ACCEPTED. *Corresponding author. Mailing address: Faculdade de Medicina Veterinária, Av. da

Transcription

1 AAC Accepts, published online ahead of print on 10 November 2008 Antimicrob. Agents Chemother. doi: /aac Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Detection of the Pandemic O25 ST131 Human Virulent Escherichia coli CTX-M- 15-producer Clone Harbouring the qnrb2 and aac(6 )-Ib-cr Genes in a Dog Constança Pomba 1*, Joana Diniz da Fonseca 1, Bruno Coelho Baptista 1, José Duarte Correia 1, and Luis Martínez-Martínez 2,3 1 CIISA, Faculty of Veterinary Medicine, Universidade Técnica de Lisboa, Av. da Universidade Técnica, Lisboa, Portugal 2 Service of Microbiology, University Hospital Marqués de Valdecilla, Santander, Spain 3 Department of Molecular Biology, University of Cantabria, Santander, Spain Keywords: qnrb2, aac(6 )-Ib-cr, clonal spread, E. coli, dog *Corresponding author. Mailing address: Faculdade de Medicina Veterinária, Av. da Universidade Técnica, LISBOA, Portugal. Phone: Fax: cpomba@fmv.utl.pt 1

2 The recent worldwide emergence of plasmid-mediated quinolone resistance (PMQR) due to qnr and aac(6 )-Ib-cr genes is a concerning fact among Gram-negative human and animal pathogens (9, 14). The association of PMQR with the production of extended-spectrum β-lactamases (ESBLs), including the human widespread CTX-M-15 enzyme, has been reported (8, 9). Very recently, an internationally disseminated Escherichia coli clone (O25:H4-ST131) with CTX-M-15 ESBL was described (12). Furthermore, representative isolates of this global diffusing clone were characterized as highly virulent and belonging to the B2 phylogenetic group (3). The aim of this study was to determine the prevalence of plasmid-mediated qnr genes among 61 consecutive nonrepetitive clinical strains of E. coli and to subsequently characterize positive isolates. These strains were isolated from dogs (n=41) and cats (n=20) with urinary tract infection (UTI) at the Faculty of Veterinary Medicine in Lisbon, from 2004 to Thirty-four and 26% of the strains were resistant to nalidixic acid and ciprofloxacin, respectively. Two were CMY-2 producers and only one was an ESBL-producer strain. The presence of the qnra, qnrb and qnrs genes was investigated by dot-blot DNA hybridization assays (ECL Direct Nucleic Acid labeling and Detection System 3000). qnra and qnrs genes were not found. qnrb gene was detected in one (1,6%) of 61 E. coli isolates. Full qnrb2 gene nucleotide sequence was then determined. The QnrB2 positive strain, FMV5825, was isolated in Portugal from a fifteen years old female dog with a chronic recurrent cystitis, after prolonged antimicrobial therapy, and was considered resistant to different antibiotics. These included: ampicillin and cephalosporins (except 7-α-methoxy-cephalosporins), aztreonam, piperacillin, ticarcillin, trimethoprim-sulfamethoxazole, aminoglycosides (amikacin, gentamicin, tobramycin), nalidixic acid, and fluoroquinolones (ciprofloxacin, norfloxacin, 2

3 levofloxacin) (Table 1). MICs were determined by DADE MicroScan or by standard broth microdilution assays, performed and interpreted according to the Clinical and Laboratory Standards Institute (5). Quinolone chromosomal encoded resistance of FMV5825 strain was attributed to Ser83 Ile and Asp87 Asn substitutions in GyrA, and Ser80 Ile and Glu84 Val in ParC, both previously associated with high-level fluoroquinolone resistance in E. coli (7). The transferability of the qnrb2 gene was initially assessed by transformation. Plasmid DNA, extracted with QIAGEN plasmid mini kit, was transferred by electroporation into E. coli DH5α cells. Transformants were selected with 10 µg/ml ceftazidime. To avoid the nalidixic acid chromosomal encoded resistance of DH5α cells, further conjugation experiments were performed at 35 C using the E. coli DH5 α transformant as the donor and E. coli J53 Azi r as the recipient (11). Resistance to β- lactams, amikacin, tobramycin, ciprofloxacin, norfloxacin but not to levofloxacin was co-transferred (Table 1). This phenotype raised the possibility of the association of the aminoglycoside acetyltransferase variant - AAC(6 )-Ib-cr - capable of acetylating ciprofloxacin and norfloxacin, as well as the presence of an ESBL enzyme. Indeed, we identified the following co-transferred genes: qnrb2, aac(6 )-Ib-cr, bla CTX-M-15, bla TEM- 1B, and bla OXA-1. Insertion sequence ISEcp1 was found upstream of bla CTX-M-15 gene. These genes and ISEcp1, except qnrb2, have also been detected in E. coli strains from humans with UTI in Portugal (10). To our knowledge, this is the first description of a canine uropathogenic CTX-M-15 E. coli producer strain containing the qnrb2 and aac(6 )-Ib-cr fluoroquinolone resistance genes. In this study, the plasmid encoding for the multidrug resistance (MDR) of FMV5825 strain belonged to the narrow host range incompatibility group IncFII, and had the FII replicon and the FIA replicon, according to a PCR-based replicon-typing 3

4 scheme (2). The association of the bla CTX-M-15 gene with IncFII plasmids also containing bla TEM-1B, bla OXA-1, and aac(6 )-Ib-cr from human E. coli strains has been reported in different continents, suggesting a common origin for all of them (1, 6). A comparable MDR region was found in our animal IncFII plasmid except for the presence of qnrb2. Our E. coli strain of animal origin shares features identical to an intercontinental human clone (12), also belonging to the B2 phylogenetic group, O25 serogroup and ST131 [determined by phylogenetic group multiplex PCR assay (4), O typing by the traditional antiserum method, and MLST scheme ( (15), respectively]. Representative isolates of this global diffusing clone constitute the B2 phylogenetic subgroup I, characterized by producing biofilm and being highly virulent in mice despite lacking classical extraintestinal pathogenicity islands and their adhesion factors (3). The absence of pap, sfa, afa fimbriae and hly operons was also noted in our E. coli strain. The presence of the iutd gene (representative of the siderophore aerobactin operon), the biofilm producing ability, evaluated using a fluorescent in situ hybridization method (13), and the similarity with O25:H4-ST131 TNN (TE2) French E. coli isolate (3, 17), raises the possibility that the E. coli strain isolated from our canine patient belongs to the same phylogenetic B2 subgroup. This is the first report of the presence of the O25 ST131 human virulent E. coli CTX-M-15-producer clone harbouring the disseminated incompatibility group IncFII plasmid with a multidrug resistance region containing bla CTX-M-15, bla TEM-1B, bla OXA-1, aac(6 )-Ib-cr and qnrb2 genes in animals. It is possible that human-to-animal or animal-to-human transmission of the O25-ST131 clone has occurred. 4

5 We are thankful to Dr Jorge Machado from the Portuguese National Health Institute for his contribution to this study. We are also grateful to Dr Manuela Oliveira for scientific and expert technical assistance in the biofilm assays. This work was supported by CIISA/82/2006 grant from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal. References 1. Boyd, D. A., S. Tyler, S. Christianson, A. McGeer, M. P. Muller, B. M. Willey, E. Bryce, M. Gardam, P. Nordmann, M. R. Mulvey, and Canadian Nosocomial Infection Surveillance Program, Health Canada Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum β- lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob. Agents Chemother. 48: Carattoli, A., A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods. 63: Clermont, O., M. Lavollay, S. Vimont, C. Deschamps,C. Forestier, C. Branger, E. Denamur, and G. Arlet The CTX-M-15-producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup. J. Antimicrob. Chemother. 61: Clermont, O., S. Bonacorsi, and E. Bingen Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66: Clinical and Laboratory Standards Institute Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI 5

6 document M100-S17. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA. 6. Coque, T. M., A. Novais, A. Carattoli, L. Poirel, J. Pitout, L. Peixe, F. Baquero, R. Cantón, P. Nordmann Dissemination of clonally related Escherichia coli strains expressing extended-spectrum β-lactamase CTX-M-15. Emerg. Infect. Dis. 14: Everett, M. J., Y. F. Jin, V. Ricci, and L. J. V. Piddock Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob. Agents Chemother. 40: Iabadene, H., Y. Messai, H. Ammari, N. Ramdani-Bouguessa, S. Lounes, R. Bakour, G. Arlet Dissemination of ESBL and Qnr determinants in Enterobacter cloacae in Algeria. J. Antimicrob. Chemother. 62: Jacoby, G. A., K. E. Walsh, D. M. Mills, V. J. Walker, H. Oh, A. Robicsek, and D. C. Hooper qnrb, another plasmid mediated gene for quinolone resistance. Antimicrob Agents Chemother. 50: Machado, E., T. M. Coque, R. Cantón, F. Baquero, J. C. Sousa, L. Peixe, and The Portuguese Resistance Study Group Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-Producing Enterobacteriaceae strains containing the aac(6 )-Ib-cr Gene, which encodes an aminoglycoside- and fluoroquinolonemodifying enzyme. Antimicrob. Agents Chemother. 50: Martínez-Martínez, L., A. Pascual, and G. A. Jacoby Quinolone resistance from a transferable plasmid. Lancet. 351: Nicolas-Chanoine, M. H., J. Blanco, V. Leflon-Guibout, R. Demarty,M. P. Alonso, M. M. Caniça, Y. J. Park, J. P. Lavigne, J. Pitout, and J. R. Johnson. 6

7 Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J. Antimicrob. Chemother. 61: Oliveira, M., S. F. Nunes, C. Carneiro, R. Bexiga, F. Bernardo, and C. L. Vilela Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet. Microb. 124: Robicsek, A., J. Strahilevitz, G. A. Jacoby, M. Macielag, D. Abbanat, C. H. Park, K. Bush, and D. C. Hooper Fluoroquinolone modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat. Med. 12: Wirth, T., D. Falush, R. Lan, F. Colles, P. Mensa, L.H. Wieler, H. Karch, P. R. Reeves, M. C. Maiden, H. Ochman, and M. Achtman Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60:

8 TABLE 1. Antimicrobial susceptibilities of E. coli FMV5825 uropathogenic canine clinical isolate and E. coli J53 transconjugant and recipient strains Antimicrobial MIC (µg/ml) agent E. coli FMV5825 E. coli J53 Azi r transconjugant E. coli J53 Azi r recipient Ampicillin >16 >16 4 Amoxicillinclavulanic acid 16/8 16/8 4/2 Ticarcillin >64 >64 16 Piperacilin >64 > Cefazolin >16 >16 16 Cefuroxime >16 >16 1 Cefotetan Cefoxitin Cefotaxime >64 >64 1 Piperacilintazobactam Cefotaximeclavulanic acid Ceftazidime Ceftazidimeclavulanic acid Cefpodoxime >4 >4 1 Cefepime >16 >16 1 Aztreonam >16 >16 1 Imipenem Meropenem Ertapenem Amikacin Gentamicin Tobramycin >8 >8 2 Nalidixic acid >

9 Ciprofloxacin Norfloxacin Levofloxacin Trimethoprimsulfamethoxazole >2/38 2/38 2/38 9