Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli Isolated from School Children in Ondo State, Nigeria

Molecular techniques were used for studying the epidemiology of Escherichia coli from five major towns in Ondo state, namely; Akure and Ondo, Owo, Okiutipupa and Ikare-Akoko. In total, 807 urine and faecal samples of apparently healthy primary school pupils were gathered between December 2012 and March 2013. Withal, 206 E . coli strains were isolated and characterized by biochemical tests. Polymerase chain reaction (PCR) assay was performed to determine the presence of the genes-encoding virulence factors. A total of 22 (12 male, 10 female) isolates out of 28 subjected to PCR amplification were found to possess only stx1 virulence gene associated with Enterohaemorrhagic E. coli strains. PCR assay was demonstrated to be a useful technique for the epidemiological work of E . coli w here this organism is a major cause of infection.


INTRODUCTION
Molecular epidemiology is a branch of medical science that focuses on the contribution of potential genetic and environmental risk factors, which are identified at the molecular level, to the aetiology, distribution and prevention of diseases within families and across populations. This area of medical science improves our understanding of the pathogenesis of diseases by identifying specific pathways and molecular factors which influence the risk of contracting the disease [1].
Escherichia coli is a very adaptive enteric Gramnegative bacillus, which is easily recognized as non-invasive commensal. Most strains of E. coli live as commensals; many perhaps are opportunistic pathogens of human and even animals [2]. Certain E. coli isolates have been demonstrated to produce a toxin, which was initially called verotoxin because of its distinct effect on vero-cells. This family of toxins was recently called Shiga toxins (stx), owing to its close relation to the stx of Shigella dysenteriae type 1. Enterohaemorrhagic E. coli (EHEC) is the main group of verotoxigenic strains which has emerged as the leading cause of haemorrhagic colitis and haemolytic uremic syndrome (HUS) in man.
EHEC can be communicated in the first place through the ingestion of faecal contaminated foods, particularly undercooked beef. Nevertheless, a big number of outbreaks of EHEC have also been linked with ingestion of contaminated drinking water or contact with recreational water [3]. Infection with Shiga toxin (stx) -producing Escherichia coli (STEC) can ensue in a spectrum of examples; ranging from asymptomatic carriage to uncomplicated diarrhoea, haemolytic uraemic syndrome (HUS), bloody diarrhoea, haemolytic anaemia, thrombocytopenia, and acute renal failure. High fatality and morbidity rates have been reported for HUS, which could occur from infection with STEC strains. The pathogenesis of STEC had been observed to be linked with several bacterial virulence genes. More or less of these virulence factors include the intimin protein, two Shiga toxins called stx1 and stx2 and the plasmidencoded protein known as haemolysin [4].
Information on the distribution of virulence genes of STEC strains isolated from children is rare in Ondo state. Thus, this work was designed to determine the molecular epidemiology of Shiga toxin-producing Escherichia coli isolated from school children in five major towns of Ondo State, Nigeria.

Collection of Samples
A total of 807 urine and faecal samples of apparently healthy children were collected from five major towns in Ondo state, Nigeria between December, 2012 and March, 2013. The towns include Akure, Owo, Ondo, Okitipupa and Ikare-Akoko, covering the three senatorial districts of the state. Samples were collected inside wellcovered and sterile universal bottles, kept under cold condition and then transferred to the laboratory within 2 h of collection for culturing. Questionnaire containing information about age (2-15 years), gender, and health history was filled for each pupil.

DNA Extraction
Bacterial strains were grown overnight in Luria broth medium at 37°C. Ten millilitre (10 ml) of overnight culture of E. coli on Luria broth (LB) medium was pipetted into a 15 ml disposable polypropylene tube. The tube was capped tightly and centrifuged at 14,000 rpm for 10 minutes to pellet the cell debris. The supernatant was used as a template for PCR amplification [6].

Statistical Analyses
Data obtained are presented as mean ± SE (standard error). The significance of difference between different treatment groups was tested using one-way analysis of variation (ANOVA) with the aid of Statistical Package for Social Sciences (SPSS, IL) version 17 at P≥0.05.

Isolation and Characterization of E. coli from Urine and Faecal Samples
The result of isolation of E. coli from urine and faecal samples collected from pupils aged 2-15 years in the five major towns in Ondo state, Nigeria is presented in Tables 1 and 2

Polymerase Chain Reaction of E. coli Isolated from Primary School Pupils in Ondo State
The result of polymerase chain reaction (PCR) amplification of E. coli isolates is presented in

DISCUSSION
This epidemiological survey is out to characterize a population of E. coli isolated from urine and faecal samples of school children in five major towns in Ondo State, Nigeria. It was found that a population of E. coli was present in the samples collected but sparingly diverse epidemiologically across the communities where sampling was carried out. Nevertheless, E. coli isolates were more in female than male urine and stool samples respectively. The investigation conducted by other workers showed that E. coli of different strains have been isolated from urine samples of patients suffering from cystitis and pylonephratitis [7] and from faecal samples of diarrhoaeic and non-diarrhoaeic patients [8,9].
From the statistics of samples collected and analyzed by [10] from South-Stockholm General Hospital and Rosen-Lund Hospital in Sweden following an outbreak of epidemic, more female patients suffered from E. coli infection most especially those with genes that code for TEM, OXA and CTX-M-1-type than male patients. Besides, a research conducted by [11] on both community and hospital care settings revealed that, there was a significant difference between the prevalence of E. coli in male (50/117) and female (100/133) cultures (P<0.001). Additionally, the female anatomical configuration also makes them to be more prone to urinary tract infection, due to the fact that the female urethra is much shorter and closer to the anus than in male. From these various points of view, it is evident that the female is more prone to urinary tract infection than male and this invariably showed the reason for a higher percentage of E. coli isolates in urine of females than males in this study. In the same vein, the study also showed that E. coli isolated from faecal samples were more in female (70/124) than in male (55/107). E. coli, besides being a cause of diarrhoae, it's also a microflora in the gastrointestinal tract (GIT), where its isolation may be possible irrespective of any outbreak of epidemic. The evidence of the incidence of E. coli isolates in female more than male faeces, vice versa, may not be too necessary because of its niche (GIT), unlike urinary tract where female anatomy might be reasonable. Thus, this study is in agreement with other works [12,13].  The incidence of E. coli carrying shiga toxin gene (stx1) in these pupils may be associated with the nature of food and water they take. Most foodstuffs and water may be grossly polluted due to an array of sources, including domestic and/or wild animals, malfunctioning septic systems, industrial outlets, combined sewer overflows, and wastewater effluents, as well as ineffectively controlled treatment stages and, to an extent, the persistence of bacteria after disinfection [14,15]. In general, the less sanitized environment has largely rendered our intakes unfit, thereby increasing the prevalence of this toxin.
The stx1 gene of Shiga toxin happens to be a major virulence factor of Enterohaemorrhagic E. coli (EHEC) strains detected in this study and it is known for producing life-threatening disease in humans with two key clinical microbiological observations: Haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS) [16,17]. However, in recent times, Shiga toxin-producing E. coli strains have been implicated in the . [18] reported two incidences of urinary tract infection caused by STEC in elderly women, though both strains belonged to the O157:H7 serotype and carried genes associated with severe illness. Besides, urine samples' data from the work done by [19,20], demonstrated that E. coli O157:H7 were isolated (2.3%) and (1.5%) respectively from urine. In addition, three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs were geno-and phenotypically characterized according to [21]. These isolates carried typical EHEC virulence markers and one of them exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. E. coli O157:H7 serotype was also isolated from urine samples of adult males and females suffering from severe UTI in Nigeria. From the report, a very high prevalence of this E. coli serotype (46.4%) among tested patients was observed; their study cited that renal diseases associated with severe UTI were mainly caused by E. coli O157:H7 [22]. These and other works [13] showed that STEC strains are implicated in UTI and in support of this study.
Enterohaemorrhagic E. coli strains are also known to be involved in diarrhoea in both male and female. From our result, stx1 virulent gene was identified and this was consistent with what other researchers have got. Results from other researchers have shown that Shiga toxinproducing E. coli causes diarrhoae. It was observed that the genes encoding Shiga toxins and intimin were the most commonly detected virulence factors isolated from diarrhoaeic and non-diarrhoaeic stools of infants [13,4,23]. Although samples in this present study were collected from non-diarrhoaeic children, but there might be an occurrence of watery diarrhoae in the population studied, now or later [24].
Studies had shown that Shiga toxin family with related structural and similar biological activity is composed of stx1 which is essentially identical to the toxin of Shigella dysenteriae differing in a single amino acid and, stx2 with less than 60% amino acid homology to stx1 [25,26]. This indicates that there is possibility of gene transfer between Enterohaemorrhagic E. coli and Shigella dysenteriae strains [27]. Owing to these vital observations, and because man cannot exist without his environment, the interaction most times result into infection caused by these pathogens. It had also been observed that the presence of stx1 and stx2 alone may not be sufficient to cause disease in humans except when other virulent genes such as eaeA, ehxA or saa are present [16]. Thus, since the samples were collected from apparently healthy children, detection of stx1 gene, which had been observed not capable of causing a disease condition alone, may further contribute to the fact that the E. coli strain isolated from these children were not carrying other genes.

CONCLUSION
In conclusion, there is a strong indication of the presence of E. coli in both urine and faecal samples collected from apparently healthy children in Ondo state. In the same vein, the molecular study of these isolates showed that, stx1 gene was predominant among the isolated E. coli, which suggested that most of them belong to the Enteroheamorrhagic E. coli group. The presence of the stx1 gene alone has been proven to be incapable of causing an infection, and thus further strengthened the fact that the samples were collected from apparently healthy pupils.