Molecular Epidemiology of Nosocomial Infection: Analysis of chromosomal Restriction Fragment Patterns by Pulsed-Field Gel Electrophoresis

Abstract

Acinetobacter baumannii is a species of non-fermentative gram-negative coccobacilli commonly found in soil, water and other environmental samples. This bacterium is defined as being strict aerobes, nonmotile, catalase-positive and oxidase-negative. This organism was susceptible to most antibiotics in the 1970s. A. baumannii is an opportunistic pathogen that may be an important threat due to its increasing multidrug resistance and is involved in nosocomial infections that are often severe. The objective of this study was undertaken to elucidate the molecular epidemiology of A. baumannii using the most widely applicable DNA – based typing methods namely Pulsed-Field Gel Electrophoresis (PFGE). These strains comprised isolates from environmental samples, blood, wound, urine, cerebrospinal fluid and tracheal aspirates. PFGE analysis of 81 clinical isolates has been carried out by using CHEF–DR III systems from Bio – Rad and following the protocol of Gautom with some modifications. A 2.00% band tolerance and an optimization of 4.00% were selected for use during comparisons of generated fingerprints or pulsotypes after digestion with Apa I restriction enzyme. Similarity values have been generated using BioNumerics software, cluster analysis was performed by the unweighted pair – group method using arithmetic averages and DNA relatedness was calculated based on Dice coefficient. An interlinkage homology level of 80% between patterns was assumed as the cutoff for defining a close genetic relationship between strains and was used to define the cluster. As per the generated dendogram, isolates were categorized into 18 major groups designated as Strain I to Strain xvIII. Overall, PFGE was able to discriminate the 81 different Acinetobacter baumannii isolates with similarity levels of 63.63%.