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Antimicrobial Agents and Chemotherapy, April 2006, p. 1525-1527, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1525-1527.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Selection of Fluoroquinolone-Resistant Escherichia coli Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum ß-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 Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275, K.-Bicêtre, France,1 Division of Microbiology, Calgary Laboratory Services,2 Departments of Pathology and Laboratory Medicine,3 Microbiology and Infectious Diseases,4 Medicine, University of Calgary, Alberta, Canada,5 University Hospital Virgen Macarena, University of Sevilla, Sevilla, Spain6

Received 20 September 2005/ Returned for modification 10 November 2005/ Accepted 2 February 2006


   ABSTRACT
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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.


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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 ß-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 ß-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 for qnrA (6) and qnrS (primers QnrS-A2 [5'-AGTGATCTCACCTTCACCGC-3'] and QnrS-B2 [5'-CAGGCTGCAATTTTGATACC-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.


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  		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

 
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 topoisomerase 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 ß-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 µg/ml) and kanamycin (30 µg/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).


Figure 1
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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.

 
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 ESBL- and 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.


   ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France, and mostly by the European Community (6th PCRD, LSHM-CT-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.


   FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr. Back


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Antimicrobial Agents and Chemotherapy, April 2006, p. 1525-1527, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1525-1527.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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