Identification of PSE and OXA beta-lactamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism

Abstract

Objective: A method using PCR-restriction fragment length polymorphism was developed to identify Pseudomonas aeruginosa beta-lactamase genes.

Methods: Two hundred and fifty-nine P. aeruginosa isolates were screened by PCR with 11 primer pairs designed to detect genes encoding PSE, OXA, TEM and SHV enzymes. PSE and OXA gene variants were distinguished by restriction of PCR products with endonucleases recognizing sites involved in point mutations. Nucleotide sequences were verified for a few isolates by sequencing the PCR products.

Results: Four isolates produced extended-spectrum beta-lactamases (ESBLs) according to the double disc synergy test. PCR detecting bla(PSE) genes was positive in 162 (62.5%) isolates: 151 carried bla(PSE-1) and 11 carried a variant encoding an enzyme differing from PSE-1 by a single amino acid substitution (Pro102 to Ser). PCR detecting sequences for enzymes of the OXA-10 group was positive in 68 (26.3%) isolates: 31 carried bla(OXA-10), one carried bla(OXA-14) and 36 carried a new variant intermediate between bla(OXA-13) and bla(OXA-19). The bla(OXA-2) gene was identified in 13 (5%) isolates. Two other isolates carried bla(OXA-2) variants encoding ESBLs differing from OXA-2 by a single amino acid substitution (Asp150 to Tyr and Trp159 to Cys, respectively). PCR detecting sequences for enzymes of the OXA-1 group was positive in 12 (4.6%) isolates. A bla(TEM) gene was identified in five (1.9%) isolates (three bla(TEM-1), one bla(TEM-2), one bla(TEM-4)).

Conclusion: This approach is effective for screening P. aeruginosa for beta-lactamase gene carriage in epidemiological studies and for detecting new variants.