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Journal of Antimicrobial Chemotherapy (2002) 50, 11-18
© 2002 The British Society for Antimicrobial Chemotherapy

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

Frédéric Bert*, Catherine Branger and Nicole Lambert-Zechovsky

Department of Microbiology, Hospital Beaujon, 100 boulevard du Général Leclerc, 92110 Clichy, France

Received 3 September 2001; returned 17 January 2002; revised 6 February 2002; accepted 27 March 2002

Objective: A method using PCR–restriction fragment length polymorphism was developed to identify Pseudomonas aeruginosa ß-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 ß-lactamases (ESBLs) according to the double disc synergy test. PCR detecting blaPSE genes was positive in 162 (62.5%) isolates: 151 carried blaPSE-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 blaOXA-10, one carried blaOXA-14 and 36 carried a new variant intermediate between blaOXA-13 and blaOXA-19. The blaOXA-2 gene was identified in 13 (5%) isolates. Two other isolates carried blaOXA-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 blaTEM gene was identified in five (1.9%) isolates (three blaTEM-1, one blaTEM-2, one blaTEM-4).

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

* Corresponding author. Tel: +33-1-40-87-54-42; Fax: +33-1-40-87-05-50; E-mail: bertfrederic{at}hotmail.com


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