JAC Advance Access published online on June 8, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm204
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Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates
1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 78, rue du Général Leclerc, 94275 K.-Bicêtre, France 2 Service de Bactériologie-Virologie-Hygiène, Hôpital Henri Mondor, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine de Créteil, Université Paris XII, Créteil, France 3 Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.aphp.fr
Received 21 February 2007; returned 11 April 2007; revised 17 April 2007; accepted 11 May 2007
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Abstract |
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Objectives: To develop a rapid and reliable single-tube-based PCR technique for detecting simultaneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes.
Methods: After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum ß-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to identify qnr and associated ESBL genes.
Results: In optimized conditions, all positive controls (used separately or mixed) confirmed the specificity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-12+) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-12+) and Citrobacter freundii K70 (VEB-1b+) isolates, respectively.
Conclusions: We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates.
Key Words: qnrA , qnrB , qnrS , Kuwait , Middle East
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Plasmid-mediated quinolone resistance was originally reported in a Klebsiella pneumoniae clinical isolate from the USA in 1998.1 This qnrA gene responsible for this resistance (now termed qnrA1) codes for a 218 amino acid protein belonging to the pentapeptide family that protects DNA from quinolone binding to gyrase and topoisomerase IV.2 QnrA confers resistance to quinolones such as nalidixic acid and increases MIC values of fluoroquinolones up to 20-fold.2 The QnrA determinant has been reported worldwide from unrelated enterobacterial species and six variants of QnrA are known (QnrA1 to QnrA6).2 Recently, two other plasmid-mediated quinolone resistance genes, namely, qnrB and qnrS, have been identified that code for QnrB (six variants) and QnrS (two variants) belonging also to the pentapeptide repeat family and sharing 41% and 60% amino acid identity with QnrA, respectively.2
Several surveys, based on molecular approaches consisting of PCR and sequencing, indicate a high rate of the association between Qnr-positive and ESBL-positive isolates.2,3 However, in view of the genetic heterogeneity of those genes encoding Qnr determinants (94% to 99%, 85% to 99% and 90% nucleotide identity for qnrA-, qnrB- and qnrS-like genes, respectively), it is likely that their prevalence may be underestimated, as a consequence of a lack of sensitivity of the molecular tools that are used. In addition, performing such studies by amplifying separately the qnrA-, qnrB- and qnrS-like genes is time-consuming and expensive. A first multiplex PCR-based method has been described by Robicsek et al.4 and was applied for screening a collection of ceftazidime-resistant Enterobacteriaceae clinical isolates from the USA. The primers used for the detection of qnrB-like genes did not fully match all six variants, particularly for the reverse primer that mismatched at the 3' end with qnrB5 and qnrB6 genes.
The purpose of the present work was to design an updated, simple and rapid multiplex PCR technique for the detection of the qnrA-, qnrB- and qnrS-like genes. It was applied to screen a collection of ESBL-producing enterobacterial isolates from Kuwait, a region of the world with unknown distribution of Qnr-like determinants.
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Bacterial isolates
For optimization of the multiplex PCR technique, well-characterized Qnr-positive strains were used as positive controls: Escherichia coli Lo qnrA1+,3 Shewanella algae KB-1 qnrA3+, S. algae KB-2 qnrA4+ and S. algae KB-3 qnrA5+5; K. pneumoniae B1 qnrB1+, Enterobacter cloacae B2 qnrB2+, E. coli B4 qnrB4+, E. coli B5 qnrB5+ and E. coli B6 qnrB6+ (P. Nordmann, unpublished results); E. coli S7 qnrS1+3 and E. coli A37 qnrS2+ (P. Nordmann, unpublished results); and E. cloacae S1 carrying both qnrB4+ and qnrS1+.3
ESBL-producing enterobacterial isolates (n = 64), collected from the University Hospital of Kuwait City from 2002 to 2004, were identified by using the Vitek2 Analyzer (bioMérieux SA, Marcy-l'Étoile, France). They included 29 E. coli, 19 K. pneumoniae, 6 Proteus mirabilis, 4 E. cloacae, 3 Enterobacter aerogenes, 2 Citrobacter freundii and 1 Serratia marcescens clinical isolates. These isolates were non-repetitive and only a single isolate per patient was retained.
ESBL production was suggested by results of the Vitek2 Analyzer and confirmed by disc diffusion and synergy tests performed on Mueller–Hinton agar-containing plates. MICs of quinolones, fluoroquinolones and ß-lactams were determined using the Etest method, according to the manufacturer's recommendations (AB Biodisk, Solna, Sweden). MIC breakpoints used for susceptibility and resistance to nalidixic acid and ciprofloxacin were 
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32 mg/L and 1 and 4 mg/L, respectively, as recommended by the CLSI (formerly NCCLS).6
Rapid DNA preparation was performed by a boiling technique that includes a heating step at 100°C of a single colony in a total volume of 100 µL of distilled water followed by a centrifugation step of the cell suspension. On the basis of a sequence alignment of the qnrA-, qnrB- and qnrS-like genes, pairs of primers were designed to amplify internal fragments with sizes of 580, 264 and 428 bp, respectively (Table 1). A pair of degenerated primers was specifically designed to amplify the six variants of qnrB, despite the high polymorphism of this gene (Table 1). Total DNA (2 µL) was subjected to multiplex PCR in a 50 µL reaction mixture containing 1x PCR buffer [10 mM Tris–HCl (pH 8.3), 50 mM KCl], 1.5 mM MgCl2, 200 µM each deoxynucleotide triphosphate, 20 pmol of each of the six primers (Table 1) and 2.5 U of Taq polymerase (Applied Biosystems, Courtaboeuf, France). Amplification was carried out with the following thermal cycling profile: 10 min at 95°C and 35 cycles of amplification consisting of 1 min at 95°C, 1 min at 54°C and 1 min at 72°C and 10 min at 72°C for the final extension. DNA fragments were analysed by electrophoresis in a 2% agarose gel at 100 V for 1 h in 1x TAE [40 mM Tris–HCl (pH 8.3), 2 mM acetate and 1 mM EDTA] containing 0.05 mg/L ethidium bromide.
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PCR amplification and sequencing of qnr and ESBL genes
To further characterize the qnrB-like genes, additional PCR experiments were performed by using primers described by Jacoby et al.7 followed by direct sequencing of both strands. Molecular identification of the ESBL genes was carried out for the isolates found to be Qnr-positive, as described previously.3
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Positive controls (used separately or mixed) yielded expected bands and confirmed the specificity of the PCR primers used (Figure 1). Both the qnrB4 and qnrS1 genes were unambiguously detected in E. cloacae S1 clinical isolate (Figure 1, lane 8). Given the high genetic diversity between the different qnr genes and the sequence variability within the qnrA (94% to 99%), qnrB (85% to 99%) and qnrS genes (90%), the use of degenerate primers (especially for the detection of qnrB-like genes) might be preferred and therefore amplification of unknown variants could be obtained.
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Only three isolates (two E. cloacae and one C. freundii) isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like or qnrS-like gene was detected. The qnrB-like gene from E. cloacae K34 isolate was qnrB2 (445/445 bp), which was mostly identified from the QnrB-positive enterobacterial isolates from the USA.4 The qnrB-like genes from E. cloacae K37 and C. freundii K70 isolates coded for proteins showing 97% and 96% amino acid identity with QnrB1 and QnrB6, respectively (corresponding to nucleotide identity of 434/445 and 397/447, respectively). These two novel QnrB variants were designated QnrB7 and QnrB8, respectively (GenBank accession no. EF026242 and EF026243, respectively). Only QnrB determinants were identified from that collection from Kuwait, whereas QnrA has been reported worldwide.2
Both E. cloacae K34 and K37 were of intermediate susceptibility to nalidixic acid and remained susceptible to fluoroquinolones, whereas C. freundii K70 was fully susceptible to nalidixic acid and fluoroquinolones (Table 2). Interestingly, no mutation was detected in the quinolone-resistance-determining regions of gyrA and parC genes for these isolates that may have led to additional chromosome-encoded resistance to fluoroquinolones (data not shown). Therefore, other resistance mechanisms may be associated in these strains, such as membrane impermeability and/or efflux pumps overexpression.
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E. cloacae K34 and E. cloacae K37 isolates produced ESBL SHV-12, whereas C. freundii K70 isolate produced ESBL VEB-1b. Although an association between the QnrA and VEB-1 determinants has been identified, we found here a qnrB-like gene associated with the blaVEB-1b gene.8 Association between QnrB-like determinants (QnrB1, QnrB2 and QnrB5) and ESBLs (SHV-12 and CTX-M-15) has been recently reported from enterobacterial clinical isolates as well as association between QnrB4 variant and plasmid-mediated AmpC DHA-1 in E. coli and K. pneumoniae clinical isolates.7,9
This study shows that the multiplex PCR technique is a fast (<3 h) and reliable tool for rapid screening of Qnr-positive strains. The technique used will identify all so-far known qnr genes, including qnrB-like genes by using degenerate primers. Finally, our study showed a low prevalence of QnrB determinants among ESBL-producing enterobacterial isolates and the lack of detection of QnrA and QnrS determinants in that collection from Kuwait. It further identified the spread of Qnr-like determinants in Enterobacteriaceae from the Middle-East.
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None to declare.
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Acknowledgements |
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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).
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References |
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1 . Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet (1998) 351:797–9.[CrossRef][Web of Science][Medline]
2 . Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis (2006) 6:629–40.[CrossRef][Web of Science][Medline]
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Poirel L, Leviandier C, Nordmann P. Prevalence and genetic analysis of plasmid-mediated quinolone resistance determinants QnrA and QnrS in Enterobacteriaceae isolates from a French university hospital. Antimicrob Agents Chemother (2006) 50:3992–7.
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6 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15 (2005) Wayne, PA, USA: CLSI.
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Jacoby GA, Walsh KE, Mills DM, et al. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother (2006) 50:1178–82.
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Poirel L, Van De Loo M, Mammeri H, et al. Association of plasmid-mediated quinolone resistance with extended-spectrum ß-lactamase VEB-1. Antimicrob Agents Chemother (2005) 49:3091–4.
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