Isolation and characterization of Escherechia coli 0157 from colonic fecal samples of pigs in selected slaughter houses in Cavite / by Gladys M. Lontoc.

Thesis (Doctor of Veterinary Medicine) Cavite State University

Includes bibliographical references.

College of Veterinary Medicine and Biomedical Sciences (VETMET)

LONTOC, GLADYS M. Cavite State University. Indang, Cavite. April 2006. Isolation and Characterization of Shiga (Vero) Toxin-Producing Escherichia coli O\57 trom Colonic Fecal Samples of Pigs in Selected Slaugterhouses in Cavite. Undergraduate Thesis. Doctor of Veterinary Medicine. Adviser: Ma. Cynthia N. Rundina, DVM, MS.

This study was done to isolate and characterize shiga (Vero) toxin-producing Escherichia coli 0157:H7 from 169 colonic fecal samples of pigs obtained in Mahogany Slaughterhouse and Indang Slaughterhouse in Cavite.

All of the fecal samples were subjected to primary cultivation in Brilliant Green Bile Broth or BGB (Acumedia®). Majority of the samples fermented lactose indicated by cloudy yellow appearance of the medium and gas formation in the durham tubes indicating growth of coliforms. These presumptive coliforms were then subjected to direct culture using Sorbitol MacConkey Agar or SMAC (Acumedia®) and were characterized culturally on the basis of its inability to ferment sorbitol i.e., colorless appearance of colonies with smoky centers. A total of 73 colonies were isolated and were considered as presumptive Enterohemorrhagic Escherichia coli (EHEC). These isolates were further characterized morphologically and biochemically. All of the isolates were gram-negative and rod-shaped organisms. Biochemically, the isolates produced the following reactions: oxidase negative (-), Acid slant/Acid butt with gas production (A/A, gas) in Triple Sugar Iron (TSI) Agar, no hydrogen sulfide production, indole positive (+) and motile but some were non-motile in Sulfide Indole Motility (SIM) Medium, Methyl red test positive (+), Voges Proskauer test negative (-), Citrate utilization test negative (-), Gelatin utilization test negative (-), Nitrate reduction test positive (+) and Urease test negative (-).

In sorbitol utilization test, only 7 (22.6%) did not oxidize nor ferment the sugar (O/F’).These isolates were considered as the presumptive Escherichia coli 0157 and were further tested serologically using the O157 and H7 antisera. Two (28.6%) out of the seven isolates produced granular agglutination patterns, therefore these two were considered as likely possessing the 0157 antigen whereas the five isolates, which did not produce agglutination with the said antiserum, may possibly belong to non-O157 EHEC Strains (3%). However, upon subjecting these 0157 positive isolates in the H7 agglutination test, none of these agglutinated the H7 antiserum.

The seven isolates that did not utilize the sugar sorbitol were further subjected to the Polymerase Chain Reaction (PCR) for amplification of the SLT II primer to detect six 2 gene. The result showed highly similar sizes of estimated amplified product i.e2.1 kb, 1.5 kb, and 1.3 kb. However, these differed from the sizes of the positive control strain, which were 1.2 kb and 1.8 kb. Amplification of the primers targeting the six 2 gene revealed that none of the isolates possessed the said gene.

Results of the study showed that the presumptive E. coli 0157 isolates do not possess the six 2 gene for SLT II. However, these could not completely rule out the absence of SLT II since the Vero cell assay was not done, which is considered as the gold. Standard for detection of the toxin. The prevalence of E. coli O157 in Mahogany Slaughterhouse and Indang Slaughterhouse in Cavite was 1.2%, which is similar with other studies on STEC O157:H7 in other countries. Moreover, further study on E. coli O157:H7 in pork carcasses in different areas in the Philippines are warranted, as these may also possess the pathogenic SLT II. Presence of other EHEC strains in swine should also be evaluated.

Submitted to the University Library 07/18/2007 T-3232