol. 78, 45524560. Nicolas-Chanoine, M.H., Blanco, J., Leon-Guibout, V., Demarty, R., Alonso, M.P., Canica, M.M., Park, Y.J., Lavigne, J.P., Pitout, J., Johnson, J.R., 2008. Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J. Antimicrob. Chemother. 61, 273281. Novais, A., Rodrigues, C., Branquinho, R., Antunes, P., Grosso, F., Boaventura, L., Ribeiro, G., Peixe, L., 2012. Spread of an OmpK36-modied ST15 Klebsiella pneumoniae variant during an outbreak involving multiple carbapenem-resistant Enterobacteriaceae species and clones. Eur. J. Clin. Microbiol. Infect. Dis., http://dx.doi.org/10.1007/ s10096-012-1665-z. Oteo, J., Diestra, K., Juan, C., Bautista, V., Novais, A., Perez-Vazquez, M., Moya, B., Miro, E., Coque, T.M., Oliver, A., Canton, R., Navarro, F., Campos, J., 2009. Extended-spectrum beta-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2. Int. J. Antimicrob. Agents 34, 173176. Shibata, N., Kurokawa, H., Doi, Y., Yagi, T., Yamane, K., Wachino, J.-I., Suzuki, S., Kimura, K., Ishikawa, S., Kato, H., Ozawa, Y., Shibayama, K., Kai, K., Konda, T., Arakawa, Y., 2006. PCR classication of CTX-M-type beta-lactamase genes identied in clinically isolated gram-negative bacilli in Japan. Antimicrob. Agents Chemother. 50, 791795. Stokes, M.O., Cottell, J.L., Piddock, L.J., Wu, G., Wootton, M., Mevius, D.J., Randall, L.P., Teale, C.J., Fielder, M.D., Coldham, N.G., 2012. 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. J. Antimicrob. Chemother. 67, 16391644.

Botrel, M.A., Haenni, M., Morignat, E., Sulpice, P., Madec, J.Y., Calavas, D., 2010. Distribution and antimicrobial resistance of clinical and subclinical mastitis pathogens in dairy cows in Rhone-Alpes, France. Foodborne Pathog. Dis. 7, 479487. Boyd, D.A., Tyler, S., Christianson, S., McGeer, A., Muller, M.P., Willey, B.M., Bryce, E., Gardam, M., Nordmann, P., Mulvey, M.R., 2004. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob. Agents Chemother. 48, 37583764. Brisse, S., Fevre, C., Passet, V., Issenhuth-Jeanjean, S., Tournebize, R., Diancourt, L., Grimont, P., 2009. Virulent clones of Klebsiella pneumoniae: identication and evolutionary scenario based on genomic and phenotypic characterization. PLoS ONE 4, e4982. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L., Threlfall, E.J., 2005. Identication of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63, 219228. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 45554558. Cloeckaert, A., Praud, K., Lefevre, M., Doublet, B., Pardos, M., Granier, S.A., Brisabois, A., Weill, F.X., 2010. IncI1 plasmid carrying extendedspectrum beta-lactamase gene blaCTX-M-1 in Salmonella enterica isolates from poultry and humans in France, 2003 to 2008. Antimicrob. Agents Chemother. 54, 44844486. Cremet, L., Caroff, N., Giraudeau, C., Dauvergne, S., Lepelletier, D., Reynaud, A., Corvec, S., 2010. Occurrence of ST23 complex phylogroup A Escherichia coli isolates producing extended-spectrum AmpC betalactamase in a French hospital. Antimicrob. Agents Chemother. 54, 22162218. Dahmen, S., Haenni, M., Madec, J.Y IncI1/ST3 plasmids contribute to the dissemination of the blaCTX-M-1 gene in Escherichia coli from several animal species in France. J. Antimicrob. Chemother., http://dx.doi.org/ 10.1093/jac/dks308. Deng, Y., He, L., Chen, S., Zheng, H., Zeng, Z., Liu, Y., Sun, Y., Ma, J., Chen, Z., Liu, J.H., 2011. F33:A-:B- and F2:A-:B- plasmids mediate dissemination of rmtB-blaCTX-M-9 group genes and rmtB-qepA in Enterobacteriaceae isolates from pets in China. Antimicrob. Agents Chemother. 55, 49264929. Diancourt, L., Passet, V., Verhoef, J., Grimont, P.A., Brisse, S., 2005. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J. Clin. Microbiol. 43, 41784182. Dierikx, C., van Essen-Zandbergen, A., Veldman, K., Smith, H., Mevius, D., 2010. Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry. Vet. Microbiol. 145, 273278. Dolejska, M., Jurcickova, Z., Literak, I., Pokludova, L., Bures, J., Hera, A., Kohoutova, L., Smola, J., Cizek, A., 2011. IncN plasmids carrying bla CTX-M-1 in Escherichia coli isolates on a dairy farm. Vet. Microbiol. 149, 513516. Eckert, C., Gautier, V., Saladin-Allard, M., Hidri, N., Verdet, C., Ould-Hocine, Z., Barnaud, G., Delisle, F., Rossier, A., Lambert, T., Philippon, A., Arlet, G., 2004. Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob. Agents Chemother. 48, 12491255. Garcia-Fernandez, A., Chiaretto, G., Bertini, A., Villa, L., Fortini, D., Ricci, A., Carattoli, A., 2008. Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal origin. J. Antimicrob. Chemother. 61, 12291233. Geser, N., Stephan, R., Hachler, H., 2012. Occurrence and characteristics of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in food producing animals, minced meat and raw milk. BMC Vet. Res. 8, 21. Haenni, M., Galofaro, L., Ponsin, C., Bes, M., Laurent, F., Madec, J.Y., 2011. Staphylococcal bovine mastitis in France: enterotoxins, resistance and the human Geraldine methicillin-resistant Staphylococcus aureus clone. J. Antimicrob. Chemother. 66, 216218. Haenni, M., Ponsin, C., Metayer, V., Medaille, C., Madec, J.Y., 2012. Veterinary hospital-acquired infections in pets with a ciprooxacin-resistant CTX-M-15-producing Klebsiella pneumoniae ST15 clone. J. Antimicrob. Chemother. 67, 770771. Hartmann, A., Locatelli, A., Amoureux, L., Depret, G., Jolivet, C., Gueneau, E., Neuwirth, C., 2012. Occurrence of CTX-M producing Escherichia coli in soils, cattle, and farm environment in France (Burgundy Region). Front. Microbiol. 3, 83. Hasman, H., Mevius, D., Veldman, K., Olesen, I., Aarestrup, F.M., 2005. Beta-Lactamases among extended-spectrum beta-lactamase (ESBL)resistant Salmonella from poultry, poultry products and human patients in The Netherlands. J. Antimicrob. Chemother. 56, 115121.

S. Dahmen et al. / Veterinary Microbiology 162 (2013) 793799 Teale, C.J., Barker, L., Foster, A.P., Liebana, E., Batchelor, M., Livermore, D.M., Threlfall, E.J., 2005. Extended-spectrum beta-lactamase detected in E. coli recovered from calves in Wales. Vet. Rec. 156, 186187. Valat, C., Auvray, F., Forest, K., Metayer, V., Gay, E., Peytavin de Garam, C., Madec, J.Y., Haenni, M., 2012. Phylogenetic grouping and virulence potential of extended-spectrum-beta-lactamase-producing Escherichia coli strains in cattle. Appl. Environ. Microbiol. 78, 46774682. Villa, L., Garcia-Fernandez, A., Fortini, D., Carattoli, A., 2010. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J. Antimicrob. Chemother. 65, 25182529. Woodford, N., Carattoli, A., Karisik, E., Underwood, A., Ellington, M.J., Livermore, D.M., 2009. Complete nucleotide sequences of plasmids

799

pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrob. Agents Chemother. 53, 44724482. Wu, G., Ehricht, R., Mafura, M., Stokes, M., Smith, N., Pritchard, G.C., Woodward, M.J., 2012. Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages. Vet. Microbiol. 160, 197206. Zheng, H., Zeng, Z., Chen, S., Liu, Y., Yao, Q., Deng, Y., Chen, X., Lv, L., Zhuo, C., Chen, Z., Liu, J.H., 2012. Prevalence and characterisation of CTX-M beta-lactamases amongst Escherichia coli isolates from healthy food animals in China. Int. J. Antimicrob. Agents 39, 305310.