Almulhim, Ahlam Fahad2022-05-182022-05-186669https://drepo.sdl.edu.sa/handle/20.500.14154/158Background: E. coli O157 is the most remarkable of the five known diarrheagenic E. coli (DEC). The global spread of E. coli O157:H7 among children has caused the Food and Agriculture Organization of the United Nations (FAO/UN) to improve standards for food security and quality. This bacterium is the most notorious because its potentially harbors virulence genes, which can result in kidney failure, particularly hemolytic uremic syndrome (HUS).Aims: This study aims to investigate the presence of E. coli O157 in imported frozen beef marketed in Saudi Arabia by using immunomagnetic beads, multiplex-PCR, and ERIC-PCR DNA fingerprinting.Methods: In this study, 201 imported frozen beef samples were purchased from different supermarkets in the Eastern Province of Saudi Arabia. All samples were analyzed by using immunomagnetic beads (IMB), CHROMagar O157, and SMAC agar. Virulence gene markers (stx-1, stx-2, and eae) were screened by using multiplex-PCR. ERIC-PCR DNA fingerprinting was used to identify the clonal relationship among the strains isolated from different countries.Results: The overall occurrence rate of presumptive E. coli was 43.8% in examined samples, obtained by using chromogenic agar without IMB and yielding 89 isolates. In chromogenic agar with IMB, the overall occurrence rate was 52.7% in examined samples from India, Australia, Brazil, and New Zealand, with a yield of 111 isolates. Three isolates were found to be positive for the stx-1 gene while no single isolate was detected for the stx-2 and eae genes among presumptive isolates that were recovered on SMAC agar without IMB. Meanwhile, on SMAC agar with IMB,xix8 isolates were found to be positive for the stx-1 gene in samples that were imported from Australia, India, and Brazil. No samples were positive for eae from New Zealand. The highest isolates harboring the stx-1 gene were detected in beef samples imported from Australia, for which the stx-1 gene was detected in 9 isolates out of 89 recovered on chromogenic agar without IMB. Two isolates were detected for the eae gene in beef samples imported from India, while no single isolate was detected for the stx-2 gene. The overall distribution rate of the stx-1 gene among the presumptive isolates of E. coli O157 which were examined from imported frozen beef were positive in 37 (12.4%) out of 298 isolates. The stx-2 gene was detected only in a single sample from Brazil and New Zealand. ERIC-PCR primers generated different DNA polymorphisms among 42 isolates of E. coli pathotypes and were able to group all confirmed 42 isolates of E. coli into 4 clusters (A, B, C, and D) by using the UPGMA algorithm and gel J software. Therefore, 85.7% of E. coli pathotype isolates were typeable with ERIC-PCR, while 14.3% produced a single cluster. ERIC-PCR was able to group all strains according to their countries of origin except for one isolate of Australian beef, which was grouped with E. coli isolates from Indian beef. In addition, one isolate of Brazilian beef was grouped with E. coli isolates from Australian beef.Conclusion: This study confirmed that the imported frozen beef marketed in the Eastern Province of Saudi Arabia was harboring virulence gene markers of E. coli O157. The used methods of IMB and multiplex-PCR proved to be more reliable and adequate for the detection of E. coli O157 in imported frozen beef. This study recommends that food authorities such as the Saudi Food and Drug Authority (SFDA) set adequate measures to monitor imported frozen beef.enEnteropathogenic E coliVirulence genesEpidemiologyMolecular techniquesDNA extractionThesis