Shiga toxin-producing Escherichia coli (STEC) infection in humans is estimated to cause around 2.8 million cases of severe illness annually worldwide (Withenshaw et al. 2022). Hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) infections in humans are linked to consumption of contaminated bovine food sources with Shiga toxin-producing E. coli (STEC). Among the foodborne diseases, STEC-O157:H7 has been documented as a leading cause of severe outbreaks in areas where the consumption of bovine products is high. Although serogroup O157 is the most frequently identified STEC in humans, there has been a significant rise in cases associated with non-O157 STEC serogroups as a result of enhanced surveillance and advancements in detection techniques (Withenshaw et al. 2022). Since cattle are a significant reservoir for both STEC O157 and possibly non-O157 STEC, lowering their prevalence in cattle could lower the risk of human infection. The essential virulence markers of STEC are the ability of these organism to produce one or both shiga toxins (stx1 and stx2). However, presence of them both or one of the stx and eae genes has been considered an important and enhanced virulence trait of this organism (Mathusa et al. 2010). This study focuses on the detection of STEC-O157 and non-O157 in imported frozen beef marketed in the Eastern Province of Saudi Arabia using immunomagnetic separation and multiplex-PCR.
In this study, the overall reported strains harboring stx1 gene were isolated from examined frozen beef imported from India and Australia and the overall frequencies of stx1 gene among the 298 isolates were 12.7%. Whereas the overall frequency rate of stx2 gene was 0.7% and detected in two strains isolated from beef imported from Brazil and New Zealand. Additionally, the distribution rate of eae+ gene among the overall isolates was 4.7% and reported in 6, 4, 3, and one strains isolated from examined frozen beef imported from India, Brazil, Australia and New Zealand, respectively. Therefore, the isolates that were tested positive for the combined presence of stx1+ and eae+ or stx2+ and eae+ reported and categorized as E. coli O157 with prevalence rate of 3.7%, whereas those isolates reported positive for stx1+ or stx2+ gene regardless of absence of eae gene were reported as E. coli non-O157 with prevalence rate 10.4%. Furthermore, E. coli non-O157 was detected in examined frozen beef imported from Australia, India and Brazil, but none from New Zealand. In this study, the isolation of STEC in the examined imported frozen beef samples marketed in the Eastern Province of Saudi Arabia were reported in 26 strains and is considered as a high detection level of stx1+ in non-O157. A similar study was conducted in the Southern region of Thailand were reported as having higher levels of stx1+ non-O157 detection among examined marketed beef and reported large number of isolates carrying stx1 gene (Kayali 2015; Sirikaew et al. 2016). Our findings were similar with a recent study conducted in 2017, by Saudi Food and Drug Authority (SFDA) that reported the presence of E. coli O157:H7 in 6.8% and 2.2% of the examined beef meat samples imported from India and Brazil, respectively. Moreover, the study concluded that the imported meat can serve as a carrier of E. coli O157:H7, which may lead to epidemics throughout regions of Kingdom of Saudi Arabia (Alhadlaq et al. 2023).
In the present study, the IMB was used via an enrichment treatment technique for detection of E. coli O157 from imported frozen beef samples. Enrichment using IMB conjugated with coated antibodies in beads against E. coli O157 antigen after pre-enrichment yielded a better detection on SMAC agar and CHROMagar O157 medium. Subsequently, all examined beef samples were plated on similar selective culture mediums after pre-enrichment without IMB revealing lower detection. Therefore, best isolation was observed when using IMB enrichment and CHROMagar O157 medium as a selective medium for isolation of E. coli O157 from frozen beef samples. Several published studies reported that application of IMB to the in enrichment process after sample pre-enrichment, will modify the protocol making it more rapid and sensitive for separation and detection of E. coli O157 in comparison to the conventional culturable method (Dego 2024; Lewis et al. 2020; Mengistu and Mengesha 2023; Sarimehmetoglu et al. 2009). In this study, the examined samples were pre-enriched for six hours and incubated at 42°C before the use of IMB separation. Hence, it is noteworthy to mention that the six hours pre-enrichment has been reported to be more sensitive and effective rather than just one enrichment process for 24 hrs.
Despite a variety of available techniques in molecular biology, for instance, random amplified polymorphism deoxyribonucleic acid (RAPD), amplified fragment length polymorphism (AFLP), pulsed field gel electrophoresis (PFGE), microarray and restriction fragment length polymorphism (RFLP), the techniques propose a number of challenges including the requirement of high budget (Adzitey et al. 2013; Ramadan 2022; Simar et al. 2021). From published literature, several studies used ERIC-PCR fingerprints to type bacterial strains isolated from clinical, food and environmental samples, and was found to be cost-effective and faster than other typing techniques such as PFGE (Alsultan and Elhadi 2022; Elhadi 2018; Yamani and Elhadi 2022). Among PCR based typing techniques, ERIC-PCR has frequently been used and proven a strong discriminatory power for a wide range of organisms in the Enterobacteriaceae family, such as E. coli (Ranjbar et al. 2014). In this study, ERIC-PCR was able to type all 42 isolates of E. coli O157 and non-O157 into four clusters (A, B, C and D), where 36 isolates were genetically related considering the genetic similarity value (> 90% cut-off). On the other hand, 6 out of 42 isolates showed single lineages ERIC type (ET) patterns [ET-1, ET-2, ET-4, ET-5, ET-6 and ET-9] below 90% genetic similarity. These were considered genetically unrelated in accordance with a study published by Szczuka & Kaznowski (2004). In this study, ERIC-PCR demonstrated that these strains were genetically related and was able to group the isolated strains into distinctive clusters according to their imported country of origin based on ERIC DNA fingerprints. Among the phylogeny of four clusters of ERIC-PCR constructed by UPGMA, cluster-B comprised the highest number of E. coli O157 and non-O157 isolated from frozen beef imported from Australia and, interestingly, one strain of E. coli non-O157 (46-A3) isolated from frozen beef imported from Brazil shared 96% genetic similarity with these strains in this cluster. On the other hand, one strain (82A-1) of E. coli O157 (stx2+/eae+) in cluster-A was isolated from imported frozen beef imported from New Zealand shared a 100% genetic similarity with strain (60A-1) of E. coli O157 (stx2+/eae+) isolated from imported beef imported from Brazil. Moreover, cluster-C and D comprised 13 strains of E. coli O157 and non-O157, 12 strains were isolated from frozen beef imported from India and one strain (26B-3) was isolated from frozen beef imported from Australia sharing a 100% genetic similarity. Therefore, the results reported in this study are in concordance with our previous published study which evaluated ERIC-PCR method for determining genetic diversity among E. coli isolated from human and retail imported frozen shrimp and beef, when the study concluded that ERIC-PCR is a useful molecular typing tool to discriminate pathogenic E. coli (Alsultan and Elhadi 2022).