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Antimicrob Agents Chemother. 2010 July; 54(7): 3068–3069.
Published online 2010 April 26. doi:  10.1128/AAC.01339-09
PMCID: PMC2897306

Novel Variants of the qnrB Gene, qnrB22 and qnrB23, in Citrobacter werkmanii and Citrobacter freundii[down-pointing small open triangle]

Il Kwon Bae and Indal Park
Research Institute for Antimicrobial Resistance and Department of Microbiology
Kosin University College of Medicine
Busan, Republic of Korea
Jae Jin Lee, Ha Ik Sun, Kwang Seung Park, Jeong Eun Lee, Ji Hye Ahn, and Sang Hee Lee*
Drug Resistance Proteomics Laboratory
Department of Biological Sciences
Myongji University
San 38-2 Namdong, Yongin
Gyeonggido 449-728, Republic of Korea

Resistance to quinolones in Gram-negative bacteria is usually mediated by the following: (i) chromosomal mutations that alter the target enzymes, DNA gyrase and topoisomerase IV, in their quinolone resistance-determining regions (QRDR), (ii) changes in drug entry (loss of porin channels), and (iii) the presence of plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qnrC, and qnrD, coding for Qnr proteins that protect DNA gyrase from quinolone attack; aac(6′)-Ib-cr, coding for a protein that acetylates quinolones; and qepA, coding for a quinolone efflux pump] (2, 12). The recent worldwide emergence of PMQR due to the qnr and aac(6′)-Ib-cr genes is a concerning fact among human and animal Gram-negative pathogens (8).

The aim of this study was to determine the prevalence of qnr genes among 93 consecutive nonrepetitive Enterobacteriaceae of animal origin and to characterize positive isolates. These isolates were collected from chickens (n = 37) and pigs (n = 56) at five farms near the city of Seoul (South Korea) in 2007.

The presence of PMQR determinants and QRDR mutations was investigated by PCR-based detection and sequencing (2, 5, 6). The qnrA, qnrS, qnrC, qnrD, aac(6′)-Ib-cr, and qepA genes were not found. Two isolates (2.2%) were found to carry qnr-like genes (Citrobacter werkmanii PS012 and Citrobacter freundii S008). Sequence analysis identified two novel qnrB variants, qnrB22 and qnrB23, in C. werkmanii PS012 (isolated from a pig at the Daeyoung Farm) and C. freundii S008 (isolated from a chicken at the Hanmi Farm), respectively. These new variants were assigned according to the qnr numbering scheme shown in the Lahey website ( The qnrB22 gene had 99.7% nucleotide identity with qnrB4. The qnrB23 gene had 99.9% nucleotide identity with qnrB9. The deduced QnrB22 product had two amino acid substitutions (Ser36Cys and Gly188Val) compared with the amino acid sequence of QnrB4. Compared with the amino acid sequence of QnrB9, QnrB23 showed one amino acid substitution (Asn27Tyr).

C. werkmanii PS012 showed a reduced susceptibility (MIC > 0.125 μg/ml) to fluoroquinolones (ofloxacin, norfloxacin, levofloxacin, and ciprofloxacin) (Table (Table1).1). C. freundii S008 was nonsusceptible (resistant or intermediate) to the fluoroquinolones (Table (Table1).1). The MICs were determined by E-test (AB Biodisk, Solna, Sweden) and interpreted according to Clinical and Laboratory Standards Institute guidelines (4). The QRDR mutations associated with fluoroquinolone resistance were not detected in the two isolates (Table (Table11).

Characteristics of the two Citrobacter isolates, their transconjugants, Escherichia coli DH5α transformants, and reference (recipient or host) strains

The transfer of qnrB22- and qnrB23-harboring plasmids to Escherichia coli J53 AzideR was accomplished through mating experiments described previously (9). Transconjugants were selected on Mueller-Hinton agar plates containing sodium azide (150 μg/ml) and ciprofloxacin (0.125 μg/ml). Fluoroquinolone (or nalidixic acid) MICs of the two transconjugants (TrcPS012 and TrcS008) were similar to those of the donor strains (Table (Table1).1). Strain TrcS008, carrying qnrB23, had MIC values for nalidixic acid and fluoroquinolones that were higher than those of TrcPS012, harboring qnrB22 (Table (Table11).

The PCR amplicons of the qnrB22 and qnrB23 genes were cloned into the vector pCR-BluntII-TOPO and transformed into the E. coli DH5α host strain (Invitrogen, Karlsruhe, Germany). Primers used were as follows: for cloning of qnrB22, 5′-ATGACTCTGGCGTTAGTTGG-3′ and 5′-TTAACCCATGACAGCGATACCAA-3′; and for cloning of qnrB23, 5′-ATGACGCCATTACTGTATAAAAAAACA-3′ and 5′-CTAGCCAATAATCGCGATGCC-3′. A decrease in quinolone susceptibility was observed with both transformants, even though the qnrB23-carrying transformant showed higher MICs than that carrying qnrB22 (Table (Table1).1). Fluoroquinolone (or nalidixic acid) MICs of two transformants (TrfPS012 and TrfS008) were lower than those of two transconjugants (TrcPS012 and TrcS008), which was compatible with a recent finding (11). The differences observed between transconjugants and transformants might be related to recipient susceptibility (E. coli DH5α was more susceptible than E. coli J53 AzideR), plasmid copy number, and/or the presence of additional PMQR determinants in the two plasmids.

The conjugative plasmids of C. werkmanii PS012 and C. freundii S008 showed identical patterns (showing 13 distinct bands) and similar molecular sizes (about 23 kb) in restriction fragment length polymorphism analysis after digestion with BglII, as described previously (1). qnrB22- and qnrB23-harboring plasmids belonged to an incompatibility group, IncL/M, according to a PCR-based replicon-typing scheme (3). These results suggest that conjugative IncL/M plasmids might play a role in the dissemination and evolution of qnrB genes. The association of various antibiotic resistance genes, including PMQR determinants with conjugative IncL/M plasmids from human isolates of the Enterobacteriaceae, has been described in several reports (7, 10, 13, 14). Despite the currently low prevalence (2.2%) of qnrB22 and qnrB23, surveillance for bacterial isolates carrying these resistance determinants in animals is warranted.

Nucleotide sequence accession numbers.

The nucleotide sequences of qnrB22 and qnrB23 have been submitted to the GenBank database and assigned the accession numbers FJ981621 and FJ981622, respectively.


We acknowledge the financial support of a National Research Foundation of Korea Grant funded by the Korean Government (KRF-2008-313-C00790 and KRF-2007-359-E00006), a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean Government (MEST), and funding from the Marine & Extreme Genome Research Center Program of the Ministry of Land, Transport, and Maritime Affairs in the Republic of Korea and the Second-Phase of Brain Korea 21 Project.


[down-pointing small open triangle]Published ahead of print on 26 April 2010.


1. Andrysiak, A. K., A. B. Olson, D. M. Tracz, K. Dore, R. Irwin, L.-K. Ng, M. W. Gilmour, and Canadian Integrated Program for Antimicrobial Resistance Surveillance Collaborative. 2008. Genetic characterization of clinical and agri-food isolates of multi drug resistant Salmonella enterica serovar Heidelberg from Canada. BMC Microbiol. 8:89. [PMC free article] [PubMed]
2. Cano, M. E., J. M. Rodríguez-Martínez, J. Agüero, A. Pascual, J. Calvo, J. M. García-Lobo, C. Velasco, M. V. Francia, and L. Martínez-Martínez. 2009. Detection of plasmid-mediated quinolone resistance genes in clinical isolates of Enterobacter spp. in Spain. J. Clin. Microbiol. 47:2033-2039. [PMC free article] [PubMed]
3. Carattoli, A., A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63:219-228. [PubMed]
4. Clinical and Laboratory Standards Institute. 2010. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. CLSI document M100-S20. Clinical and Laboratory Standards Institute, Wayne, PA.
5. Eaves, D. J., L. Randall, D. T. Gray, A. Buckley, M. J. Woodward, A. P. White, and L. J. L. J. Piddock. 2004. Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica. Antimicrob. Agents Chemother. 48:4012-4015. [PMC free article] [PubMed]
6. Jeong, S. H., I. K. Bae, S. B. Kwon, J. H. Lee, H. I. Jung, J. S. Song, B. C. Jeong, S. J. Kim, and S. H. Lee. 2004. Investigation of extended-spectrum β-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea. Lett. Appl. Microbiol. 2004:41-47. [PubMed]
7. Kim, S.-Y., Y.-J. Park, J. K. Yu, Y. S. Kim, and K. Han. 2009. Prevalence and characteristics of aac(6′)-Ib-cr in AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens: a multicenter study from Korea. Diagn. Microbiol. Infect. Dis. 63:314-318. [PubMed]
8. Martínez-Martínez, L., M. E. Cano, J. M. Rodríguez-Martínez, J. Calvo, and A. Pascual. 2008. Plasmid-mediated quinolone resistance. Expert Rev. Anti Infect. Ther. 6:685-711. [PubMed]
9. Pai, H., M.-R. Seo, and T. Y. Choi. 2007. Association of QnrB determinants and production of extended-spectrum β-lactamases or plasmid-mediated AmpC β-lactamases in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 51:366-368. [PMC free article] [PubMed]
10. Park, Y.-J., S.-Y. Kim, J. K. Yu, S. I. Kim, Y. Uh, S. G. Hong, J. Lee, and H.-S. Kwak. 2009. Spread of Serratia marcescens coharboring aac(6′)-Ib-cr, blaCTX-M, armA, and blaOXA-1 carried by conjugative IncL/M type plasmid in Korean hospitals. Microb. Drug Resist. 15:97-102. [PubMed]
11. Quiroga, M. P., P. Andres, A. Petroni, A. J. C. Soler Bistué, L. Guerriero, L. J. Vargas, A. Zorreguieta, M. Tokumoto, C. Quiroga, M. E. Tolmasky, M. Galas, and D. Centrón. 2007. Complex class 1 integrons with diverse variable regions, including aac(6′)-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina. Antimicrob. Agents Chemother. 51:4466-4470. [PMC free article] [PubMed]
12. Robicsek, A., G. A. Jacoby, and D. C. Hooper. 2006. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect. Dis. 6:629-640. [PubMed]
13. Tosini, F., P. Visca, I. Luzzi, A. M. Dionisi, C. Pezzella, A. Petrucca, and A. Carattoli. 1998. Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype Typhimurium. Antimicrob. Agents Chemother. 42:3053-3058. [PMC free article] [PubMed]
14. Villa, L., C. Pezzella, F. Tosini, P. Visca, A. Petrucca, and A. Carattoli. 2000. Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended-spectrum β-lactamase gene and a class 1 integron. Antimicrob. Agents Chemother. 44:2911-2914. [PMC free article] [PubMed]

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