Esherichia coli O157:H7 Serotypes Isolation from Children in Stool Samples

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. This work was therefore aimed at isolating Esherichia coli O157 from human stool. A total of 100 stool samples were collected from patients with age ranging from (0-05) years, statistical analysis of the risk factors showed that only zero to five years age range of the respondents had a significant statistical difference of 0.012 (P<0.05). The presumptive Esherichia coli isolates that appeared as pink flat lactose fermenting on MacConkey Agar and green metallic sheen on Eosin Methylene Blue agar were picked and confirmed biochemically as Esherichia coli using biochemical test kit. The confirmed E. coli isolates were then cultured on Sorbitol MacConkey Agar which shows non sorbitol fermenting. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7. Although 1.39% prevalence rate of Esherichia coli O157 was obtained it is pertinent to note that, Esherichia coli O157 is becoming a public health threat because of the debilitating effects it has on humans and also due to its low infectivity dose. There is therefore, the need for more public awareness to educate our citizens on ways of improving on the unsanitary environment.

Enteric pathogens are gastrointestinal organisms known to cause gastrointestinal infection. Gastrointestinal infection also known as gastroenteritis is any infection caused by Viruses, Bacteria or Parasites and is characterised by excessive watery diarrhoea and stomach pain. Acute diarrhoea is a second most common cause of infant deaths worldwide and is a common cause of mortality in developing countries 1 . It is estimated that 1.3 billion episodes of diarrhoea occur in children below five years of age with about 760,000 deaths occurring yearly 2 .
Escherichia coli is a common inhabitant of the human and animal gut, but can also be found in the physical environment such as; water, soil and vegetation and are thus referred to as being ubiquitous. Many Escherichia coli strains are usually not harmful and act as commensals in the intestine of warm blooded animals, but some few strains have been found to cause mild to severe disease in man. Pathogenic strain of Escherichia coli that is Escherichia coli O157:H7 is responsible to cause diarrhoea and other severe complications such as haemolytic colitis, haemolytic uraemic syndrome and thrombotic thrombocytopenic pupura in humans. The majority of E. coli O157:H7 strains can be distinguished from most E. coli by their inability to ferment sorbitol rapidly and by their lack of production of glucuronidase [3][4] . They also differ from other E. coli because of their ability to produce verocytotoxins (VT) or shiga toxins (ST). Escherichia coli O157:H7 is a zoonotic food borne and waterborne pathogens with cattle serving as the main reservoir for this organism which they shed in their faeces and is often times used as manure by farmers. Transmission of this organism is usually through faecal oral route and Humans become infected with this pathogen through consumption of faecally contaminated fruits, vegetables and water or through person to person contact and direct contact with infected faeces.
Escherichia coli O157:H7 causes approximately 70,000 illnesses and 60 deaths annually in the United States and is a cause of several outbreaks of gastroenteritis around the world 5 . In developing countries where diarrhoeal disease and associated mortality are much more pervasive there is very limited information about E. coli 0157:H7 prevalence 6 . The first major outbreak of bloody diarrhoea in the developing world associated with E. coli 0157:H7 occurred in Swaziland in 1992 7 and infection with this pathogenic strain may have accounted for tens of thousands of cases during this epidemic [8][9][10] Due to the low infective dose of E. coli O157:H7, the potential severity of the infection and the possibility of laboratory-acquired infections 11 , an inoculation of fewer than 10 to 100 colony forming units (CFU) of E. coli O157:H7 is sufficient to cause infection, compared to over onemillion CFU for other pathogenic E. coli strains 12 . Their ability to survive in the environment and the environmental contamination with Escherichia coli O157:H7 may be an important public health problem [13][14] . Also another major problem with E. coli O157:H7 is that it is not detected by the usual methods used to isolate and identify "traditional" enteric bacterial pathogens therefore, most microbiology laboratories in many countries of Africa do not routinely test for E.coli O157:H7, hence many infections may go unrecognized 15 This work therefore, sought to isolate and characterise Escherichia coli O157 from human stool.

Isolation of and Identification of E. coli Isolates
The colony appeared as green with black metallic sheen of the stool samples which were cultured on Eosin Methylene Blue agar , and again were picked and sub cultured on fresh EMB agar plates to obtain presumptive E. coli isolates. These presumptive E. coli isolates were confirmed by the conventional biochemical test for E. coli (IMViC)

Isolation of E. coli O157:H7
The confirmed E. coli isolates were cultured on Sorbital MacConkey Agar plates (SMAC), the colonies that appeared colourless on SMAC were tagged as presumptive E. coli O157.

Ethical consideration
The study was ethically approved by the institutional ethics review board of Rama Medical College, Kanpur. Written consent was obtained from parents/guardians of the children before enrolment into the study.

RESULTS
In 100 fecal samples, E. coli O157:H7 was isolated and collected in Rama Medical College Hospital, Kanpur which is summarized in Table 1. The E. coli O157:H7 appears as sorbitolnonfermented colonies on MacConkey agar (whitegray) as shown in Figure 1. The confirmed E. coli isolates were cultured on Sorbital MacConkey Agar plates (SMAC), the colonies that appeared colourless on SMAC were tagged as presumptive E. coli O157. Analysis of the risk factors associated with diarrhea was carried out using the Fischer Exact Test and the result shows that only age of the respondents had a statistical significant difference of p<0.05 (p=0.012). The other risk factors that were analyzed had p values greater than 0.05 which means they had no statistical significant difference.

DISCUSSION
The findings in this study indicates that age remains a major risk factor in diarrhoea disease, children between the ages of 0-5 are highly vulnerable to diarrhoea as this study has shown. The prevalence of 9.8% diarrhoea in respondents 0-5 years in this work, is higher than the 2.6% obtained by Yilgwan and Okolo,17 in Jos Plateau State and lower than the 43.1% obtained by Ifeanyi et al. 18 in Abuja. These differences might be due to breaches in sanitation and hygiene infrastructure of the respondents from these cities. The high occurrence rate of diarrhoea among children 0-5 years in this study may be due to the fact that children within this age group on their own cannot differentiate between what to eat and what not to eat; they have not learnt the rudiment of adherence to aseptic or hygienic practices. Another reason for their high vulnerability to diarrhoea may be due to weaker immunity as a result of them having lost their inborn immunity after being weaned from breast milk. Young children use more water over the course of a day given their higher metabolic rates, also their kidneys are less able to conserve water compared to older children and adults as such diarrhoea is usually prevalent and often life threatening too. In this study, it was observed that the number of diarrhoeic stool gotten from adults was quite small compared to that obtained from children and this might not be unrelated to the fact that, adults in the locality rarely visit health institutions when they have diarrhoea unless they perceive the diarrhoea as being serious, usually if blood is present as reported by Okeke et al. 19 .
The 1.39% prevalence rate of E. coli O157in this study is lower than the 6% prevalence by Olorunshola et al. 10 in Lagos and the 20% prevalence recorded by Esumeh et al. 16 in Benin. Escherichia coli O157 remains an aetiological agent for diarrhoea in Nigeria, although there are differences in prevalence rate of Escherichia coli O157 in the stool samples in different parts of Nigeria, this result however shows that The presence of Escherichia coli O157 in stool samples might not be unconnected to the fact that patients have been exposed to unsanitary conditions such as consumption of contaminated water, food, fruits and vegetables. 4