[Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR].
We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-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 bla(OXA18) and bla(SHV) 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 bla(SHV-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 bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-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.
Unité de recherche, laboratoire de bactériologie médicale, centre national de greffe de moelle osseuse de Tunis, Tunis, Tunisie. souadkalai@yahoo.fr
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Kalai Blagui
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Achour
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Abdeladhim
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Ben Hassen
D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics*; D015139:Blotting, Southern; D003428:Cross Infection / Q000453:epidemiology / Q000382:microbiology; D004866:Equipment Contamination; D005798:Genes, Bacterial; D006801:Humans; D016867:Immunocompromised Host; D008403:Mass Screening; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011552:Pseudomonas Infections / Q000453:epidemiology / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014416:Tunisia / Q000453:epidemiology; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics*
D001426:Bacterial Proteins; D001618:beta-Lactamases
Identification des gènes de beta-lactamase à spectre étendu de type SHV chez Pseudomonas aeruginosa par PCR-restriction fragment lengthpolymorphism et insertion site restriction-PCR.