Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool L7 8XP, UK
Received 29 July 2005; returned 20 September 2005; revised and accepted 28 September 2005
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Abstract |
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Methods: Over a 29 month period, a total of 47 non-repetitive isolates of Enterobacteriaceae resistant to both ciprofloxacin and cefotaxime were identified. Isolates were screened for the presence of the qnrA gene, class I integrons and blaESBL by PCR. Transferability was examined by conjugation with the sodium azide-resistant Escherichia coli J53. All qnrA-positive isolates were examined for DNA-relatedness by PFGE.
Results: A total of 15 of the 47 test isolates (32%) were positive for the qnrA gene, and included single isolates of E. coli and Citrobacter freundii, 4 Klebsiella pneumoniae and 9 Enterobacter cloacae. All 15 qnrA-positive isolates carried class 1 integrons, and 11 the extended-spectrum ß-lactamase gene blaSHV-12. By PFGE two K. pneumoniae and three E. cloacae, respectively, were considered clonally but not temporally related. Plasmid transfer of quinolone resistance was only achieved with single isolates of K. pneumoniae and E. cloacae. Both plasmids carried class 1 integrons with a pSAL-1-like gene cassette arrangement intl1-aadA2-qacE-sul1.
Conclusions: In this selected group of ciprofloxacin- and cefotaxime-resistant bacteria, carriage of the qnrA gene was high (32%). This compares with <2.0% as demonstrated in worldwide studies of laboratory collections of ciprofloxacin-resistant bacteria. The majority of qnrA-positive isolates in our study originated from high-dependency care units within our hospital, but were shown not to be clonal by PFGE. This is the first report of qnrA-positive Enterobacteriaceae in the United Kingdom.
Keywords: fluoroquinolones , class 1 integrons , molecular epidemiology
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Introduction |
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The gene responsible for plasmid-mediated resistance qnrA (GenBank accession number AY070235) encodes the protein QnrA, a 218 amino acid pentapeptide that binds to subunits of topoisomerase IV, preventing further binding of quinolones. Introduction of plasmid pMG252 into E. coli strains with differing underlying mechanisms of quinolone insusceptibility increased quinolone resistance 4- to 128-fold.8 Furthermore, plasmid carriage by E. coli facilitated the selection of chromosomal resistance mutations by significantly raising the level at which mutants could be selected.1
Most qnrA-positive enterobacterial isolates have been associated with the plasmid-mediated AmpC-type ß-lactamase (pACBL) FOX-5 or clavulanic acid-inhibited extended-spectrum ß-lactamases (ESBLs). As a departmental policy we retain all bacteraemic isolates, therefore we were able to undertake a study to determine the prevalence of qnrA-positive isolates from a collection of Enterobacteriaceae resistant to both ciprofloxacin and cefotaxime isolated from episodes of bacteraemia at the Royal Liverpool University Teaching Hospital (RLUH) over the period January 2003 to May 2005.
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Material and methods |
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A computerized search of blood culture isolates was undertaken for Enterobacteriaceae reported as being both cefotaxime (potential ESBL producers) and ciprofloxacin resistant. A total of 47 non-repetitive isolates were identified and included, 1 C. freundii, 16 E. cloacae, 13 E. coli, 15 K. pneumoniae and 2 Morganella morganii. E. coli J53 (sodium azide-resistant) was used as a conjugation recipient, and E. coli 39R861 (NCTC 50192) and V517 (NCTC 50193) as standards for plasmid analysis.
Susceptibility testing
Disc susceptibility testing was performed according to BSAC guidelines9 on Iso-Sensitest agar (Oxoid Ltd, Basingstoke, UK). Detection of ESBLs was achieved with a combination of the Mast ESBL set (Mast Laboratories Ltd, Bootle, Merseyside, UK) and cefpirome discs with and without clavulanic acid (Oxoid Ltd). MICs of nalidixic acid and ciprofloxacin were determined by the Etest method (AB Biodisk, Solna, Sweden).
Plasmid analysis and conjugation experiments
Conjugal transfer of resistance determinants was performed by both broth culture and on 0.45 µM nitrocellulose membranes (Millipore Corporation, Billerica, MA, USA) with E. coli J53 as recipient. After 24 h of incubation, mating mixtures were plated onto agar containing sodium azide (100 mg/L) supplemented with either trimethoprim (10 mg/L) or chloramphenicol (30 mg/L). Plasmid DNA was prepared from donors and transconjugants using a commercial kit (Plasmid Mini Kit, Qiagen GmbH, Hilden, Germany).
Genomic analysis
PFGE was performed with a CHEF DR III system (Bio-Rad, Hemel Hempstead, UK). DNA insert blocks were digested overnight with XbaI at 37°C.
PCR and sequencing
Total DNA for PCR was extracted by suspending bacteria in 5% Chelex-100 resin slurry (Bio-Rad) in injection grade water followed by boiling for 10 min. PCR was performed to detect the following: qnrA,2 blaSHV,10 TEM, CTX-M, VEB-1, PER-1, pACBL and class 1 integron cassette structures 3'-CS and 5'-CS conserved segments (integron variable region containing gene cassettes), intI1 integrase gene and sul1 conferring resistance to sulphonamides.11 PCR products were detected by electrophoresis on 1.0% (w/v) agarose gels and extracted with a commercial kit (QIAquick gel extraction kit, Qiagen GmbH). Sequence determination of amplicons for both parents and transconjugants was performed on both strands using the respective PCR primers. Nucleotide sequence structures were compared with the following DDBJ/EMBL/GenBank accession numbers: qnrA, AY070235; SHV-1 X98100; aadA2, AJ517791, dfr16-aadA2, AY259085 and aacA4-aadA2, L06822.
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Results |
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Plasmid transfer was achieved for five isolates but quinolone resistance was only detected in the E. coli J53 transconjugants (TCJs) crossed with K. pneumoniae QNR1 (E. coli TCJ-QNR1, plasmid 120 kb) and E. cloacae QNRQ3 (E. coli TCJ-QNRQ3, plasmid
160 kb). Both qnrA-positive plasmids carried blaSHV-12 and class 1 integrons with a pSAL-1-like gene cassette arrangement intl1-aadA2-qacE
-sul1. Quinolone resistance in E. coli TCJ-QNR1 increased 6-fold for nalidixic acid (MIC 4.024 mg/L) and 12.5-fold for ciprofloxacin (MIC 0.0080.1 mg/L) and in the E. coli TCJ-QNRQ3 12-fold for nalidixic acid (MIC 4.048 mg/L) and 24-fold for ciprofloxacin (MIC 0.0080.19 mg/L). Resistance to amoxicillin, trimethoprim, gentamicin, kanamycin, cefotaxime, ceftazidime, aztreonam, sulphonamide and chloramphenicol was co-transferred on both plasmids. However, tetracycline resistance was only transferred to the E. coli TCJ-QNRQ3.
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Conclusions |
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By using PCR, a high prevalence of the plasmid-mediated quinolone resistance determinant qnrA was detected [15 of 47 (32%) ciprofloxacin- and cefotaxime-resistant Enterobacteriaceae isolated from bacteraemic patients at the RLUH]. The majority of isolates originated from high-dependency care units. Despite the international distribution of the qnrA determinant, our sequence analyses confirmed the maintained homogeneity of this gene.
PFGE has shown that most of our isolates were not clonal, yet 11 isolates, including K. pneumoniae, E. cloacae and C. freundii, were characterized by carriage of class 1 integrons with a similar gene cassette (aadA2) and expressing the ESBL blaSHV-12. Other studies have demonstrated the association of ESBLs and pACBLs with the qnrA determinant, but this is the first report of association with SHV-12. Transferability of plasmid-mediated resistance was low, with only 2 of the 15 isolates producing quinolone-resistant transconjugants. Nalidixic acid resistance increased 6- to 12-fold and ciprofloxacin resistance increased 12- to 24-fold. The plasmids involved were large (120 and 160 kb), and carried class 1 integrons and multiple resistance determinants including blaSHV-12. Other studies have also shown that not all qnrA-positive isolates were able to transfer quinolone resistance.5,7
This combined carriage of the qnrA determinant with blaSHV-12 on similar class 1 integrons and its dissemination among differing Enterobacteriaceae in our institution may reflect a localized event. However, in the absence of other reports it may herald the unrecognized spread of integron-carrying bacteria in high-dependency units within UK hospitals.
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Transparency declarations |
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Acknowledgements |
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