Identification des gènes de β-lactamase à spectre étendu de type SHV chez Pseudomonas aeruginosa par PCR-restriction fragment length polymorphism et insertion site restriction-PCR

Abstract

We propose a simple and rapid method to discriminate SHV-type extended spectrum β-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for blaOXA18 and blaSHV genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the blaSHV-1 negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied blaSHV genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in blaSHV-1 gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the blaSHV-1. PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants. © 2008 Elsevier Masson SAS. All rights reserved.