JAC Advance Access originally published online on July 27, 2007
Journal of Antimicrobial Chemotherapy 2007 60(4):868-871; doi:10.1093/jac/dkm266
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Prevalence and diversity of qnr alleles in AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens: a multicentre study from Korea
1 Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Kangnam St Mary's Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea 2 Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Holy Family Hospital, Sosa-dong, Wonmi-gu, Bucheon, Kyunggi-do 420-717, Korea 3 Korea Food and Drug Administration, 231 Jinheungno, Eunpyeong-gu, Seoul 122-704, Korea
* Correspondence address. Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Kangnam St Mary's Hospital, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea. Tel: +82-2-590-1604; Fax: +82-2-590-2547; E-mail: yjpk{at}catholic.ac.kr
Received 12 April 2007; returned 27 April 2007; revised 23 June 2007; accepted 25 June 2007
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Abstract |
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Objectives: To investigate the prevalence of qnr determinants, their influence on quinolone susceptibility and their association with other plasmid-mediated genes in AmpC-producing Enterobacteriaceae without any selection criteria.
Methods: A total of 644 consecutive, non-duplicate isolates of Enterobacter cloacae (186), Enterobacter aerogenes (154), Citrobacter freundii (138) and Serratia marcescens (166) were examined. We performed antimicrobial susceptibility testing and PCR for qnr determinants (qnrA, qnrB and qnrS), extended-spectrum ß-lactamase (ESBL) (blaTEM, blaSHV and blaCTX-M), orf513, orf1005 and blaDHA-1. To differentiate qnr subtypes, restriction enzyme analysis and sequencing was performed.
Results: The prevalence of qnr determinants was high in C. freundii (38.4%) and E. cloacae (28.5%), but low in E. aerogenes (3.2%) and S. marcescens (2.4%). qnrA1 was most frequent in E. cloacae, and qnrB was prevalent in C. freundii. All the qnrA- and qnrB4-positive isolates showed ciprofloxacin MICs 
0.5 mg/L and nalidixic acid MICs 16 mg/L. However, the B1 and B2 subtypes showed a wide range of quinolone MICs. In relation to ESBLs, we found that qnrA1, qnrB2 and qnrB4 producers were significantly more frequent among ESBL producers (P < 0.05). Twelve of 13 qnrB4 producers harboured blaDHA-1. orf513 was detected in 43 isolates of the 47 isolates with co-resident qnr and ESBL genes. None of the qnr producers harboured orf1005.
Conclusions: The prevalence of qnrA and qnrB was high among C. freundii and E. cloacae in Korea and there were characteristics unique to the qnr subtypes. Quinolones should be used cautiously in these species, especially when they are ESBL producers.
Keywords: quinolones , resistance , orf513 , ESBLs
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Introduction |
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The qnr genes (qnrA, qnrB and qnrS) encode pentapeptide repeat proteins that block the action of ciprofloxacin on bacterial DNA gyrase and topoisomerase IV1 and, most importantly, facilitate the selection of chromosomal mutants in the presence of a quinolone.2
The qnrA1 allele has been found in plasmids with a variety of other resistance determinants, but always as part of a complex sul1-type integron containing orf513.3 qnrB is associated with extended-spectrum ß-lactamases (ESBLs) such as SHV-12 and is often located near orf1005.1 The qnrB4 allele has been associated with a plasmid-mediated AmpC-type ß-lactamase, DHA-1, in Klebsiella pneumoniae.4 qnrS was first detected in Shigella flexneri from Japan5 but, in contrast to qnrA and qnrB, has been found only rarely and mainly identified in non-ESBL strains.6
Although there are a few studies of qnr prevalence, they were performed with selected isolates showing decreased susceptibility to quinolones or ceftazidime resistance.7,8 In this study, we investigated the prevalence of qnr determinants (qnrA, qnrB and qnrS), the correlation with quinolone (ciprofloxacin and nalidixic acid) MICs and their association with ESBLs, orf513 and orf1005 in AmpC-producing strains of Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens collected from various parts of Korea without any selection criteria.
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Materials and methods |
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Bacterial isolates and susceptibility testing
A total of 644 consecutive, non-duplicate isolates, including E. cloacae (186), E. aerogenes (154), C. freundii (138) and S. marcescens (166), were collected from clinical specimens at 12 clinical laboratories in Korea between March and July 2005.
The MICs of ciprofloxacin (0.06–64 mg/L), nalidixic acid (0.5–256 mg/L) and cefepime (0.125–2 mg/L) were determined by an agar dilution method.9
PCR amplification of qnr determinants and associated genes
The presence of qnrA, qnrB, qnrS, orf513 and orf1005 was detected by PCR as described previously.1,7 For isolates showing cefepime MICs 0.5 mg/L, PCR for blaTEM, blaSHV and blaCTX-M-1, -M-2, -M-9 was performed with the following sets of primers: TEM-1F, 5'-ATA AAA TTC TTG AAG ACG AAA-3' and TEM-1B, 5'-GAC AGT TAC CAA TGC TTA ATC A-3' for blaTEM; SHV-1F, 5'-TGG TTA TGC GTT ATA TTC GCC-3' and SHV-1B, 5'-GGT TAG CGT TGC CAG TGC T-3' for blaSHV; CTX-M-1F, 5'-AAA AAT CAC TGC GCC AGT TC-3' and CTX-M-1B, 5'-AGC TTA TTC ATC GCC ACG TT-3', CTX-M-2F, 5'-CGA CGC TAC CCC TGC TAT T-3' and CTX-M-2B, 5'-CCA GCG TCA GAT TTT TCA GG-3', CTX-M-9F, 5'-CAA AGA GAG TGC AAC GGA TG-3' and CTX-M-9B, 5'-ATT GGA AAG CGT TCA TCA CC-3' for blaCTX-M-1, -M-2, -M-9.
For blaTEM PCR, plasmids were extracted with a Solgent DNA extraction kit (Solgent, Seoul, Korea), and PCR products were purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced on a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
To differentiate qnrA1 from the qnrA2–5 alleles, the 516 bp PCR products were digested with PstI (A1, expected products of 82 and 434 bp; A2–A5, remain undigested). To discriminate between qnrB alleles, the 469 bp PCR products were digested with DsaI (New England Biolabs, Beverly, MA, USA) (B1 and B3, 73, 76 and 320 bp; B2, 76 and 393 bp; B4, 50, 76 and 343 bp; B5, 149 and 320 bp; B6, 469 bp) and then run on a 10% (w/v) polyacrylamide gel. To differentiate B1 from B3, the PCR products were also digested with BglII (B1, 146 and 323 bp; B3, 469 bp). Selected qnrA, qnrB and all the qnrS PCR products were purified and sequenced on a 3730 DNA Analyzer (Applied Biosystems). For qnrB4-harbouring isolates, PCR for blaDHA-1 was also performed with the primers DHAM-F, 5'-AAC TTT CAC AGG TGT GCT GGG T-3' and DHAM-B, 5'-CCG TAC GCA TAC TGG CTT TGC-3'. The nucleotide and deduced protein sequences were analysed with software available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The PCR-NheI method10 and sequencing was used to discriminate between blaSHV ESBL and blaSHV non-ESBL genes.
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Results |
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Prevalence of qnr determinants and their distribution according to quinolone MICs
Of the 644 isolates, 89/186 (47.8%) E. cloacae, 26/154 (16.9%) E. aerogenes, 65/138 (47.1%) C. freundii and 110/166 (66.3%) S. marcescens isolates showed ciprofloxacin MICs of 0.25 mg/L and/or nalidixic acid MICs of 16 mg/L. Among them, 51 (57.3%) E. cloacae, 5 (19.2%) E. aerogenes, 39 (60%) C. freundii and 2 (1.8%) S. marcescens isolates harboured qnr determinants. Of the remaining 354 isolates, 0/225 Enterobacter spp. harboured qnr alleles, but 14/73 (19.2%) isolates of C. freundii and 2/56 (3.6%) isolates of S. marcescens harboured qnrB. qnr-positive isolates of E. cloacae, E. aerogenes and C. freundii were obtained from 10, 2 and 12 clinical laboratories, respectively. In S. marcescens, only one isolate harboured qnrA and the three that harboured qnrB were from three different hospitals.
The prevalence of qnr determinants was highest among C. freundii (38.4%), followed by E. cloacae (28.5%), E. aerogenes (3.2%) and S. marcescens (2.4%) (Table 1). All qnrA PCR products were digested by PstI, indicating qnrA1, which was confirmed by sequencing. This was the most frequently detected qnr allele in E. cloacae, whereas in C. freundii all but one of the qnr determinants was a qnrB variant (alleles B2, B1 and B4 in order of frequency). Among qnrB producers, the B2 and B4 alleles were found in E. cloacae, B4 and B6-like (Asp202Asn) in E. aerogenes, B1, B2 and B4 in C. freundii and B1 and B4 in S. marcescens. Overall, qnrB2 was the most frequent, followed by the B1, B4 and B6-like alleles. All but one of the qnrB4 producers also harboured blaDHA-1. One E. cloacae harboured both qnrA and qnrB alleles, and two E. cloacae isolates harboured qnrS1.
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All qnrA1- and qnrB4-positive isolates showed ciprofloxacin MICs of
0.5 mg/L and/or nalidixic acid MICs of 16 mg/L, but qnrB1- and qnrB2-positive isolates had a wider range of quinolone MICs, including full susceptibility (ciprofloxacin, 0.06–32 mg/L; nalidixic acid, 0.5–256 mg/L) (Figure 1).
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Association of qnr determinants with ESBLs, orf513 and orf1005
The ESBL production rates among E. cloacae, E. aerogenes, C. freundii and S. marcescens were 32.8%, 10.4%, 2.9% and 12.7%, respectively (Table 1). Among the ESBL producers, 62.3% of E. cloacae, 31.3% of E. aerogenes and 75% of C. freundii harboured qnr determinants. In contrast, only 4.8% of ESBL-producing S. marcescens harboured qnr. Among the qnr subtypes, qnrA1, qnrB2 and qnrB4 were significantly more frequent among ESBL producers (P < 0.05), whereas for qnrB1, no significant difference was observed between ESBL producers and non-producers (P = 0.12). Examining the ESBL types associated with qnr subtypes, the qnrA1 type was most frequently associated with CTX-M-9 (62.5%), and qnrB4 and qnrB2 with SHV types (38.5% and 26.1%). Regarding the association with orf513, it was high in qnrA1 and qnrB4 producers (93.8% and 92.3%, respectively) but lower (34.8%) in qnrB2 producers, and 0% in the B1 subtype. orf513 was detected in 43 of the 47 isolates co-producing qnr and ESBLs. None of the qnr producers harboured orf1005.
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Discussion |
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In this study, the occurrence of isolates with ciprofloxacin MICs of
0.25 mg/L and/or nalidixic acid MICs of 16 mg/L was highest in S. marcescens (66.3%), followed by E. cloacae (47.8%), C. freundii (47.1%) and E. aerogenes (16.9%), while the prevalence of qnr determinants was highest in C. freundii (38.4%) and E. cloacae (28.5%), but low in E. aerogenes (3.2%) and S. marcescens (2.4%). The occurrence of qnr carriage was high among ESBL-producing E. cloacae (62.3%) and C. freundii (75.0%), but lower in E. aerogenes (31.3%) and S. marcescens (4.8%). In addition, the occurrence of qnr determinants among ESBL-non-producers was higher (12.5%) compared with a French study (0%),8 which might be related to the fact that most of them produced qnrB, which the other authors did not seek.
Based on our results, there were some characteristics associated with particular qnr subtypes. First, there was a difference in the distribution of ciprofloxacin and nalidixic acid MICs; all the qnrA1 and qnrS1 producers showed ciprofloxacin MICs of 0.5 mg/L and nalidixic acid MICs of 16 mg/L (regardless of species). This is in line with the French study where none of the qnrA-positive isolates was susceptible to nalidixic acid.8 In contrast, the distribution of MICs of ciprofloxacin and nalidixic acid among qnrB producers varied. Examining the qnrB subtypes, the distribution of quinolone MICs varied among qnrB1- and qnrB2-positive isolates, but all the qnrB4-positive isolates showed ciprofloxacin MICs of 0.5 mg/L and nalidixic acid MICs of 16 mg/L.
Secondly, qnrA1 and B4 were both highly associated with ESBLs and orf513, but, unlike a previous report,6 not all of the qnrA1 producers were associated with orf513. In qnrB1-positive isolates, orf513 was not detected. The qnrB2-positive isolates showed an intermediate association. All but one of the qnrB4 producers also harboured blaDHA-1, as was reported in K. pneumoniae.4 Although only two E. cloacae isolates were found to produce qnrS1, neither of them produced an ESBL, which coincides with the finding of Poirel et al.6
In summary, in Korean isolates, qnrA and qnrB were prevalent in C. freundii and E. cloacae. Quinolones should be used more cautiously in these species, especially when they are ESBL producers.
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This work was supported by a grant from the Korea Food and Drug Administration in 2006 (FD100-06042).
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None to declare.
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Acknowledgements |
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We wish to thank all the contributing laboratories that provided isolates for this study. We are especially grateful to Prof. Nordmann (Service de Bactériologie-Virologie, Hôpital de Bicêtre, France), Prof. Kim (University of Ulsan College of Medicine, Korea) and Prof. Jacoby (Lahey Clinic, USA) for providing us with the qnr-positive strain, and are also indebted to Jung Jun Park and Kang Gyun Park for valuable technical assistance.
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References |
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