Plasmid-mediated quinolone resistance is increasingly reported worldwide for Enterobacteriaceae (4). This resistance is related to Qnr-like proteins belonging to the pentapeptide repeat family that protects DNA from quinolone binding to type II topoisomerases (9, 10). The three main groups of Qnr determinants, QnrA, QnrB, and QnrS, are known. QnrA (six variants) has been identified worldwide, whereas QnrB (six variants) and QnrS (two variants) have been reported in a few countries (7) and QnrB has not yet been identified in Europe. The aim of this study was to determine the prevalence of QnrB in extended-spectrum β-lactamase (ESBL)-producing enterobacterial isolates already tested for QnrA and QnrS (6).
(The results of this study were presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2006.)
A total of 186 ESBL-producing enterobacterial isolates were collected from the Bicêtre hospital, in a suburb of Paris, France, from January to June 2005 (5). The presence of the qnrB gene was investigated by a PCR assay as previously described (2). A single Enterobacter cloacae isolate, S1, recovered from an ascites of a 2.5-year-old child in May 2005, was positive for qnrB. This isolate was shown to be qnrS1 positive also (5). E. cloacae S1 was resistant to β-lactams via production of the ESBL SHV-12, the penicillinase TEM-1, and the novel penicillinase LAP-1 (5). In addition, this isolate was resistant to fluoroquinolones with a single amino acid change in the quinolone resistance-determining region (QRDR) of gyrA (S83F) and none in the QRDR of parC. QnrS1-mediated quinolone and LAP-1-mediated β-lactam resistance determinants were self-transferable and located onto a 100-kb conjugative plasmid termed pS1A.
Conjugation and transformation experiments using azide-resistant Escherichia coli J53 and E. coli TOP10 as recipient strains, respectively, were performed as previously described (5). QnrB-like protein-mediated quinolone resistance and SHV-12-mediated β-lactam resistance determinants were transferred only by transformation. Analysis of plasmid content in E. cloacae S1 and its transformants performed by using the Kieser technique (3) identified a 160-kb plasmid (termed pS1B) that hybridized to a qnrB-specific probe. Antibiotic susceptibility testing was carried out according to the guidelines of the CLSI (1). MICs of quinolones and fluoroquinolones were determined for E. coli TOP10 and the corresponding qnrB4- and qnrS1-positive transformants by using the E-test technique according to the manufacturer's recommendations. The qnrB-positive transformant expressed an ESBL phenotype (the blaSHV-12+ phenotype), together with resistance to aminoglycosides (except amikacin), chloramphenicol, and rifampin. In addition, it showed reduced susceptibilities to quinolones and fluoroquinolones, as observed for the qnrS-positive transformant (Table 1).
TABLE 1.
MICs of different quinolones and fluoroquinolones for E. cloacae S1, its E. coli TOP10 transformants harboring pS1A and pS1B (expressing QnrB4 and QnrS1 determinants, respectively), and reference strain E. coli TOP10
Antibiotic | MIC (μg/ml) of:
|
|||
---|---|---|---|---|
E. cloacae S1 | E. coli TOP10 (pS1A) | E. coli TOP10 (pS1B) | E. coli TOP10 | |
Nalidixic acid | >256 | 2 | 4 | 1 |
Norfloxacin | >32 | 0.25 | 0.5 | 0.03 |
Ofloxacin | >32 | 0.12 | 0.25 | 0.01 |
Ciprofloxacin | >32 | 0.06 | 0.12 | <0.01 |
Moxifloxacin | >32 | 0.12 | 0.25 | <0.01 |
Sparfloxacin | >32 | 0.03 | 0.25 | <0.01 |
Cloning experiments with total DNA of E. cloacae S1 allowed the sequence of the qnrB-like gene, which had perfect nucleotide identity with the qnrB4 variant reported for an E. coli isolate from the United States, to be determined (8). The genetic environment of the qnrB4 gene was bracketed at its 5′ extremity by a psp operon coding for putative phage shock proteins and at its 3′ extremity by a sap operon coding for a putative peptide transport system permease, sharing 87% and 80% amino acid identities, respectively, with similar proteins identified in E. coli K-12 (GenBank accession no. U00096).
This report identified the first QnrB-like determinant from Europe and the first coproduction of two Qnr-like determinants in a single isolate. This finding underlines that these plasmid-mediated quinolone resistance determinants may accumulate in enterobacterial clinical isolates, which could very likely further increase resistance to quinolones.
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 a grant from the European Community (6th PCRD, LSHM-CT-2005-018705). L.P. is a researcher from the INSERM (Paris, France).
Footnotes
Published ahead of print on 14 May 2007.
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