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

In Pseudomonas aeruginosa, resistance to cefclidin is usually associated wi th resistance to another third-generation cephalosporin, ceftazidime. In th is study we analysed 22 isolates of P. aeruginosa, collected at Showa Unive rsity Fujigaoka Hospital between 1992 and 1993, which were resistant to cef clidin 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 beta-lactamase were 0.84 kb in length. The isoelectr ic points of the beta-lactamases produced by these isolates were typical of the OXA-4 type of beta-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 aci d substitution of OXA-1 beta-lactamase to form OXA-4 beta-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 ca n be classified by SSCP analyses based on the PCR method. Although OXA-4 be ta-lactamase is generally plasmid-mediated, the chromosomal DNA of these is olates, but not their plasmids, hybridized with the OXA-4 gene amplified by the PCR method. Based on these results, we suspected that the plasmids enc oding OIXA-4 beta-lactamase had been spontaneously cured, or that the gene had been deleted from the plasmid. The distribution of P. aeruginosa produc ing OXA-4 beta-lactamase amongst hospital wards and clinical specimens demo nstrated that the OXA-4 enzyme in this collection period was representative of hospital P. aeruginosa.