Novel Variants of the qnrB Gene, qnrB22 and qnrB23, in Citrobacter werkmanii and Citrobacter freundii

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 (http://www.lahey.org/qnrStudies). 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 1). C. freundii S008 was nonsusceptible (resistant or intermediate) to the fluoroquinolones (Table 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 1).

TABLE 1.

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

Strain	QRDR mutationa	PMQRb determinant	MIC (μg/ml) of drugc
			NAL	OFX	NOR	LVX	CIP
Citrobacter field strainsd
C. werkmanii PS012	ND	qnrB22	32	1	0.5	0.5	0.25
C. freundii S008	ND	qnrB23	>256	8	8	3	2
Transconjugantse
TrcPS012		qnrB22	16	1	0.25	0.5	0.25
TrcS008		qnrB23	>256	8	8	3	2
E. coli J53 AzideR			3	0.064	0.064	0.023	0.016
Transformantsf
TrfPS012		qnrB22	4	0.064	0.064	0.004	0.016
TrfS008		qnrB23	16	0.25	0.25	0.094	0.064
E. coli DH5α			2	0.023	0.032	<0.002	0.003

^aQRDR mutation, quinolone resistance-determining region mutation associated with fluoroquinolone resistance; ND, not detected.

^bPMQR, plasmid-mediated quinolone resistance.

^cNAL, nalidixic acid; OFX, ofloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin.

^dThe two Citrobacter strains were isolated at two farms near the city of Seoul, the capital of South Korea, in 2007. Species assignment was confirmed biochemically and by 16S rRNA gene sequence analysis.

^eTrcPS012 and TrcS008, E. coli J53 Azide^R (recipient strain) transconjugants of C. werkmanii PS012 and C. freundii S008, respectively.

^fTrfPS012 and TrfS008, E. coli DH5α (host strain) transformants harboring pCR-BluntII-TOPO::qnrB22 and pCR-BluntII-TOPO::qnrB23 (expressing QnrB22 and QnrB23 determinants, respectively), respectively.

The transfer of qnrB22- and qnrB23-harboring plasmids to Escherichia coli J53 Azide^R 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 1). Strain TrcS008, carrying qnrB23, had MIC values for nalidixic acid and fluoroquinolones that were higher than those of TrcPS012, harboring qnrB22 (Table 1).

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 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 Azide^R), 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.