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JAC Advance Access originally published online on November 20, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):291-295; doi:10.1093/jac/dkm448
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Prevalence of qnr genes among extended-spectrum β-lactamase-producing enterobacterial isolates in Barcelona, Spain

S. Lavilla1, J. J. González-López1, M. Sabaté1, A. García-Fernández2, M. N. Larrosa1, R. M. Bartolomé1, A. Carattoli2 and G. Prats1,*

1 Department of Microbiology, Hospital Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain 2 Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy

* Corresponding author. Tel: +34-93-2746817; Fax: +34-93-2746801; E-mail: gprats{at}vhebron.net

Received 1 August 2007; returned 18 August 2007; revised 22 October 2007; accepted 22 October 2007


   Abstract
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Objectives: To evaluate the presence of qnr genes among enterobacterial isolates carrying extended-spectrum β-lactamases (ESBLs) in Barcelona, Spain.

Methods: Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification with specific primers in 305 non-duplicate, clinically relevant ESBL-producing enterobacterial isolates obtained from February 2003 to August 2004. ESBLs from all qnr-positive isolates were characterized by isoelectric focusing, PCR amplification and DNA sequencing. Plasmid analysis was performed by S1 digestion and hybridization with specific probes for the qnr and bla genes. Plasmids containing qnr genes were transferred by conjugation or transformation. The genetic environment of qnrA1 in selected isolates was characterized by cloning experiments.

Results: Fifteen isolates, each from a different individual, carried qnr. Among them, 14 had qnrA1 (6 Klebsiella pneumoniae, 6 Enterobacter cloacae and 2 Escherichia coli isolates) and 1 had qnrS1 (K. pneumoniae). None of the isolates carried qnrB. Among the qnrA1-carrying isolates, 10 possessed both blaCTX-M-9 and blaSHV-12, 2 had both blaCTX-M-9 and blaSHV-92 and 2 had blaCTX-M-9 alone. The isolate with qnrS1 possessed blaSHV-12. The qnrA1 and ESBL genes were located together on plasmids ranging in size from 40 to 320 kb. qnrS1 and blaSHV-12 were not located on the same plasmid. Transfer of quinolone resistance was successfully achieved from all but three isolates. The cloned region surrounding qnrA in two K. pneumoniae isolates revealed a novel genetic organization.

Conclusions: The prevalence of qnr among enterobacterial clinical isolates carrying ESBLs between 2003 and 2004 in Barcelona was 4.9%. qnrA1 was the most prevalent, whereas only one qnrS and no qnrB were detected.

Keywords: ESBLs , quinolones , enterobacteria


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In 1998, the first plasmid-mediated quinolone resistance (Qnr) was reported, in a Klebsiella pneumoniae isolate.1 Qnr proteins belong to the pentapeptide repeat family and are able to bind to DNA gyrase and topoisomerase IV, protecting them from the inhibitory activity of quinolones.2 The first Qnr detected, a protein of 218 amino acids, was named QnrA. Since then, two more Qnr determinants have been described, QnrB and QnrS, which share 40% and 59% amino acid identity, respectively, with QnrA.3

Qnr has been detected in several members of the Enterobacteriaceae family, mainly in K. pneumoniae, Escherichia coli and Enterobacter spp., in different countries.2 Plasmids harbouring qnr may also carry extended-spectrum β-lactamasesz (ESBLs).3–5 The aim of this study was to evaluate the presence of qnr genes among enterobacterial isolates carrying ESBLs in our area.


   Materials and methods
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Bacterial isolates

During the period February 2003 to August 2004, 305 non-duplicate clinically relevant ESBL-producing enterobacterial isolates obtained at Hospital Vall d’Hebron (Barcelona, Spain) were analysed for the presence of qnr genes. These included 247 E. coli, 33 K. pneumoniae, 9 Klebsiella oxytoca, 8 Enterobacter cloacae and 1 each of Hafnia alvei, Raoultella ornithinolytica, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Salmonella enterica serovar Concord, S. enterica serovar Enterica and S. enterica serovar Virchow. Only one representative isolate among all those from a single individual that shared the same enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) profile was included (see below).

Antimicrobial susceptibility testing

Susceptibility to antimicrobials was determined by disc diffusion, following the CLSI recommendations. Suggestive evidence of ESBL production was defined as synergy between amoxicillin/clavulanate and at least one of the following antibiotics: cefotaxime, ceftazidime, aztreonam or cefepime, and confirmed by the Etest ESBL strip (AB Biodisk, Solna, Sweden). Nalidixic acid and ciprofloxacin MICs were determined using Etest (AB Biodisk).

PCR amplification and sequencing

Screening for the qnrA, qnrB and qnrS genes was carried out by PCR amplification using specific primers (Table 1). All positive results were confirmed by direct sequencing of both strands of amplicons. The quinolone-resistance-determining region (QRDR) of the gyrA and parC genes was sequenced directly from PCR-amplified DNA.


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  		Table 1. Primers used in this study

 
Characterization of ESBLs

ESBLs of all qnr-positive isolates were characterized further by isoelectric focusing. On the basis of these results, PCR amplification and DNA sequencing of different bla genes were done using specific primers (Table 1).

Plasmid DNA analysis

Plasmid number and sizing was performed on all qnr-positive isolates by S1 nuclease digestion as previously described.6 PFGE gels were transferred onto positively charged nylon membranes and hybridized with specific probes for qnrA1, qnrS1, blaCTX-M-9 and blaSHV-12/92 obtained by PCR with primers mentioned above.

Transfer of quinolone resistance

Conjugation experiments involving the qnr-positive isolates were performed by the liquid mating assay as previously described,6 using a rifampicin-resistant derivative of E. coli HB101 as the recipient isolate and selecting on LB agar supplemented with 100 mg/L rifampicin, 3 mg/L nalidixic acid and/or 100 mg/L ampicillin.

The plasmid DNA of the qnrS-positive isolates and of the isolates for which no transconjugants were obtained was electroporated into the kanamycin-resistant E. coli AG100A (hypersusceptible {Delta}acrAB mutant). Transformants were selected on LB agar plates supplemented with 50 mg/L kanamycin and 3 mg/L nalidixic acid and/or 100 mg/L ampicillin.

Cloning experiments

On selected isolates, the region surrounding qnr was cloned by ligating HindIII-digested total DNA fragments into the vector pBC-SK+ (Stratagene, La Jolla, CA, USA). DNA sequencing was carried out with a series of outward-facing primers starting from both ends of the qnr gene.

Molecular typing

To elucidate the clonality of qnr-carrying isolates, PFGE analysis of XbaI-digested DNA was done as previously described.6 Clonal relationship of the isolates that could not be typed by PFGE was assessed by studying ERIC-PCR genomic DNA profiles, as generated using primers ERIC1 and ERIC2 (Table 1).

Nucleotide sequence accession number

The described sequences for SHV-92, pQKp274H and pQKp331H have been assigned GenBank accession numbers DQ836922, EF682136 and EF682137, respectively.


   Results and discussion
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Fifteen out of the 305 isolates studied, each from a different individual, carried qnr (4.9%). Among the qnr-positive isolates, 14 had qnrA1 (6 K. pneumoniae, 6 E. cloacae and 2 E. coli isolates) and one had qnrS1 (K. pneumoniae). No isolate carried qnrB (Table 2).


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  		Table 2. Characteristic of isolates with quinolone-resistant determinants (qnr) and the qnr-positive transconjugants (Tc) obtained

 
Among the total isolates studied, 39.7% and 69.2% were susceptible to nalidixic acid and ciprofloxacin, respectively. Four of the 15 qnr-positive isolates (26.7%) were nalidixic acid-resistant, with MICs ranging from 32 to ≥256 mg/L, and one was of intermediate susceptibility (MIC 24 mg/L). For ciprofloxacin, all but one of the qnr-positive isolates (93.3%) were susceptible, with MICs ranging from 0.06 to 0.75 mg/L. The remaining isolate was intermediate to ciprofloxacin (MIC 1.5 mg/L) according to the CLSI. PCR amplification and sequencing of the QRDR of the gyrA and parC genes of the qnr-positive isolates identified an amino acid change in gyrA in E. cloacae isolates 154 and 834 (Ser-83->Tyr and Ser-83->Phe, respectively).

Among the 14 qnrA1-carrying isolates, 10 possessed both blaCTX-M-9 and blaSHV-12, 2 both blaCTX-M-9 and blaSHV-92, a novel SHV variant which showed three amino acid changes compared with SHV-1 (Leu-35->Gln, Met-69->Ile, Thr-141->Ile; pI 7.6; SHV number assigned by G. A. Jacoby, Lahey Clinic, MA, USA, personal communication), and 2 blaCTX-M-9 alone. The isolate with qnrS1 possessed blaSHV-12 and a bla gene, which shares 100% identity with the bla gene found in pK245 and differs in only one amino acid (Gly-193->Glu) from the recently described blaLAP-1.4,7 No blaTEM β-lactamase was found in any of the qnr-positive isolates.

Plasmid analysis of all qnr-positive isolates showed that qnrA1 was located in plasmids ranging in size from 40 to 320 kb (Table 2). All but 1 of the 14 qnrA1-positive isolates harboured ESBL genes and qnrA1 located in the same plasmid. In the remaining qnrA1-positive isolate (K. pneumoniae 331), blaCTX-M-9 was located in a plasmid of 250 kb, whereas blaSHV-12 and qnrA1 were present together on a different plasmid of 40 kb. K. pneumoniae 529 carrying qnrS1 was not possible to digest with S1 nuclease because of DNA self-degradation.

Conjugation experiments involving the qnr-positive isolates revealed that Qnr was successfully transferred from all but three qnr-positive donor isolates (Table 2). No transformants were obtained after electroporation of the plasmid DNA from those isolates for which no transconjugants were obtained. Transconjugants carrying qnrA1 also carried blaCTX-M-9 and blaSHV-12/92. Transconjugants carrying qnrS1 did not possess blaSHV-12.

qnrA1 variants are typically found in a sul1-type integron that contains ISCR1 upstream of the qnr gene and either qacE{Delta}1-sul1 or ampR-qacE{Delta}1-sul1 downstream.3 Analysis of the regions surrounding qnrA1 by PCR showed that all isolates had ISCR1 immediately upstream of qnrA1 and all but two isolates (K. pneumoniae 274 and K. pneumoniae 331) had an ampR gene between qnrA1 and qacE{Delta}1-sul1.

The region surrounding qnrA1 in K. pneumoniae 274 and K. pneumoniae 331 cloned by ligating HindIII-digested total DNA fragments into the vector pBC-SK+ resulted in recombinant qnrA1 plasmids pQKp274H and pQKp331H, respectively. Sequence analysis of these constructs revealed that the ampR gene, located downstream of qnrA1, was truncated by a composite transposon consisting of two inverted repeat IS26 elements flanking a tetR and a tetA gene [Figure S1, available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].

qnrS1 has been described downstream of two different structures, including: (i) a Tn3 element which contains blaTEM-1; and (ii) blaLAP-1.4,7,8 DNA sequence analysis of a 5 kb region surrounding qnrS1 revealed 100% identity with qnrS1-containing plasmid pK245, including the blaLAP-like gene. However, unlike pK245, the present plasmid harbouring qnrS1 did not contain a blaSHV-2 gene and could be self-transferred by conjugation (Table 2).

PFGE analysis of XbaI-digested DNA of the qnr-positive isolates showed that all the E. cloacae isolates had a different PFGE pattern. The two E. coli were obtained from two brothers and shared the same PFGE profile. Likewise, two K. pneumoniae isolates had the same PFGE profile. These isolates had been obtained from different patients who shared the same paediatric intensive care unit room. K. pneumoniae isolate 529 could not be typed by PFGE because of DNA self-degradation; however, by ERIC-PCR it showed a different pattern from the other K. pneumoniae isolates, suggesting a unique clonal background (data not shown) (Table 2).

In this work, four qnr-positive isolates were resistant to nalidixic acid. Two of these exhibited high-level resistance (MIC ≥256 mg/L) presumably due to the associated mutation in the QRDR of gyrA and two isolates exhibited low-level resistance (MIC 32 mg/L) Regarding ciprofloxacin, all isolates except one were susceptible, but showed decreased susceptibility compared with those isolates without any resistance mechanism. Moreover, the two isolates with the highest MICs of ciprofloxacin were those with mutations in the QRDR. These data agree with previous reports showing that the presence of qnr does not necessarily lead to MICs above CLSI breakpoints for resistance to ciprofloxacin.1 The basis for concern regarding the low-level resistance conferred by this mechanism remains the increment this causes in the selection window to high-level quinolone resistance.9

Our results differ from those of a recent Spanish study where 11% of 202 Enterobacter spp. clinical isolates carried qnrS but none carried qnrA.10 However, our results are similar to those of a recent report in France, which showed that, in a collection of 186 ESBL-producing bacteria obtained from 2002 to 2005 in a hospital in Paris, 2.2% and 1.6% of the isolates carried qnrA1 and qnrS1 (qnrB was not evaluated), respectively.5

Our findings show that in our locale qnr variants are variably prevalent among ESBL-producing enterobacterial and are associated with reduced quinolone susceptibility in the absence of other explanatory mechanisms. Further studies in more heterogeneous bacterial populations are necessary to comprehensively assess the prevalence of Qnr and its clinical significance.


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This work was supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008) and by the ‘Fondo de Investigación Sanitaria (PI 020358 and PI 050289)’. S. Lavilla received a grant from the ‘Institut de Recerca Hospital Universitari Vall d’Hebron’ and J. J. Gonzalez-Lopez a grant from the Spanish Network for the Research in Infectious Diseases.


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None to declare.


   Supplementary data
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Figure S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).


   Acknowledgements
 
We are grateful to P. Nordmann (Hôpital de Bicêtre, Paris, France) for providing the E. cloacae S4 that carries the qnrS gene. We thank L. Martinez (Hospital Universitario Marqués de Valdecilla, Santander, Spain) for providing K. pneumoniae UAB1 harbouring the qnrA1 gene and E. coli J53 pMG298 harbouring the qnrB1 gene, and P. Courvalin (Institut Pasteur, Paris, France) for providing E. coli AG100A used in cloning experiments. We extend appreciation to J. R. Johnson (Minneapolis VA Medical Center, Minneapolis, MN, USA) for critically reading and providing helpful comments during the writing of this manuscript.


   References
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1 Martínez-Martínez 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]

3 Nordmann P, Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother (2005) 56:463–9.[Abstract/Free Full Text]

4 Poirel L, Cattoir V, Soares A, et al. Novel Ambler class A β-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1. Antimicrob Agents Chemother (2007) 51:631–7.[Abstract/Free Full Text]

5 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.[Abstract/Free Full Text]

6 García A, Navarro F, Miró E, et al. Acquisition and diffusion of blaCTX-M-9 gene by R478-IncHI2 derivative plasmids. FEMS Microbiol Lett (2007) 271:71–7.[CrossRef][Web of Science][Medline]

7 Chen YT, Shu HY, Li LH, et al. Complete nucleotide sequence of pK245, a 98-kilobase plasmid conferring quinolone resistance and extended-spectrum-β-lactamase activity in a clinical Klebsiella pneumoniae isolate. Antimicrob Agents Chemother (2006) 50:3861–6.[Abstract/Free Full Text]

8 Kehrenberg C, Friederichs S, de Jong A, et al. Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis. J Antimicrob Chemother (2006) 58:18–22.[Abstract/Free Full Text]

9 Rodríguez-Martínez JM, Velasco C, García I, et al. Mutant prevention concentrations of fluoroquinolones for Enterobacteriaceae expressing the plasmid-carried quinolone resistance determinant qnrA1. Antimicrob Agents Chemother (2007) 51:2236–9.[Abstract/Free Full Text]

10 Cano ME, Rodríguez-Martínez JM, Agüero J, et al. Detection of qnrS in clinical isolates of Enterobacter cloacae in Spain. Abstracts of the Sixteenth European Congress of Clinical Microbiology and Infectious Diseases, 2006: Nice, France. Taufkirchen, Germany: European Society of Clinical Microbiology and Infectious Diseases. Abstract O52.


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