Detection and molecular characterization of shiga-like toxin producing escherichia coli and escherichia coli 0157 : H7 isolated from raw beef marketed in east Malaysia

Chang, Pheh Ping (2003) Detection and molecular characterization of shiga-like toxin producing escherichia coli and escherichia coli 0157 : H7 isolated from raw beef marketed in east Malaysia. Masters thesis, Universiti Malaysia Sarawak, (UNIMAS).

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Abstract

A total of 88 raw beef samples marketed in Sarawak and Sabah, East Malaysia, were investigated for the presence of Shiga-like toxin producing coli, . coli serogroup 0157: H7 and E. coli 0157. Identification of the bacterial species were based on morphological and biochemical tests, followed by immunological test. The presence off. coli 0157. -H7 and its virulence properties, Shiga-like toxins, Stxl and Stx2 were confirmed by multiplex polymerase chain reaction (PCR) using four PCR primer pairs that simultaneously amplified segments of sal, stx2, rE and iC 7 genes in a single reaction. Five isolates of Shiga-like toxin producing E. coli 0157: H7, four non Shiga-like toxin producing E. coli 0157 and two non-0157 Shiga-like toxin producing E. coli were isolated from 1.1%, 2.3%, and 2.3% of raw beef samples respectively. The prevalence of E. coli 0157: H7 and STEC in East Malaysia were found to have a link with the location, with 54.5% (6/11) isolated from locations situated in the central region of Sarawak. The STEC 0157: H7 isolates were detected only in frozen imported beef whereas non-0157 STEC in local beef samples. In an attempt to improve PCR-based assay to allow rapid detection, multiplex PCR targeting on stxl, stx2, rE and fl- iCK genes were conducted directly in primary enriched beef samples. The multiplex PCR could detect 51 organisms in one gram of beef sample, demonstrating a higher sensitivity of the assay as compared to the conventional culture method with cefixime-tellurite Sorbitol MacConkey agar, CTSMAC. The use of multiplex PCR was shown to provide rapid and sensitive identification of E. coli 0157. H7 in raw beef. Analysis of 33 beef samples marketed in East Malaysia with multiplex PCR and conventional culture method has indicated that the detection of the simultaneous presence of two target genes, rE and iC 7 genes was sufficient for a 0157: H7 positive diagnosis in a beef sample. The inclusion of primer sets for stxl and stx2 genes in the same reaction provided additional information on the toxin profiles of a sample and were useful for detecting STEC 0157: H7 and other STEC. Pulsed-field gel electrophoresis (PFGE) with restriction enzyme, Xbal was used to assess the genetic relatedness of the isolated E. coli 0157: H7, E. coli 0157, non-0157 STEC and other E. coli strains. The PFGE profiles indicated that five E. coli 0157: H7 and three E. coli 0157 isolates presumably represented a single strain of E. toll 0157: H7 and E. coli 0157 respectively. Identical PFGE profiles found in the same E. coli serotype of the same beef sample indicated that each individual beef marketed in East Malaysia harboured only one type of STEC 0157: H7 or E. coli 0157. A large variety of PFGE patterns (45 patterns) were found among E. coli isolates demonstrating a high E. coli diversity in the beef marketed in East Malaysia. From the dendrogram generated, there was no close relation observed between STEC 0157: H7 and non-0157 STEC to the nonpathogenic E. coli strains. In this study, PFGE typing method was shown to possess high discriminatory power and proved its usefulness in differentiating among E. coli 0157: H7 and STEC. The PFGE profiles obtained from the E. coli beef isolates could provide a database that would aid in the epidemiological investigation of the study area in future.

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