AN INVESTIGATION ON CLONAL DIVERSITY OF ESCHERICHIA COLI FROM CATTLE IN SMALLHOLDINGS WHILE USING SELECTIVE MEDIA FOR O157 STRAINS

Abstract

An investigation was carried out to verify the clonal diversity of enterohemorrhagic Escherichia coli (EHEC) O157 strains isolated from fecal materials originated from recto anal junction (RAJ) of 518 smallholders’ cattle while using cefixime-tellurite added sorbitol MacConkey (CT-SMAC) agar as a selective growth medium. Any EHEC O157 is sorbitol non-fermenter and therefore yields colorless colonies on CT-SMAC, opposed to non-O157 ones that produce pink colonies. The diversities among the colorless-colony producing isolates on CT-SMAC from the said materials were verified by conventional cultural methods and application of some molecular diagnostic tools – polymerase chain reaction (PCR), plasmid profiling and pulsed field gel electrophoresis (PFGE). The presence of any of the three virulent genes – Stx1, Stx2 and hly of the probable EHEC O157 isolates was diagnosed based on PCR. The diversity among the isolates lacking any of the said genes was established by plasmid profiling and PFGE. The variations in the antimicrobial resistance profiles of the colorless colony producing isolates on CT-SMAC to 11 selected isolates were also investigated, by disk-diffusion method. In primary screening, RAJ-originated fecal samples from 142 (27%) cattle yielded colorless colonies on CT-SMAC. While 5 cross-sectional such colonies from each positive sample was homogenized and re-verified by giving passage for the second time on CT-SMAC, only 57 produced pure colorless colonies. These (57) were verified further by culturing on eosin methylene blue (EMB) agar; of them 39 were finally found to be E. coli and based on their repeated positive sorbitol non-fermenting fidelity they were considered as probable EHEC O157. Thirty one (~6%) of the 39 isolates were diagnosed positive with ≥ 1 virulent genes of EHEC O157; 16 (3.1%) isolates had hly gene and 28 (5.4%) isolates were shiga toxin producing because they harbored either Stx1 or Stx2 or the both. Six genotypes were identified by PFGE in 7 isolates that lacked any of the three virulent genes; plasmid profiling identified variable number of different sizes plasmids in them. Variable resistance profiles were seen among the isolates tested. In conclusion, the probable EHEC O157 strains isolated from smallholder’s cattle by using CT-SMAC were not clonally related rather different clones of EHEC O157 that are circulating in the population.