Emergence of Oxacillinase-Mediated Resistance to Imipenem in Klebsiella pneumoniae

Bacterial strains and plasmids.K. pneumoniae 11978 was a clinical isolate from the Istanbul Faculty Hospital, Istanbul, Turkey. It was identified with the API 32GN system (bioMérieux, Marcy l'Etoile, France). Escherichia coli reference strain DH10B and plasmid pBK-CMV (Stratagene, Amsterdam, The Netherlands) were used for cloning experiments. K. pneumoniae CIP53153 and E. coli K-12 (a gift from P. Plésiat) were used as reference strains in endonuclease I-CeuI digestion experiments. K. pneumoniae CIP53153 was also used in transformation experiments.

Antimicrobial agents and MIC determinations.The antimicrobial agents and their sources have been described elsewhere (29). Antibiotic-containing disks were used for detection of antibiotic susceptibility with Mueller-Hinton agar plates by a disk diffusion assay (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). MICs were determined by an agar dilution technique, as reported previously (31), and the results were interpreted in accordance with the guidelines of the National Committee for Clinical Laboratory Standards (24).

Cloning experiments.Whole-cell DNA of K. pneumoniae 11978 was extracted as described previously (29). Cloning experiments were performed with BamHI- or BssHII-digested DNA of K. pneumoniae 11978 and BamHI- or BssHII-restricted plasmid pBK-CMV, followed by expression of recombinant plasmids in E. coli DH10B, as described previously (31). Antibiograms were performed with E. coli DH10B harboring recombinant plasmids, and the sizes of the plasmid inserts were determined by restriction analysis (35). Recombinant plasmids pVT-1 to pVT-5 were retained for further analysis. In addition, since part of the IS1999 element was present in pVT-1, it was amplified from whole-cell DNA of K. pneumoniae 11978 by PCR with primers IS1999B and OXA-TKext (Table 1).

DNA sequencing and protein analysis.Both strands of the cloned DNA fragments were sequenced with an Applied Biosystems sequencer (ABI 373). The nucleotide and deduced protein sequences were analyzed with software available over the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).

Plasmid analysis and hybridizations.Extraction of plasmid DNA from K. pneumoniae 11978 and the electroporants was performed by the method of Kieser (14). Analysis was performed on a 0.7% agarose gel with ethidium bromide staining after migration. Southern transfer was performed on a nylon membrane (Hybond N⁺; Amersham Pharmacia Biotech, Orsay, France) as described previously (33). The plasmid extract of K. pneumoniae 11978 was also used for transformation experiments with a Gene Pulser II electroporator (Bio-Rad, Ivry-sur-Seine, France) and selection on amoxicillin (100 μg/ml)-containing plates.

The endonuclease I-CeuI (New England Biolabs, Saint-Quentin-en-Yvelines, France), which digests a 26-bp sequence in rrn genes for 23S large-subunit rRNA (17), was used to identify the genetic locations of the β-lactamase genes. After digestion, the DNA fragments were separated by pulsed-field gel electrophoresis (PFGE) with a CHEF-DRII apparatus (Bio-Rad) (11). The sizes of the endonuclease I-CeuI-generated fragments were determined by comparison with those generated from E. coli K-12 (17). The I-CeuI-generated DNA fragments of K. pneumoniae 11978 and CIP53513 and plasmid DNA of K. pneumoniae 11978 and the transformants analyzed by PFGE were transferred onto a nylon membrane by the Southern technique and subsequently UV cross-linked (Stratalinker; Stratagene) for 2 min. The membranes were successively hybridized with several probes, including a 743-bp PCR-generated probe specific for the bla_OXA-48 gene (primers OXA-48A and OXA-48B [Table 1]), a 609-bp PCR-generated probe specific for the bla_OXA-47 gene (primers OXA-1A and OXA-1B [Table 1]), a 300-bp PCR-generated probe specific for bla_SHV genes (primers SWSHV-A and SWSHV-B [Table 1]), a 850-bp PCR-generated probe specific for the bla_TEM-1 gene (primers TEMalpha and TEMbeta [Table 1]), a 985-bp PCR-generated probe specific for the IS1999 transposase gene (primers IS1999A and IS1999B [Table 1]), and a 1,504-bp PCR-generated probe specific for 16S and 23S rRNA genes (primers A and B [Table 1]). Southern hybridizations were performed with the ECL nonradioactive labeling and detection kit (Amersham Pharmacia Biotech) as described by the manufacturer.

IEF analysis.Isoelectric focusing (IEF) analysis was performed with an ampholine polyacrylamide gel (pH 3.5 to 9.5) as described previously (31) with culture extracts of K. pneumoniae 11978 and E. coli DH10B harboring recombinant plasmids.

β-Lactamase purification.A culture of E. coli DH10B harboring recombinant plasmid pVT-1 that produced OXA-48 was grown overnight at 37°C in 4 liters of tryptic soy broth containing 100 μg of amoxicillin per ml and 30 μg of kanamycin per ml. The protein extracts obtained were purified as described previously (32). Briefly, the extracts were subjected to purification steps, including ion-exchange chromatography with Q-Sepharose and 20 mM Tris-HCl buffer (pH 9.0), followed by chromatography with S-Sepharose columns and 100 mM sodium phosphate buffer (pH 5.8). Elution of the β-lactamase was performed with a K₂SO₄ gradient in order to prevent any potential inhibition by NaCl. Peaks of β-lactamase activity were concentrated by using Centrisart-C30 spin columns (Sartorius, Göttingen, Germany) and were dialyzed with 100 mM phosphate buffer (pH 7.0).

Kinetic studies.Purified β-lactamases were used for kinetic measurements, which were determined at 30°C in 100 mM sodium phosphate (pH 7.0) (34). The k_cat and K_m values were determined by analyzing β-lactam hydrolysis under initial-rate conditions with a UV spectrophotometer, as described previously (31). The 50% inhibitory concentrations (IC₅₀s) of clavulanic acid, tazobactam, sulbactam, and NaCl were determined as described previously (31). The specific activities of the protein extracts and purified β-lactamase from the culture of E. coli DH10B(pVT-1) were determined as described previously (3). The protein purification rate was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein content was measured by the Bio-Rad DC protein assay. The specific activity of the β-lactamase for imipenem was also determined by UV spectrophotometry (wavelength, 297 nm) with a culture extract of K. pneumoniae 11978 from 10 ml of tryptic soy broth with 100 μM imipenem, as described previously (3). One unit of enzyme activity was defined as the activity that hydrolyzed 1 μmol of imipenem per min per mg of protein.

OMP analysis.OMPs were isolated by sarcosyl extraction of total membrane preparations, as described previously (13). Briefly, cell cultures were harvested in logarithmic phase and lysed by sonication. OMPs were obtained after treatment of the cell membranes with sodium lauryl sarcosylate (Sigma, Saint-Quentin-Fallavier, France) and subsequent ultracentrifugation. The proteins were examined by SDS-PAGE on 10% polyacrylamide gels.

Nucleotide sequence accession numbers.The nucleotide sequences of bla_OXA-48 and bla_OXA-47 reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been given accession nos. AY236073 and AY237830, respectively.

Origin of K. pneumoniae isolate and preliminary antibiotic susceptibility testing.K. pneumoniae 11978 was isolated in September 2001 at the Istanbul Faculty Hospital from the urinary tract of a 54-year-old man with a urinary tract infection and skin burns. An antibiotic regimen consisting of vancomycin and meropenem had been given for 1 month before strain isolation. Antibiotic susceptibility testing suggested an uncommon mechanism of resistance since the isolate was resistant to all β-lactams tested (Table 2). Addition of clavulanic acid and tazobactam did not significantly decrease the MICs of ticarcillin, piperacillin, and ceftazidime (Table 2). K. pneumoniae 11978 was also resistant to aminoglycosides, chloramphenicol, nalidixic acid, rifampin, sulfonamides, and tetracycline and of intermediate susceptibility to ciprofloxacin (data not shown). Culture extracts of K. pneumoniae 11978 analyzed by IEF gave five β-lactamases with pIs of 5.4, 7.2, 7.4, 7.6, and 7.7 (data not shown). To determine whether resistance to carbapenems was due to at least one of these β-lactamases, preliminary hydrolysis experiments were performed with culture extracts of K. pneumoniae 11978, which showed significant imipenem-hydrolyzing activity (22 mU/mg of protein).

Cloning and sequencing of β-lactamase content.Cloning of BssHII- or Sau3AI-restricted DNA of K. pneumoniae 11978 into pBK-CMV and expression in E. coli DH10B gave recombinant plasmids expressing different β-lactam resistance phenotypes. E. coli DH10B with recombinant plasmid pVT-1 had an oxacillinase (pI 7.2) phenotype, consistent with a carbapenem-hydrolyzing activity, which confers resistance to most penicillins and reduced susceptibility to imipenem (Table 2).

DNA sequence analysis of the 1.5-kb insert of pVT-1 identified a 798-bp open reading frame (ORF) for bla_OXA-48 that encoded a 265-amino-acid protein (Fig. 1). A serine-threonine-phenylalanine-lysine tetrad (S-T-F-K) was found at positions 70 to 73 in the deduced protein encoded by this ORF. All four structural elements characteristic of Ambler class D β-lactamases (DBLs) were found: W-X-X-X-X-X-X-I-X at DBL positions 164 to 172, Q-X-X-X-L at DBL positions 176 to 180, K-T-G at positions 216 to 218, and YGN at DBL positions 144 to 146 (8, 16, 21). A K-T-G element (positions 216 to 218) was found, as in the carbapenem-hydrolyzing OXA-23 and OXA-27 β-lactamases, whereas it was replaced by a K-S-G motif in the second group of carbapenem-hydrolyzing oxacillinases, consisting of OXA-24, OXA-25, OXA-26, and OXA-40. The fourth structural element of oxacillinases, YGN, was not replaced by an FGN motif in OXA-48 as it was in the other carbapenem-hydrolyzing oxacillinases (34). OXA-48 was weakly related to other oxacillinases and shared 46, 36, 32, and 21% amino acid identities with OXA-10; OXA-23 and OXA-27; a cluster consisting of OXA-24, OXA-25, OXA-26, and OXA-40; and OXA-1, respectively (Fig. 2).

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FIG. 1.

Nucleotide sequence of a 2,622-bp fragment of recombinant plasmid pA-1 containing the bla_OXA-48 gene. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The start codons of the ORFs are indicated by horizontal arrows. The left inverted repeat (IRL) of IS1999 is shaded in gray, and the putative −35 and −10 promoter sequences (P_out) provided by the insertion sequence element are underlined. The tnpA transposase gene is indicated. The asterisk indicates the stop codon, and the vertical arrow indicates the Sau3AI cloning insertion site in pVT-1.

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FIG. 2.

Comparison of the amino acid sequence of OXA-48 to those of OXA-23, OXA-40, OXA-1, and OXA-10. The shading indicates conserved residues of the oxacillinases, and asterisks indicate highly conserved residues. The numbering of the β-lactamases is according to DBL (8).

Part of insertion sequence element IS1999 was found upstream of the bla_OXA-48 gene in pVT-1 (Fig. 1). Since only 96 bp upstream of the bla_OXA-48 was cloned in pVT-1, PCR experiments were performed with combinations of primers specific for the IS1999 transposase gene and for the 5′ part of bla_OXA-48 and with whole-cell DNA of K. pneumoniae 11978. An entire IS1999 element was found. This element is also found upstream of the class A ESBL bla_VEB-1 gene of Pseudomonas aeruginosa isolates from Thailand (23). The IS1999 element that was located 26 bp upstream of the start codon of bla_OXA-48 likely provided promoter sequences. Indeed, DNA sequences typical of −35 and −10 enterobacterial promoter regions were found inside the IS1999 transposase-coding sequence (Fig. 1; P_out). No transposon or integron structure was found downstream of the bla_OXA-48 gene. Thus, unlike most of the oxacillinase genes (21, 34), bla_OXA-48 was not in the form of a gene cassette.

A second recombinant strain, E. coli DH10B(pVT-2), expressed another oxacillinase (pI 7.4) phenotype and was resistant to penicillins and β-lactamase inhibitors and susceptible to most cephalosporins and carbapenems (Table 2). Analysis of part of the 8-kb cloned fragment of pVT-2 identified the bla_OXA-47 gene encoding a novel oxacillinase that had seven amino acid substitutions compared to the sequence of OXA-1, i.e., Pro34Gln, Thr65Ala, Thr98Ile, Lys191Arg, Glu200Asp, Asp208Glu, and Asn254Ser (DBL numbering [8]). None of these changes was located inside the conserved motifs of DBLs (16, 21). The bla_OXA-47 gene was in the form of a gene cassette in a class 1 integron structure. The 59-bp-element sequence associated with this β-lactamase gene was 108 bp long and had a 1-bp change compared to the sequence of the oxa-1 cassette (25). The oxa-47 gene cassette was located downstream of an aadA-like gene cassette that was truncated in recombinant plasmid pVT-2 and downstream of a qacF gene cassette identical to that previously reported from Enterobacter aerogenes (30).

Another recombinant plasmid, pVT-3, gave an ESBL (pI 7.7) phenotype to E. coli DH10B due to the encoded β-lactamase SHV-2a. E. coli DH10B(pVT-3) was resistant to penicillins and expanded-spectrum cephalosporins but not to carbapenems (Table 2). Its activity was inhibited by clavulanic acid and tazobactam (Table 2). As is observed in P. aeruginosa RP-1 (22), the bla_SHV-2a gene was preceded by an IS26 insertion sequence element that gave rise to a likely hybrid promoter with a −35 promoter sequence provided by the insertion sequence element and a −10 promoter sequence of the bla_SHV-2a gene (data not shown).

E. coli DH10B harboring recombinant plasmid pVT-4 had a penicillinase (pI 5.4) phenotype corresponding to TEM-1. The bla_TEM-1 gene was preceded by a tnpR resolvase gene, which was preceded by the bla_OXA-9 β-lactamase gene (data not shown). This structure was identical to that of Tn1331, a plasmid-located transposon that has also been identified in K. pneumoniae (36, 37). However, a single base substitution inside the coding sequence for bla_OXA-9 resulted in a stop codon that truncated the last 163 carboxy-terminal amino acids. This observation was consistent with the β-lactam resistance phenotype of E. coli DH10B(pVT-4), which was susceptible to β-lactamase inhibitors (the lack of expression of oxacillinase OXA-9 likely had no effect on the β-lactam resistance phenotype).

The last recombinant plasmid, pVT-5, expressed a penicillinase (pI 7.6) phenotype in E. coli DH10B that corresponded to SHV-1. This β-lactamase gene corresponded to the naturally occurring SHV enzyme found in K. pneumoniae (4).

Biochemical properties of OXA-48 β-lactamase.After purification from culture extracts of E. coli DH10B(pVT-1), the specific activity of the OXA-48 β-lactamase against cephalothin was 17 U/mg of protein, and its purification factor was 75-fold. Protein purity was estimated to be >90% by SDS-PAGE analysis (data not shown). The OXA-48 β-lactamase had a narrow-spectrum hydrolysis profile that included penicillins, cephalothin, and imipenem and, to a lesser extent, cefotaxime, ceftazidime, cefepime, and cefpirome (Table 3). The catalytic activity (k_cat/K_m) of OXA-48 for imipenem (Table 3) was 10- and 3-fold higher than those reported for OXA-40 and the class A carbapenemase KPC-1, respectively (39). Hydrolysis of meropenem was detectable at a very low level, as is the case for the carbapenem-hydrolyzing β-lactamases OXA-24, OXA-25, and OXA-26 (1, 6). OXA-48 had hydrolytic activity against ceftazidime, although at a lower level than that of OXA-40 (Table 3). Oxacillin was significantly hydrolyzed by OXA-48, whereas it was slightly hydrolyzed by the other carbapenem-hydrolyzing oxacillinases, including OXA-40 (Table 3) (12).

Studies of activity inhibition, as measured by determination of IC₅₀s, showed that OXA-48 was weakly inhibited by clavulanic acid (IC₅₀, 16 μM), tazobactam (IC₅₀, 1.7 μM), and sulbactam (IC₅₀, 50 μM), as has been found for most of the oxacillinases (24). Nevertheless, these IC₅₀s were lower than those reported for OXA-40 (12). OXA-48 activity was inhibited by NaCl (IC₅₀, 7 mM), which is a property commonly observed for oxacillinases except those possessing an FGN motif at DBL positions 144 to 146 (12).

Genetic support of the β-lactamase genes.Analysis of plasmid extracts of K. pneumoniae 11978 identified a 70-kb plasmid (pA-1) and a 140-kb plasmid (pA-2) (data not shown). After electroporation into E. coli DH10B, three β-lactam-resistant phenotypes were obtained. Transformants contained either pA-1 or pA-2, or both. E. coli DH10B(pA-1) expressed an oxacillinase phenotype with reduced susceptibility to imipenem (Table 2), since pA-1 had the bla_OXA-48 gene. E. coli DH10B(pA-2) expressed an ESBL phenotype that corresponded to SHV-2a (data not shown). PCR experiments indicated that E. coli DH10B(pA-2) possessed the bla_SHV-2a, bla_OXA-47, and bla_TEM-1 genes and a nonexpressed bla_OXA-9 gene. Transformants harboring pA-1 and pA-2 expressed a broad-spectrum β-lactam resistance phenotype, including resistance to ceftazidime and aztreonam and reduced susceptibility to imipenem (Table 1).

Although most of the β-lactamase genes identified were located on plasmids, the I-CeuI digestion technique was used to search the K. pneumoniae 11978 chromosome for an additional chromosomal location of these novel bla_OXA-48 and bla_OXA-47 genes. Whereas a DNA probe specific for rRNA genes hybridized with five of six fragments of whole-cell DNA of K. pneumoniae 11978 generated with I-CeuI, the bla_OXA-48- and bla_OXA-47-specific probes did not hybridize with any of these fragments, indicating that these genes are located only on plasmids.

OMPs of K. pneumoniae 11978.Because natural plasmid pAT-1 conferred decreased susceptibility to imipenem once it was transformed into E. coli DH10B, this plasmid was also used to transform reference strain K. pneumoniae CIP53153 to evaluate its effect on the susceptibility of a wild-type K. pneumoniae strain. Similar β-lactam MICs were obtained for the reference strain, indicating that the type of species of the family Enterobacteriaceae by itself does not explain the high imipenem MIC for K. pneumoniae 11978 (Table 2). OMP analysis of K. pneumoniae 11978 was performed, and the results were compared to that for a K. pneumoniae reference strain. K. pneumoniae 11978 lacked a 36-kDa porin, whereas the 35- and 37 kDa porins were found in cell extracts of both strains in the form of a doublet (Fig. 3).

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FIG. 3.

OMP profiles of K. pneumoniae 11978 (lane A) and reference strain K. pneumoniae CIP53153 (lane B). Lane M, molecular size marker, with sizes (in kilodaltons) indicated on the left. The arrow indicates the position of the 36-kDa protein that was missing from K. pneumoniae 11978, whereas the 35- and 37-kDa proteins likely appeared in the form of a doublet.