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Shigella species are a major cause of diarrheal diseases and mortality in developing countries (4), and the frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, and streptomycin is causing growing concern. -Lactamases are primarily responsible for -lactam resistance in gram-negative bacteria. The presence of TEM-, OXA-1, and OXA-3 -lactamases in S. flexneri and S. sonnei has been previously reported (21). However, detailed characterization of these enzymes in S. dysenteriae is lacking. We report a clinical strain of S. dysenteriae PB-10 (obtained from the National Institute of Cholera and Enteric Diseases [NICED], Calcutta, India) harboring the SHV-11 -lactamase capable of hydrolyzing oxacillin, cloxcillin, and oxyimnocephalosporins.

Bacteria were routinely grown in tryptic soy broth on a rotary shaker at 37°C. The -lactamase was partially purified from sonic extracts of cells by size-exclusion chromatography on Sephadex G-75, followed by successive chromatography on a Q-Sepharose anion exchanger at pH 7 (at which time -lactamase activity remained in the flowthrough) and then at pH 10 (where -lactamase activity eluted at a salt concentration of 0.1 M NaCl in 20 mM ethanolamine containing 5% glycerol and 5% ethylene glycol). During purification of the enzyme, -lactamase activity was routinely determined at 30°C by monitoring the hydrolysis of nitrocefin spectrophotometrically at 482 nm (17). One unit of -lactamase activity is the amount of enzyme hydrolyzing 1 µmol of nitrocefin per min.

Hydrolysis of -lactams was monitored at 30°C in 100 mM sodium phosphate buffer, pH 7.2, at wavelengths that gave a maximum in the difference spectrum of the hydrolyzed antibiotic against the unhydrolyzed antibiotic. The relative V_max values of the substrates were obtained after absorbance data were fit to the integrated Michaelis-Menten equation (7). The rate of hydrolysis of benzylpenicillin was set at 100.

Conjugative transfer of S. dysenteriae PB-10 antibiotic resistance plasmid into Escherichia coli HB-101 was performed as described previously by Philippon et al. (18). Transconjugants were selected by plating on medium containing 25 µg of ampicillin/ml and 10 µg of rifampin/ml.

For cloning of the -lactamase gene, plasmid DNA from the transconjugant was partially digested with BamHI and ligated to the BamHI-digested vector pK19 (20). Transformants were selected on Luria-Bertani agar supplemented with 1 mM isopropyl--D-thiogalactopyranoside (IPTG), 50 µg of ampicillin, and 50 µg of kanamycin per ml. Plasmid DNA containing an 9-kb insert was subsequently subcloned in several steps by using successively the enzymes SstI, SaII, and AvaI and selecting each time for Amp Kan^r recombinants. The plasmid obtained in the final step, pMK105, contained a 1.8-kb insert. A library of nested deletions of the clone pMK105 was finally generated in the vector pK19 to sequence the entire cloned DNA, by using the double-stranded nested deletion kit from Amersham Pharmacia Biotech. DNA sequencing was done by using the Thermosequenase cycle sequencing kit (Amersham Pharmacia Biotech). Both strands of DNA were sequenced from two clones. The nucleotide sequence and the deduced protein sequence were analyzed by using the Genetics Computer Group software package. Similarity searches were performed by using the Gap BLAST algorithm (1).

The -lactam resistance of strain PB10 could be transferred to a rifampin-resistant strain of E. coli, HB101, by transconjugation. MICs were determined on Mueller-Hinton agar plates containing serial dilutions of antibiotics by inoculating with 10⁴ CFU per spot and reading after 18 h of growth at 37°C. The clinical isolate PB-10 was resistant toward all the penicillins and some of the cephalosporins tested (Table 1). Cefoxitin and imipenem appeared to be the most effective antibiotics. The MICs of selected antibiotics were determined in combination with that of clavulanic acid (MIC, 32 µg/ml) at a concentration of 4 µg/ml. The decreased MICs indicated the involvement of a transferable -lactamase resistant to strain PB-10. The cefotaxime and ceftazidime MICs are high compared to data reported earlier for Shigella species (6, 21). For cefotaxime, this could be due, at least in part, to the presence of the -lactamase reported here, since a decrease in the MIC was observed in the presence of clavulanic acid for strain PB-10. The higher ceftazidime MIC could not be correlated with -lactamase activity, particularly since there was no decrease in the MIC in the presence of clavulanate.

View this table: [in this window] [in a new window]   TABLE 1.   In vitro susceptibilities of S. dysenteriae and transconjugant to -lactam antibiotics

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell lysate and the partially purified -lactamase of strain PB-10, followed by renaturation and overlay of gels with nitrocefin (12), showed the presence of a single band of -lactamase activity, with an approximate M_r of 29,000 ± 2,000 (mean ± standard deviation). Isoelectric focusing of the reference SHV-1 enzyme as well (3) as crude and partially purified -lactamase of PB-10, followed by overlay with nitrocefin, showed in each case a single band with -lactamase activity at an isoelectric point of 7.6 ± 0.2, which corresponded to that reported for SHV enzymes (2, 5).

The hydrolysis of cephaloridine and of almost all other cephalosporins tested was much slower than that of benzylpenicillin, nitrocefin being the only exception (Table 2). The oxyiminocephalosporins cefotaxime, cefuroxime, and ceftizoxime were hydrolyzed at measurable rates. No detectable hydrolytic activity was observed for ceftazidime, cefoxitin, cefsulodin, aztreonam, or imipenem. Also, these antibiotics failed to inhibit the enzyme. Unlike other SHV enzymes, the PB-10 enzyme hydrolyzed oxacillin and cloxacillin at appreciable rates. Comparison of relative rates of hydrolysis of -lactams by a reference SHV-1 enzyme and the -lactamase harbored by pMK105 demonstrate that, unlike the reference SHV-1 enzyme, the -lactamase produced by pMK105 hydrolyzed oxacillin. Cefotaxime was hydrolyzed by this enzyme, whereas its hydrolysis could not be measured with the reference SHV-1 enzyme. Sensitivity to clavulanic acid is characteristic of TEM and SHV enzymes. The 50% inhibitory concentrations (IC₅₀) of clavulanic acid and sulbactam were determined by incubating the enzyme with either inhibitor for 10 min prior to the addition of benzylpenicillin (1 mM). The IC₅₀ was determined graphically. For clavulanic acid, the values were 2 and 40 nM, while for sulbactam, the values were 17,000 and 30,000 nM for the reference strain SHV-1 and the S. dysenteriae enzyme, respectively. The S. dysenteriae enzyme was therefore less susceptible to clavulanic acid and sulbactam than the SHV-1 enzyme.

View this table: [in this window] [in a new window]   TABLE 2.   Hydrolysis parameters for SHV-1 and SHV-11 -lactamases for selected -lactamsc

The nucleotide sequence (EMBL nucleotide sequence database, accession no. Y18299) of the plasmid pMK105 revealed a 286-amino-acid open reading frame. The nucleotide sequence corresponding to positions 1 through 286 of the deduced amino acid sequence was identical to that reported for the Klebsiella pneumoniae SHV-1a (or SHV-11) (GenBank accession no. X98101), except for a silent CAC-to-CAT mutation encoding histidine 108. Mutations of the plasmid-determined TEM, SHV, and OXA -lactamases that enhance their affinity for expanded-spectrum cephalosporins usually occur between positions 104 and 240. These mutations have resulted in roomier active-site cavities that permit expanded-spectrum -lactams with bulky side chains to enter and bind to the active site serine-70 (10, 13). With site-directed mutants it has been reported that the leugln substitution at position 35 on the protruding NH₂ terminus (16) increases resistance to ceftazidime but reduces the MICs of all other cephalosporins tested compared to SHV-2. In our case, the situation was not comparable, since the SHV-11 enzyme has glycine at position 238 rather than serine, as in SHV-2. (8) A survey of SHV -lactamases in Switzerland previously led to the identification of the variant enzyme SHV-11 (15) harboring the LeuGln substitution at position 35. In this report, K. pneumoniae KPZU-12, harboring the SHV-11 -lactamase, showed -lactam susceptibilities virtually identical to K. pneumoniae KPAA-I harboring the SHV-1 -lactamase. We demonstrate that the SHV-11 enzyme from S. dysenteriae hydrolyzes oxyiminocephalosporins. The hydrolysis of oxacillin and cloxacillin was intriguing.

There was little similarity of the nucleotide sequence upstream of the 10 with that of the prototype SHV-1 bla gene (14). Changes in the 35 and 10 regions are a powerful influence on promoter strength (11). In E. coli, the closer the sequence is to the consensus, the stronger the promoter (35 consensus sequence, TTGACA; 10 consensus sequence, TATAAT) (9). The promoter comprising 5'TTGCAA'3' (35 box) and 5'TATTCT3' (10 box) identified in the pMK105 bla gene has been reported to increase -lactam resistance when coupled to the SHV-2 gene. (19). The association of the bla gene with a strong promoter likely influenced susceptibilities to -lactams in the present study as well.

The continued emergence of -lactam-resistant S. dysenteriae is of particular concern to developing countries. Surprisingly, reports of noteworthy efforts to characterize the -lactamases most likely to be associated with this resistance phenomenon are sparse in the literature, particularly in terms of nucleotide sequence information. Our report describes perhaps the first serious attempt both to characterize the structural bla gene of a -lactamase from a clinical isolate of S. dysenteriae and to biochemically characterize the elaborated enzyme. Although the leu-35gln substitution had been reported while this work was in progress (15), its effect on substrate profile had not been investigated. Rather, conclusions had been drawn merely from an evaluation of MIC data, which may be influenced by an interplay of factors, including the permeability of the outer membrane of the hosts, the amount of -lactamase produced, the affinities of the target penicillin-binding proteins for the tested -lactam, and the involvement of efflux pumps. We demonstrate, using the SHV-1 J35R1010 as a reference enzyme, that the SHV-11 enzyme hydrolyzes oxacillin, cloxacillin, and oxyiminocephalosporins such as cefotaxime and is less sensitive to clavulanic acid. The results from our biochemical characterization of this enzyme suggest that an amino acid substitution in the protruding amino terminus of the -lactamase likely alters the overall conformation of the molecule and, consequently, its substrate profile. The structural basis for this needs to be investigated further.