Isolation and characterization E. coli O157:H7 from meat &cheese and detection of Stx1, Stx2, hlyA, eaeA by using multiplex PCR


total of 189 samples were collected from 74 raw uncooked minced beef meat, 115 local white cheeses from 3 different areas in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identified by using morphological diagnostic methods; Samples were cultured on liquid enrichment medium, incubated at 41.5Cº for 6 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 66 non-sorbitol fermenting bacterial isolates were obtained of which 13 were identified as Escherichia coli from (6 meat and 7 cheese samples). By using traditional biochemical tests and Api20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates. Four specific biochemical tests (Cellobiose fermentation, β-Glocuronidase production, KCN and Enterohemolysin production) were done to differentiate serotype O157 differentiation from other NSF bacteria. Only 2 isolates belonging to the serotype O157 were obtained of which one isolate from meat and other isolate from cheese. Latex agglutination test for O157 and H7 showed that the 5 isolates gave positive results with both kits. The Bacterial isolates were identified by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated from various sources by using specific primers in mPCR. The result showed that gene content variety in two E. coli O157:H7 isolates, 1 from meat contain all 4 genes and other isolate from cheese contains 2 genes: Stx1 and hlyA .