A study of clinical strains of Pseudomonas Aeruginosa and the investigation of antibiotic resistance mechanisms in the multidrug resistant strain PA13

Abstract

Thirteen clinical strains of bacteria from two Irish hospitals were identified as Pseudomonas aeruginosa using classical methods, API 20NE and Biolog GN. Their identification was confirmed by 16S rRNA gene sequencing. The antibiotic resistance profiles of the isolates were determined against forty-one antibiotics belonging to eleven distinct classes. All the isolates were resistant to penicillin G, ampicillin, cephalothin, cloxacillin, oxacillin, amoxicillin, cefotaxime, moxalactam, sulphatriad cotrimoxazole, chloramphenicol and tetracycline. All were sensitive to ceftazidime, piperacillin-tazobactam, cefepime, ceftriaxone, meropenem, aztreonam, amikacin, apramycin, butirosin A, lividomycin and colistin sulphate. One of the isolates, PA13, was resistant to a further fourteen antibiotics and was identified as a multidrug resistant strain. A 2.2 kbp PCR product was amplified from P. aeruginosa PA13. When this product was sequenced it was found to contain four open reading frames. BLASTN analysis identified these as being an integrase gene (ORF1), an aminoglycoside acetyltransferase gene, aac(6’)-Ib (ORF2), an oxacillinase gene (ORF3) and a quaternary ammonium compound resistance gene (ORF4). The presence of the integrase gene and the quaternary ammonium compound gene suggested that the genes were on a Class 1 integron. The acetyltransferase aac(6’)-Ib gene contained the mutant type of the enzyme with a leucine substitution by serine at position 119. Two expression vectors were chosen to investigate the novel oxacillinase gene. One was a commercially available vector, pET-28a (Novagen) and the other was an in-house vector, pPC. The gene was successfully cloned into both vectors. Following induction the desired protein was not expressed in either the soluble or insoluble fractions