Analysis of oprD gene in imipenem-intermediate clinical isolates of Pseudomonas aeruginosa / 中国感染与化疗杂志

ABSTRACT

Objective To analyze the mechanism of imipenem resistance in Pseudomonas aeruginosa clinical isolates. Methods? Antibiotic?resistance?was?analyzed?using?VITEK32?system.?Metallo?β-lactamase?activity?was?determined?by?double-disc?synergy?test.?Amp?C?β-lactamase?activity?was?determined?by?Kirby-Bauer?disc?method.?OprD?protein?was?analyzed?by?sodium?dodecyl sulphate-polyacrylamide gel electrophoresis. PCR was performed to amplify gene oprD. The amplified products were subject?to?sequencing?analysis.?The?phylogenetic?relationship?was?determined?using?random?amplified?polymorphic?DNA?(RAPD)?method. Results Membrane protein OprD was analyzed in 7 clinical isolates of imipenem-intermediate P. aeruginosa. Two strains were devoid of OprD proteins and the corresponding oprD genes were found disrupted by the insertion element ISRP10 in the coding regions. Five strains had OprD proteins with different sizes. Sequence analysis showed that the peptides ranged from 427 to 443 amino acids. Multiple amino acid substitutions and / or deletions were found within the Loop 1 through Loop 8 of the OprD secondary structures. Conclusions ISRP10 inactivation and amino acid substitutions in oprD gene confer imipenem resistance in the clinical isolates of P. aeruginosa.