Detection of OXA-4 β-lactamase in Pseudomonas aeruginosa isolates by genetic methods

In Pseudomonas aeruginosa, resistance to cefclidin is usually associated with resistance to another third-generation cephalosporin, ceftazidime. In this study we analysed 22 isolates of P. aeruginosa, collected at Showa University Fujigaoka Hospital between 1992 and 1993, which were resistant to cefclidin but susceptible to ceftazidime. All polymerase chain reaction (PCR) products amplified by a primer pair covering the full-length gene of OXA-4 (also OXA-1) precursor β-lactamase were 0.84 kb in length. The isoelectric points of the β-lactamases produced by these isolates were typical of the OXA-4 type of β-lactamase (pl 7.5) rather than the OXA-1 type (pl 7.4). All PCR products at 216 bp were amplified by the primer pair covering the A928→T point mutation, which corresponds to the Asp48→Val amino acid substitution of OXA-1 β-lactamase to form OXA-4 β-lactamase. These single-strand conformation polymorphism (SSCP) patterns are typical of the OXA-4 gene, rather than the OXA-1 gene, demonstrating that these enzymes can be classified by SSCP analyses based on the PCR method. Although OXA-4β-lactamase is generally plasmid-mediated, the chromosomal DNA of these isolates, but not their plasmids, hybridized with the OXA-4 gene amplified by the PCR method. Based on these results, we suspected that the plasmids encoding OXA-4 β-lactamase had been spontaneously cured, or that the gene had been deleted from the plasmid. The distribution of P. aeruginosa producing OXA-4 β-lactamase amongst hospital wards and clinical specimens demonstrated that the OXA-4 enzyme in this collection period was representative of hospital P. aeruginosa.