Emerging Plasmid-Mediated Quinolone Resistance Associated with the qnr Gene in Klebsiella pneumoniae Clinical Isolates in the United States

Abstract

Although quinolone resistance commonly results from chromosomal mutation, recent studies indicate that such resistance can also be transferred on plasmids carrying the gene responsible, qnr. One hundred ten ciprofloxacin-resistant clinical isolates of Klebsiella pneumoniae and Escherichia coli from the United States were screened for the qnr gene by PCR and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as the recipient and selection with azide and sulfonamide, a resistance frequently linked to qnr. EcoRI and BamHI digests of qnr-hybridizing plasmids were subjected to electrophoresis on agarose gels and probed with qnr by Southern hybridization. qnr was detected in 8 (11.1%) of 72 K. pneumoniae strains. These eight positive strains were from six states in the United States. qnr was not found in any of the 38 E. coli strains tested. Quinolone resistance was transferred from seven of the eight probe-positive strains. Transconjugants with qnr-hybridizing plasmids had 32-fold increases in ciprofloxacin MICs relative to E. coli J53. For all eight strains, the sequence of qnr was identical to that originally reported. By size and restriction digests, four plasmids were related to the first-reported plasmid, pMG252, and three were different. Five new qnr plasmids encoded FOX-5 β-lactamase, as did pMG252, but two others produced SHV-7 extended-spectrum β-lactamase. Transferable plasmid-mediated quinolone resistance associated with qnr is now widely distributed in quinolone-resistant clinical strains of K. pneumoniae in the United States. Plasmid-determined quinolone resistance contributes to the increasing quinolone resistance of K. pneumoniae isolates and to the linkage previously observed between resistance to quinolones and the latest β-lactam antibiotics.

Quinolone resistance is usually caused by chromosomal mutations, but plasmid-mediated quinolone resistance has been discovered recently. The gene responsible for plasmid-mediated resistance, qnr, was found on plasmids varying in size from 54 to ≥180 kb in clinical isolates of Klebsiella pneumoniae and Escherichia coli from which low-level quinolone resistance could be transferred to a sensitive recipient by conjugation (11, 19). Although our previous study showed that qnr was present in 8% of quinolone-resistant clinical isolates of E. coli from Shanghai, China (19), surveys for the presence of qnr are few, and in those few surveys qnr has rarely been found in clinical strains from other areas of the world (6, 15). More information on the prevalence of qnr in clinical strains is needed to understand its importance and contribution to the development of quinolone resistance.

Our previous study found that qnr is located in complex In4 family class 1 integrons In36 and In37 (19), which are also known as complex sul1-type integrons because of the presence of duplicate qacEΔ1 and sul1genes. This relationship is supported by in vitro susceptibility results on transconjugants containing plasmids carrying qnr, showing that all transconjugants were resistant to sulfamethoxazole (19).

Quinolone resistance is found with surprisingly high frequency (18% to 56%) in extended-spectrum β-lactamase (ESBL)-producing isolates (4, 8, 14). Eighty-six percent of the ESBL-producing E. coli strains were resistant to levofloxacin in Shanghai, China (20), where the ciprofloxacin resistance rate of all clinical isolates of E. coli exceeds 50% (19). Plasmid pMG252, the first plasmid found that carried qnr, also encodes the AmpC-type β-lactamase FOX-5 (6). In our previous study, the MIC of cefotaxime was 2 to 32 μg/ml for four of the nine qnr transconjugants from clinical strains of E. coli, an MIC 60- to 1,000-fold higher than that of the recipient E. coli, suggesting the possibility of ESBL production (19). Thus, the plasmids in these strains could provide a genetic linkage between resistance to expanded-spectrum cephalosporins and quinolones.

In this study, we investigated the frequency of qnr in recent clinical isolates of K. pneumoniae and E. coli from the United States, determined the transferability of quinolone resistance, characterized the β-lactamases encoded by the plasmids hybridizing with a qnr probe, and analyzed the relatedness of these plasmids to previously known qnr-carrying plasmids.

MATERIALS AND METHODS

Bacterial strains.

Seventy-two clinical isolates of K. pneumoniae and 38 of E. coli that were resistant to quinolone (ciprofloxacin MIC, ≥2 μg/ml) and also to ceftazidime (MIC, ≥16 μg/ml) collected from around the United States between 1999 and 2002 were chosen for study. For both K. pneumoniae and E. coli, these isolates included all but one of the isolates with the designated phenotype in the Focus Technologies collection. During the same period in this collection, the overall rates of ciprofloxacin resistance (defined as an MIC of ≥4 μg/ml) were 4.1% (164 of 4,002) for K. pneumoniae and 4.2% (376 of 9,023) for E. coli, and for K. pneumoniae 210 of 4,002 isolates (5.2%) had an MIC of ≥2 μg of ciprofloxacin per ml. Thus, slightly less than one third of all the K. pneumoniae isolates with ciprofloxacin MICs of ≥2 μg/ml in the collection met the additional selection criterion of an MIC of ≥16 μg of ceftazidime per ml. Each strain studied came from a unique patient.

Additional strains used were E. coli V517 (9); E. coli J53 containing plasmid R1 (5), Plac (5), or R27 (17) as standards for plasmid size; E. coli J53Az^R (resistant to azide) as a recipient for conjugation (11); K. pneumoniae UAB1 (11) as a positive control for qnr hybridization; and E. coli J53/pMG252 (11), a transconjugant of UAB1. Strains were routinely grown at 37°C in Luria-Bertani (LB) medium except as noted. Stock cultures were stored at −80°C in 10% glycerol-brain heart infusion broth.

Screening for the qnr gene in clinical strains.

The qnr gene was screened by PCR and Southern hybridization with the ECL direct nucleic acid labeling and detection system (Amersham Biosciences Corp., Piscataway, N.J.). qnr (657 bp) was amplified with primers 5′-TCAGCAAGAGGATTTCTCA and 5′-GGCAGCACTATTACTCCCA to produce a 627-bp product. DNA templates for PCR were prepared by boiling. PCR conditions were 94°C for 45 s, 48°C for 45 s, and 72°C for 45 s for 30 cycles. Strains positive and negative for qnr were included in each batch of strains tested. Amplification products were detected by electrophoresis on a 1% agarose gel with ethidium bromide and photographed under UV light. The qnr probe was made from plasmid pMG254 (18) by PCR amplification with the above primers. Plasmid DNAs were isolated with an alkaline extraction method (16). DNAs were subjected to electrophoresis in 0.5% Certified Megabase agarose (Bio-Rad Laboratories, Hercules, Calif.) gel without ethidium bromide at 70 V for 3.5 h. After depurination, denaturation, and neutralization of the gel, DNAs were transferred to a Hybond-N+ membrane (Amersham Biosciences Corp.) by capillary blotting overnight. The membrane, which was fixed by UV exposure, was hybridized with the qnr probe, and signals were detected by exposure of the membrane to Hyperfilm ECL (Amersham Biosciences Corp.).

Conjugation.

Conjugation experiments were carried out in LB broth with E. coli J53Az^R as the recipient. Cultures of donor and recipient cells in logarithmic phase (0.5 ml each) were added to 4 ml of fresh LB broth and incubated overnight without shaking. Transconjugants were selected on Trypticase soy agar (TSA) plates containing sodium azide (100 μg/ml; Sigma Chemical Co., St. Louis, Mo.) for counterselection and sulfamethoxazole (300 μg/ml) to select for plasmid-encoded resistance. To determine if quinolone resistance was cotransferred, colonies were replica-plated onto TSA with and without ciprofloxacin (0.06 μg/ml). MICs for the donors, recipient, and transconjugants were measured by agar dilution, with the guidelines of the National Committee for Clinical Laboratory Standards (13). The antimicrobial agents tested were ciprofloxacin (Bayer Corporation Pharmaceutical Division, West Haven, Conn.), ampicillin, cefotaxime, cefoxitin, chloramphenicol, gentamicin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole (Sigma Chemical Co.).

Estimation of plasmid size.

Plasmid size was estimated as previously described (19).

Sequencing of qnr genes and DNA adjacent to qnr.

The qnr genes from qnr hybridization-positive strains were sequenced directly from PCR-amplified DNA, and the sequences were compared with the sequence of the qnr gene from K. pneumoniae UAB1 (GenBank AY070235). The primers used were 5′-GGGTATGGATATTATTGATAAAG and 5′-CTAATCCGGCAGCACTATTA. From selected plasmids, the sequences of DNA adjacent to qnr were determined with plasmid DNA purified from transconjugants and a series of outward-facing primers starting from both ends of the qnr gene.

Digestion of plasmid DNA with restriction enzymes.

Plasmids were extracted with the Qiagen plasmid midi kit (Qiagen, Valencia, Calif.) and digested separately with EcoRI and BamHI (New England Biolabs, Beverly, Mass.). Reactions for digestion with EcoRI were carried out in buffer that contained 33 μl of DNA, 4 μl of 10× EcoRI buffer, and 3 μl (60 U) of EcoRI. Reactions for digestion with BamHI were carried out in buffer containing 29 μl of DNA, 4 μl of 10× BamHI buffer, 4 μl of 10× bovine serum albumin, and 3 μl (60 U) of BamHI, both at 37°C for 2.5 h; 20 μl of each digestion mixture was subjected to electrophoresis on 0.7% agarose gels, stained with 0.5 μg of ethidium bromide per ml, and photographed under UV light. The digested DNAs were transferred to a Hybond-N+ membrane and probed with qnr by Southern hybridization. The restriction digestion patterns were compared to each other and with that of pMG252.

β-Lactamase characterization.

β-Lactam resistance in J53Az^R transconjugants was characterized further by isoelectric focusing with a lysozyme extract (1) that was concentrated with a Micron YM-10 device (Millipore Corp., Bedford, Mass.), applied to a precast polyacrylamide gel with a pH 3 to 9 gradient, and subjected to electrophoresis with the PhastSystem (Amersham Biosciences, Piscataway, N.J.) with enzyme localization with nitrocefin as described by Huovinen (3). K. pneumoniae strain 48188, producing β-lactamases with isoelectric points of 5.4 (TEM-1), 7.6 (SHV-1), and 8.2 (SHV-12), was used as a standard. Suspected FOX-5 genes were amplified by PCR with primers 5′-ATGCCAATTTCATTCACCAC and 5′-ATKTGGAMGCCTTGAACTCG and sequenced with the same and internal primers. Suspected SHV and TEM genes were amplified and sequenced with specific primers as previously described (7).

RESULTS

Screening for the qnr gene.

The qnr gene was detected in 8 (11.1%) of the 72 K. pneumoniae strains by PCR. Plasmid DNA from these strains also yielded strong hybridization signals with a qnr gene probe. These eight positive strains were isolated from six states in the United States, including Alabama, Arizona, Delaware, Kentucky (two strains), New York (two strains), and Tennessee. qnr was not found in any of the 38 quinolone-resistant E. coli strains tested. The qnr probe hybridized with plasmids with a range of sizes in the eight probe-positive strains and with plasmid pMG252 from K. pneumoniae UAB1, but not with plasmids from other strains. In six strains (54, 77, 78, 79, 83, and 109), the qnr probe hybridized to plasmids of 180 kb or greater (the size could not be estimated more accurately with the techniques used), in strain 39 to a plasmid of 110 kb, and in strain 68 to a plasmid of 78 kb (Fig. 1a and b).

FIG. 1.

Plasmid DNAs from clinical strains of K. pneumoniae and reference strains of E. coli (a) and Southern hybridization of plasmid DNAs from clinical strains of K. pneumoniae with the qnr probe (b). Lanes: A, E. coli V517; B, E. coli J53/R1; C, E. coli J53/Plac; D, E. coli J53/R27; E, UAB1 (used as a positive control); F, E. coli J53 (used as a negative control); 39, 54, 68, 77, 78, 79, 83, and 109, K. pneumoniae clinical strains.

Transfer of quinolone resistance.

Quinolone resistance could be transferred by conjugation from seven of the eight qnr-positive donors. The seven transconjugants were selected with sulfamethoxazole at a conjugation frequency of 2 × 10⁻² to 8 × 10⁻⁴ (number of transconjugants divided by the number of donor cells). Ciprofloxacin resistance was found in 83% to 100% of sulfamethoxazole-resistant transconjugants tested. These transconjugants contained single plasmids hybridizing with the qnr probe. The qnr-positive plasmids in transconjugants were the same size as those in their respective donors (Fig. 2a and b). Transfer from the eighth donor strain was not successful despite conjugation experiments in broth and on filter surfaces and including separate selections with each of the antibiotics to which the donor was resistant (except quinolones). The reasons for the lack of detectable transfer of quinolone resistance from this donor strain are not known, but possible explanations include that in this strain qnr may have been present on a plasmid without other linked resistance determinants and that qnr was poorly expressed from the plasmid in which it was found by Southern hybridization.

FIG. 2.

Plasmid DNAs of donors and transconjugants (a) and Southern hybridization of plasmid DNAs from donors and transconjugants with the qnr probe (b). Strain designations are shown above the lanes, and those of the transconjugants end in the letter T.

Antimicrobial resistance in transconjugants.

The MIC of ciprofloxacin against the transconjugants was uniformly 0.25 μg/ml, representing an increase of 32-fold relative to that of the recipient E. coli J53Az^R (Table 1). Various resistances to other antimicrobial agents were also transferred with the plasmid, but all were resistant to ampicillin and sulfamethoxazole (Table 1). All transconjugants had elevated MICs for cefotaxime, and five were resistant to cefoxitin.

TABLE 1.

Resistance profile of donor strains and transconjugants^a

Group and strain	MIC (μg/ml)
	CIP	AMP	CTX	FOX	GEN	SXT	SMZ	TMP	CHL	TET
Donor (state of origin)
39 (Arizona)	2	≥256	8	8	16	≥512	≥512	≥256	16	≥256
54 (Delaware)	8	≥256	16	16	16	≥512	≥512	32	128	≥256
68 (Kentucky)	2	≥256	16	2	8	8	≥512	0.5	2	1
77 (New York)	4	≥256	8	128	8	≥512	≥512	≥256	64	4
78 (Alabama)	8	≥256	4	128	4	≥512	≥512	≥256	64	4
79 (New York)	2	≥256	8	128	8	≥512	≥512	≥256	64	4
83 (Kentucky)	16	≥256	8	≥256	0.125	128	≥512	32	≥256	16
109 (Tennessee)	32	≥256	16	≥256	0.125	8	≥512	2	128	16
UAB1 (Alabama)	8	≥256	8	128	8	≥512	≥512	≥256	128	4
Recipient E. coli J53	0.008	16	0.03	2	0.125	0.5	16	0.125	4	1
J53 transconjugants (plasmids)
54-3 (pMG291)	0.25	≥256	2	2	8	1	≥512	0.125	4	1
68-45 (pMG292)	0.25	≥256	1	2	2	1	≥512	0.125	4	1
77-6 (pMG293)	0.25	≥256	4	64	2	≥512	≥512	≥256	32	1
78-38 (pMG294)	0.25	≥256	4	64	2	≥512	≥512	≥256	32	1
79-66 (pMG295)	0.25	≥256	4	64	2	≥512	≥512	≥256	32	1
83-2 (pMG296)	0.25	≥256	4	64	0.125	0.5	≥512	0.125	32	1
109-55 (pMG297)	0.25	≥256	4	64	0.125	0.5	≥512	0.125	32	1
pMG252	0.5	≥256	4	64	2	≥512	≥512	≥256	32	1

^aCIP, ciprofloxacin; AMP, ampicillin; CHL, chloramphenicol; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; SMZ, sulfamethoxazole; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline; TMP, trimethoprim.

DNA sequencing.

All eight strains contained qnr with a nucleotide sequence identical to that of the originally reported qnr sequence (18) except for strain 39, which had a single functionally silent nucleotide change, CTA→CTG, at position 537. The DNA sequence adjacent to qnr in transconjugants of donors 54 and 68 (plasmids pMG291 and pMG292) indicated the presence of ORF513 upstream and of qacEΔ1 and sul1 downstream from qnr, suggesting that qnr is located in a genetic environment similar to that in the original qnr plasmid pMG252 (18).

Plasmid relatedness.

Seven plasmid DNAs isolated from transconjugants were digested singly with EcoRI and BamHI. For plasmids pMG294 and pMG295 (from strains 78 and 79, respectively), the EcoRI digestion patterns were identical to that of pMG252, while two other plasmids, pMG293 and pMG296 (from strains 77 and 83, respectively), showed a pattern similar to that of pMG252 (Fig. 3a). For these four plasmids and pMG252, qnr was located on a 4.0-kb EcoRI fragment (Fig. 3b). For plasmids pMG291, pMG292, and pMG297 (from strains 54, 68, and 109, respectively), qnr was located on EcoRI fragments of 12.5, 6.5, and 5.8 kb, respectively (Fig. 3a and b). Strains 54, 68, and 109 were obtained from Delaware, Kentucky, and Tennessee, respectively. In addition, the sizes of the qnr plasmids in strains 77, 78, 79, and 83 were similar to that of pMG252 (≥180 kb) (Fig. 1a and b). Thus, the plasmids in strains 77, 78, 79, and 83 were related to the first-reported plasmid, pMG252, by size and restriction digests. These four strains were isolated from Alabama (strain 78), Kentucky (strain 83), and New York (strains 77 and 79) and also showed similar patterns when the plasmids were digested with BamHI (data not shown).

FIG. 3.

Digestion of plasmid DNAs containing qnr isolated from transconjugants with EcoRI (a) and Southern hybridization of the digestions of plasmid DNAs containing qnr isolated from transconjugants (b). Lanes: A, 1-kb ladder; B, HindIII DNA size markers; others are transconjugant designations (strain numbers ending in the letter T).

β-Lactamase characterization.

All seven transconjugants produced β-lactamases. On isoelectric focusing, strains with plasmids pMG293, pMG294, pMG295, pMG296, and pMG297 demonstrated multiple bands characteristic of FOX-5 β-lactamase, encoded also by plasmid pMG252. These strains gave an identical PCR product with FOX-specific primers, and the amplification product with plasmid pMG293 proved to be identical to that of FOX-5 on sequencing. These five strains also made a second β-lactamase of pI 5.7 that failed to give a product with TEM-specific primers and has been identified as PSE-1 (data not shown). The remaining two transconjugants had an ESBL phenotype and gave a product with SHV-specific primers that proved on sequencing to be SHV-7.

DISCUSSION

In this study, qnr was detected in 8 (11%) of 72 clinical strains of K. pneumoniae with ciprofloxacin MICs of ≥2 μg/ml and ceftazidime MICs of ≥16 μg/ml from the United States by PCR and Southern hybridization. These strains represented all but one of those available with the selected phenotypes for each of the two species in Focus Technologies’ collection from 1999 through 2001. The eight positive strains were isolated from Alabama, Arizona, Delaware, Kentucky, New York, and Tennessee, states in different regions of the country, indicating that qnr has emerged in ciprofloxacin-resistant clinical strains of K. pneumoniae and may be spreading in the United States. Rodríguez-Martínez et al. recently also reported three qnr-positive K. pneumoniae isolates from Delaware and North Carolina (15).

qnr was located on plasmids ranging in size from 78 to ≥180 kb; five of eight were ≥180 kb. Four plasmids (pMG293, pMG294, pMG295, and pMG296) were highly similar to pMG252, the first plasmid found containing qnr, which also isolated from a clinical strain of K. pneumoniae. These plasmids had similar EcoRI and BamHI restriction digestion patterns and produced FOX-5 and PSE-1 β-lactamases, suggesting the spread of a similar plasmid to different states. qnr was not found in 38 quinolone-resistant clinical strains of E. coli from the United States, in contrast to our former finding that qnr existed in 8% of resistant E. coli isolates from Shanghai (19). The number of E. coli strains tested in this study was small, however.

Quinolone resistance was transferred from seven of eight qnr-positive strains by conjugation. The MICs of ciprofloxacin against transconjugants were uniform (0.25 μg/ml), suggesting similar expression of the qnr gene. In contrast, qnr-containing transconjugants of E. coli from Shanghai exhibited a 16-fold range of MICs of ciprofloxacin, from 0.125 to 2 μg/ml (19), suggesting differences in the level of expression of qnr. The U.S. plasmids all conferred resistance to sulfonamides, and for pMG291 and pMG292 the DNA sequences flanking the qnr gene suggested that qnr may be located in a class 1 integron.

All seven transconjugants encoded β-lactamases. Five encoded the AmpC-type β-lactamase FOX-5, which has been found in pMG252 (6); the other two of the new qnr plasmids produced ESBL SHV-7 rather than FOX-5. Several of the qnr plasmids from Shanghai also encoded an ESBL, CTX-M-9 (unpublished observations). The linkage between qnr and genes encoding ESBLs and AmpC-type β-lactamases may explain, in part, the association between resistance to quinolones and resistance to expanded-spectrum cephalosporins that has been noted in several previous studies (4, 8, 14).

Although plasmids containing qnr have been associated with only low-level resistance to fluoroquinolones in transconjugants, donor strains have in most cases had high levels of quinolone resistance, presumably associated with additional chromosomal mutations, to which qnr has been shown to contribute additively (10). Previous studies have also indicated that the presence of qnr facilitates the selection of chromosomal mutations causing quinolone resistance (6, 11). Thus, qnr likely acts in facilitating clinically important levels of quinolone resistance.

Understanding of the prevalence and clinical importance of plasmid-mediated resistance to quinolones should be further enhanced by future studies of the presence of qnr or its variants in a broader range of strains of K. pneumoniae and other gram-negative bacteria known to commonly contain integrons, including those with the lower MICs of fluoroquinolones found in transconjugants. This information may also be useful in defining the origin of the qnr gene, the DNA sequence of which is remarkably conserved in isolates from both China and various regions of the United States. Since quinolones are synthetic antimicrobial agents for which selection pressures for resistance likely began only in the 1960s with the introduction of the first quinolone, nalidixic acid, into clinical use, we speculate that qnr may have other functions that contributed to its evolution and emergence. Currently the closest known relatives of qnr with related functions, mcbG (encoding protection of DNA gyrase from microcin B19) (2) and mfpA (encoding reduced susceptibility to quinolones in Mycobacterium smegmatis) (12), show only 19.6% and 18.9% amino acid identity, respectively (18).

The repeating nature of amino acid sequences in the pentapeptide repeat family of proteins to which Qnr belongs and the lack of known function of other homologs (18) add further difficulty in assessing the evolutionary origin of qnr. In any case, current antibiotic or other selection pressures seem now to be sufficient for the broad geographic distribution of qnr, likely aided by its genetic linkage to resistance determinants for multiple other antibiotics.