Detection and molecular characterization of verotoxin gene in non-O157 diarrheagenic Escherichia coli isolated from Miri hospital, Sarawak, Malaysia M.N.H. Maziz1,∗, S. ismail

Background: Non-O157 diarrheagenic Escherichia coli are typical Escherichia coli. Infections with non-O157 Escherichia coli are now increasingly recognized in many countries. The virulence profiles of most of the non-O157 E. coli are unknown. Therefore, easy detection, isolation, and characterization of non-O157 Escherichia coli isolates are essential for improving our knowledge of these organisms. Methods: A total of 32 non-O157 diarrheagenic Escherichia coli isolated (Miri hospital, Sarawak, Malaysia, 2001) from the patients with diarrhea were examined for the detection of verotoxin (VT) gene. For this purpose, two sets of primers (VT1 and VT2) were used for PCR method. VT1 probe was prepared which was used for the Southern hybridization study. Bacteriophage induction was carried out using mitomycin C. Nucleotide sequencing was made from the VT1 gene fragment isolated from an E. coli isolates using PCR protocol. Results: The PCR protocol used here produced a fragment of 348 bp of VT1 gene and 584 bp of VT2 gene, respectively. It was shown that 13 E. coli isolates (40%) carried VT1 gene whereas none was found to carry VT2 gene using PCR protocols. Plasmids were detected in all the VT1-positive isolates. From the Southern hybridization study, VT1 probe showed homology with the chromosome of all 13 VT1-positive isolates indicating the VT1 gene to be located on chromosome. Bacteriophage induction as carried out using mitomycin C showed that none of the VT1-positive isolates harbor any lysogenic bacteriophage. However, nucleotide sequencing made from the VT1 gene fragment isolated from an E. coli isolates using PCR protocol showed 97% homology with the known VT1 gene which proves to be the similar gene carried by E. coli O157 and other ancestors. Conclusion: The PCR method used here was sensitive, specific and reliable. VT1 was found to be the most common verotoxin among the E. coli strains isolated from clinical sources in Malaysia and this VT1 gene is located in the chromosome of the E. coli isolates. This study improves the knowledge of a highly significant emerging pathogen nonO157 E. coli.