Prevalence of Escherichia coli Pathotypes among Children with Diarrhoea in Zaria, Nigeria

Diarrhoea is one of the foremost public health problems worldwide especially among Children under five years in developing countries. Only few studies have investigated the epidemiology and virulence of Escherichia coli pathotypes in South-Eastern and South-Western Nigeria leaving the Northern part of the country unstudied. In this study, a total of 100 isolates of E. coli (45%) were obtained from the stool of 222 diarrhoea patients who were children below five (5) years attending an annex The isolation and biochemical identification of the E. coli isolates were performed using standard microbiological procedures. A multiplex polymerase chain reaction (PCR) technique was used to differentiate the five (5) major diarrhoeagenic Escherichia coli pathotypes (EHEC, ETEC, EPEC, EIEC and EAEC) in one reaction condition, by using different diarrhoeagenic E. coli primers for different virulent genes found in E. coli . From the result obtained, only one (1) percent of the isolates was found to harbor the virulence gene out of the 100 E. coli isolated from the diarrhoea stools of children employed in this study.


INTRODUCTION
Escherichia coli (E. coli), documented as the most-studied bacterium, colonizes the gastrointestinal tract of most warm-blooded animals within hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant [1]. E. coli is easily cultured in the clinical laboratory, but the identification of the different pathogenic genotypes requires virulence gene detection methods.
Based on pathogenesis, clinical manifestations and presence of specific virulence factors, there are five major diarrhoeagenic E. coli groups/pathotypes [2].
Enterohaemorrhagic E. coli (EHEC) strains are implicated in food-borne diseases principally due to ingestion of uncooked minced meat, raw milk or through ingestion foods contaminated with cattle faeces. These strains produce verotoxins 1 and 2 (Shiga-like-toxin 1 (stx1) and Shiga-like toxin 2 (stx2)) and variants thereof. They are involved in episodes of diarrhoea with complications. Serotype O157:H7 is the prototype of increasing importance and is associated with hemorrhagic colitis, bloody diarrhoea and hemolytic uremic syndrome (HUS) in about 10% of patients [3].
Enteropathogenic E. coli (EPEC) strains are a significant cause of infant diarrhoea in developing nations. EPEC were historically recognized on the basis of serotypes such as O55:H6 and O127:H6. EPEC (a non invasive and non toxin producing) is an established etiological agent of human infantile diarrhoea. Its pathogenicity (localized adhesion) destabilises intestinal epithelial cell function to produce distinctive "attaching and effacing" (A/E) lesions. EPEC cause a watery diarrhoea that may contain mucus but typically does not have blood in it.
Vomiting, fever, malaise and dehydration are the symptoms, which may last for several days, resulting to chronic EPEC disease [1].
Enterotoxigenic E. coli (ETEC) produce toxins which are heat-labile (LT) and/or heat-stable (STa and STb) that also cause diarrhoea in humans and animals [4]. ETEC cause watery diarrhoea that can be mild in nature or in some instances can be a severe, cholera-like illness where rapid dehydration can be life-threatening. In endemic areas of ETEC-mediated diarrhoea, infants and children under the age of 5 are the most commonly affected. ETEC exposure in endemic areas is one of the most common causes of traveler's diarrhoea.
Enteroinvasive E. coli (EIEC) cause a broad spectrum of human's diseases. They are biochemically, genetically and pathogenetically closely related to Shigella spp., both characteristically cause an invasive inflammatory colitis, but either may also elicit a watery diarrhoea syndrome indistinguishable from that caused by other E. coli pathogens. The pathogenesis of disease caused by EIEC and Shigella involves cellular invasion and spread, and requires specific chromosomal and plasmid borne virulence genes [5].
Enteroaggregative E. coli (EAEC) are pathogens associated with persistent diarrhoea in the developing world and have been implicated recently in the developed world as causes of both outbreaks and sporadic diarrhoea among AIDS patients. Enteroaggregative E. coli (EAEC) are a heterogeneous group of bacteria that display a wide array of virulence factors [6]. EAEC disease, as described by human volunteers, is a watery diarrhoea that occurs in some cases with abdominal cramps, but no fever and there is no invasion of the bloodstream [1].

a. Samples outside Ahmadu Bello University
Teaching Hospital, Shika-Zaria were not used. b. Non diarrheic stool samples were not analysed. c. Diarrhoeic patients above five (5) years were not included.

Sample Collection and Treatment
The stool samples were cultured within 2-4 hours of collection. About one gram of stool sample was suspended in sterile nutrient broth and incubated at 37ºC for 24 hours. The concentrated isolates from the nutrient broth were then used to inoculate MacConkey agar plate and incubated at 37ºC for 24 hours. Colonies that showed typical characteristics of E. coli morphology were subcultured into eosinmethylene blue agar and incubated at 37ºC for 24 hours. The colonies that showed green metallic sheen, were further subjected to biochemical tests for confirmation.

Biochemical Tests
The biochemical tests that were used to identify the E. coli isolates are Indole test, Methyl red test, Voges-Proskauer test, Citrate utilization test and finally confirmed using Eijkmann test (fermentation of lactose and gas production at 44.5ºC for 24-48 hours).

Molecular Characterization of the E. coli Pathotypes
Molecular assay was carried out at Molecular Diagnostic Laboratory, Veterinary Teaching Hospital, Ahmadu Bello University, Zaria, Nigeria.

Genomic DNA extraction
All isolates were inoculated into 5 ml Luria-Bertani liquid medium and incubated for 24 hrs at 37ºC. DNA extraction was carried out using the ZR Fungal/Bacterial DNA MiniPrep™ ZRD6005 (Zymo research, CA, USA). About 1.5 ml of the bacterial cell suspension was added into a microcentrifuge tube and centrifuged at 10,000 x g for 1 minute. It was then decanted and 400 µl of lysis buffer was added to the pellets. This was incubated at 70ºC for 30 mins and then centrifuged at 10,000 x g for 1 minute. The pellets were then transferred into a Zymo-Spin™ IIC Column in a new collection tube and centrifuged at 10,000 x g for 1 minute. Then 200 μl DNA Pre-Wash Buffer was added to the Zymo-Spin™ IIC Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. This was followed by adding 500 μl Fungal/Bacterial DNA Wash Buffer to the Zymo-Spin™ IIC Column and centrifuge at 10,000 x g for 1 minute. The Zymo-Spin™ IIC Column was transfered to a clean 1.5 ml microcentrifuge tube and added 100 μl DNA Elution Buffer directly to the column matrix, and centrifuged at 10,000 x g for 30 seconds to elute the DNA. This was then used as template for the PCR. Amplification conditions include 94 (initial template denaturation), 35 cycles at 94 for 50s (final denaturation), primer annealing at 57ºC for 40s, primer extension at 72 and finally at 72ºC for 3 minutes (final extension).

Agarose gel electrophoresis
Amplicons were analysed by electrophoresis on agarose 1.5% w⁄v gels prepared under standard conditions, followed by staining with 20 µl of ethidium bromide and allowed to solidify. Thereafter, 25 µl of the sample (Amplicons) was then loaded onto the gel wells and allowed to run for 30 minutes at 120 v. Amplification conditions include 94ºC for 6 min ion), 35 cycles at 94ºC for 50s (final denaturation), primer annealing at C for 40s, primer extension at 72ºC for 50s, C for 3 minutes (final extension).

gel electrophoresis
Amplicons were analysed by electrophoresis on ⁄v gels prepared under standard conditions, followed by staining with 20 µl of ethidium bromide and allowed to solidify. Thereafter, 25 µl of the sample (Amplicons) was lls and allowed to run for 30 minutes at 120 v. Amplicons were illuminator UV light of wavelength 302 nm. This was then photographed with a Polaroid camera and documented using gel electrophoresis documentation system.

RESULTS
A total of 100 isolates of Escherichia coli recovered from 222 stool samples of Children under five (5) years of age within the period of the study. From the result obtained, only one (1) percent of the isolates was found to harbor the virulence gene out of the 100 E. coli from the diarrhoea stools of children employed in this study. Plate 2, lane 14 showed a band which is interpreted to be antiaggregation protein ( a characteristic of enteroaggregative (EAEC) at a base pair of 232.

Genomic DNA Extraction and PCR Analysis
The results of the genomic DNA extraction is as shown in plate 1 below

DISCUSSION AND CONCLUSION
In this study, a total of 100 isolates of E. coli (45%) were obtained from the stool of 222 diarrhoea patients who were children below five (5) years attending Ahmadu Bello University Teaching Hospital, Shika, and Institute of Child Health, Banzazzau; an annex of Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria. The isolation and biochemical identification were performed using standard microbiological procedures. The low percentage of E. coli obtained (45%) from children with diarrhoea may be due to the fact that antimicrobial therapy previous to sample collection might have been given in some cases and it is a known fact that this can reduce the percentage of bacterial enteropathogens isolation [9].
Multiplex PCR was performed to characterize the E.
coli isolates into their respective pathotypes/diarrhoeagenic groups as described by Persson et al. [7]. From the result obtained, only one (1) percent of the isolates was found to harbor the virulence gene out of the 100 E. coli, isolated from the diarrhoea stools of children employed in this study. Plate 2, lane 14 showed a band which is interpreted to be antiaggregation protein (aap), a characteristic of enteroaggregative E. coli (EAEC) at a base pair of 232. The very low percentage (1%) prevalence of diarrhoeagenic E. coli obtained in this study is in close agreement with the study conducted and reported by Chigor et al. [10], who reported a prevalence of E. coli 0157: H7 in children with diarrhoea as 5.4% in Zaria, Nigeria. Also, Ogunsanya et al. and Olorunshola et al. [11,12], reported a prevalence of 5% EHEC 0157:H7 in humans, in Lagos, Nigeria. But it is in contrast with the study conducted by Nweze, who reported 19.6% prevalence of diarrhoeagenic E. coli in a study conducted in Southeastern Nigeria [13], an incidence higher than 40% has been reported in Bangladesh [14]. A lower incidence of about 30% has been reported in Jordan [15]. The very low (1%) prevalence of diarrhoeagenic E. coli obtained in this study might be attributed to encouragement of breast feeding and educating the mothers on proper personal hygiene by the dieticians both in the main Teaching Hospital and the Institute of Child Health. The subjects employed in this study may be infected by other pathogens other than diarrhoeagenic E. coli since there are different pathogens that can cause diarrhoea in children, including Rotavirus, Salmonella spp., Shigella spp., Campylobacter jejuni, Entamoeba histolytica, and Giardia lamblia [16,17].  [7,8] Antimicrobial therapy previous to sample collection might have been given in some cases where it was recommended, and it is known that this can reduce the percentage of bacterial enteropathogens isolation [9]. Finally, the direct effect of other pathologies, such as malaria, cannot be discarded. It has been previously described that between 5% and 38% of malaria cases present diarrhoea as one of the main symptoms [18].
In conclusion, molecular characterization of the E. coli isolates showed that, only 1% of the isolates carried a virulence gene (antiaggregation protein/dispersin), a characteristic of enteroaggregative E. coli. This report opens the door to further studies addressed to analyze the specific epidemiology, resistance patterns, and virulence of diarrhoea-causing pathogens in Zaria, and the whole country in general.