Improved isolation and detection of shiga toxin-producing Escherichia coli O157 and non-O157 from retail imported frozen beef marketed in Saudi Arabia using immunomagnetic separation and multiplex-PCR

Abstract

Abstract Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 are responsible for a wide range of foodborne outbreaks worldwide. Both E. coli O157 and non-O157 are causing life-threatening complications, such as hemolytic colitis and hemolytic uremic syndrome, which can result in kidney failure. This study was conducted to determine the presence of both STEC and E. coli O157 in imported frozen beef marketed in the Eastern Province of Saudi Arabia using immunomagnetic beads (IMB), CHROMagr O157 medium, and multiplex-PCR. A total of 201 frozen beef samples were purchased from different supermarkets in the Eastern Province of Saudi Arabia and enriched in tryptic soy broth (TSB). One-milliliter portions from enriched samples in TSB were mixed with immunomagnetic beads (IMB) coated with specific antibodies against E. coli O157 O antigen, separated beads with captured bacteria streaked on CHROMagar O157, and Sorbitol MacConkey (SMAC) agar. The extracted DNA template was examined for potential virulence gene markers (stx-1, stx-2, and eae) using multiplex-PCR. ERIC-PCR DNA fingerprinting was used to identify the clonal relationship among the strains isolated from frozen beef imported from different countries. Among 201 examined frozen beef samples, 88 (43.8%) and 106 (52.7%) were positive for E. coli and produced colorless and mauve colonies on SMAC agar and CHROMagar O157, respectively. Out of 298 isolates in total, 124 and 174 isolates of E. coli were isolated without and with IMB enrichment, respectively. The highest detection rate of virulence gene markers were found among isolates that had been isolated using IMB enrichment, where 25 (8.4%), 2 (0.7%) and 12 (4%) isolates tested positive for stx1, stx2, and eae genes respectively. The least number of virulence gene markers were found among isolates that were isolated without using IMB enrichment with a rate of 12 (4%) and 2 (0.7%) isolates testing positive for stx1 and eae genes, respectively. Among 42 isolates harboring potential virulence gene markers, 11 isolates were identified as E. coli O157 (stx1+/eae+ or stx2+/eae+). ERIC-PCR DNA fingerprinting was able to type the 42 identified isolates of E. coli O157 and E. coli non-O157 into 10 types with four identical related clusters and a genetic similarity rate above 90% homology from the identified isolates. This study confirmed that the imported frozen beef marketed in the Eastern Province of Saudi Arabia harbored virulence gene markers of E. coli O157. IMB and multiplex-PCR methods proved to be more reliable and adequate for detecting shiga toxin-producing E. coli O157 in imported frozen beef.