Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type ß-lactamase

J. M. Rodríguez-Martínez1,*, A. Pascual1,2, I. García1 and L. Martínez-Martínez1,2

1 University Hospital Virgen Macarena and 2 School of Medicine, University of Seville, Seville, Spain

Received 5 March 2003; returned 22 April 2003; revised 25 June 2003; accepted 25 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Plasmid pMG252 contains the qnr locus, which is responsible for low-level resistance to quinolones by protecting the DNA gyrase. pMG252 also encodes the AmpC-type ß-lactamase (pACBL), FOX-5. The aim of this study was to determine the prevalence of qnr in strains from different geographical locations in America and Europe.

Methods: Four hundred and twenty-five (159 Klebsiella pneumoniae and 266 Escherichia coli) clinical isolates were studied. The detection of qnr was by PCR using specific primers for an internal fragment of 543 bp.

Results: qnr was detected in three cefoxitin-resistant K. pneumoniae strains, which also produced a pACBL. None of the E. coli isolates tested contained qnr. The three qnr-positive K. pneumoniae came from the USA, and all transferred a conjugative plasmid coding for cefoxitin resistance to E. coli J53. qnr was also transferred by the same plasmid in two out of the three strains. The sequences of amplified qnr fragments from the three strains were identical to the qnr sequence from pMG252.

Conclusions: The qnr determinant is uncommon among clinical isolates of K. pneumoniae and E. coli, but its identification in three pACBL(+) K. pneumoniae from the USA indicates the emergence of this quinolone resistance mechanism.

Keywords: Escherichia coli, fluoroquinolones, cephamycinase, molecular epidemiology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmid-mediated resistance to quinolones in clinical isolates was first reported in 1998 in a Klebsiella pneumoniae strain isolated in 1994 in Birmingham, AL, USA.1 Plasmid pMG252 contains the locus qnr (quinolone resistance), and also codes for the plasmid-mediated AmpC-type ß-lactamase (pACBL), FOX-5.2 qnr is included in an integron-like element upstream from qacE{Delta}1 and sulI, and codes for a 218-amino-acid protein of the pentapeptide repeat family that protects DNA from quinolone attack.2 qnr increases resistance to nalidixic acid and fluoroquinolones by four- to eight-fold.1

High prevalences of quinolone resistance in enterobacteria, particularly in Escherichia coli, have been described in Europe, South America and Asia, and are increasing in regions that until now maintained low rates of resistance (i.e. the USA).3 In K. pneumoniae and E. coli, quinolone resistance has been more frequently found among strains producing plasmid-mediated extended-spectrum ß-lactamase (ESBL) than among ESBL-negative strains, which may be related to the coordinated expression of several mechanisms of resistance.4 pACBL has been described in AmpC-negative organisms (K. pneumoniae), but also in E. coli.5 pACBLs commonly cause resistance to oxyimino-cephalosporins and cephamycins.5 Plasmids coding for pACBL vary in size (from 7 to 180 kb),5 and are usually conjugative, although non-self-transmissible plasmids, transferable by transformation or mobilization, have also been described.5

The frequency of the qnr determinant in clinical isolates is largely unknown. In a recent study of 275 strains with pACBL or ESBL,6 this determinant was only detected among FOX-5-producing strains isolated in Birmingham, AL, USA, between July 1994 and January 1995, being absent after that time, or in FOX-5-producing organisms from other locations. In a more recent study transferable plasmid-mediated quinolone resistance associated with qnr was reported in quinolone-resistant clinical strains of E. coli from Shanghai, China.7

The objective of this study was to determine the prevalence of qnr in clinical isolates of K. pneumoniae and E. coli strains from different geographical locations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Four hundred and twenty-five clinical isolates were evaluated, including 266 E. coli and 159 K. pneumoniae. Strains were from the SENTRY study (75 E. coli, 75 K. pneumoniae), the GEIH-BLEE 2000 study (108 E. coli, 10 K. pneumoniae), the University Hospital Virgen Macarena (70 E. coli, 45 K. pneumoniae), a multicentre study in the USA (13 E. coli, 11 K. pneumoniae), the ICARE project (five K. pneumoniae) and other sources.812 Fifty-seven isolates produced a pACBL or (in the case of K. pneumoniae) presented a phenotype compatible with pACBL production, which includes resistance to cefoxitin (NCCLS breakpoints),13 porin production (as determinated by SDS acrylamide gel electrophoresis) and expression of a ß-lactamase inhibited by cloxacillin, but not by clavulanic acid14 (using in situ inhibition on isoelectric focusing gels) (data not shown). Three hundred and eighty-one strains were resistant to ciprofloxacin (NCCLS breakpoints).

Plasmid extraction

Plasmid DNA was extracted with a Qiagen Plasmid Purification kit (Izasa, Barcelona, Spain), following the manufacturer’s recommendations.

PCR detection of qnr and DNA sequencing

Plasmid DNA was amplified by PCR. Primers 5'-ATCCAGATCGGCAAAGGTTA-3' and 5'-GATAAAGTTTTTCAGCAAGAGG-3' (sequences kindly provided by G. A. Jacoby) were used for qnr detection. They amplify a 543 bp fragment. The amplification reaction consisted of 30 cycles of 90 s of denaturation at 94°C, 30 s of hybridization at 64°C, and 90 s of extension at 72°C, with a final extension cycle of 7 min at 72°C.

Purified plasmid pMG252 was used as a positive control for PCR detection of qnr.

Sequencing was performed at the CIB-CSIC laboratory (Madrid, Spain) using the Big Dye terminator v. 3.0 sequencing kit and a 3700 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Molecular fingerprinting

Analysis of total DNA from three isolates in which qnr-detection was positive (see below) was performed by pulsed-field gel electrophoresis (PFGE). PFGE analysis of XbaI-restricted bacterial DNA was performed using a CHEF-DR II System (Bio-Rad Laboratories, Hercules, CA, USA).

Conjugation assays

Conjugation experiments were done to determine whether the qnr determinant was transferable to E. coli J53 RifR. Transconjugants were selected on Mueller–Hinton agar plates containing rifampicin (100 mg/L) and either ampicillin (100 mg/L) or ceftazidime (4 mg/L).

Susceptibility testing

The Etest method was used to determine MICs for the parental clinical isolates, their derived transconjugants and E. coli J53-RifR, following the manufacturer’s recommendations.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
qnr was detected in three out of the 159 K. pneumoniae isolates (strains N5, 1960 and 1132) and in none of the 266 E. coli isolates evaluated (Figure 1a, lanes 4, 5 and 6). All three strains were resistant to nalidixic acid (MIC 32 mg/L), but only one (strain 1132) was resistant to ciprofloxacin (MIC 4 mg/L).



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Figure 1. (a) PCR of plasmid DNA from K. pneumoniae strains that contain the qnr determinant and their transconjugants qnr+ and qnr–. Lanes 1 and 13, molecular markers (100 bp); lane 2, E. coli J53 RifR; lane 3, E. coli J53 RifR (pMG252); lane 4, K. pneumoniae N5; lane 5, K. pneumoniae 1960; lane 6, K. pneumoniae 1132; lane 7, qnr-positive transconjugant from K. pneumoniae N5; lane 8, qnr-negative transconjugant from K. pneumoniae N5; lane 9, qnr-positive transconjugant from K. pneumoniae 1960; lane 10, qnr-negative transconjugant from K. pneumoniae 1960; lane 11, transconjugant (qnr-negative) from K. pneumoniae 1132; lane 12, negative control. (b) PFGE patterns of three K. pneumoniae strains containing the qnr determinant. Lane 1: molecular marker (phage {lambda} digest); lane 2, K. pneumoniae N5; lane 3, K. pneumoniae 1960; lane 4, K. pneumoniae 1132; lane 5, molecular marker.

 
K. pneumoniae N5 was isolated during the SENTRY Study in Wilmington, DE, USA in 1997 (D. Biedenbach, The JONES Group/JMI Laboratories, North Liberty, IA, USA, personal communication). Strains K. pneumoniae 1960 and 1132 were isolated during the ICARE project from the same institution in North Carolina in 1995 and 1996, respectively (F. Tenover, Centers for Disease Control and Prevention, Atlanta, GA, USA, personal communication). As shown in Figure 1(b), PFGE indicated that the three qnr-positive K. pneumoniae strains were not clonally related. All of these produced a pACBL (data not shown), and were able to transfer ß-lactam resistance to E. coli J53 RifR. PCR allowed detection of qnr in transconjugants derived from strains K. pneumoniae N5 and 1960 and from plasmid DNA isolated from them (Figure 1a). As observed in Table 1, the qnr-positive transconjugants were resistant to cefoxitin, ampicillin and piperacillin, and showed decreased susceptibility to both nalidixic acid (eight-fold) and ciprofloxacin (16-fold) compared with E. coli J53-RifR. MICs obtained by microdilution of fluoroquinolones and ß-lactams agreed with those obtained by the Etest method.


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Table 1. MICs (mg/L) by Etest of antimicrobial agents against three K. pneumoniae (Kp) strains containing qnr and their derived transconjugants (TC)
 
Some of the transconjugants from these two strains, as well as all transconjugants evaluated from strain K. pneumoniae 1132, were resistant to cefoxitin, ampicillin and piperacillin, but presented unchanged susceptibility to both nalidixic acid and ciprofloxacin (data not shown), and PCR detection of qnr was negative (Figure 1a).

Sequences of the 543 bp fragment of qnr from the three clinical isolates were identical to those of the original plasmid pMG252.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1998 a multiresistant isolate of K. pneumoniae was reported from urine collected in Birmingham, AL, USA. It contained a broad host-range plasmid (pMG252) contributing to decreased in vitro activity of nalidixic acid and ciprofloxacin.1 In the presence of plasmid, resistance to quinolones due to defined DNA gyrase alterations, porin loss or efflux pump expression increased four- to eight-fold. The gene for quinolone resistance, named qnr, was found in an integron-like environment upstream from qacE{Delta}1 and sul1. Its product protects DNA gyrase from quinolone action.2

Three K. pneumoniae strains containing qnr are reported here. These strains were isolated in two different states of the USA and were not clonally related (Figure 1b). It is possible that qnr was acquired by conjugation, but its actual origin in these three strains is currently unknown, as is the relationship between the new plasmids encoding the qnr determinant and pMG252.1 Studies are in progress to clear up these aspects.

It should be emphasized that two of our three K. pneumoniae strains were susceptible to fluoroquinolones, according to NCCLS breakpoints. This may make the identification of qnr in clinical strains difficult, as no phenotypic marker is currently available for detecting its presence. Additional studies using PCR or a probe-based approach may help clarify the actual prevalence and dissemination of plasmid-mediated fluoroquinolones resistance.

The three qnr-positive strains from this study produced a pACBL, as does K. pneumoniae UAB1, the FOX-5-producing strain in which qnr was first detected.2 Sequences of the genes coding for the corresponding pACBL in the strains reported here will aid understanding of the relationship between qnr and genes coding for pACBLs.

In K. pneumoniae 1132, the plasmid coding for pACBL did not transfer the qnr determinant by conjugation. Similarly, transconjugants from strains N5 and 1960, selected in the presence of ß-lactams (and coding for pACBL) were also qnr-negative and did not affect quinolone susceptibility of the recipient E. coli (data not shown). Additionally, in a study on E. coli from China, transfer of plasmids carrying qnr was observed for five out of the six strains in which qnr was detected.7 The exact location of qnr in K. pneumoniae 1132 has not yet been determined, but in the case that it is plasmid-mediated, it is possible that the plasmid is not conjugative, that the plasmid coding for qnr is different from that coding for the pACBL or that during the process of conjugation qnr is lost. The fact that qnr from K. pneumoniae UAB1 is contained in an integron-like element may be of relevance in understanding the possible movement of this genetic determinant within the same host and between different microorganisms.2

In conclusion, the identification of the qnr determinant among K. pneumoniae strains producing pACBL and the recent finding of qnr in organisms from the Far East indicate the emergence of this new mechanism of resistance to quinolones in clinical isolates.7


    Acknowledgements
 
We thank G. A. Jacoby (Lahey Clinic, Burlington, MA, USA) for providing the sequences of the primers for qnr detection. Strains were kindly provided by R. Jones (SENTRY Project; The JONES Group/JMI Laboratories, North Liberty, IA, USA), F. Tenover (ICARE Project), the SEIMC-BLEE 2000 Study, P. Bradford (Rutgers University, Piscataway, NJ, USA), G. A. Jacoby, J. Liñares (Hospital de Bellvitge, Barcelona, Spain) and L. Tzouvelekis (University of Athens, Greece). PFGE analysis was performed by M. A. Dominguez (Hospital de Bellvitge, Barcelona, Spain). This work was supported by grant 00/0242 from the Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo, Spain (to L.M.-M.). J.M.R.-M. received a grant from Asociación Virgen Macarena (Seville, Spain).


    Footnotes
 
* Correspondence address. Department of Microbiology, School of Medicine, University of Seville, C/Sanchez Pizjuan s/n, 41009, Seville, Spain. Tel: +34-9545-52862; Fax: +34-9541-77413; E-mail: jmrodriguez{at}us.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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4 . Martínez-Martínez, L., Pascual, A., Conejo, M. C. et al. (2002). Energy-dependent accumulation of norfloxacin and porin expression in clinical isolates of Klesiella pneumoniae and relationship to extended-spectrum beta-lactamase production. Antimicrobial Agents and Chemotherapy 46, 3926–32.[Abstract/Free Full Text]

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