ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2006, p. 1525–1527 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.50.4.1525–1527.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 4
In Vivo Selection of Fluoroquinolone-Resistant Escherichia coli Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum -Lactamase Laurent Poirel,1 Johann D. D. Pitout,2,3,4 Lucy Calvo,1 Jose-Manuel Rodriguez-Martinez,1,6 Deirdre Church,2,3,5 and Patrice Nordmann1* Service de Bacte´riologie-Virologie, Ho ˆpital de Biceˆtre, Assistance Publique/Ho ˆpitaux de Paris, Faculte´ de Me´decine Paris-Sud, Universite´ Paris XI, 94275, K.-Biceˆtre, France1; Division of Microbiology, Calgary Laboratory Services,2 and Departments of Pathology and Laboratory Medicine,3 Microbiology and Infectious Diseases,4 and Medicine,5 University of Calgary, Alberta, Canada; and University Hospital Virgen Macarena, University of Sevilla, Sevilla, Spain6 Received 20 September 2005/Returned for modification 10 November 2005/Accepted 2 February 2006
A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase. for qnrA (6) and qnrS (primers QnrS-A2 [5⬘-AGTGATCTCA CCTTCACCGC-3⬘] and QnrS-B2 [5⬘-CAGGCTGCAATTTT GATACC-3⬘]). Total DNAs of E. coli Lo (qnrA) (6) and plasmid PBC-H2.6 (qnrS) (gift from M. Hata) were used as controls. PCR and sequencing revealed that isolates E. coli 1A and 1B possessed the qnrA1 gene (9), being the first identification of plasmid-mediated quinolone resistance in Canada and also the first evidence of such a determinant in a clear community-acquired isolate. To explain the difference in fluoroquinolone resistance between the two isolates, the quinolone resistance-determining regions of subunit gyrA of the DNA gyrase gene (primers gyrA6 and gyrA631R) and of subunit parC of the topoisomer-
Plasmid-mediated resistance to the quinolones was reported first in 1998 from a Klebsiella pneumoniae isolate from the United States (7). The plasmid-encoded protein QnrA protects DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones (15, 16). Another plasmid-mediated quinolone resistance determinant, QnrS, sharing 59% amino acid identity with QnrA, has been identified in Japan from a single Shigella flexneri isolate (3). These Qnr determinants confer resistance to quinolones and reduced susceptibility to fluoroquinolones (9). Several studies showed a worldwide dissemination of QnrA determinants among enterobacterial isolates that often also produced extended-spectrum -lactamases (ESBLs), such as SHV-5, SHV-7, CTX-M-9, and VEB-1 (9, 12, 13). Our study was initiated by the observation of in vivo selection of fluoroquinolone resistance in Escherichia coli. Strains 1A and 1B were isolated in December 2000 and April 2001, respectively, from urine samples from a patient with community-acquired urinary tract infections in Calgary, Canada. After isolation of strain 1A, the patient received norfloxacin for five days. Resistance to quinolones and fluoroquinolones and production of ESBL were evaluated as previously described and interpreted according to Clinical and Laboratory Standards Institute guidelines (1, 10). Both isolates had an identical ESBL-mediated -lactam resistance phenotype, whereas isolate 1A had reduced susceptibility to nalidixic acid and ciprofloxacin and isolate 1B was resistant to nalidixic acid and ciprofloxacin (Table 1). Pulsed-field gel electrophoresis analysis showed that strains 1A and 1B were clonally related (data not shown). Since isolate 1A had reduced susceptibility to quinolones, the qnrA and qnrS genes were searched for by using a PCR protocol as described previously (6) with specific primers
TABLE 1. Antibiotic susceptibility profiles of E. coli 1A and 1B clinical isolates, the E. coli J53 (p1) transconjugant, and the E. coli J53 Azr reference strain MIC (g/ml) for strain Antibiotic
NAL CIP OFX NOR MXF SPX AMX AMC PIP TZP CAZ CTX FEP IPM
* Corresponding author. Mailing address: Service de Bacte´riologieVirologie, Ho ˆpital de Biceˆtre, 78 rue du Ge´ne´ral Leclerc, 94275 Le Kremlin-Biceˆtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-4521-63-40. E-mail:
[email protected].
a
E. coli 1A (p1) qnrA⫹
E. coli 1B (p1) qnrA⫹
E. coli J53 transconjugant (p1) qnrA⫹
E. coli J53 Azr
16 0.5 3 2 1.5 1.5 ⬎512 6 32 4 512 8 2 0.12
⬎256 ⬎32 ⬎32 ⬎256 ⬎32 ⬎32 ⬎512 16 32 4 512 8 2 0.12
64 0.25 1.5 1.5 1.5 1.5 512 8 16 2 512 4 1 0.12
3 0.12 0.1 0.06 0.06 0.02 4 4 2 1 0.06 0.06 0.01 0.06
a NAL, nalidixic acid; CIP, ciprofloxacin; OFX, ofloxacine; NOR, norfloxacine; MXF, moxifloxacine; SPX, sparfloxacine; AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; PIP, piperacillin; TZP, piperacillin-tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IPM, imipenem.
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FIG. 1. Schematic structure of the sul1-type integron that contains the qnrA and blaVEB-1 genes identified in Canadian isolates. Open reading frames are indicated by horizontal arrows and 59-bp elements by circles.
ase IV gene (primers ParCF43 and ParCR981) (6) were sequenced. E. coli isolate 1A had wild-type sequences, whereas isolate 1B harbored three amino acid substitutions, Ser83Leu and Asp87Asn, in the GyrA gyrase subunit and Ser80Ile in the ParC topoisomerase IV subunit, already known to be responsible for resistance to quinolones and fluoroquinolones (14). PCR and sequencing performed as described previously (6) indicated that isolates 1A and 1B harbored the ESBL blaVEB-1 gene. Whereas VEB-1 had been reported mostly from Asian isolates (2), this is the first identification of that ESBL in the Americas. This finding underlines association between VEB-1 and QnrA determinants, as already noticed (12). Transfer of quinolone and -lactam resistance determinants attempted by conjugation for E. coli isolates 1A and 1B using azide-resistant E. coli J53 as a recipient strain as described previously (6) was successful. Transconjugants had an identical resistance profile, with an ESBL phenotype related to VEB-1 expression and with reduced susceptibility to quinolones (Table 1), aminoglycosides, sulfonamides, chloramphenicol, and rifampin. Plasmid content analysis performed by the Kieser method (5) identified a similar 180-kb plasmid that cohybridized with blaVEB-1 and qnrA-specific probes using Southern hybridization (data not shown), as previously described (12). In order to further identify the genetic environment of the qnrA1 gene, cloning experiments were performed using total DNA of an E. coli transconjugant restricted with XbaI and BamHI enzymes and the pBK-CMV recipient plasmid as described previously (11). Selection of E. coli recombinant strains was performed on nalidixic acid (8 g/ml) and kanamycin (30 g/ml)-containing trypticase soy plates. Sequence analysis of a recombinant plasmid harboring a ca. 10-kb BamHI insert, pLCB1, identified the qnrA1 gene in the same sul1-type class 1 integron structure that possessed blaVEB-1 (Fig. 1). The 3⬘ part of that integron that included blaVEB-1 was almost similar to the blaVEB-1-positive integron In53 from E. coli MG-1 (8). Next, a study was designed to investigate the spread of qnrA/ qnrS-like genes in a collection of enterobacterial isolates recovered from the Calgary Health Region, Canada. It consisted in 139 ESBL-negative and ciprofloxacin-resistant clinical strains isolated from December 2004 to February 2005 and in 101 ESBL-positive strains recovered during a three-year period (2000 to 2002). In the latter collection, 54% of the strains were resistant to nalidixic acid and ciprofloxacin and 29% additional strains were resistant to nalidixic acid. No qnrS gene was identified, whereas the qnrA1 gene was detected in two additional ESBL-positive E. coli isolates (3% of ESBL-positive strains). Strains 2 and 3, which were from urines of patients with community-onset urinary tract infections, had been isolated in March 2000 and October 2002, respectively, and were resistant to fluoroquinolones. PCR indicated that these QnrA1-positive isolates also harbored the blaVEB-1 gene. PFGE showed that isolates 2 and 3 exhibited similar patterns,
but these patterns were different from those of strains 1A and 1B (data not shown). Conjugation assays and plasmid analysis from isolates 2 and 3 revealed very similar plasmids compared to isolates 1A and 1B (identical size, same coresistance markers transferred). Our study revealed key features reported here for the first time: (i) in vivo selection of ciprofloxacin-resistant strains in a qnr-positive background; (ii) physical linkage between ESBLand QnrA-encoding genes in the same integron; (iii) identification of qnrA in Canada and blaVEB-1 in North America; and (iv) first systematic survey for identification of qnrS-positive isolates. Nucleotide sequence accession number. The nucleotide sequences reported here have been assigned GenBank accession number DQ091243. This work was funded by a grant from the Ministe`re de l’Education Nationale et de la Recherche (UPRES-EA3539), Universite´ Paris XI, France, and mostly by the European Community (6th PCRD, LSHMCT-2003-503-335 and LSHM-2005-018705). L.P. is a researcher from the INSERM, Paris, France. J.M.R.M. was a recipient of a travel grant from the Spanish Society for Clinical Microbiology and Infectious Diseases in 2004. We thank Luisa Peixe for support of L.C. REFERENCES 1. Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing; 15th informational supplement. M100–S15. Clinical and Laboratory Standards Institute, Wayne, Pa. 2. Girlich, D., L. Poirel, A. Leelaporn, A. Karim, C. Tribuddharat, M. Fennewald, and P. Nordmann. 2001. Molecular epidemiology of the integronlocated VEB-1 extended-spectrum -lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand. J. Clin. Microbiol. 39:175–182. 3. Hata, M., M. Suzuki, M. Matsumoto, M. Takahashi, K. Sato, S. Ibe, and K. Sakae. 2005. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob. Agents Chemother. 49: 801–803. 4. Jeong, J. Y., H. J. Yoon, E. S. Kim, Y. Lee, S. H. Choi, N. J. Kim, J. H. Woo, and Y. S. Kim. 2005. Detection of qnr in clinical isolates of Escherichia coli from Korea. Antimicrob. Agents Chemother. 49:2522–2524. 5. Kieser, T. 1984. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12:19–36. 6. Mammeri, H., M. Van De Loo, L. Poirel, L. Martinez-Martinez, and P. Nordmann. 2005. Emergence of plasmid-mediated quinolone resistance in Escherichia coli in Europe. Antimicrob. Agents Chemother. 49:71–76. 7. Martinez-Martinez, L., A. Pascual, and G. A. Jacoby. 1998. Quinolone resistance from a transferable plasmid. Lancet 351:797–799. 8. Naas, T., Y. Mikami, T. Imai, L. Poirel, and P. Nordmann. 2001. Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli which carries an unusual array of gene cassettes. J. Bacteriol. 183:235–249. 9. Nordmann, P., and L. Poirel. 2005. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J. Antimicrob. Chemother. 56: 463–469. 10. Pitout, J. D., M. D. Reisbig, E. C. Venter, D. L. Church, and N. D. Hanson. 2003. Modification of the double-disk test for detection of enterobacteriaceae producing extended-spectrum and AmpC -lactamases. J. Clin. Microbiol. 41:3933–3935. 11. Poirel, L., T. Naas, M. Guibert, E. B. Chaibi, R. Labia, and P. Nordmann. 1999. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum -lactamase encoded by an Escherichia coli integron gene. Antimicrob. Agents Chemother. 43:573–581. 12. Poirel, L., M. Van De Loo, H. Mammeri, and P. Nordmann. 2005. Association of plasmid-mediated quinolone resistance with extended-spectrum -lactamase VEB-1. Antimicrob. Agents Chemother. 49:3091–3094.
VOL. 50, 2006 13. Robicsek, A., D. F. Sahm, J. Strahilevitz, G. A. Jacoby, and D. C. Hooper. 2005. Broader distribution of plasmid-mediated quinolone resistance in the United States. Antimicrob. Agents Chemother. 49:3001–3003. 14. Ruiz, J. 2003. Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection. J. Antimicrob. Chemother. 51:1109–1117.
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15. Tran, J. H., G. A. Jacoby, and D. C. Hooper. 2005. Interaction of plasmid-encoded quinolone resistance protein Qnr with Escherichia DNA gyrase. Antimicrob. Agents Chemother. 49:118–125. 16. Tran, J. H., G. A. Jacoby, and D. C. Hooper. 2005. Interaction of plasmid-encoded quinolone resistance protein QnrA with Escherichia topoisomerase IV. Antimicrob. Agents Chemother. 49:3050–3052.
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