MOLECULAR EPIDEMIOLOGY OF MULTIDRUG RESISTANT ESCHERICHIA COLI ISOLATED FROM PRIMARY SCHOOL PUPILS IN ONDO STATE, NIGERIA

Abstract

Investigations were conducted to determine the molecular epidemiology of multidrug resistant Escherichia coli isolates from urine and faeces of apparently healthy pupils in five major communities namely; Akure, Owo, Ondo, Okitipupa and Ikare-Akoko in Ondo state, Nigeria. Questionnaire containing information about age, sex, and health history was completed for the pupils. The samples collected were kept under cold chain and then analyzed in the laboratory within 2h of collection. The following analyses were carried out on the samples; isolation and identification of the E. coli strains, antibiotic sensitivity testing, DNA extraction, and amplification of the isolates using polymerase chain reaction (PCR) assay. A total number of 807 urine and stool samples were collected from 399 male and 408 female pupils, out of which 206 E. coli strains were isolated. Each of the isolates was tested for susceptibility to different classes of antibiotics, namely; penicillin combinations [augumentin-30μg (AUG)]; nitrofurans [nitrofurantoin-300μg (NIT)]; second generation fluoroquinolones [ciprofloxacin-5μg (CPR) and ofloxacin-5μg (OFL)]; second generation cephalosporins [cefuroxime-30μg (CRX)]; third generation cephalosporins [ceftazidime-30μg (CAZ) and cefixime-30μg (CXM)]; aminoglycosides [gentamicin-10μg (GEN)]; tetracyclines [tetracycline-30μg (TET)]; chlormphenicols [chloramphenicol-30μg (CHL)]; and sulfonamides [trimethoprim-5μg (TRM)] by the disc diffusion method. The antibiotic-resistant patterns of E. coli isolates were in decreasing order of CAZ (100%), CRX (100%), AUG (98.1%), TRM (97.1%), TET (94.7%,), CXM (94.7%,), GEN (83.5%) and CHL (67.5%) while E. coli had less than 50% resistance to NIT, CPR and OFL. However 6.79% of the E. coli isolates was resistant to all the eleven antibiotics tested while 0.97% was resistant to at least four antibiotics. The molecular pattern of these strains was determined using PCR assay. DNA was extracted from 50 E. coli strains from which 28 (14 male and female each) that are resistant to multiple drugs were further subjected to amplification by uniplex PCR to detect the presence of virulence genes using 13 primers namely; LT, stII, ipaH, eae, stx1, stx2, E-hly, virF, daaE, EAF, bfp, uidA, aafII usually found in E. coli strains causing infections. A total of 22 (78.57%) isolates were found to possess stx1 virulence gene associated with Enterohaemorrhagic E. coli strains, while the remaining 6 (21.43%) isolates showed a false or no band. In conclusion, there was a clear indication of the presence of E. coli in urine and faecal samples collected. Additionally, high resistance to antibiotics was observed and stx1 gene was predominantly found in the E. coli isolates.