A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections

Muhammad Salman,Abdul Haque,Aamir Ali

doi:10.12669/pjms.294.3652

Abstract

Wound infections are often difficult to treat due to various bacterial pathogens. Pseudomonas aeruginosa is one of the common invaders of open wounds. Precise diagnosis of this etiological agent in wound infections is of critical importance particularly in treatment of problematic cases. The existing diagnostic methods have certain limitations particularly related to specificity. Our objective was to to establish a comprehensive and reliable multiplex PCR to confirm diagnosis of P. aeruginosa. A multiplex PCR test was developed for rapid and comprehensive identification of P. aeruginosa. Four highly specific genes were targeted simultaneously for detection of genus, species and exotoxin production (16S rDNA, gyrB, oprL and ETA) in P. aeruginosa; additionally one internal control gene (invA) of Salmonella was used. The specificity of the multiplex PCR was confirmed using internal and negative controls. Amplified fragments were confirmed by restriction analysis and DNA sequencing. The developed method was applied on 40 morphologically suspected P. aeruginosa isolates (from 200 pus samples) and 18 isolates were confirmed as P. aeruginosa. In comparison, only 12 could be identified biochemically. Combination of the four reported genes in multiplex PCR provided more confident and comprehensive detection of P. aeruginosa which is applicable for screening of wound infections and assisting treatment strategy.

Highlights

Wound infections are complications caused by bacteria which result in belated healing and can sometimes be even life-threatening.1These infections considerably contribute to increased health care1
Molecular techniques, such as polymerase chain reaction (PCR) are rapid and reliable for the identification of microbial pathogens,[6] many PCR based diagnostic methods have been developed for P. aeruginosa.[7,8,9]
Identification of P. aeruginosa: From 200 clinical pus samples, 40 isolates were suspected as P. aeruginosa on the basis of formation of large, translucent, pale, mucoid colonies on MacConkey agar plates

Summary

Introduction

Correspondence: May 21 , 2013 costs.[2] Pseudomonas aeruginosa (P. aeruginosa) has been recognized as a frequent inhabitant of chronic non-healing wounds[3] and is one of the foremost opportunistic bacteria isolated from wounds which cause high morbidity and mortality despite antimicrobial therapy.[4] P. aeruginosa infections are generally detected by standard microbiological techniques such as phenotypic and biochemical profiles,[5] these commercial tests tend to be lengthy and unreliable.[5,6] Molecular techniques, such as polymerase chain reaction (PCR) are rapid and reliable for the identification of microbial pathogens,[6] many PCR based diagnostic methods have been developed for P. aeruginosa.[7,8,9]. Methods: A multiplex PCR test was developed for rapid and comprehensive identification of P. aeruginosa. Conclusions: Combination of the four reported genes in multiplex PCR provided more confident and comprehensive detection of P. aeruginosa which is applicable for screening of wound infections and assisting treatment strategy

Objectives

Methods

Results

Conclusion