MOLECULAR CHARACTERISATION OF EXTENDED SPECTRUM BETA-LACTAMASE IN HUMAN AND BOVINE ESCHERICHIA COLI ISOLATES

Abstract

Extended Spectrum β-Lactamase (ESBL) is an enzyme capable of hydrolysing wide range of extended spectrum β-lactam antibiotics leading to antibiotic resistance. In Nigeria, there is a dearth of information on the genetic characterisation of ESBL in clinical human and bovine microbial isolates. This study was undertaken to genetically characterise ESBL in human and bovine Escherichia coli isolates and determine possible dissemination of the resistant genes. Fifty-seven E. coli isolates were collected from University College Hospital, Ibadan, Oluyoro Catholic Hospital, Ibadan, General Hospital Adeoyo, Ibadan, Bowen University Teaching Hospital, Ogbomoso and the same number of E. coli were isolated from 100 faecal samples of healthy bovine animals using conventional isolation methods between August 2010 and August 2011. Antibiotic susceptibility profile of the Human Bacterial (HB) and Bovine Bacterial (BB) isolates was determined by disc-diffusion. ESBL was determined in all the isolates by double-disc synergy test (DDST). Detection of ESBL genes in DDST positive isolates, exploration of upstream sequence of detected ESBL gene as well as detection of class 1 integron and gene cassettes were determined by PCR and DNA sequencing. Genetic relatedness among ESBL producers was determined by repetitive-PCR genomic fingerprinting. Plasmid DNA was isolated by alkaline lysis. Horizontal resistance transfer by conjugation and plasmid replicon typing were carried out on ESBL positive isolates. Two ESBL isolates having successful resistance transfer were typed by multilocus sequencing. The data were analysed using coefficient of correlation. Thirty (53%) of the HB isolates were found resistant to amoxicillin, trimetoprim, nalidixic acid, gentamicin, ciprofloxacin and amoxicillin-clavulanic acid while nine (16%) of the BB isolates were resistant to trimetoprim and amoxicillin. There was no relationship in the antibiotic susceptibility patterns of the BB and HB isolates. ESBL was detected in eight HB isolates, all of which were resistant to Cefotaxime and had the CTX-M-15 enzyme while six ESBL producers had aac(6’)-lb-cr gene and two, qnrB gene. One BB isolate had the qnrS gene. Class I integron was demonstrated in 75% of ESBL isolates with aadA1, aadA5 and dfrA17 as the gene cassettes identified. The insertion sequence (ISEcp1) element was detected upstream of the blaCTX-M genes in all ESBL isolates. The IS26 element was identified in three ESBL isolates while one isolate had both the IS26 and IS903 elements. Plasmid replicon types FIA, FIB, F, HI2, and K were found in HB isolates while HII, Y and also FIB occurred in BB isolates. Horizontal transfer of ESBL resistance gene was detected in two ESBL isolates of the sequence types ST2695 and ST131. The ST2695 exhibited a unique plasmid DNA sequence, representing a novel report. Three DNA fingerprint patterns were identified among the ESBLs isolates. Most of the plasmids extracted were >10kb. The prevalent clone of extended spectrum β-lactamase (CTX-M-15) and five different plasmid replicon types in human bacterial isolates are reported. The detailed and reliable knowledge on the antibiotic resistance of human and bovine pathogens will improve the controlled and safe use of antibiotics in human and veterinary medicine. Keywords-Escherichia coli, Extended-spectrum β-lactamases, Antibiotic resistance Word count: 492