Association of QnrB Determinants and Production of Extended-Spectrum β-Lactamases or Plasmid-Mediated AmpC β-Lactamases in Clinical Isolates of Klebsiella pneumoniae

Abstract

Clinical isolates of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases or plasmid-mediated AmpC β-lactamases were screened for qnrA and qnrB genes. QnrB was present in 54 of 54 DHA-1-producing K. pneumoniae isolates and 10 of 45 SHV-12-producing ones, suggesting that the distribution of plasmids conferring resistance to extended-spectrum cephalosporins and quinolones in clinical isolates of K. pneumoniae is widespread.

Since the first plasmid-mediated quinolone resistance-conferring gene (qnr) was discovered in a Klebsiella pneumoniae isolate from Alabama (7), Qnr determinants have been identified worldwide (2, 9, 13, 15). A frequent association of quinolone resistance with the production of extended-spectrum β-lactamases (ESBLs) has been noticed and was explained by the selective pressures of multiple antibiotics and plasmid-mediated qnr (3, 9, 12, 15, 17). At present, several qnrA-positive isolates have been found to express ESBLs, such as SHV-5, SHV-7, CTX-M-15, and VEB-1, or plasmid-mediated AmpC enzymes, such as DHA-1 (9, 17). The association of qnrB and SHV-12 was recently described (3).

In order to study the prevalence of Qnr determinants and to identify ESBLs or plasmid-mediated AmpC β-lactamases associated with qnr genes, we screened 239 isolates of Escherichia coli and K. pneumoniae producing variable ESBLs or plasmid-mediated AmpC β-lactamases for qnrA and qnrB.

(This study was presented at the 44th IDSA meeting, Toronto, Ontario, Canada, 2006.)

Twenty-seven E. coli strains and 62 K. pneumoniae strains were obtained from the blood isolate collection of Seoul National University Children's Hospital (SNUCH). These isolates were collected from 1993 to 2005 and were identified as producers of an ESBL or a plasmid-mediated AmpC β-lactamase (part of these data were published previously [6]). Forty-nine isolates of K. pneumoniae known to produce ESBLs or to have a plasmid-mediated AmpC enzyme were recruited from the blood isolate collection of Seoul National University Hospital (SNUH) during the 5 years from 1998 to 2002 (10). Fifty-four E. coli strains and 23 K. pneumoniae strains, selected for resistance to cefoxitin and extended-spectrum cephalosporins (ESCs), were obtained from Hanyang University Hospital (HUH), a tertiary-care teaching hospital in Seoul, from 2003 to 2005. Twenty-four K. pneumoniae isolates were collected from Korea University Hospital (KUH), Seoul, in 2005 and were chosen because of their ESC resistance. Only one isolate from each patient was included in all collections. Thus, a total of 81 E. coli strains and 158 K. pneumoniae strains which produced an ESBL or a plasmid-mediated AmpC were included in this study.

Isoelectric focusing (IEF) was performed by the method of Mathew et al. (8) by using a Mini IEF cell system (Bio-Rad, Hercules, CA). Strains carrying plasmids encoding β-lactamases TEM-1 (R1), TEM-4 (pUD16), SHV-2 (pMG229), SHV-5 (pAFF2), and CMY-1 (pMVP-1) served as the IEF standards (6). The β-lactamase genes from the clinical isolates were amplified and sequenced as described previously (5, 10, 11). The qnrA gene was detected by PCR with primers QP1 and QP2 under the PCR conditions described previously (4). For the detection of qnrB, PCR was performed with primers FQ1 and FQ2 (3, 13) and primers MFQ1 (5′-GATCGTGAAAGCCAGAAAGG-3′) and MFQ2 (5′-ACGATGCCTGGTAGTTGTCC-3′), as described previously (13). Plasmid pMG252 and the K. pneumoniae 35 strain were used as the positive controls for qnrA and qnrB, respectively (4, 7).

Products were amplified from 54 isolates of K. pneumoniae and 2 isolates of E. coli by PCR with primers MFQ1 and MFQ2, and the nucleotide sequences of the PCR products from 3 isolates were identical to the nucleotide sequence of qnrB4. One E. coli isolate and 10 K. pneumoniae isolates were positive for amplification with both primers MFQ1 and MFQ2 and primers FQ1 and FQ2. The PCR products obtained from five isolates with primers FQ1 and FQ2 and with primers MFQ1 and MFQ2 were sequenced, which revealed that the nucleotide sequences were identical to that of qnrB2 in two isolates but to that of qnrB5 in three isolates. In order to differentiate between qnrB2 and qnrB5, the products amplified with primers FQ1 and FQ2 were restricted with ApaI or HindIII. The results showed that seven and three isolates contained qnrB2 (restricted into 467- and 95-bp DNA fragments with ApaI) and qnrB5 (restricted into 333- and 229-bp DNA fragments with HindIII), respectively. One isolate whose PCR product was partially restricted with both ApaI and HindIII but which showed a nucleotide sequence identical to that of qnrB2 was considered to have both qnrB2 and qnrB5 concurrently. The nucleotide sequences of the PCR products from one E. coli isolate and three of six K. pneumoniae isolates obtained by qnrA-specific PCR were identical to the nucleotide sequence of qnrA1.

A conjugation experiment was performed for 10 of 54 K. pneumoniae isolates containing qnrB4, 10 of 10 K. pneumoniae isolates with qnrB2 or qnrB5, and 1 E. coli isolate with qnrB2 by using E. coli J53 Azi^r as a recipient, as described previously (3). The selective medium used contained ceftazidime (10 μg/ml), sodium azide (100 μg/ml), and nalidixic acid (12 μg/ml) or ciprofloxacin (0.125 μg/ml). One of 10 DHA-1-producing K. pneumoniae isolates, 3 of 10 SHV-producing K. pneumoniae isolates, and a CMY-1-producing E. coli isolate transferred resistance to ESCs and quinolones. Table 1 shows the susceptibilities of the wild types and E. coli J53 Azi^r transconjugants to several β-lactam and quinolone antibiotics.

TABLE 1.

Susceptibilities of wild types and E. coli J53 transconjugants to β-lactam and quinolone antibiotics

Straina	MIC (μg/ml)b							qnrB allele	β-Lactamase
	NA	CIP	LEVO	MOXI	FOX	CTX	CAZ
E. coli J53 Azir	8	0.03	0.06	0.06	4	<0.25	1
12-1183	>64	>16	>16	>8	>128	16	128	B4	DHA-1
T12-1183	32	0.25	0.5	1	32	4	128
35	>64	16	4	>8	32	>128	>128	B5	SHV-12
T35	32	0.5	0.25	0.5	4	16	128
07-006	>64	16	8	8	16	64	128	B2	SHV-12
T07-006	32	0.5	0.25	1	4	16	128
03-222	>64	16	16	8	16	4	128	B2	SHV-12
T03-222	32	1	0.25	0.5	4	2	64
12-1502	16	0.5	0.25	0.5	>128	64	128	B2	CMY-1
T12-1502	32	1	0.25	0.5	>128	64	64

^aT, an E. coli J53 Azi^r transconjugant of each isolate. Isolate 12-1502 was E. coli, and all other clinical isolates were K. pneumoniae.

^bNA, nalidixic acid; CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin; FOX, cefoxitin; CTX, cefotaxime; CAZ, ceftazidime.

Table 2 shows the distribution of qnrB among the clinical isolates of E. coli and K. pneumoniae according to their β-lactamase subtypes.

TABLE 2.

Distribution of Qnr determinants according to ESBL or plasmid-mediated AmpC β-lactamases among clinical isolates of Klebsiella pneumoniae and Escherichia coli^a

Strain and ESBL(s) or plasmid-mediated AmpC β-lactamase(s)	No. of isolates with:
	Total	qnrA1	qnrB4	qnrB2	qnrB5	qnrB2 and qnrB5
Klebsiella pneumoniae (n = 158)
CTX-M-14	7	2
DHA-1	39		39
DHA-1, CTX-M-14	1		1
DHA-1, TEM-52	2		2
SHV-12	41	4		5	3	1
SHV-12, DHA-1	12		12
SHV-12, pI 7.8	1			1
Total		6	54	6	3	1
Escherichia coli (n = 81)
CMY-1	3			1
CMY-2, CTX-M-14	2	1
DHA-1	1		1
DHA-1, TEM-52	1		1
Total		1	2	1

^aThe TEM-1 β-lactamase is not described in this table.

The distribution of qnrB was closely related to the presence of certain β-lactamases; all 54 K. pneumoniae isolates and 2 E. coli isolates producing DHA-1 with or without other enzymes contained qnrB4 (100%). Among 45 K. pneumoniae isolates that produced SHV-12 with or without other enzymes, excluding 12 isolates that coproduced SHV-12 and DHA-1, qnrB2 was present in 6 strains (13.3%), qnrB5 was present in 3 strains (6.7%), and both qnrB2 and qnrB5 were present in 1 strain (2.2%). No isolate of E. coli or K. pneumoniae harbored qnrA and qnrB concurrently.

Blood isolates from SNUH and SNUCH were proved to be epidemiologically not related by pulsed-field gel electrophoresis in previous studies (6, 10). Enterobacterial repetitive intergenic consensus sequence (ERIC) PCR was performed with the DHA-1-producing K. pneumoniae isolates (23 and 16 isolates from HUH and KUH, respectively), as described previously (1). Isolates from HUH and KUH showed 11 and 6 distinct ERIC patterns, respectively. However, six isolates from KUH revealed indistinguishable ERIC patterns.

This study shows the absolute association between qnrB4 and DHA-1 determinants in isolates of the family Enterobacteriaceae, which was explained by the partial nucleotide sequences of pTN60013 from K. pneumoniae containing bla_DHA-1 (16). The nucleotide sequences of pTN60013 from nucleotides 1225 to 1586 completely match the partial nucleotide sequences of qnrB4 (14). The nucleotide sequences of our purified products obtained from three clinical isolates by PCR with primers MFQ1 and MFQ2 were identical to those of pTN60013 from nucleotides 1196 to 1609. Thus, we considered them to be qnrB4 (G. A. Jacoby, personal communication).

Although the association of SHV-12 and qnrB was not as close as that of DHA-1 and qnrB, 20% of the SHV-12-producing K. pneumoniae isolates evaluated in this study were positive for qnrB. Further spread of the resistance plasmid encoding qnrB as well as bla_SHV-12 may occur in the near future, and strains should be carefully observed for this.

The increase in the prevalence of the DHA-1 β-lactamase in Korea was recently reported (10). We suspect that qnrB possibly contributes to the widespread distribution of DHA-1 in areas where ESCs and fluoroquinolone are widely used.