PHENOTYPIC AND MOLECULAR DETECTION OF OXA-48 GENE IN ENTEROBACTERIACEAEا

Abstract

SUMMARY C arbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Several resistance mechanisms have been reported to circumvent the efficacy of carbapenems, and carbapenemases. A large variety of carbapenemases belonging to three molecular classes of β-lactamases have been identified in Enterobacteriaceae: the Ambler class A e.g (KPC), class B e.g (NDM and IMP) and class D e.g (OXA 48) β-lactamases. The OXA-48 is an active-serine-site enzyme like Ambler class A and class C β-lactamases, differing from class A and C enzymes in amino acid structure and is usually not inhibited by clavulanic acid, tazobactam, and sulbactam. It was first identified from a carbapenem-resistant Klebsiella pneumoniae isolate in Turkey 2001 and was found to be multidrug resistant and exhibited a high level of resistance to all β-lactams, including broad-spectrum cephalosporins, cephamycins, monobactams and carbapenems. During this study, 50 specimens were submitted to the Central Microbiology Laboratory of Ain Shams University Hospitals for routine culture and susceptibility testing. Specimens were subjected to identification of isolates by using conventional methods including morphology on MacConkey agar, Gram stained film and biochemical reactions. Antibiotic susceptibility testing for isolates was done by Kirby Bauer disk diffusion method according to CLSI (2014) and they were resistant to carbapenem. Confirmation of carbapenem resistance was done by performing broth microdilution method for MIC detection. Identification of carbapenem resistant classes using carbapenemase detection set by (MastDiagnostics) and temocillin disk was done followed by singleplex real time PCR to detect OXA 48, KPC, NDM, VIM and IMP genes. For carbapenamase detection set, 41(82%) specimens were positive for MBL disk and 9(18%) were negative. For KPC disk 17 (34%) specimens were positive and 33(66%) were negative and all samples were negative for AmpC. For OXA 48 (temocillin disk), 48 (96%) specimens were positive and 2(4%) were negative. Some specimens showed two or more positive results in combination. 23 specimens were positive for both OXA 48 and MBL together (46%). 16 specimens showed positive results for OXA 48, MBL and KPC together (32%). Only one sample showed OXA 48 and KPC together (2%). By PCR, OXA 48 gene was detected in (96%) then VIM (94%) followed by NDM (54%), KPC (46%) and finally IMP (40%).The whole detection set was evaluated according to PCR and the results were that 33 out of 40 specimens were true positive (82.5%) and 7 were false negative (17.5%) while 7 out of 10 specimens were true negative (70%) and 3 were false positive (30%). For Klebsiella isolates, 97% were positive for OXA 48, MBL (VIM, NDM, IMP) and 55% were positive for KPC. For E.coli, 92% were positive for OXA 48, MBL (VIM, NDM, IMP) and 33% were positive for KPC. All five Serratia isolates (100%) were positive for OXA 48, 80% were positive for MBL and 40% were positive for KPC. The two Citrobacter isolates were positive for OXA 48 and MBL and were negative for KPC.