qnrB, Another Plasmid-Mediated Gene for Quinolone Resistance
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《抗菌试剂及化学方法》
1.Lahey Clinic, Burlington,2.Massachusetts General Hospital, Boston, Massachusetts
ABSTRACT
A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 ?-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to 95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 μg/ml (6 μM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 ?-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.
INTRODUCTION
The first plasmid-mediated quinolone resistance gene (qnr) was discovered in a Klebsiella pneumoniae isolate from Birmingham, Alabama, collected in 1994 (10). It occurred in a multiresistance plasmid, pMG252, in an integron-like structure near Orf513 (17). Qnr, the gene product, is a member of the pentapeptide repeat family of proteins and has been shown to block the action of ciprofloxacin on purified DNA gyrase and topoisomerase IV (17, 19). In Escherichia coli pMG252 determines low-level quinolone resistance but facilitates the selection of higher-level resistance mutations (10). qnr plasmids have been found in clinical isolates of Citrobacter freundii, Enterobacter spp., E. coli, K. pneumoniae, Providencia stuartii, and Salmonella spp. from the United States, Europe, and the Near and Far East (1, 13). Another qnr gene, qnrS, has also recently been found in a plasmid from a strain of Shigella flexneri isolated in Japan (2).
While investigating strains of K. pneumoniae from India, some of which contained qnr, it was realized that several could transfer low-level quinolone resistance but were negative by PCR for qnr. The new plasmid-mediated quinolone resistance gene has been termed qnrB, and the original gene is now designated qnrA. qnrB has been cloned and sequenced. Purified QnrB protects DNA gyrase from quinolone action like QnrA does. A PCR assay for qnrB indicates that it is as common as qnrA in samples from the United States and has greater amino acid variability.
(The results of this study were presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, 30 October to 2 November 2004, Washington, D.C.)
MATERIALS AND METHODS
Strains, plasmids, and growth conditions. K. pneumoniae strains 17, 19, 21, 24, and 25 were isolated from blood, a catheter tip, pus, and sputum in 2002 and 2003 at Coimbatore in South India. Citrobacter koseri 3699-1 was isolated at Long Beach, CA, in 2000. Enterobacter cloacae 3668 came from Philadelphia, PA, in 2000-2001 (9). E. cloacae 78149 and Escherichia coli EC-35 were acquired in 1996 from St. Louis, MO. Matings were performed with E. coli J53 Azir (met pro; azide-resistant) as a recipient (6). E. coli TOP10 (Invitrogen, Carlsbad, CA) was used in cloning. Strains were routinely grown in Luria-Bertani broth. Culture plates contained tryptic soy agar (TSA) or Mueller-Hinton agar (Becton, Dickinson and Co., Sparks, MD). Selective media contained ampicillin (100 μg/ml), cefotaxime (10 μg/ml), chloramphenicol (25 μg/ml), ciprofloxacin (0.015 μg/ml), kanamycin (25 μg/ml), or nalidixic acid (12 μg/ml) as required.
Susceptibility testing. Disk and agar dilution susceptibility testing was performed as described in CLSI (formerly NCCLS) publications, using Mueller-Hinton agar and 16 to 20 h of incubation at 37°C (11, 12).
Cloning and nucleotide sequence analysis. Plasmid DNA was isolated from an E. coli J53 derivative by using the Large-Construct kit (QIAGEN, Valencia, CA), digested with one of several endonucleases, ligated to similarly restricted phagemid pBC SK (Stratagene), and introduced into E. coli TOP10 with selection on TSA plates containing chloramphenicol and nalidixic acid. Using the GPS-1 genome priming system (NE Biolabs, Ipswich, MA), a kanamycin resistance transposon was inserted to inactivate the quinolone resistance gene and allow sequencing using primerN and primerS from the transposon ends. Cycle sequencing was carried out with an ABI Prism 3100 genetic analyzer (Tufts University Core Facility) and was continued by primer walking on both DNA strands. For sequence comparisons, the NCBI BLAST program and facilities of the TIGR Comprehensive Microbial Resource (www.tigr.org) were utilized.
Overexpression and purification of QnrB. The qnrB1 gene from pMG298 was cloned into expression vector pQE-60 (QIAGEN) at its NcoI and BamHI sites. An internal NcoI site was first removed from qnrB1 by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with primers 5'-GTGATTTATCGATGGCGGATTTTCGC and 5'-GCGAAAATCCGCCATCGATAAATCAC, which cause no change in the amino acid sequence. The gene was then amplified by PCR using primers 5'-GGCCATGGCGCCATTACTGTATAAA and 5'-GCGCGGATCCACCAATCACCGCGATGCC and ligated after digestion with NcoI and BamHI into pQE-60. In the process, the amino acid following the initial methionine was changed from threonine to alanine and a tag of six histidines was added to the C terminus of the protein. Proper construction was confirmed by sequencing, and the pQE60-QnrB1 plasmid was transformed into E. coli M15(pREP4) (QIAGEN) to place expression of qnrB1 under the control of induction by isopropyl-1-thio-?-D-galactopyranoside (IPTG).
E. coli M15 (pREP4)(pQE-60-QnrB1) was grown in LB medium with 100 μg/ml ampicillin and 25 μg/ml kanamycin until the cell density reached an optical density at 600 nm of 0.6 to 0.8, induced with IPTG at a final concentration of 1 mM, and allowed 4 h of further growth until being harvested by centrifugation. The pellet was suspended in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 150 mM NaCl and lysed with 0.02% lysozyme and 1x EDTA-free protease inhibitor mix (Roche Diagnostics, Mannheim, Germany) on ice for 0.5 to 2 h. The lysate was centrifuged at 25,000 x g for 90 min at 4°C. The supernatant containing the soluble protein was filtered through a 0.2-μm membrane with a Nalgene filter unit (Nalgene, Rochester, NY) and loaded onto a preequilibrated HiTrapChelating HP column (Amersham Biosciences, Piscataway, NJ), and the histidine-tagged protein was eluted with increasing concentrations of imidazole (50 to 300 mM) and collected in 1-ml fractions. The eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using precast 12% Ready Tris-HCl gels (Bio-Rad, Hercules, CA). Fractions containing a single protein band of 25 kDa were dialyzed against 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 150 mM NaCl, 10% glycerol overnight at 4°C with several buffer changes. Protein concentrations were determined using the Bradford assay (Bio-Rad).
Gyrase supercoiling assay. DNA supercoiling assays were performed as described previously, using DNA gyrase reconstituted from cloned E. coli GyrA and GyrB subunits (18) and relaxed pBR322 plasmid DNA (Topogen Inc., Port Orange, FL).
?-Lactamase characterization. The presence of an extended-spectrum ?-lactamase (ESBL) was suspected from cefotaxime and ceftazidime resistance and confirmed by repeat testing in the presence of clavulanic acid (11). Transmissibility of resistance was tested by mating clinical isolates with E. coli J53 Azir with selection on TSA agar plates containing 10 μg/ml cefotaxime and 200 μg/ml sodium azide. ?-Lactamase isoelectric focusing was carried out using the PhastSystem (Amersham Biosciences, Piscataway, NJ) as described by Huovinen (4). bla genes were amplified using primers 5'-TTTCCCCATTCCGTTTCCGC and 5'-TTCGTATCTTCCAGAATAAG for blaCTX-M-15 amplification and previously described primers S1 and S2 for blaSHV amplification (20). The amplified genes were sequenced using the same primers and additional primers designed from the known bla gene sequence.
PCR conditions. Detection of qnrA utilized primers QP1 and QP2 with PCR conditions as previously described (5). For qnrB, primers FQ1 (5'-ATGACGCCATTACTGTATAA) and FQ2 (5'-GATCGCAATGTGTGAAGTTT) were used. PCR conditions were 94°C for 45 s, 53°C for 45 s, and 72°C for 1 min for 32 cycles. The presence of Orf513 was tested with primers 5'-AAGGAACGCCACGGCGAGTCAA and 5'-TGCAAAGACGCCGTGGAAGC, and the presence of Orf1005 was evaluated with primers 5'-CTTGGTATTCGAAGCTGGTC and 5'-CTACCGTTTGCAACAGTAAG.
Nucleotide sequence accession numbers. The qnrB1 and qnrB2 sequences have been submitted to GenBank with accession numbers DQ351241 and DQ351242.
RESULTS
Discovery of qnrB. The K. pneumoniae strains from India were originally studied to determine the basis of their ESBL phenotype. Transconjugants of strains 17, 19, 24, and 25, selected for cefotaxime resistance, were noted to have low-level quinolone resistance, but when tested by PCR with primers QP1 and QP2, only K. pneumoniae strains 19 and 24 were positive for the known qnr gene.
Accordingly, DNA of plasmid pMG298 from strain 17 was digested with PstI endonuclease and ligated into vector pBC SK (determining chloramphenicol resistance), selecting for simultaneous resistance to ciprofloxacin and chloramphenicol. A recombinant plasmid containing a 15.3-kb insert was obtained. Quinolone resistance was also expressed from pBC SK containing a 4.8-kb BamHI fragment from plasmid pMG299 originating in strain 24 and from a 5.9-kb PstI fragment from plasmid pMG300 transferred from strain 25.
To facilitate DNA sequencing, a Tn7-based transposon carrying a kanamycin resistance gene was inserted into the recombinant plasmids, and colonies were screened for loss of nalidixic acid resistance. Using primers that matched sequence at the ends of the inserted transposon, sequencing was initiated and continued by primer walking with the original unmodified recombinant plasmid. A new quinolone resistance gene, qnrB1, was discovered. It had 49.5% nucleotide identity and 39.5% amino acid identity with qnrA and 49.3% nucleotide identity and 37.4% amino acid identity with qnrS (Fig. 1) and had the potential to code for a 226-amino-acid protein, which belonged to the pentapeptide repeat family.
Distribution of qnrB. Using PCR with primers derived from the qnrB1 sequence, the gene was found also in K. pneumoniae strains 21, 24, and 25 from southern India, but the plasmids involved differed in resistance properties (Table 1). Plasmid pMG299 from strain 24, like plasmid pMG298 from strain 17, carried blaCTX-M-15, but the qnrB1 plasmid pMG300 from strain 25 encoded SHV-12 and had no CTX-M gene by PCR. The blaSHV-12 gene was also present in strain 21, but neither ?-lactam nor other antibiotic resistances could be transferred from it by conjugation, implying the presence of a Tra– plasmid.
Over 100 other plasmid-carrying strains were screened for qnrB. A strain with another CTX-M-15 plasmid was negative, but 4 of about 20 independently derived plasmids encoding SHV-12 were positive, including plasmids found in C. koseri, E. cloacae, and E. coli (Table 1). All had identical sequences which differed from that of qnrB1 by 26 of 680 nucleotides, including an additional nucleotide prior to a second potential ATG start codon at position 36. The extra nucleotide would cause a frameshift if translation began at the first site, so that the qnrB2 gene codes for a 215-amino-acid protein that in addition differs from QnrB1 in 5 amino acids (Fig. 1). The qnrB2 plasmids all encode SHV-12 but differ in other associated resistances (Table 1). Attempts to clone qnrB2 with BamHI or PstI failed, but the gene was cloned into pBC SK as a 2.4-kb EcoRI fragment.
Genetic environment of qnrB. The immediate genetic environments of qnrB1 in plasmids pMG298, pMG299, and pMG300 were similar (Fig. 2). qnrB1 was found downstream from Orf1005, which encodes a putative transposase and is bracketed by imperfect 83-bp inverted repeat segments (16). In pMG298 a tnpA gene was found downstream from Orf1005, but this gene was absent in pMG300. Between Orf1005 and qnrB1 was a 383-bp open reading frame, Orf1, which is >70% identical to a truncated pspF gene coding for the transcriptional activator of the stress-inducible psp operon (7). Orf1 contains an EcoRI site, and a shorter segment of Orf1 was found upstream from the qnrB2 gene, which was cloned on an EcoRI fragment (Fig. 2). Downstream from qnrB2 was an open reading frame (Orf2), with >60% identity to hypothetical proteins of several gram-negative species, and Orf3, with 83% identity to the sapA gene, which encodes a peptide transport periplasmic protein in gram-negative bacteria. By a PCR assay Orf1005 was present in K. pneumoniae strains 17, 24, and 25 but not in any of the strains containing qnrB2. Orf512 was not detected in any of the strains.
Effect of QnrB on quinolone susceptibility. Like QnrA, QnrB provided low-level resistance to all quinolones tested (Table 2).
Effect of QnrB on quinolone inhibition of gyrase. To investigate the mechanism of quinolone protection, qnrB was cloned into an expression vector that attached a C-terminal polyhistidine tag, which facilitated purification of QnrB-His6 protein by Ni affinity chromatography. His-tagged QnrB appeared to be 95% homogenous by gel assay.
QnrB-His6 demonstrated a concentration-dependent protection of purified gyrase from ciprofloxacin inhibition of DNA supercoiling (Fig. 3). With 2 μg/ml (6 μM) ciprofloxacin, the concentration of QnrB-His6 required for half protection was about 0.5 nM, and a protective effect was seen with as little as 5 pM. The highest concentration of QnrB-His6 tested (25 μM) inhibited gyrase-mediated DNA supercoiling, but inhibition was not seen with 5 μM QnrB-His6, a concentration still 750 times higher than that of DNA gyrase (Fig. 3, compare lanes 4 and 5).
DISCUSSION
QnrB, like QnrA and QnrS (2), provides low-level resistance to quinolones and belongs to the pentapeptide repeat family of proteins, one member of which has recently been shown to have a DNA-like structure which would allow it to mimic DNA as a substrate for DNA gyrase (3). For a protein in which overall structure is important rather than catalytic activity, considerable amino acid variability may be permissible. Thus, QnrB and QnrA have only 39.5% of their amino acids in common, while QnrB and QnrS share 37.4%, but all are pentapeptide repeat proteins with two domains joined by a glycine residue (17).
Purified QnrB, like QnrA, protected DNA gyrase from quinolone action. It seems to be even more potent than QnrA in blocking the action of ciprofloxacin (17). At a concentration almost 4,000 times that of DNA gyrase, QnrB inhibited the enzyme, but this effect disappeared at a fivefold lower concentration of QnrB. Thus, in contrast to MfpA, another pentapeptide repeat protein that blocks ciprofloxacin action (3), QnrB did not inhibit gyrase-mediated DNA supercoiling over a wide range of quinolone-protective concentrations. Accordingly, models of Qnr action that do not require direct gyrase inhibition must be considered.
A close relative and likely progenitor of QnrA, differing in only 1 to 2% amino acids, has recently been found in the commensal water organism Shewanella algae (15). The origin of QnrB is not yet known, since the closest relative currently disclosed by a BLAST search is a hypothetical protein from Photobacterium profundum with only 44.5% amino acid identity. The qnrA gene has been found in plasmids with a variety of other resistance determinants but always as part of a sul1-type integron (13). qnrB1 is located near a putative transposase, Orf1005, in plasmids pMG298 and pMG299, but Orf1005 was absent from QnrB2 plasmids from the United States. The linkage of qnrB to Orf1, Orf2, and Orf3, which resemble known chromosomal genes, suggests that all were acquired from a chromosomal source. The homologues of Orf1 and Orf3 are adjacent to each other on the P. profundum chromosome, but the homologues of qnrB and Orf2 occupy separate and distant locations. The mechanism of qnrB acquisition, like that of qnrS, remains to be elucidated.
In both India and the United States qnrB has been found on plasmids also encoding ESBLs: CTX-M-15 and SHV-12 in India and SHV-12 in the United States. The frequent association of quinolone resistance with ESBL production has been noted in several studies (8, 14). The presence of qnr and blaESBL genes on the same plasmid is one of several possible explanations for this association.
qnrB2 has been found on plasmids collected in 1996, and the association of qnrB with SHV-12 on plasmids found in both India and the United States suggests that this resistance mechanism has been present long enough to disseminate widely. Preliminary surveys indicate that in ceftazidime-resistant Enterobacter and Klebsiella isolates from the United States qnrB is as common as qnrA (Robicsek et al., unpublished observations). It would not be surprising if even more members of the qnr family were discovered.
ACKNOWLEDGMENTS
This study was supported in part by grants AI43312 (to G.A.J.) and AI57576 (to D.C.H.) from the National Institutes of Health, U.S. Public Health Service.
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ABSTRACT
A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 ?-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to 95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 μg/ml (6 μM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 ?-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.
INTRODUCTION
The first plasmid-mediated quinolone resistance gene (qnr) was discovered in a Klebsiella pneumoniae isolate from Birmingham, Alabama, collected in 1994 (10). It occurred in a multiresistance plasmid, pMG252, in an integron-like structure near Orf513 (17). Qnr, the gene product, is a member of the pentapeptide repeat family of proteins and has been shown to block the action of ciprofloxacin on purified DNA gyrase and topoisomerase IV (17, 19). In Escherichia coli pMG252 determines low-level quinolone resistance but facilitates the selection of higher-level resistance mutations (10). qnr plasmids have been found in clinical isolates of Citrobacter freundii, Enterobacter spp., E. coli, K. pneumoniae, Providencia stuartii, and Salmonella spp. from the United States, Europe, and the Near and Far East (1, 13). Another qnr gene, qnrS, has also recently been found in a plasmid from a strain of Shigella flexneri isolated in Japan (2).
While investigating strains of K. pneumoniae from India, some of which contained qnr, it was realized that several could transfer low-level quinolone resistance but were negative by PCR for qnr. The new plasmid-mediated quinolone resistance gene has been termed qnrB, and the original gene is now designated qnrA. qnrB has been cloned and sequenced. Purified QnrB protects DNA gyrase from quinolone action like QnrA does. A PCR assay for qnrB indicates that it is as common as qnrA in samples from the United States and has greater amino acid variability.
(The results of this study were presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, 30 October to 2 November 2004, Washington, D.C.)
MATERIALS AND METHODS
Strains, plasmids, and growth conditions. K. pneumoniae strains 17, 19, 21, 24, and 25 were isolated from blood, a catheter tip, pus, and sputum in 2002 and 2003 at Coimbatore in South India. Citrobacter koseri 3699-1 was isolated at Long Beach, CA, in 2000. Enterobacter cloacae 3668 came from Philadelphia, PA, in 2000-2001 (9). E. cloacae 78149 and Escherichia coli EC-35 were acquired in 1996 from St. Louis, MO. Matings were performed with E. coli J53 Azir (met pro; azide-resistant) as a recipient (6). E. coli TOP10 (Invitrogen, Carlsbad, CA) was used in cloning. Strains were routinely grown in Luria-Bertani broth. Culture plates contained tryptic soy agar (TSA) or Mueller-Hinton agar (Becton, Dickinson and Co., Sparks, MD). Selective media contained ampicillin (100 μg/ml), cefotaxime (10 μg/ml), chloramphenicol (25 μg/ml), ciprofloxacin (0.015 μg/ml), kanamycin (25 μg/ml), or nalidixic acid (12 μg/ml) as required.
Susceptibility testing. Disk and agar dilution susceptibility testing was performed as described in CLSI (formerly NCCLS) publications, using Mueller-Hinton agar and 16 to 20 h of incubation at 37°C (11, 12).
Cloning and nucleotide sequence analysis. Plasmid DNA was isolated from an E. coli J53 derivative by using the Large-Construct kit (QIAGEN, Valencia, CA), digested with one of several endonucleases, ligated to similarly restricted phagemid pBC SK (Stratagene), and introduced into E. coli TOP10 with selection on TSA plates containing chloramphenicol and nalidixic acid. Using the GPS-1 genome priming system (NE Biolabs, Ipswich, MA), a kanamycin resistance transposon was inserted to inactivate the quinolone resistance gene and allow sequencing using primerN and primerS from the transposon ends. Cycle sequencing was carried out with an ABI Prism 3100 genetic analyzer (Tufts University Core Facility) and was continued by primer walking on both DNA strands. For sequence comparisons, the NCBI BLAST program and facilities of the TIGR Comprehensive Microbial Resource (www.tigr.org) were utilized.
Overexpression and purification of QnrB. The qnrB1 gene from pMG298 was cloned into expression vector pQE-60 (QIAGEN) at its NcoI and BamHI sites. An internal NcoI site was first removed from qnrB1 by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with primers 5'-GTGATTTATCGATGGCGGATTTTCGC and 5'-GCGAAAATCCGCCATCGATAAATCAC, which cause no change in the amino acid sequence. The gene was then amplified by PCR using primers 5'-GGCCATGGCGCCATTACTGTATAAA and 5'-GCGCGGATCCACCAATCACCGCGATGCC and ligated after digestion with NcoI and BamHI into pQE-60. In the process, the amino acid following the initial methionine was changed from threonine to alanine and a tag of six histidines was added to the C terminus of the protein. Proper construction was confirmed by sequencing, and the pQE60-QnrB1 plasmid was transformed into E. coli M15(pREP4) (QIAGEN) to place expression of qnrB1 under the control of induction by isopropyl-1-thio-?-D-galactopyranoside (IPTG).
E. coli M15 (pREP4)(pQE-60-QnrB1) was grown in LB medium with 100 μg/ml ampicillin and 25 μg/ml kanamycin until the cell density reached an optical density at 600 nm of 0.6 to 0.8, induced with IPTG at a final concentration of 1 mM, and allowed 4 h of further growth until being harvested by centrifugation. The pellet was suspended in 20 mM Tris-HCl (pH 7.5), 10% glycerol, 150 mM NaCl and lysed with 0.02% lysozyme and 1x EDTA-free protease inhibitor mix (Roche Diagnostics, Mannheim, Germany) on ice for 0.5 to 2 h. The lysate was centrifuged at 25,000 x g for 90 min at 4°C. The supernatant containing the soluble protein was filtered through a 0.2-μm membrane with a Nalgene filter unit (Nalgene, Rochester, NY) and loaded onto a preequilibrated HiTrapChelating HP column (Amersham Biosciences, Piscataway, NJ), and the histidine-tagged protein was eluted with increasing concentrations of imidazole (50 to 300 mM) and collected in 1-ml fractions. The eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using precast 12% Ready Tris-HCl gels (Bio-Rad, Hercules, CA). Fractions containing a single protein band of 25 kDa were dialyzed against 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 150 mM NaCl, 10% glycerol overnight at 4°C with several buffer changes. Protein concentrations were determined using the Bradford assay (Bio-Rad).
Gyrase supercoiling assay. DNA supercoiling assays were performed as described previously, using DNA gyrase reconstituted from cloned E. coli GyrA and GyrB subunits (18) and relaxed pBR322 plasmid DNA (Topogen Inc., Port Orange, FL).
?-Lactamase characterization. The presence of an extended-spectrum ?-lactamase (ESBL) was suspected from cefotaxime and ceftazidime resistance and confirmed by repeat testing in the presence of clavulanic acid (11). Transmissibility of resistance was tested by mating clinical isolates with E. coli J53 Azir with selection on TSA agar plates containing 10 μg/ml cefotaxime and 200 μg/ml sodium azide. ?-Lactamase isoelectric focusing was carried out using the PhastSystem (Amersham Biosciences, Piscataway, NJ) as described by Huovinen (4). bla genes were amplified using primers 5'-TTTCCCCATTCCGTTTCCGC and 5'-TTCGTATCTTCCAGAATAAG for blaCTX-M-15 amplification and previously described primers S1 and S2 for blaSHV amplification (20). The amplified genes were sequenced using the same primers and additional primers designed from the known bla gene sequence.
PCR conditions. Detection of qnrA utilized primers QP1 and QP2 with PCR conditions as previously described (5). For qnrB, primers FQ1 (5'-ATGACGCCATTACTGTATAA) and FQ2 (5'-GATCGCAATGTGTGAAGTTT) were used. PCR conditions were 94°C for 45 s, 53°C for 45 s, and 72°C for 1 min for 32 cycles. The presence of Orf513 was tested with primers 5'-AAGGAACGCCACGGCGAGTCAA and 5'-TGCAAAGACGCCGTGGAAGC, and the presence of Orf1005 was evaluated with primers 5'-CTTGGTATTCGAAGCTGGTC and 5'-CTACCGTTTGCAACAGTAAG.
Nucleotide sequence accession numbers. The qnrB1 and qnrB2 sequences have been submitted to GenBank with accession numbers DQ351241 and DQ351242.
RESULTS
Discovery of qnrB. The K. pneumoniae strains from India were originally studied to determine the basis of their ESBL phenotype. Transconjugants of strains 17, 19, 24, and 25, selected for cefotaxime resistance, were noted to have low-level quinolone resistance, but when tested by PCR with primers QP1 and QP2, only K. pneumoniae strains 19 and 24 were positive for the known qnr gene.
Accordingly, DNA of plasmid pMG298 from strain 17 was digested with PstI endonuclease and ligated into vector pBC SK (determining chloramphenicol resistance), selecting for simultaneous resistance to ciprofloxacin and chloramphenicol. A recombinant plasmid containing a 15.3-kb insert was obtained. Quinolone resistance was also expressed from pBC SK containing a 4.8-kb BamHI fragment from plasmid pMG299 originating in strain 24 and from a 5.9-kb PstI fragment from plasmid pMG300 transferred from strain 25.
To facilitate DNA sequencing, a Tn7-based transposon carrying a kanamycin resistance gene was inserted into the recombinant plasmids, and colonies were screened for loss of nalidixic acid resistance. Using primers that matched sequence at the ends of the inserted transposon, sequencing was initiated and continued by primer walking with the original unmodified recombinant plasmid. A new quinolone resistance gene, qnrB1, was discovered. It had 49.5% nucleotide identity and 39.5% amino acid identity with qnrA and 49.3% nucleotide identity and 37.4% amino acid identity with qnrS (Fig. 1) and had the potential to code for a 226-amino-acid protein, which belonged to the pentapeptide repeat family.
Distribution of qnrB. Using PCR with primers derived from the qnrB1 sequence, the gene was found also in K. pneumoniae strains 21, 24, and 25 from southern India, but the plasmids involved differed in resistance properties (Table 1). Plasmid pMG299 from strain 24, like plasmid pMG298 from strain 17, carried blaCTX-M-15, but the qnrB1 plasmid pMG300 from strain 25 encoded SHV-12 and had no CTX-M gene by PCR. The blaSHV-12 gene was also present in strain 21, but neither ?-lactam nor other antibiotic resistances could be transferred from it by conjugation, implying the presence of a Tra– plasmid.
Over 100 other plasmid-carrying strains were screened for qnrB. A strain with another CTX-M-15 plasmid was negative, but 4 of about 20 independently derived plasmids encoding SHV-12 were positive, including plasmids found in C. koseri, E. cloacae, and E. coli (Table 1). All had identical sequences which differed from that of qnrB1 by 26 of 680 nucleotides, including an additional nucleotide prior to a second potential ATG start codon at position 36. The extra nucleotide would cause a frameshift if translation began at the first site, so that the qnrB2 gene codes for a 215-amino-acid protein that in addition differs from QnrB1 in 5 amino acids (Fig. 1). The qnrB2 plasmids all encode SHV-12 but differ in other associated resistances (Table 1). Attempts to clone qnrB2 with BamHI or PstI failed, but the gene was cloned into pBC SK as a 2.4-kb EcoRI fragment.
Genetic environment of qnrB. The immediate genetic environments of qnrB1 in plasmids pMG298, pMG299, and pMG300 were similar (Fig. 2). qnrB1 was found downstream from Orf1005, which encodes a putative transposase and is bracketed by imperfect 83-bp inverted repeat segments (16). In pMG298 a tnpA gene was found downstream from Orf1005, but this gene was absent in pMG300. Between Orf1005 and qnrB1 was a 383-bp open reading frame, Orf1, which is >70% identical to a truncated pspF gene coding for the transcriptional activator of the stress-inducible psp operon (7). Orf1 contains an EcoRI site, and a shorter segment of Orf1 was found upstream from the qnrB2 gene, which was cloned on an EcoRI fragment (Fig. 2). Downstream from qnrB2 was an open reading frame (Orf2), with >60% identity to hypothetical proteins of several gram-negative species, and Orf3, with 83% identity to the sapA gene, which encodes a peptide transport periplasmic protein in gram-negative bacteria. By a PCR assay Orf1005 was present in K. pneumoniae strains 17, 24, and 25 but not in any of the strains containing qnrB2. Orf512 was not detected in any of the strains.
Effect of QnrB on quinolone susceptibility. Like QnrA, QnrB provided low-level resistance to all quinolones tested (Table 2).
Effect of QnrB on quinolone inhibition of gyrase. To investigate the mechanism of quinolone protection, qnrB was cloned into an expression vector that attached a C-terminal polyhistidine tag, which facilitated purification of QnrB-His6 protein by Ni affinity chromatography. His-tagged QnrB appeared to be 95% homogenous by gel assay.
QnrB-His6 demonstrated a concentration-dependent protection of purified gyrase from ciprofloxacin inhibition of DNA supercoiling (Fig. 3). With 2 μg/ml (6 μM) ciprofloxacin, the concentration of QnrB-His6 required for half protection was about 0.5 nM, and a protective effect was seen with as little as 5 pM. The highest concentration of QnrB-His6 tested (25 μM) inhibited gyrase-mediated DNA supercoiling, but inhibition was not seen with 5 μM QnrB-His6, a concentration still 750 times higher than that of DNA gyrase (Fig. 3, compare lanes 4 and 5).
DISCUSSION
QnrB, like QnrA and QnrS (2), provides low-level resistance to quinolones and belongs to the pentapeptide repeat family of proteins, one member of which has recently been shown to have a DNA-like structure which would allow it to mimic DNA as a substrate for DNA gyrase (3). For a protein in which overall structure is important rather than catalytic activity, considerable amino acid variability may be permissible. Thus, QnrB and QnrA have only 39.5% of their amino acids in common, while QnrB and QnrS share 37.4%, but all are pentapeptide repeat proteins with two domains joined by a glycine residue (17).
Purified QnrB, like QnrA, protected DNA gyrase from quinolone action. It seems to be even more potent than QnrA in blocking the action of ciprofloxacin (17). At a concentration almost 4,000 times that of DNA gyrase, QnrB inhibited the enzyme, but this effect disappeared at a fivefold lower concentration of QnrB. Thus, in contrast to MfpA, another pentapeptide repeat protein that blocks ciprofloxacin action (3), QnrB did not inhibit gyrase-mediated DNA supercoiling over a wide range of quinolone-protective concentrations. Accordingly, models of Qnr action that do not require direct gyrase inhibition must be considered.
A close relative and likely progenitor of QnrA, differing in only 1 to 2% amino acids, has recently been found in the commensal water organism Shewanella algae (15). The origin of QnrB is not yet known, since the closest relative currently disclosed by a BLAST search is a hypothetical protein from Photobacterium profundum with only 44.5% amino acid identity. The qnrA gene has been found in plasmids with a variety of other resistance determinants but always as part of a sul1-type integron (13). qnrB1 is located near a putative transposase, Orf1005, in plasmids pMG298 and pMG299, but Orf1005 was absent from QnrB2 plasmids from the United States. The linkage of qnrB to Orf1, Orf2, and Orf3, which resemble known chromosomal genes, suggests that all were acquired from a chromosomal source. The homologues of Orf1 and Orf3 are adjacent to each other on the P. profundum chromosome, but the homologues of qnrB and Orf2 occupy separate and distant locations. The mechanism of qnrB acquisition, like that of qnrS, remains to be elucidated.
In both India and the United States qnrB has been found on plasmids also encoding ESBLs: CTX-M-15 and SHV-12 in India and SHV-12 in the United States. The frequent association of quinolone resistance with ESBL production has been noted in several studies (8, 14). The presence of qnr and blaESBL genes on the same plasmid is one of several possible explanations for this association.
qnrB2 has been found on plasmids collected in 1996, and the association of qnrB with SHV-12 on plasmids found in both India and the United States suggests that this resistance mechanism has been present long enough to disseminate widely. Preliminary surveys indicate that in ceftazidime-resistant Enterobacter and Klebsiella isolates from the United States qnrB is as common as qnrA (Robicsek et al., unpublished observations). It would not be surprising if even more members of the qnr family were discovered.
ACKNOWLEDGMENTS
This study was supported in part by grants AI43312 (to G.A.J.) and AI57576 (to D.C.H.) from the National Institutes of Health, U.S. Public Health Service.
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