Effective Application of RAPD Method to DNA Typing Analysis of Pseudomonas aeruginosa

Introduction Genetic polymorphism analysis has been widely utilized to clarify the infection routes and the origins of pathogenic bacteria in-hospital infection. Pulsed-field gel electrophoresis (PFGE) is a standardized method of analyzing DNA fragments digested with restriction enzymes. In addition to PFGE, The random amplified polymorphic DNA (RAPD) method, composed of polymerase chain reaction (PCR) and gel electrophoresis, has also started to be utilized to analyze pathogenic DNA. In this study, the authors performed comparative analysis of genetic polymorphisms in Pseudomonas aeruginosa (P. aeruginosa) strains isolated at Nippon Medical School Hospital and demonstrated that the RAPD findings were comparable PFGE. Materials and Methods Twenty clinically isolated P. aeruginosa strains (12 metallo-β-lactamase producing strains and 8 non-producing strains) and 1 strain derived from an outside environment were tested. Drug-resistant genes were detected by amplifying metallo-β-lactamase (blaIMP-1) and aminoglycoside acetyltransferase genes [aac (6')-1b] by PCR. PFGE was performed by embedding the P. aeruginosa bacterial bodies in agarose gels, followed by lysis, deproteinization, and restriction digestion with Spe-1 enzyme. The gel was electrophoresed with the Genofield system (ATTO, Tokyo, Japan). For RAPD, the extracted DNA was amplified according to the Mahenthralingham's method using 2 primers. Results The blaIMP-1 genes were detected in all 12 metallo-β-lactamase producing strains. The aac (6')-1b genes were identified in 2 of these 12 strains. There were blaIMP-1 or aac (6')-1b genes detected in strains that did no produce metallo-β-lactamase. As shown in Fig. 1, 8 strains of blaIMP-1 negative were categorized to 6 types (Fig. 1a).