Microbes and Infectious Diseases Detection of virulence genes in Shiga toxigenic Escherichia coli isolated from diarrhoeic and non-diarrhoeic pediatric patients in Ondo State, Nigeria

Background: Shiga toxigenic Escherichia coli (STEC) is one of the major causes of intestinal infection, bloody and non-bloody diarrhoea and extra-intestinal complications in humans. It is therefore necessary to enumerate the virulence genes present in isolated STEC strains. Methods: This research investigated the prevalence of STEC and identified some virulence genes (stx1, stx2, and eaeA) from diarrhoeic and non-diarrhoeic pediatric patients of age 1 to 16 years. A total of 243 stool samples were collected from diarrhoeic and non-diarrhoeic pediatric patients (in ratio 43:200) in four different State Specialist Hospitals in Ondo State, Nigeria. Polymerase chain reaction (PCR) assay was used for detection of Shiga toxin-producing E. coli . The virulence genes (stx1, stx2, and eaeA) were detected by PCR assay. Results: Out of the 243 samples, 163 (67%) were positive for E. coli and specifically 6 of them were positive for E. coli O157 (3.7%). Exactly 50% of the 6 E. coli O157 contained eaeA while all the strains contained Stx1 and Stx2. The results obtained also revealed that 6 strains that were non-O157:H7 STEC also contained Stx1 and four strains contained Stx2 genes. Conclusion: The results obtained from this study revealed that Stx1


Introduction
Escherichia coli (E. coli) is popular for its ability to cause diarrhoea [1]. So far, E. coli has remained separated into 6 different classes, which are: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), diffusely adherent E. coli (DAEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC or STEC) [2]. Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria classified by their ability to produce one or more types of shiga toxin (Stx) [3]. Shiga toxin-producing Escherichia coli are associated with a disease spectrum ranging from diarrhoea and haemorrhagic colitis (HC) to the potentially fatal haemolytic ureamic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) [4]. According to Rahal et al. [5], STEC infections are characteristically food-borne and the ability to produce Shiga toxins (Stx1, Stx2 or a variant) is believed to be essential to the pathogenesis of these organisms. Shiga toxins hinder production of protein in the host cell, which causes cell killing. Shiga toxin-producing Escherichia coli strains arise as a result of the insertion of one of a group of lysogenic lambdoid bacteriophages that holds an Stx1/2-encoding gene into the genome of E. coli [5].
The clinical symptoms, pathogenic characteristics and the pathobiology of these organisms and the toxins they produce was reviewed by Kruger and Lucchesi [6]. According to Quinn et al. [7], over 30% of cows infected with STEC does not show any symptoms. Another study established that it causes mortality in 3-5% of patients with long-term complications in about 30% of patients [8]. Little infectious dose of O157 EHEC increases the virulence and risk of infection [9]. The use of effective antibiotics in treatment of EHEC infection results in the release of verotoxin and also the death of bacteria [10].
The aim of this study is therefore, to isolate and characterize STEC strains and to identify the virulence genes (stx1, stx2 and eaeA) from stool samples of diarrhoeic and non-diarrhoeic pediatric patients in Ondo State, Nigeria.

Ethical approval
Ethical approval to conduct this research was obtained from the Health Research Ethics Committee, Ondo State Ministry of Health (OSHREC/20/01/2018/036), while informed consent was obtained from each patient before sampling and strict confidentiality of the participated patients was maintained.

Sample collection
A total of 243 stool samples were collected from diarrhoeic (43) and non-diarrhoeic (200) pediatric patients in State Specialist Hospitals in Akure, Ikare, Ondo and Okitipupa, Ondo State. Patient demographic characteristics including age and gender, socioeconomic and clinical characteristics were recorded.

Preparation and identification of STEC
The stool samples were cultured onto MacConkey's agar (Merck, Germany) and Sorbitol MacConkey agar (Merck, Germany) and then incubated for 24 hours at 37°C. Lactose positive colonies were selected from each sample, streaked onto Eosin Methylene Blue (Merck, Germany) plates and incubated for 18 h at 37°C. Green colonies with a metallic luster identified as E. coli colonies were further confirmed using standard biochemical tests such as indole, methyl red, Voges-Proskauer, Indole, H2S production, citrate utilization tests and urease production test [11,12]. Colonies were confirmed as E. coli by PCR as described by Madic et al. [13]. The E. coli isolates were stored in tryptic soy broth (Merck, Germany) which contained 20% glycerol at 70°C for more investigation.

DNA extraction
Bacterial isolates were cultured in trypticase soy agar (Merck, Germany) overnight at 37°C. A single colony was suspended in 100μL of sterile distilled water. The suspension was boiled for 13 min, frozen and centrifuged at 14,000 rpm for 15 min to pellet the cell debris according to Schoppee and Wamhoff [14]. The filtrate was then used as a template for polymerase chain reaction (PCR) amplification.

Detection of virulence genes by PCR
Polymerase chain reaction assay was carried out according to the method described by Schouler et al. [15]. The set of primers in table (1) were used for identification of E. coli O157 and the stx1, stx2, eaeA, for the presence of virulence genes. Polymerase chain reaction assay was carried out in a volume of 25 μL, so that 21 μL of Master Mix (CinnaGen, Iran), containing 400 mM of deoxy nucleoside triphosphates, 0.05 U/μL of Taq DNA polymerase, 4 mM of MgCl2, 1 μL of each primer, and 2 μL of the DNA extracted. The PCR was carried out in 35 cycles using the thermocycler (Biorad, USA), One hundred microlitre aliquots of the suspension was then transferred to PCR tubes and heated at 95-99°C for 10 minutes on a thermocycler. Then, the tubes were subjected to the protocol mentioned in table (2). The tubes were centrifuged at 10,000 rpm for 3 minutes to pellet cellular debris. The supernatant was used as the PCR positive control for all PCR assays [16].

DNA electrophoresis
Agarose gel electrophoresis was used to determine the quality and integrity of the DNA by size fractionation on 1.0% agarose gels. Agarose gels were prepared by dissolving and boiling 1.0g agarose in 100ml 0.5X TBE buffer solution and stained with 10µl of 5mg mL-1 ethidium bromide (Merck, SA). The amplified products were visualized and photographed under the BioDoc-It System (UVP Upland, CA 91786, USA). A 100 bp DNA ladder (Promega, USA) was involved on each gel as a molecular size standard. Electrophoresis was done at 80V for 2 hours [16].  Table 5 reveals the distribution of virulence factors in Escherichia coli subtype, EHEC and EIEC were evenly distributed in all the age groups except EIEC which is absent.

Virulence genes targeted at STEC serogroups
The virulence genes stx1 and eae were found in STEC serotypes O157, 026, 0103 and 0145 while stx2 was only found in STEC serotype O157 ( The gel electrophoresis of the isolate using STX 1 to 3 and EAEAF are shown in Plates 2 to 4 as the molecular characterization of the isolated E. coli was determined and the E. coli possesses the aligned nucleotide sequence.

Discussion
Diarrhoea due to bacterial infections is an important cause of morbidity and mortality in infants and young children in most developing countries including Nigeria [18][19][20]. Generally, the etiological factors of acute diarrhoea in developing and developed countries are different. In developing countries, Shigella and E. coli are the dominant bacteria causing diarrhoea. Studies have shown that STEC strain are responsible for important human diseases including HUS, HC and TTP. In extreme cases, rate of death is closely associated with the presence of the virulence genes. Patients with nonbloody diarrhoea show milder symptoms and are less expected to develop HUS. However, HUS has also been reported in cases of non-bloody diarrhoea.
One hundred and sixty-three (163) out of 243 samples (78%) were positive for E. coli strains in stool culture. From the result six E. coli strains were identified in plate (1). Plate 2, 3 and 4 showed E. coli strain which harbored stx1 and stx2 gene identified as E. coli O157, and eaeA gene. Findings from this current study indicates that STEC strains and non-O157 strains, contributed to 6% of all diarrhoeal infections which were able to produce shiga-like toxins. Prior research studies have shown the low occurrence of E. coli O157: H7 strain in people residing in developing countries. The result gotten from this research study showed that only 2.5% of sampled patients were infected with this strain. This result is in agreement with the result of a research study conducted in Iran. Taghadosi et al.
[21] isolated five (1.3%) STEC strains from 395 diarrheal fecal samples in Iran. Kalule et al. [22] reported similar findings of a study carried out in Kenya which indicated the low prevalence (0.2%) of E. coli O157: H7 in hospitalized diarrhoeic children.
The presence of E. coli from the population of the non-diarrhoeic patients also indicates their role as potent carrier of the organism, which may infect the other vulnerable population of the community [23]. In non-diarrhoeic group, the E. coli from both pediatric patients from hospital and the community were detected which is very less than the previous reports in India [24,25]. This might be the possible source of transmission of this pathogen among children through mother, baby sitter or other members of the family [23].

Conclusion
Comparing the stool samples from diarrhoeic patient and non-diarrhoeic pediatric children (<1 to 16years), there was a strong indication of the presence of E. coli in stool sample of non-diarrhoeic pediatric children in (<1 to 16years) in Ondo State. It is therefore recommended that: 1. Hygiene education should be addressed for diarrhoeal disease susceptible groups, such as those pre-school children, young generation, old and weak class.
2. There should be periodic enlightenment on hygiene practice and the use of hazard analysis in the preparation and processing of food.

Conflict of Interest: None.
Funding: None.