JAC Advance Access originally published online on March 20, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1234-1239; doi:10.1093/jac/dkn111
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Original research |
Prevalence of Qnr determinants among bloodstream isolates of Escherichia coli and Klebsiella pneumoniae in a Taiwanese Hospital, 1999–2005
1 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan 2 Department of Internal Medicine, National Cheng Kung University Hospital, Tainan 70428, Taiwan 3 Department of Pathology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan 4 Department of Pathology, National Cheng Kung University Hospital, Tainan 70428, Taiwan
* Correspondence address. Department of Pathology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 70428, Taiwan. Tel: +886-6-235-3535; Fax: +886-6-276-6195; E-mail: jingjou{at}mail.ncku.edu.tw
Received 2 October 2007; returned 3 December 2007; revised 14 February 2008; accepted 19 February 2008
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Abstract |
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Objectives: To determine the prevalence and characteristics of bloodstream isolates of Escherichia coli and Klebsiella pneumoniae with qnr genes in a Taiwanese hospital.
Methods: A total of 2035 E. coli and 1147 K. pneumoniae isolates collected between 1999 and 2005 were screened for qnrA, qnrB and qnrS by PCR and colony hybridization. β-Lactamase genes, genetic relatedness and transferability were examined by PCR, PFGE and conjugation, respectively.
Results: The prevalence of qnr genes was 7.8% and 0.6% for K. pneumoniae and E. coli, respectively. The prevalence rates of qnrB2, qnrB4 and qnrS1 genes for K. pneumoniae were 2.3%, 3.6% and 2.8%, respectively, and for E. coli were 0.2%, 0% and 0.4%, respectively. The prevalence of qnrB4 in K. pneumoniae increased remarkably from 0% to 7.6% over the 7 study years. qnrA was not detected. Overall, the SHV-5-related, CTX-M-14, CTX-M-3, CMY-2, DHA-1 and IMP-8 β-lactamases were detected alone or in combination in 82.0% of qnr-positive K. pneumoniae isolates and 41.7% of qnr-positive E. coli isolates. Notably, all qnrB4-positive isolates possessed the DHA-1 enzyme, and the majority of the qnrB2-positive isolates (E. coli, 100%; K. pneumoniae, 80.8%) produced SHV-5-related β-lactamases. Genetic diversity was demonstrated by PFGE. Conjugation experiments revealed co-transfer of blaSHV-12, blaDHA-1 and blaSHV-5 with qnrB2, qnrB4 and qnrS1, respectively.
Conclusions: qnr genes remained rare in E. coli but appeared to be increasing in K. pneumoniae in our hospital. Horizontal transfer may play a major role in the intra-hospital spread of qnr.
Keywords: quinolones , cephalosporinases , extended-spectrum β-lactamases , metallo-β-lactamases
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Introduction |
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Qnr-type plasmid-mediated quinolone resistance determinants belong to the pentapeptide-repeat family of proteins,1,2 and they confer low-level resistance to quinolones probably by protecting DNA gyrase directly from quinolone inhibition.2,3 Three major groups of Qnr determinants, QnrA, QnrB and QnrS, have been identified worldwide in various members of the family Enterobacteriaceae and are often linked to extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC enzymes.4–18
Our previous study showed the emergence of QnrA1, QnrB2 and QnrS1 variants in Enterobacter cloacae and the association of QnrB2 with the SHV-12 ESBL and the IMP-8 metallo-β-lactamase (MBL) in Taiwan.16 Another study revealed the coexistence of qnrS1 and blaSHV-2 on a plasmid from a Klebsiella pneumoniae strain.18 The present study was conducted to determine the prevalence of Qnr determinants among bloodstream isolates of Escherichia coli and K. pneumoniae collected over a period of 7 years and the association of Qnr determinants with various plasmid-encoded β-lactamases in a teaching hospital in Taiwan.
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Materials and methods |
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Bacterial isolates
Between 1999 and 2005, all bloodstream isolates of E. coli and K. pneumoniae isolated at National Cheng Kung University Hospital, a 1083-bed teaching hospital in southern Taiwan, were collected. One isolate per patient was selected for this study. Thus, a total of 2035 E. coli and 1147 K. pneumoniae isolates were analysed.
The presence of qnrA, qnrB and qnrS genes was detected by colony hybridization and PCR as described previously.16 The primers used for PCR are shown in Table 1. PCR detection of qnrB genes was performed with universal primers qnrB-CS-1A and qnrB-CS-1B first and then with primer pairs for qnrB2-like and qnrB4-like genes. All PCR products were purified with a commercial kit (Roche Applied Science, Mannheim, Germany) and sequenced on both strands.
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Susceptibility testing
The disc diffusion method for screening and confirming ESBL-producing isolates was performed as described by CLSI.19 Discs of ceftazidime, cefotaxime, ceftriaxone, aztreonam and cefpodoxime were used in the screening tests. Potential ESBL producers negative by the CLSI confirmatory method were examined further by the double-disc synergy test to detect false-negatives due to co-production of ESBLs and AmpC enzymes or MBLs.20,21 Isolates resistant to both ceftazidime and cefoxitin were also screened for MBL production by the 2-mercaptopropionic acid double-disc potentiation method as described previously.22 MICs of various antimicrobials for qnr-positive isolates were determined by the agar dilution method according to the CLSI guidelines.23
The expression of β-lactamases was analysed by isoelectric focusing as described previously.24,25 PCR detection of blaSHV, blaTEM and bla genes related to blaCTX-M-1, blaCTX-M-9, blaCMY-2, blaDHA-1 and blaIMP-2 was performed with primers described elsewhere.16,26,27 The PCR-NheI method was used to discriminate SHV-type ESBLs from non-ESBLs.28 All PCR products except blaSHV amplicons from clinical isolates were sequenced.
PFGE analysis of XbaI-digested genomic DNA was performed using a CHEF-DR 3 apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the instruction manual. PFGE patterns were interpreted according to the criteria of Tenover et al.29 Two isolates were considered clonally related when they had PFGE patterns that were identical or different by no more than four bands.
Conjugation experiments and plasmid analysis
The liquid mating-out assay was carried out to transfer qnr genes from K. pneumoniae isolates to streptomycin-resistant E. coli C600 as described previously.25,30 Transconjugants were selected on tryptic soy agar plates containing 512 mg/L streptomycin (Sigma Chemical Co., St. Louis, MO, USA) and 8 mg/L nalidixic acid (Sigma). Plasmid DNA samples extracted from transconjugants were digested with the endonuclease EcoRI (Roche Applied Science). The resulting fragments were loaded onto a 0.8% agarose gel and then subjected to Southern hybridization with the digoxigenin-labelled qnr probes. The sizes of the transferred plasmids were estimated by adding up the sizes of their restriction fragments.
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Results and discussion |
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Overall, 89 (7.8%) of all K. pneumoniae isolates and 12 (0.6%) of all E. coli isolates were found to carry qnrB, qnrS or both by colony hybridization and PCR; qnrA was not detected. qnrB was more prevalent than qnrS in K. pneumoniae (5.4% versus 2.8%) as was found in E. cloacae at this hospital,16 whereas qnrS was more prevalent than qnrB in E. coli (0.4% versus 0.2%) (Table 2). QnrB determinants have also been reported to be dominant in the Middle East and Korea.8,14,15,17
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All 40 sequenced qnrS genes were 100% identical to the qnrS1 allele. Among the qnrB-positive isolates, all 4 E. coli isolates and 26 of 62 K. pneumoniae isolates carried the qnrB2-like gene; 41 K. pneumoniae isolates carried the qnrB4-like gene, indicating that qnrB4 can be amplified with primers qnrB-CS-1A and qnrB-CS-1B, despite the presence of a mismatched nucleotide. It should be noted that several novel qnrB variants such as qnrB8 and qnrB9 may not be detected with the universal primers because of their high polymorphism. The whole qnrB genes from two qnrB2-like-positive and two qnrB4-like positive K. pneumoniae isolates were determined and confirmed to be qnrB2 and qnrB4 (data not shown). Five K. pneumoniae isolates carried qnrB2 and qnrB4 concurrently, and the coexistence of qnrS with qnrB2 and qnrB4 was recognized in two and three K. pneumoniae isolates, respectively. Overall, qnrB4 was more prevalent than qnrB2 in K. pneumoniae (3.6% versus 2.3%), and it was also the most common qnr variant in K. pneumoniae as was found in Korea.14 The prevalence of qnrB4-positive isolates in K. pneumoniae increased from 0% in 1999 to 7.6% in 2005; there was no evident change in the prevalence of qnrS and qnrB2 during the study period (Table 2).
Of the 12 qnr-positive E. coli isolates, 75.0% were non-susceptible (resistant or intermediately resistant) to nalidixic acid (MIC 16 to >256 mg/L; MIC90 >256 mg/L) and 66.7% to ciprofloxacin (MIC 0.13 to 256 mg/L; MIC90 128 mg/L). Of the 89 qnr-positive K. pneumoniae isolates, 95.5% were non-susceptible to nalidixic acid (MIC 16 to >256 mg/L; MIC90 >256 mg/L) and 88.8% to ciprofloxacin (MIC 0.13 to >256 mg/L; MIC90, 256 mg/L). More than 50% of the qnr-positive isolates were non-susceptible to any of ceftotaxime (62.4%), ceftazidime (66.3%) and cefepime (15.8%). Resistance to cefoxitin was noted in all qnrB4-positive isolates and less common in the qnrS-positive isolates (27.5%) and qnrB2-positive isolates (50.0%).
The phenotypic detection tests suggested among the qnr-positive isolates the production of ESBLs in 5 (41.7%) of the 12 E. coli isolates and in 47 (52.8%) of the 89 K. pneumoniae isolates and the production of MBLs in 1 (8.3%) E. coli and 7 (7.9%) K. pneumoniae isolates. By isoelectric focusing and PCR experiments, the SHV-5-related (pI = 8.2), CTX-M-3 (pI = 8.4) and CTX-M-14 (pI = 8.0) ESBLs were detected alone or in combination in all putative ESBL producers. A high prevalence of SHV ESBLs was observed among the qnrB2-positive isolates (100% for E. coli and 80.8% for K. pneumoniae) as was found in Korea,14 Kuwait8 and the USA.6 CTX-M-3 was detected in one qnrB2-positive E. coli isolate and six K. pneumoniae isolates (one qnrB2, four qnrB4 and one qnrS), and CTX-M-14 was only found in qnrB2-positive isolates (two E. coli and one K. pneumoniae). The CMY-2 AmpC enzyme (pI = 9.0) was only detected in qnrS-positive isolates (two E. coli and one K. pneumoniae), and the DHA-1 AmpC enzyme (pI = 7.8) was detected in all 41 qnrB4-positive K. pneumoniae isolates. The striking association between DHA-1 and QnrB4 has been reported in Korea.14,15 The IMP-8 MBL (pI = 8.2) was identified in all putative MBL producers (one qnrS-positive and six qnrB2-positive K. pneumoniae and one qnrB2-positive E. coli). Overall, β-lactamases that confer resistance to extended-spectrum cephalosporins were detected in 73 (82.0%) of all qnr-positive K. pneumoniae isolates and 5 (41.7%) of all qnr-positive E. coli isolates. The prevalence rates of such enzymes altogether were 100% and 37.5% among the qnrB2-positive and qnrS-positive E. coli isolates, respectively, and 84.6%, 100% and 62.5% among the qnrB2-positive, qnrB4-positive and qnrS-positive K. pneumoniae isolates, respectively. The TEM-1 narrow-spectrum β-lactamase was detected in 10 qnrS-positive, 17 qnrB2-positive and 25 qnrB4-positive K. pneumoniae isolates and in all qnr-positive E. coli isolates.
Forty-two qnr-positive K. pneumoniae isolates collected in 2004 and 2005 were subjected to PFGE typing. Thirty-three isolates gave 23 major patterns, designated patterns I–XXIII, and nine isolates were not typeable, probably due to DNA lysis. Sixteen qnrB4-positive, 8 qnrB2-positive and 11 qnrS-positive isolates gave 11 (patterns I–XI), 7 (patterns IX and XII–XVII) and 6 (patterns XVIII–XXIII) major patterns, respectively. Each of the 23 PFGE patterns represented one to three isolates, and pattern IX represented two isolates carrying both qnrB2 and qnrB4. Two, two and three subtypes were obtained among patterns I, V and XX, respectively. The PFGE analysis of representative isolates indicates that the prevalence of qnr in K. pneumoniae in the teaching hospital was mainly attributable to horizontal transfer rather than intrahospital spread of outbreak clones.
By conjugation experiments, the qnr genes were transferred from 18 of the 42 K. pneumoniae isolates collected in 2004 and 2005 to E. coli C600. The sizes of the transferred plasmids were all >80 kb, and the transfer of qnr genes was confirmed by Southern hybridization (Figure 1). One donor strain had both qnrB2 and qnrB4, but only qnrB2 was transferred (ekp-1385/05). All transconjugants revealed decreased susceptibilities to nalidixic acid (MIC 16–32 mg/L) and ciprofloxacin (MIC 0.25–0.5 mg/L). The characteristics of the transconjugants are summarized in Table 3. The coexistence of blaDHA-1, blaSHV-12 and blaSHV-5 with qnrB4, qnrB2 and qnrS, respectively, was observed. Obviously, different restriction patterns were observed among the qnr plasmids, suggesting the occurrences of horizontal mobility of the qnr genes. The qnrB2 and qnrB4 genes have been described in complex sul1-type integrons,31,32 and the qnrS1 gene has been reported in an IS2-like element.13 Genetic mechanisms responsible for the mobility of the qnr genes in Taiwan are not known and deserve further studies.
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Funding |
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This work was funded by a grant NSC 96-2320-B-006-054 from the National Science Council, Taiwan.
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Transparency declarations |
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None to declare.
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References |
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