INTRODUCTION

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Klebsiella oxytoca, like Klebsiella spp., carries a chromosomally encoded -lactamase belonging to class A. Recently, the -lactamase genes of K. oxytoca were divided into two main groups: bla_oxy-1 and bla_oxy-2 (9). These chromosomal enzymes preferentially inactivate penicillins, conferring resistance on amino- and carboxypenicillins. The ability of the K. oxytoca -lactamases to hydrolyze extended-spectrum -lactams could confer resistance to these antibiotics when the -lactamase is overproduced from a modified promoter (5, 6, 8). The synergistic effects of the three -lactamase inhibitors clavulanate, sulbactam, and tazobactam were generally poor and varied according to the type of OXY enzymes, the combination of drugs examined, and the type of inhibitor (7). Clavulanate was effective against the overproducing strains, even though it had a relatively high 50% inhibitory concentration (IC₅₀).

We report here on a clinical strain of K. oxytoca harboring an unusual resistance phenotype characterized by high-level resistance to amoxicillin, amoxicillin-clavulanate, piperacillin, piperacillin-tazobactam, and, to a lesser extent, ticarcillin and ticarcillin-clavulanate; these resistances were associated with susceptibility to all cephalosporins including cephalothin and aztreonam. This resistance phenotype was very similar to that observed when an inhibitor-resistant TEM (IRT) enzyme was produced in Escherichia coli (19).

	MATERIALS AND METHODS

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Bacterial strains. K. oxytoca KER producing a mutant of OXY-2 -lactamase was isolated from the blood of a patient hospitalized in a gastroenterology unit of the hospital of Vannes, France. Seventeen clinical K. oxytoca strains were used for comparison of MICs: 14 K. oxytoca strains that produced an OXY-1 or an OXY-2 -lactamase and that harbored the usual resistance phenotype of wild strains of this species and 3 -lactamase-overproducing K. oxytoca strains that had high levels of resistance to penicillins, cephalothin, cefuroxime, and aztreonam but that were susceptible to cefoxitin, cefotaxime, ceftazidime, and imipenem.

Susceptibility to -lactams. The MICs of amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-clavulanate, piperacillin-sulbactam, piperacillin-tazobactam, cephalothin, cefotaxime, ceftazidime, and aztreonam were determined by dilution in Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) at an inoculum of 10⁴ CFU per spot. Antibiotics were provided as powders by SmithKline Beecham, Paris, France (amoxicillin, ticarcillin, and clavulanate); Lederlé, Paris, France (piperacillin and tazobactam); Hoechst-Roussel, Paris, France (cefotaxime); Glaxo Wellcome, Paris, France (ceftazidime); Bristol-Myers-Squibb, Paris, France (aztreonam); Eli Lilly, Paris, France (cephalothin); and Pfizer, Paris, France (sulbactam).

Isoelectric focusing. Isoelectric focusing was performed with polyacrylamide gels as described previously (18). -Lactamases with known pIs (TEM-1 [pI 5.4], TEM-2 [pI 5.6], OXY-2-2 [pI 5.7], and TEM-3 [pI 6.3]) were used as standards.

Determination of -lactamase kinetic constants K_m and relative V_max. The enzyme extracts were purified by ion-exchange chromatography with AGMP-1 resin (Bio-Rad). The resin, in the chloride form, was treated with 0.1 M ammonia in water and was then washed extensively with water. After absorption of the extracts, elution was performed with a 0.1 M NaCl solution. The active fractions were pooled, dialyzed extensively, and lyophilized. The affinity (K_m) and relative V_max values were determined with purified extracts by using a computerized microacidimetric method (15). The K_m and relative V_max values of the OXY-2 -lactamase mutant (strain KER) were compared with those of the -lactamase OXY-2-2 (strain HB60) for penicillins and cephalosporins.

DNA amplification. DNA amplification with a crude extract of K. oxytoca KER as the template was performed by PCR (18) in a DNA thermal cycler (Perkin-Elmer Cetus Instruments) with consensus primers OXY-A, 5'-d(GCC GCC GCC GTT CCG CTG)-3', at position 347 and OXY-B, 5'-d (AAG CCC TTC GGT GAC GAT)-3', at position 1180 on the lagging strand of the nucleotide sequence of the bla OXY-2 region (9).

Sequencing. The sequences of the PCR products were determined directly on an automatic sequencer (ABI 377) with AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). Complete sequencing of the gene and of its promoter region was obtained with primers Q, OXY-A, and OXY-B.

	RESULTS

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Resistance phenotype of K. oxytoca KER. K. oxytoca KER expressed an unusual -lactam resistance phenotype characterized by high levels of resistance to amoxicillin (MIC, 512 µg/ml) and piperacillin (MIC, 128 µg/ml) and a moderate level of resistance to ticarcillin (MIC, 16 µg/ml) (Table 1). Only a weak synergy was observed between clavulanate and amoxicillin (MIC, 256 µg/ml) or ticarcillin (MIC, 8 µg/ml). No synergy was observed between piperacillin and the three inhibitors (clavulanate, sulbactam, and tazobactam). This resistance to penicillin-inhibitor combinations was associated with high levels of susceptibility to cephalothin, cefotaxime, ceftazidime, and aztreonam (MICs, 0.5 µg/ml).

View this table: [in this window] [in a new window]   TABLE 1.   MICs of -lactams for clinical isolate K. oxytoca KER, wild OXY-producing K. oxytoca strains, and mutant strains of K. oxytoca overproducing OXY enzymes

Isoelectric focusing. By isoelectric focusing, one band of pI 6.3 was observed in K. oxytoca KER. The pIs of the -lactamase in 17 crude extracts of K. oxytoca strains were between 5.2 and 8.8.

Enzymatic and kinetic parameters. The kinetic parameters of the new mutant enzyme IRKO-1 (pI 6.3) with regard to penicillins and cephalosporins were compared with those of the OXY-2-2 enzyme of pI 5.7 (strain HB60) (Table 2). The specific activity of the purified IRKO-1 protein is 100 U/mg, which is approximately 10-fold lower than that of the OXY-2-2 enzyme. The affinity (K_m) of mutant IRKO-1 for benzylpenicillin and amoxicillin was slightly lower than that of OXY-2-2, while the affinity of IRKO-1 for ticarcillin (K_m, 975 µM) was markedly (10-fold) lower than that of the OXY-2-2 enzyme (K_m, 85 µM). Small K_m values (25 µM) for piperacillin and cefoperazone were found for these two enzymes, with the V_max values of the mutant IRKO-1 enzyme being 2.5-fold higher than that of the parent enzyme. The hydrolytic activity of this mutant against cephalothin was drastically reduced (>100-fold), and that against cephaloridine was reduced to a lesser extent (7-fold). No hydrolytic activity of the IRKO-1 mutant against cefotaxime or aztreonam was detected.

Inhibition studies. The IC₅₀s of the three -lactamase inhibitors clavulanate, sulbactam, and tazobactam for the new mutant and OXY-2-2 enzyme were compared (Table 3). The IC₅₀s of the three inhibitors for the IRKO-1 mutant were at least 200-fold higher than those for the OXY-2-2 enzyme.

Nucleotide sequencing. As indicated in Table 4, for K. oxytoca KER, which produced the first inhibitor-resistant K. oxytoca enzyme designated IRKO-1, nucleotide sequencing revealed a bla_oxy gene which differed by four mutations from the bla_oxy-2-1 gene reported previously (9). Three mutations consisted of the nucleotide change GA, which leads to the amino acid substitution GlySer at position 20 (1) and AspAsn at positions 197 and 255, following the numbering scheme for class A -lactamases of Ambler et al. (1). A fourth mutation consisted of the nucleotide change AG, which leads to the amino acid substitution SerGly at position 130. Promoter sequencing revealed a G-to-A transition of the fifth base in the 10 consensus sequence (GATAGT).

View this table: [in this window] [in a new window]   TABLE 4.   Amino acid substitutions in the OXY-2 -lactamase group

	DISCUSSION

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Different forms of the chromosomal K1 -lactamase of K. oxytoca have been reported (2, 9-11, 16). Chromosomal -lactamase genes were recently divided into two main groups, bla_oxy-1 and bla_oxy-2 (9), each of which includes -lactamases with four different pIs. The genes for these -lactamases are being cloned and sequenced (9, 10).

K. oxytoca enzymes are penicillinases of class A that can hydrolyze cefoperazone effectively and other broad-spectrum cephems weakly (2). The OXY-2 -lactamase group hydrolyzes several -lactams including carbenicillin, cephalothin, some extended-spectrum cephalosporins (ceftriaxone), and aztreonam better than the OXY-1 -lactamase group does (10). Fournier et al. (7), in a susceptibility analysis of 167 K. oxytoca strains, clearly individualized four susceptibility pattern clusters: pattern 1 includes the susceptible strains of K. oxytoca, pattern 2 includes strains producing plasmid-mediated TEM types, pattern 3 includes the overproducer of chromosomal -lactamase, and pattern 4 includes strains producing an extended-spectrum -lactamase.

The antibiotype of K. oxytoca KER differed from those of the strains with the four different patterns by its resistance to amoxicillin-clavulanate in association with a high degree of susceptibility to cephalosporins. This resistance pattern is similar to that observed when an IRT enzyme is produced (19). However, there is no TEM derivative in strain KER, which, on the basis of the pI value (pI 6.3) and promoter and gene sequence results, can owe its resistance to -lactams only to the overproduction of a chromosomal OXY type -lactamase. As reported previously (5, 7) OXY-2 -lactamase overproduction, which is due to a mutated promoter (6, 8), increased the level of resistance to penicillins, some cephalosporins, and aztreonam. In addition, combinations of a -lactam with clavulanate seem to be effective, sulbactam alone is not effective, and the ability of tazobactam to overcome the action of the K. oxytoca -lactamase when it is overproduced in unpredictable (7). We failed to observe the expected increase in the level of resistance to penicillins, cephalosporins, and aztreonam in this mutant strain, which instead exhibited increased levels of resistance to combinations of penicillins and inhibitors.

Determination of the nucleotide sequence suggested the existence of a gene encoding a novel OXY-2-type enzyme since the enzyme included combinations of amino acid substitutions at positions 35, 197, 223, and 255 different from those observed in the OXY-2 variants reported previously. This novel variant, harboring the substitutions Asp-Asn at positions 197 and 255, differed from OXY-2-2 at position 255, from OXY-2-3 at position 223, and from OXY-2-4 at position 35. In addition, it harbored two amino acid substitutions at positions 20 and 130. The substitution GlySer 20 that occurred in the signal sequence did not affect the catalytic properties of the enzyme. However, the amino acid substitution SerGly at position 130 is very important in this mutant enzyme because the Ser-130 residue is highly conserved in class A -lactamases and has a complex role. This residue participates in the geometry of the active site through its hydrogen bond between its side chain oxygen and the terminal nitrogen of Lys-234. It is also in the close vicinity of Ser-70 and Lys-73. Ser-130 participates in the binding of substrates interacting with the carboxylates of substrates and inhibitors and participates in the catalytic process in facilitating proton transfer to the nitrogen of -lactam during its opening (12, 14).

Brown and coworkers (3) showed that Ser-130 is involved in the final step of TEM-2 -lactamase inactivation by clavulanic acid. When Ser-70 is acylated with clavulanic acid, a rapid decarboxylation of clavulanic acid is expected on the basis of mass spectroscopic evidence. Chen and Herzberg (4) have previously shown by crystallography that a decarboxylation of clavulanic acid occurred during acylation of Staphylococcus aureus PC1 -lactamase. Brown et al. (3) observed additional adducts, two of which are linked to Ser-70 by an ester bond; the first one is an aldehyde, whereas the second one is a hydrated aldehyde. Another adduct is linked to Ser-130 in the form of a -substituted acrylic acid. A fourth adduct is an acrylic acid which is linked to Ser-70 and Ser-130 by an ester bond and an ether bond, respectively.

Thus, it is obvious that substitution of Ser-130 by a glycine yields a clavulanate-resistant -lactamase, but surprisingly, the IRKO-1 -lactamase remained an efficient enzyme. Previous observations with the natural mutant SHV-10 (17) and those obtained by directed mutagenesis of the ROB-1 -lactamase (14) and the Streptomyces albus G -lactamase (12) suggested that alteration of Ser-130 would yield enzymes with dramatically decreased catalytic properties for most, if not all, substrates. Molecular modeling performed with the TEM-1 -lactamase by Jelsch et al. (13) in 1992 suggests that if Ser-130 is substituted by a glycine, a novel water molecule could take the place in the resulting cavity at a position very close to that of the oxygen of the previous serine. This water molecule could make a hydrogen bond with Lys-234 and moreover could participate in the binding of substrates and inhibitors, facilitating proton transfer to the nitrogen of a -lactam during its opening.

Our study is the first to report on a clinical inhibitor-resistant mutant from a chromosomal enzyme of K. oxytoca. Therefore, we suggest that this mutant be designated IRKO-1. The use of amoxicillin-clavulanate (3 g/day) for 10 days in the treatment of our patient 1 month before the isolation of the strain from the patient's blood was probably responsible for the emergence of the mutant. However, the good susceptibility of the mutant to all cephalosporins may limit the spread of -lactam resistance in hospitals.