Graduate School, 2 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, and 3

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1 Jpn. J. Infect. Dis., 66, , 2013 Short Communication Plasmid-Mediated Quinolone Resistance Genes, aac(6?)-ib-cr, qnrs, qnrb, andqnra, in Urinary Isolates of Escherichia coli and Klebsiella pneumoniae at a Teaching Hospital, Thailand Woravit Pasom 1, Aroonwadee Chanawong 2 *, Aroonlug Lulitanond 2, Chotechana Wilailuckana 2, Suthida Kenprom 3, and Pirom Puang-Ngern 3 1 Graduate School, 2 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, and 3 Clinical Microbiology Laboratory, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand (Received February 27, Accepted May 15, 2013) SUMMARY: A total of 121 Escherichia coli (47 extended-spectrum b-lactamase [ESBL] and 74 non- ESBL producers) and 75 Klebsiella pneumoniae isolates (49 ESBL and 26 non-esbl producers) were collected from urine samples between October 2010 and April 2011 at a university hospital and assessed for the presence of plasmid-mediated quinolone resistance (PMQR) genes. Twenty-seven E. coli (22.3z) and49k. pneumoniae (65.3z) isolates harbored PMQR genes, which mostly consisted of aac(6?)-ib-cr and qnrs, followed by qnrb and qnra.amongthe76pmqr-positiveisolates,15(19.7z) and 2 (2.6z) carried 2 and 3 different PMQR genes, respectively. However, qnrc, qnrd, andqepa were not found in any isolate. The PMQR genes were more prevalent in ESBL producers than in non- ESBL producers (42.6z versus 9.5z in E. coli and 81.6z versus 34.6z in K. pneumoniae). Approximately 35z 60z of the PMQR-positive isolates were susceptible or intermediately susceptible to fluoroquinolones. The enterobacterial repetitive intergenic consensus-pcr method revealed that most PMQR-positive isolates belonged to different strains, indicating the spread of these resistance determinants. PMQR gene transfer by conjugation was successful in 10z 25z of the test donors. This study showed a high prevalence of PMQR genes among both organisms. Clinical use of fluoroquinolones for the treatment of infections caused by fluoroquinolone-susceptible strains harboring PMQR genes may lead to decreased therapeutic efficacy. The primary mechanism of fluoroquinolone resistance in members of the family Enterobacteriaceae is mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes encoded on bacterial chromosomes (1). However, plasmidmediated quinolone resistance (PMQR) was first discovered in a Klebsiella pneumoniae isolate from the United States in 1994 (2). This protein was called the quinolone-resistance protein (later named QnrA1). It involves quinolone resistance by protecting DNA gyrase and topoisomerase IV from quinolones (3). Till date, five Qnr proteins have been identified; QnrA, QnrB, QnrS, QnrC, and QnrD (4). The second PMQR mechanism involves a variant of aminoglycoside acetyltransferase (AAC(6?)-Ib-cr), which can reduce ciprofloxacin activity by adding an acetyl group to this agent (5). The last mechanism of PMQR is the quinolone efflux pump (QepA), a proton-dependent transporter, which causes hydrophilic quinolone resistance, particularly to norfloxacin, ciprofloxacin, and enrofloxacin (6). These forms of PMQR have been reported in clinical isolates of Enterobacteriaceae worldwide (4,7). *Corresponding author: Mailing address: Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Tel & Fax: aroonwad@kku.ac.th Because PMQR confers low-level resistance to fluoroquinolones, PMQR-positive isolates remain susceptible to these agents. However, in-vitro studies have shown that the selection of isolates with greater fluoroquinolone resistance can occur in isolates carrying PMQR genes after antimicrobial exposure (2,8). Therefore, the detection of PMQR in Enterobacteriaceae is required. The fluoroquinolone disk diffusion test, which is a simple routine laboratory method, could not detect PMQRpositive isolates because of the low-level resistance. Till date, molecular techniques such as PCR assays are required as confirmatory tests for PMQR detection in epidemiological studies. In Thailand, QnrA was first reported in Escherichia coli, K. pneumoniae, Enterobacter cloacae, and Enterobacter sakazakii producing VEB-1 extended-spectrum b-lactamase (ESBL) in 1999 (9). Subsequently, QnrS1 was discovered in Salmonella enterica serovar Corvallis isolates from humans, chickens, pigs, cattle, and seafood imported from Thailand and in S. enterica serovar Stanley from Finnish tourists returning from Thailand (10 12), suggesting an international spread of these resistance determinants. In our hospital, susceptibility rates to fluoroquinolones using the disk diffusion tests in 2010 were 13z 22z and 16z 73z in ESBLand non-esbl-producing E. coli, respectively, and 24z 57z and 30z 93z in ESBL- and non-esblproducing K. pneumoniae, respectively. These ESBL- 428

2 and non-esbl-producing fluoroquinolone-susceptible isolates may contain PMQR genes, and the clinical use of these antimicrobial agents may cause treatment failure. Till date, there are few reports on PMQR genes in clinical isolates from Thailand. In addition, fluoroquinolones are drugs of choice for the treatment of urinary tract infections, of which E. coli and K. pneumoniae are common etiological agents. Therefore, we detected PMQR genes in urinary isolates of both organisms by PCR techniques and then characterized the PMQR-positive isolates by phenotypic and genotypic examination. A total of 196 non-repetitive clinical isolates of E. coli (47 ESBL and 74 non-esbl producers) and K. pneumoniae (49 ESBL and 26 non-esbl producers) were obtained from positive urine cultures (showing bacterial growth of Æ10 5 CFU/mL) of patients at the Srinagarind Hospital, Khon Kaen University, Thailand from October 2010 to April They were selected on the basis of their antimicrobial disk susceptibility patterns and then assessed for the presence of PMQR genes using two sets of multiplex PCR assays (one for qnra, qnrb, andqnrs and the other for aac(6?)-ib and qepa) and using a simplex PCR assay for the detection of either qnrc or qnrd (13 16). Clinical isolates of Klebsiella spp. carrying qnra, Salmonella spp. carrying qnrs, Proteus mirabilis carrying qnrd (kindly provided by Dr. Tanittha Chatsuwan, Chulalongkorn University, Bangkok, Thailand), K. pneumoniae carrying qnrb1- like, and E. coli carrying aac(6?)-ib-cr (obtained from this study and confirmed by nucleotide sequencing) were used as positive controls for PCR amplification. Bacterial DNA was extracted using a rapid boiling method (17). PCR amplification was performed under the conditions described by Cattoir et al. (13). The 482-bp DNA fragment of aac(6?)-ib was then digested with the BstF5I restriction endonuclease (SibEnzyme Ltd., Novosibirsk, Russia) (15). The presence of 272-bp and 210-bp DNA fragments was indicative of aac(6?)- Ib, whereas an undigested fragment was indicative of aac(6?)-ib-cr. The qnrs, qnrb, qnra, and aac(6?)-ib DNA fragments from representative isolates were sequenced using the dideoxy-chain termination method (MegaBACE 1000; Amercham Biosciences, Champaign, Ill., USA). Sequence alignment and analysis were performed online using the BLAST program of the National Center for Biotechnology Information server ( Of the 121 E. coli isolates, 17 isolates carried qnrs, 12 carried aac(6?)-ib, and 1 carried qnra (Table 1). After BstF5I digestion, the aac(6?)-ib fragments from 11 of the 12 isolates were aac(6?)-ib-cr. Therefore, 27 of the 121 E. coli isolates (22.3z) were PMQR positive. Among the 75 K. pneumoniae isolates, 49 (65.3z) carried PMQR genes: 30 isolates with aac-(6?)-ib, 21with qnrs, 11withqnrB, and4withqnra. Subsequently, the aac(6?)-ib fragments from the 30 isolates were identified as aac(6?)-ib-cr. Nucleotide sequence determination of qnr from representative samples revealed that qnrs sequences from 3 E. coli isolates and 2 K. pneumoniae isolates were qnrs1-like, whereas qnra sequences from an E. coli isolate and 3 K. pneumoniae isolates were qnra1-like and qnrb sequences from all K. pneumoniae isolates were qnrb1-like (9 isolates), qnrb4-like (1 iso- Table 1. s of nalidixic acid, ciprofloxacin, and ofloxacin for the PMQR-positive E. coli and K. pneumoniae isolates Nalidixic acid (mg/l) Ciprofloxacin (mg/l) Ofloxacin (mg/l) No. of ERIC patterns (type) PMQR gene (No. of isolates) Organism (No. of isolates) qnrs (12) 2 À À À8 0.5 À À8 1 À8 7(A,B,F,G,H,I,J) aac(6?)-ib-cr (7) 4 À512 À512 À À8 À8 À À8 À8 À8 6(C,D,E,K,L,M) E. coli (27) ESBL (20) qnra, qnrs, aac(6?)-ib-cr (1) À512 À8 À8 1 (N) qnrs (4) 16 À À À8 1 À8 2 À8 4 À8 4(H,O,P,Q) aac(6?)-ib-cr (3) À512 À8 À8 3(C,R,S) aac(6?)-ib-cr (13) 2 À512 À512 À À8 À8 À À8 À8 À8 5(B,E,F,G,H) non-esbl (7) ESBL (40) K. pneumoniae (49) qnrs (12) 8 À À À (D, H, Q, R, T, U, V, W, X, Y, Z) qnra (2) 32 À512 2 À8 2 À8 2 (K, L) qnrb, aac(6?)-ib-cr (9) 8 À À À (A,C,M,N,O) qnrs, aac(6?)-ib-cr (2) (A, AG) qnra, aac(6?)-ib-cr (2) 16 À512 2 À8 1 À8 2 (I, J) qnrs (5) 8 À À À (S, AA, AB, AC, AD) aac(6?)-ib-cr (1) (A) qnrs, aac(6?)-ib-cr (1) (AF) qnrb, aac(6?)-ib-cr (1) À512 À8 À8 1 (P) qnrb, qnrs, aac(6?)-ib-cr (1) À512 À8 À8 1 (AE) non-esbl (9) 429

3 late), and qnrb6-like (1 isolate). In addition, the nucleotide sequences of the undigested 482-bp DNA fragments from 2 E. coli isolates were identical to that of aac(6?)- Ib-cr. The PMQR genes reported in our hospital were similar to those reported from many countries, particularly the People's Republic of China (7,18,19). They were found more frequently in the K. pneumoniae isolates than in the E. coli isolates (65.3z versus 22.3z). In addition, qnrs1-like was the most prevalent in the E. coli isolates, whereas aac-(6?)-ib-cr wasamajorpmqr genefoundinthek. pneumoniae isolates. However, qnrs was the most common qnr found in both the organisms. QnrS1 was previously reported in non-typhi S. enterica isolated from humans and food animals such as chickens, pigs, and cattle imported from Thailand (10 12). Quinolone use in agriculture may be involved in the spread of qnr-mediated resistance in Enterobacteriaceae (4). Among the PMQR-positive isolates, 19.7z (15 of 76 isolates) carried 2 different PMQR genes and 2.6z (2 isolates) carried 3 different PMQR genes (Table 1). Yang et al. (18) also reported the presence of different PMQR genes in clinical isolates of Citrobacter freundii, K. pneumoniae, ande. cloacae, indicating the wide spread of qnr and aac-(6?)-ibcr among clinical isolates of Enterobacteriaceae. The present study also revealed that PMQR genes were found more frequently in the ESBL producers than in the non-esbl producers in both E. coli (42.6z versus 9.5z) andk. pneumoniae (81.6z versus 34.6z). This is because the PMQR genes are usually associated with the same mobile genetic elements as those of ESBL genes (18,20,21). This study reported the presence of qnrb and aac-(6?)-ib-cr in Thai isolates in addition to the presence of qnra in Enterobacteriaceae and qnrs1 in Salmonella, as described previously (9 12). However, qnrc, qnrd, andqepa were not detected in any isolate. PMQR gene transfer was performed by a broth culture mating method using E. coli UB1637 (streptomycin resistant) as a recipient (22). Donor and recipient strains were separately grown in Luria-Bertani (LB) broth (Hardy Diagnostics, Santa Maria, Calif., USA) at 379C for 3 4 h with shaking. Following this, the donor culture was mixed with the recipient culture (1:25) and incubated for 3 4 h at 379C with shaking. Subsequently, transconjugants were selected on MacConkey agar plates containing 1,600 mg/l of streptomycin (M & H Manufacturing, Samutprakarn, Thailand) with either 16 mg/l of nalidixic acid or mg/l of ciprofloxacin or ofloxacin. The PMQR genes from 7 of 27 (25.9z) E. coli donors and 5 of 43 (11.6z) K. pneumoniae donors were transferred to the recipients as confirmed using PCR methods (Table 2). Only qnrs could be transferred from both organisms, with the exception that one E. coli donor transferred aac-(6?)-ib-cr in addition to qnrs. This study also showed cotransfer of ESBL genes with qnrs, similar to that reported previ- Table 2. s of nalidixic acid, ciprofloxacin, and ofloxacin for the PMQR-positive isolates and their transconjugants Organism isolate no. 1) ERIC type ESBL PMQR gene (mg/l) 2) NA CIP OFX Cotransfer of other antimicrobial resistance 3) E. coli 6 B + qnrs T + qnrs ESBL, GN, NET 17 B + qnrs T + qnrs ESBL, SXT 73 A + qnrs T + qnrs ESBL, SXT 122 G + qnrs T + qnrs ESBL, GN, NET 44 O + qnra, qnrs, aac(6?)-ib-cr À512 À16 À16 44T - qnrs, aac(6?)-ib-cr SXT, TE 92 H - qnrs À512 À16 À16 92T - qnrs Q - qnrs À512 À16 À16 110T - qnrs K. pneumoniae 26 Q + qnrs T + qnrs ESBL, SXT 31 R + qnrs T + qnrs ESBL, GN, NET 5 A + qnrs, aac(6?)-ib-cr 32 À16 2 5T - qnrs GN, NET 18 AE - qnrb, qnrs, aac(6?)-ib-cr À512 À16 À16 18T - qnrs SXT, TE 35 S - qnrs T - qnrs GN, NET E. coli UB Ã1 Ã0.008 Ã ) : T, transconjugant. 2) : NA, nalidixic acid; CIP, ciprofloxacin; OFX, ofloxacin. 3) : ESBL, extended-spectrum b-lactamase; GN, gentamicin; NET, netilmicin; SXT, trimethoprim/sulfamethoxazole; TE, tetracycline. 430

4 ously (18,20,23). Resistance to other antimicrobials, including gentamicin, netilmicin, trimethoprim/sulfamethoxazole, and tetracycline, was also cotransferred (Table 2). In a study by Cai et al. (24), transfer of qnr to E. coli C600Lac-SMR was successful in 40z of isolates (10 of 25 donor strains), and qnrs (4 isolates), qnrb (4 isolates), and qnra (2 isolates) were successfully transferred. The low transfer rate in this study may have been due to the use of different recipient strains or antimicrobials and their concentrations for selecting transconjugants. In addition, conjugation using the filter mating method was more effective than that using the broth mating method (25). The ability to transfer the PMQR genes indicated the potential spread of these resistance determinants among both the organisms. Minimum inhibitory concentrations (s) of nalidixic acid (1 512 mg/l), ciprofloxacin ( mg/l), and ofloxacin ( mg/l) (Sigma-Aldrich, St. Louis, Mo., USA) for all the PMQR-positive isolates were determined using an agar dilution method, and their susceptibilities to other antimicrobial agents (Oxoid, Basingstoke, Hampshire, England) were determined using a disk diffusion test (26). E. coli ATCC was used as an antimicrobial-susceptible control. s of nalidixic acid, ciprofloxacin, and ofloxacin for the PMQR-positive E. coli isolates d from 2 to À512, to À8, and to À8 mg/l, respectively, with 50 of À512, 4, and 8 mg/l, respectively, and 90 of À512, À8, and À8 mg/l, respectively, whereas those for the PMQR-positive K. pneumoniae isolates were 2 to À512, to À8, and to À8 mg/l, respectively, with 50 of16,2,and2 mg/l, respectively, and 90 of À512, À8, and À8 mg/l, respectively (Table 1). Among the PMQR- and ESBL-positive isolates, 45z (9 of 20) of the E. coli isolates and 32.5z (13 of 40) of the K. pneumoniae isolates had s of nalidixic acid, ciprofloxacin, and ofloxacin in susceptible or intermediately susceptible s according to the CLSI guidelines (26). All the PMQRpositive E. coli and K. pneumoniae isolates were susceptible to carbapenems; 88.9z and 95.9z, respectively, were susceptible to amikacin; and 33.3z and 36.7z, respectively, were susceptible to trimethoprim/sulfamethoxazole. Although all the transconjugants had quinolone and fluoroquinolone s higher than those of E. coli UB1637 (Æ16-fold for nalidixic acid, Æ32- to 64-fold for ciprofloxacin, and Æ64- to 128-fold for ofloxacin), their s remained in susceptible s. These results revealed the low level of fluoroquinolone resistance due to PMQR, as reported previously (2,13,15,27). The treatment of infections caused by fluoroquinolone-susceptible PMQR-positive isolates may select high-level quinolone-resistant strains and lead to clinical failure. Unfortunately, a history of diagnosis, antimicrobial therapy, and outcome of treatment were not available for any patient included in this study. In case of the PMQR-positive fluoroquinolone-resistant strains, other resistance mechanisms such as mutations in the gyrase and topoisomerase genes may be involved in combination with PMQR (19,21). Unfortunately, the QRDR mutations of these resistant strains were not detected because of the limitation of the present study. The PMQR-positive isolates were typed using an enterobacterial repetitive intergenic consensus (ERIC)- Fig. 1. Enterobacterial repetitive intergenic consensus (ERIC)- PCR fingerprints of the plasmid-mediated quinolone resistance (PMQR)-positive E. coli (lanes 1 10, there are 9 different ERIC patterns among the 10 isolates) and K. pneumoniae isolates (lanes 11 19, there are 6 different ERIC patterns among the 9 isolates). Lane M, 1 kb plus DNA ladder. PCR method with the ERIC2 primer and the conditions described by Versalovic et al. (28). The ERIC-PCR fingerprints were compared by visual inspection. Variations in the band intensity or shape were not considered. The presence or absence of more than 2 distinct bands between 2 isolates is considered to indicate different strains (17). Nineteen ERIC-PCR patterns were observed among the 27 E. coli isolates, whereas 33 different patterns were obtained among the 49 K. pneumoniae isolates (Fig. 1 and Table 2). These findings indicated the dissemination of these resistance determinants as well as the clonal spread of these resistant strains within our hospital. Although chromosomal QRDR mutations in topoisomerases play an important role in conferring a high level of quinolone resistance, the occurrence of PMQR in our hospital may contribute to an increase in quinolone resistance in clinical isolates of Enterobacteriaceae. The detection of PMQR may be useful for clinicians to ensure appropriate antimicrobial therapy. Acknowledgments This work was supported by the Centre for Research and Development in Medical Diagnostic Laboratory (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand. We are grateful to thank Dr. Tanittha Chatsuwan, Division of Bacteriology, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand for kindly providing control strains; a staff of Clinical Microbiology Laboratory, Srinagarind Hospital, Khon Kaen University for collecting the clinical isolates; and Mr. Auttawit Sirichoat and Miss Pennapa Sriruksa for their technical assistance. Conflict of interest None to declare. 431

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