Prevalence of Escherichia coli Pathotypes Among Children With Diarrhea in Babol , Northern Iran

Background: Diarrheagenic Escherichia coli (DEC) are major causes of diarrhea in the world particularly among infants and young children. Objectives: The aim of this study was to determine the prevalence of DEC strains in stool samples from children under 5 years old. Patients and Methods: Stool specimens were collected from 200 children under 5 years visiting hospital due to gastroenteritis. E. coli pathotypes were detected by using conventional culture techniques and polymerase chain reaction (PCR). Results: Sixty-eight (34%) out of 200 specimens were positive for DEC. Different pathotypes would show the following profiles: 43 (21.5%) for enteropathogenic E. coli (EPEC); 18 (9%) for enterotoxigenic E. coli (ETEC) including 10 (55.5%) st positive, 6 (33.3%) lt positive and 2 (11.1%) st and lt both positive; 6 (3%) for enteroaggregative E. coli (EAEC) and 1 (0.5%) for enteroinvasive E. coli (EIEC). Enterohemorrhagic E. coli (EHEC) was not isolated from any of the E. coli strains tested. Conclusions: This study shows that DEC is a common cause of childhood diarrhea in Babol. EPEC and ETEC were the most frequent pathotypes in the population under study. *Corresponding Author: Majid Alipour, Department of Microbiology, Babol Branch, Islamic Azad University, Babol, Iran Email: alipourrna@yahoo.com Int J Enteric Pathog. 2016 August;4(3):e36326. http://enterpathog.com Copyright © 2016, Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.


Background
Escherichia coli is one of the predominant facultative anaerobes in the human gastrointestinal tract.Many strains of E. coli are harmless and even provide many health benefits to the host.However, there are small groups of E. coli that have evolved and developed pathogenic strategies that can cause a broad spectrum of disease, including severe diarrheal disease. 1 Diarrheagenic Escherichia coli (DEC) are conveniently classified into 6 major pathotypes according to their virulence genes and including enterotoxigenic E. coli (ETEC), Shiga toxin producing E. coli (STEC, also referred to as verotoxigenic E. coli (VTEC) or enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC). 2 EIEC is also the only E. coli pathotype to invade and multiply within host epithelial cells, and can cause invasive inflammatory colitis and dysentery, but most symptomatic infections are characterized by watery diarrhea indistinguishable from that produced by other diarrheagenic E. coli pathotypes. 3EHEC cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). 4ETEC is an important cause of diarrheal disease in infants (6-18 months), young children, and the elderly in the developing world.It is also known to be the major cause of "traveler's diarrhea" acquired by tourists visiting developing nations. 5The main distinguishing feature of EPEC is the ability to induce a characteristic histopathology called the attaching and effacing (A/E) lesion. 6EAEC is increasingly recognized as an emerging enteric pathogen and cause of persistent diarrhea (greater than 2 weeks duration) in children and adults in both developing and developed countries. 7The pathogenicity of DAEC strains is inadequately understood but it has been associated with diarrhea in young children under 12 months which is typically mild without blood in the feces. 8In many studies, a different prevalence of DEC strains has been reported.0][11] The identification of diarrheagenic E. coli using biochemical and serological tests is unreliable.Therefore, several polymerase chain reaction (PCR) tests have been developed to amplify the target regions present in virulence genes and identify strains of DEC. 12 Babol, Northern Iran.Since DAEC is not well defined as a distinct pathotype, its detection was not included in this study.

Isolation and Identification of Escherichia coli
Stool samples, from June to October 2014 were collected from hospitalized children under 5 years old in the Babol.A total of 200 stool samples from diarrheal patients were processed by directly inoculating the fecal matter onto MacConkey Agar and culturing overnight at 37°C.The suspected pink colonies were then cultured on eosin methylene blue (EMB) agar plates to see the metallic sheen color of the E. coli.Stool samples were a metallic green sheen colony from each plate with a typical E. coli morphology was selected and examined by biochemical tests, including indole, methyl red, Voges-Proskauer, citrate and urease tests.The isolates that were positive to indole and methyl red tests but negative to Voges-Proskauer, citrate and urease tests were identified as E. coli.

DNA Templates for PCR Reactions
All isolated E. coli strains were grown on Luria-Bertani (LB) broth at 37°C for 24 hours.Genomic DNA was extracted according to the method described by Gómez-Duarte et al. 13 Bacteria were first harvested from 1.5 mL of an overnight LB broth culture, suspended in 200 μL of sterile water, and boiled at 100°C for 10 minutes.Following centrifugation of the lysate, the supernatant containing a crude DNA extract was used as a DNA template on a multiplex PCR for identification of E. coli pathotypes.

Polymerase Chain Reaction Detection of Diarrheagenic Escherichia coli
Two multiplex PCR assays were performed for the detec-tion of five pathotypes of DEC.The oligonucleotide primers used in this study are listed in Table 1.
Multiplex PCR 1 contained primer mix 1 for the detection of elt and est for the enterotoxin of ETEC, hlyA for the plasmid encoded enterohemolysin of EHEC and, pCVD432 for the nucleotide sequence of the EcoR1-Pst DNA fragment of EAEC.[16][17][18] Multiplex Polymerase Chain Reaction PCRs were performed with a 20 L reaction mixture containing 2 L of the solution containing DNA, 2 L of 10x PCR buffer, 0.4 L of 10mM mixture of deoxynucleoside triphosphates, 0.6 L of 50mM MgCl 2 , 0.2 L of 5 U of Taq DNA polymerase, 1 L of each primer (20 pmol), and 10 L of distilled water.The reactions were performed as follows: initial denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C (for multiplex PCR 1) and 57°C (for multiplex PCR 2) 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes.Positive and negative controls were conducted for all DEC strains which were positive in the multiplex PCR.The PCR products were electrophoresed on a 2% agarose gel and visualized with UV transilluminator after EtBr staining.

Results
From the 200 stool samples, DEC strains were identified in 80 of the samples, and DEC was detected in 68 (34%) of the diarrheic children under 5 years old within the period of the study.Of the 68 DEC strains detected by PCR, 43 (21.5%) were EPEC, 18 (9%) ETEC, 6 (3%) EAEC and 1 (0.5%) EIEC isolates.Among 18 ETEC isolates, st gene was detected in 10 (55.5%), lt gene in 6 (33.3%) and both lt and st genes were present in 2 (11.1%) isolates.The frequency of each DEC pathotype is shown in Table 2. EHEC was not isolated from any of the E. coli strains tested.The most frequently isolated was EPEC.The amplified products for the elt gene and the est gene (ETEC) were of 322 bp and 170 bp, respectively.The PCR product for eae gene was of 229 bp.The amplified products for the ipaH gene and the ial gene (EIEC) were 933 bp and 320 bp, respectively.The PCR product for pCVD432 gene (EAEC) was 630 bp (Figures 1 and 2).

Discussion
Among the bacterial pathogens, E. coli plays an important role in causing diarrhea in children under 5 year old.
In the present study, 68 (34%) isolates were identified as DEC strains by multiplex PCR.Hegde et al 19 reported a prevalence of 26% DEC strains from 200 stool samples from children with diarrhea in India.In a study conducted by Gómez-Duarte et al 13 in Colombia, a lower prevalence of 14.4% has been reported.The prevalence of DEC varies in the world from region to region and even between countries.Albert et al 9 reported that in children with diarrhea, EPEC had the highest prevalence.Alikhani et al 20 identified that EPEC was the most prevalent pathotype among DEC.In another study in Iran, Alikhani et al 11 reported that the most frequently identified DEC was EPEC (47.5%).Usein et al 21 also showed that 9% of the children with diarrhea carried EPEC isolates.In this study, EPEC was the predominant E. coli pathotype (21.5%).The reason for different rates of identification of the EPEC may be due to health status in various areas.In our study, the second most common pathotype of DEC was the ETEC 18 (9%).The prevalence of ETEC was lower in our study than in some previous studies.The rate of isolation of the ETEC reported by Wolk et al 22 and Viboud et al 23 was 20.7% and 18.3%, respectively.In studies conducted by Hegde et al 19 and Nguyen et al, 24 the rate of isolation of the ETEC was 3.5% and 2.2%, respectively.In this study, the detection rate for EAEC was 3.0%.Our finding is approximately similar to those reported by Aranda et al, 25 Ifeanyi et al, 26 and Aslani et al, 27 in which they found that the rate of EAEC was 2%, 2% and 10.7%, respectively.The very low percentage (0.5%) prevalence of EIEC obtained in this study is in close agreement with the study reported by Hegde et al, 19 who reported a prevalence of EIEC in children with diarrhoea as 1.5% in India.In the present study, we did not isolate any EHEC strains.The results of current investigation are consistent with the studies reported by Moyo et al 28 and Nguyen et al. 24 In conclusion, EPEC was the most commonly identified DEC strain in the region studied.Therefore, further studies are needed to investigate virulence properties and antimicrobial resistance of EPEC strains.

Table 1 .
PCR Primers Used in This Study