PREVALENCE OF CARBAPENEM RESISTANT PSEUDOMONAS AERUGINOSA ISOLATED BY PHENOTYPIC AND GENOTYPIC (OXA-48) METHODS IN A TERTIARY CARE HOSPITAL

Abstract

Pseudomonas aeruginosa is a human pathogen, it is a normal flora, but whenever opportunity arises it will cause disease. It is a very common pathogen involved in nosocomial infections. According to WHO (2017) infections caused by this organism have to face a challenge of resistance towards antibiotic carbapanem. Carbapenems are the drug of choice for serious infections and generally used for multi-drug resistant infections. These antibiotics are resistant to hydrolysis by enzyme β-lactamases which are produced by most of these microorganisms, nowadays their use as a last-choice treatment option has been compromised by the emergence of carbapenem inactivating enzymes called carbapenemases. These enzymes are produced by Pseudomonas aeruginosa, Acinetobacter bauminnii and Enterobacteriacea. Carbapenemases are classified according to Ambler molecular classes A, B and D coded by genes residing in plasmids, integron and transposon which often carry multiple resistance determinants limiting further treatment options. The aim of our study was to detect the presence of OXA-48 which has not been previously reported in our part of the world. We also tested by phenotypic methods carbapenemase resistance in isolates recovered in our center and then looked for the presence of OXA-48 genes in those isolates exhibiting resistance. Objective The intent of our study was to determine the prevalence of carbapenem resistance in isolates of P. aeruginosa among indoor patients of a tertiary care hospital Karachi. This resistance was tested by three phenotypic methods. We also did the molecular method for detection of resistance transcribing genes, although this resistance has been reported to be transcribed by a multitude of genes encoding for different forms of carbapenemases, we only detected OXA-48 gene which has not been previously reported in Pakistan. Subject, Materials and Methods This study was carried out at microbiology department in PNS Shifa Hospital from September 2018 to May 2019. This study consisted of 140 samples of P. aeruginosa, 2 which were received from different wards [ENT (ear, nose and throat), Plastic Surgery, Burn Unit, Medical wards, Pedriatics, Family ward and ICU (intensive care unit)] of PNS Shifa. These clinical samples were cultured on blood agar and Mac Conkey agar at 37ᵒC ±2ᵒC for 24-48 hours. Subsequently, we verified the cultured isolates biochemically by testing for oxidase production. When results were positive; antimicrobial disc (Meropenem and Imipenem 10μg) (oxoid) was applied through AST (antibiotic susceptibility test). When these carbapenem discs exhibited a resistance diameter of 15mm, then carbapenemase production was checked by both phenotypic methods like Modified Hodge test (MHT) and Modified Carbapenem Inactivation Test (mCIM). In case of carbapenem resistance, OXA-48 gene was detected by Real-Time PCR (Polymerase chain reaction). Conclusion Carbapanem resistance is an alarming threat all over the world in general and in particular in our geographical area; because of rampant misuse of antibiotics. We were successful to detect carbapenemase production phenotypically by MHT (Modified Hodge test) and mCIM (modified carbapenem inactivation method). These are screening methods for detection of carbapenemase. We found that mCIM is easy, affordable and accurate method for detection of carbapenemase. We are also first time reporting the presence of OXA-48 gene in isolates of Pseudomonas aeruginosa in Pakistan. We conclude from our findings that the implementations of screening tests are effective for the detection of resistant strains and should be done as a first stage of treatment strategy. This will prevent the evolution of gram negative organisms from multidrug resistant to pan-drug resistant because their genome is very dynamic and there is a continuous acquisition of resistance genes in bacterial populations. Infection control measures may be instituted in preventing the dissemination of these resistant bacteria