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Molecular Identification of Virulence Genes of Escherichia coli Isolated from Cow Milk and Its Products in Abuja, Nigeria

Shiga toxin-producing Escherichia coli have been identified as an emerging foodborne pathogen which portends serious risk to human health. Cow milk and its products are potential sources of shiga toxin-producing Escherichia coli. A relatively small number from the family of shiga toxinproducing Escherichia coli are pathogenic. It becomes necessary that Cow milk and milk products are regularly screened for the presence of virulence genes in microbes. The study aimed to genetically determine the presence of virulence genes that are characteristic of Enterohaemorrhagic E. coli in 600 milk samples. The E. coli isolates were recovered from the milk Original Research Article Okechukwu et al.; MRJI, 30(6): 11-18, 2020; Article no.MRJI.54570 12 samples (n=35), biochemically examined and genetically screened for virulence genes by multiplex Polymerase Chain Reaction (PCR). The results of the molecular profiling revealed that (stx2) was detected in 17(60.7%), (hlyA) 11(39.3%) and eae genes 8(28.6%) of the E. coli isolates respectively, while (stx1) was not detected. The results indicated a high prevalence of virulent shiga toxin-producing Escherichia coli in the milk samples. Priority attention should be given to this microbe as it will demand stringent steps in the detection given that they are known to be rigorous in identification.


INTRODUCTION
Escherichia coli is a Gram-negative, lactose fermenting, facultative aerobic, rod-shaped and non-sporulating, flagellated bacterium in the family of Enterobacteriaceae [1,2,3,4,5,6]. The bacterium, E. coli is well recognized as a commensal inhabitant of the gastrointestinal tract [7,8] and considered as a reliable indicator of contamination by faeces [9], manure, soil, contaminated water and breach of hygiene in milking [10,11]. Strains of E. coli are characterized by their ability to produce shiga toxins and other bacterial toxins [12]. This attribute is definitive of the virulence factors of the class of E. coli, enteric pathogens known as shiga toxin-producing E. coli (STEC) or vero toxin-producing E. coli (VTEC). Transmission occurs through consumption of contaminated fruits and vegetables, undercooked meat, unpasteurized raw milk [5,13] or consumption of water directly or indirectly contaminated by faeces [14], direct or indirect contact with animals [15] or person to person contact [16,17]. The Enterohaemorrhagic Escherichia coli (EHEC), is a subgroup of shiga toxin-producing E. coli based on the clinical manifestations in humans [18]. Several virulence genes are associated with the pathogenicity of STEC, and genes encoding E. coli virulence factors are located either on plasmids on large genome regions called pathogenicity islands (10 to 200 kb), or on integrated bacteriophages [19] which can be moved by the mechanism of horizontal gene transfer. The genes encoding shiga toxins 1 and 2 (stx1 and stx2) respectively are carried by lambdoid phages and can be integrated into the bacterial host genome in multiple copies [20] also known as phage-encoded cytotoxins. Shiga toxins are A1B5 toxins that halt protein synthesis in the host cells, a process that may lead to apoptotic cell death, and cause the vascular endothelial damage observed in patients with hemorrhagic colitis and the haemolytic uraemic syndrome (HUS) [2,21]. Shiga toxin E. coli capable of tightly attaching and causing attaching and effacing lesions on the intestinal epithelial cells is the intimin factor encoded by the eae gene (E. coli attachment and effacement). These are located in the chromosomal locus of enterocyte effacement (LEE), a pathogenicity island. The plasmid-encoded enterohaemolysin (hlyA) virulence gene is found in shiga toxinproducing E. coli. It is responsible for the lysis of the blood cells after the tissue damage and thus facilitating the proliferation of organisms [22]. Raw milk and dairy products are one of the main sources of transmission of the E. coli strains to humans [13]. Milk contamination is usually due to the faecal contamination by the pathogenic E. coli that colonize the gut of dairy animals which are subclinical and remain unnoticed. Personal and environmental hygiene during the milking process contribute to the risk of contamination of these ready-to-eat dairy products. The study aimed to determine the presence of virulence genes in shiga toxin-producing E. coli from Cow milk and its products.

Bacteria Strain
Reference bacteria strains of Escherichia coli ATCC 25922, ATCC43888 and LMG 21766 were used in the study to serve as positive controls.

Isolation and Identification of Escherichia coli
Ten ml (10 ml) of milk samples were aseptically transferred into 90 ml of modified Tryptic Soy

Molecular Detection
Genomic extraction: The genomic deoxyribonucleic acid (DNA) was extracted using ZR Genomic DNA™ Miniprep Kit (ZYMO Research Corp. USA) according to manufacturer's instructions. The extracted DNA samples were stored at -20°C ready for (PCR) molecular studies [25]. Multiplex PCR was carried out on 28 of the 35 E. coli isolates.
Polymerase chain reaction: Amplification was performed in Gene Amp 9700 (Applied Biosystems) with initial denaturation at 94°C for 3 minutes followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 30 seconds and a final extension at 72°C for 8 minutes.
Electrophoresis: Five microliters of the PCR product was electrophoresed in agarose gel (1.5%) containing 5 µl of 10 mg/ml ethidium bromide at 80 V for 50 mins. Fifty (50) bp DNA marker (New England Biolabs®) was used as a molecular size marker. The genes were examined under U.V transilluminator and results documented using Gel Documentation System (BIO RAD).

DISCUSSION
The present study revealed that the positive for shiga-toxin genes (stx

Distribution of virulence genes present in the cow milk and its products
No examined Virulence genes the traditional dairy products (kindrimo, nono and manshanu). Among the two toxins, only shiga-toxin 2 (stx2) was detected while shiga toxin 1 (stx1) was not isolates subjected to E. coli isolates gene. This finding suggests that the milk samples were contaminated with capable of producing toxins hence the name (STEC).

PCR
This is one of the most important virulence characteristics of STEC strains. The high contamination of milk and milk products by STEC could be attributed to the traditional methods of milking which increases the risk of transmission of the food pathogens into the milk samples, lack of storage facility to maintain the temperature of the milk products will increase the rate of survival and multiplication of pathogens in these milk samples, the unpasteurized milk product itself and the unhygienic status of the milkers. The result is consistent with the reports [26,27,28,29,30,31] with the following prevalence (0.87%, 11.11%, 5.7%, 11.29%, 17.47%, 67.44%) at different places (Ontario, Belgium, Italy, Egypt, Iran, Saudi Arabia) respectively. Shiga-toxin 2 (stx2) in beef, 94.21% of STEC was reported in Saudi Arabia [31], 3.5% in Brazil [32] and 4.4% in Japan [33]. The variation in the distribution of the shiga-toxins at the different places could be as a result of the several factors such as geographical location, the season of the year, farm management practices, variation in sampling, variation in the type of samples evaluated, hygiene status and the methods of detection [30,34].
Escherichia coli from other sources (faecal, pork and poultry samples) that were positive for (stx2) gene have been reported in China [35], faecal samples in Bangladesh [36] with the prevalence of (40% and 50%) respectively. Shiga-like toxin E. coli strains could be detected alone, or in combination, either stx1, or stx2 and stx2 variants toxins [27]. The combination of the two shiga-toxins (stx1 and stx2) was observed in the report [37] from raw milk (madara) samples in South Africa. Momtaz et al. [13] reported that only shiga-toxin 1 (stx1) was detected in milk samples. In contrast, previous studies reported that none of the shiga-toxin genes was detected in the E. coli isolated from the milk sample that was screened by PCR [38]. It is possible that the stx genes are present in the samples but not expressed due to the isolation methodologies of the very scanty cells of STEC and stx-carrying phages [39,40]. In this study, the predominant virulence gene was stx2 which have been reported to be more virulent than shiga-toxin1 (stx1) as it poses more health risk to humans through the consumption of the high-risk foods [37].
Moreover, stx1 and stx2 sequences are located on mobile genetic elements and might have different mobility patterns. Therefore, the transference of stx2 from non-pathogenic to pathogenic serotypes may happen more frequently [41]. It has been shown that accessory virulence factors such as intimin eae, and enterohaemolysin, hlyA genes are important in the pathogenicity of STEC [42]. In this study, the intimin eae gene was detected in 8(28.6%) of the E. coli isolates recovered from the milk samples. The finding is consistent with the reports [13,17,31,38,43] with prevalence (100%, 36%, 44.19%, 4.05-13.4%, 18.0%-24.3%) in these locations (Iran, South Africa, USA, Saudi Arabia) respectively. Al-zogibi et al. [31] reported eae gene of 10(58.82%) in E. coli isolates from raw beef, E. coli isolates from milk and dairy products in Iran were positive for eae genes [4], E. coli isolates from faecal samples of diarrheic calves [2] and chicken meat [44] were positive for eae gene in Iran. The variation of the prevalence of eae genes could be due to the geographical location, sample types and methods of detection of STEC positive for eae genes. This finding suggests that the presence of intimin eae gene in the isolates may increase the risk of infection in humans. It is considered as a marker for Enterohemorrhagic Escherichia coli [45]. The eae gene is capable of initiating pathogenesis in cell host by inducing attaching and effacing" (A/E) lesions, known to be a virulence factor of diarrhoea in children [46]. It has been reported that there is an association between STEC infection and the presence of the eae gene [47].
On the contrary, a previous study [30] reported that no eae gene was detected in the E. coli isolates from the milk and dairy samples in Iran. However, absence of eae gene does not completely eliminate virulence potentials in E. coli. Though some studies reported that eae gene is not essential in the pathogenicity in humans [48]. It was reported with evidence that etiologic agent STEC lacking eae gene was responsible for the outbreaks of human illness in the United States and Australia [49].
In this study enterohaemolysin hlyA gene was detected in 11 (39.3%) of E. coli isolates. These findings agree with the reports [17,37] in Iran and South Africa with prevalence (28.0%, and 2.4%) respectively.
The presence of hlyA gene expressed by the E. coli isolates from milk and dairy samples was reported in Iran [4,13]. This finding suggests that the E. coli producing-shiga toxin which expressed the enterohaemolysin gene (hlyA) is potentially harmful pathogen in humans and its presence in food portends risk on public health. Studies have shown that enterohaemolysin production hlyA gene might have a direct association with the pathogenicity of a given STEC strain to cause more serious disease in humans [21].

CONCLUSION
The result of this study showed that shiga-toxin 2 (stx2), intimin (eae) and enterohaemolysin (hlyA) virulence genes were present in E. coli strains isolated from Cow milk and its products sold in Abuja (Nigeria). This indicates that the ready -toeat dairy products are contaminated with virulent E. coli strains with potential to cause Enterohaemorrhagic food-borne diseases. It is important that standard hygiene practices are adopted at all stages of Cow milk processing. and