Molecular Diagnosis of Diarrheagenic E. coli Infections Among the Pediatric Patients in Wasit Province, Iraq

Diarrheagenic Escherichia coli still an important pathogen that cause diarrhea which lead to hospital admissions and death specially in children. In order to identify the common pathotypes of E. coli via investigate different virulence genes. A total of 210 stool samples were collected from children under five years presented with diarrhea from different hospitals and private clinics in Wasit province, Iraq, on the other hand, 40 stool samples were collected from healthy children considered as control group. regarding to culture, biochemical tests and API 20E results 100 isolates were supposed to be E. coli. The DNA were extracted to that 100 isolates from diarrheal cases and for 40 isolates of control, concentration of DNA samples were between (50-360 mg/μl ) and the purity between (1.8-2). All isolates studied for detectionvirulence gene of five Diarrheagenic Escherichia Coli strains based on using multiplex Polymerase Chain Reaction technique, by amplified 13 primer (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2, invE, ipaH, elt, estIa, estIb), and showed the distribution of the strains and its susceptibility to antibiotics. The most frequent pathotypes was Enteropathogenic E.coli 19/42 (45.3%) ) with 9 typical and 10 atypical, followed by Enteroaggregative E. coli 17/42 (40.5%), Enterotoxigenic E. coli 3/42 (7.1%), Enteroinvasive E. coli 3/42 (7.1% ), and 0/42 (0%) in Shigatoxin producing E .coli and no DEC in all control patients. The highest resistance to antibiotics was (95.2%) to Amoxicillin and Ampicillin, respectively, Sulfa-Trimethoprim 92.9%, followed by 85.7% for Tetracycline and Cephalothin, Ceftriaxone 81% and Cefotaxim “clavulanic acid 71.4%. While the lowest resistance was to Chloramphenicol (19 %), Ciprofloxacin (16.7%), Amikacin (7.1%) and no resistance was detected toward Imipenem. We can conclude in this study, multiplex PCR is a swift, and accurate procedure can be used for Diarrheagenic E.coli identification and isolation successfully of strains.


INTRODUCTION
Diarrheal disease is still a global problem around the world specially in children under five years in developing countries 1 . According to the World Health Organization (WHO), diarrheal diseases are the second leading cause of death (~760,000 per year) in children 2,3 . The microbial causes of diarrhea are variety of bacterial, viral and parasite 4 , Among these pathogens, diarrheagenic E. coli play a major role in causing diarrhea in children under 5 years 5,6,7 .
When the microbial agent is bacteria, E. coli consider one of the major causes, specially to infantile diarrhea 8,9,10 . Depending on specific virulence gene, clinical features, and serotypes diarrheagenic E.coli divided into 6 stains: Enteropathogenic E. coli, Enteroaggregative E. coli, Enteroinvasive E. coli, Enterotoxigenic E. coli, Shiga toxin-producing E. coli and Diffusely Adherence E. coli 11 . Culture and biochemical test can't distinguished between commensal or pathogenic strains of E. coli in stool, therefor PCR used to detect the virulence genes in pathogenic strains 12 , multiplex PCR provide detection to many Diarrheagenic E.coli strains virulence genes with high sensitivity, specificity 13 .
The aim of thisstudy was detecting the distribution of diarrheagenic E. coli pathotypes among children with diarrhea in Wasit province, Iraq by multiplex PCR, and assessing the antimicrobial susceptibility profile of diarrheagenic E. coli, in order to contribute to the establishment of a more effective empirical antibiotic therapy for the disease.

Collection of samples
During period from middle of September 2017 to middle of December 2017 a total of 210 stool samples were collected from children The amplification products were electrophoresed through a 2 % agarose gel and visualized with UV transilluminator after ethidium bromide staining. A 100 bp DNA ladder was used as a molecular size marker in gel. The statistical analysis of all the evidence was done using the system SPSS IBM version 20 software, Chi-squire test. P-value ≤ 0.05 was considered statistically significant.

RESULTS
E. coli were isolated in 100 (47.6 %) of 210 collected samples followed by 78 (37.2%) of other gram negative bacteria (Salmonella, Klebsiella, Proteus, Pseudomonas) and 32 (15.2 %) samples that were no growth. The results of primary diagnosis to these 100 E. coli isolates by selective and differential culture media were consistent with the microscopic and biochemical tests results.
From 19 isolates detected as EPEC which was watery diarrhea 10 (52.6%) isolates of them are atypical EPEC showed eaeA gene found without bfpB gene, and 9 (47.4 %) were typical EPEC which showed eaeA gene together with bfpB gene. All 19 isolates in our study don't produce nether stx1 or stx2, in addition one of aEPEC showed astA gene.
Enteroaggregative E. coli 17 (40.5%) isolates came second after Enteropathogenic E.coli as causative agent of diarrhea among Diarrheagenic E. coli pathotypes in our study, aggR gene was appeared in all EAEC isolates detected in our study that mean all of them were typical EAEC. Enterotoxigenic E.coli account 3 isolates (7.1%) and Enteroinvasive E. coli was detected in 3 isolates (7.1%) that suggested these pathotype maybe play a lessimportant role in childhood diarrhoea in developingcountries. When age stratification was analysed high incidence of DEC E. coli recorded in first and second age group flowed by third and fourth age group, and there were no cases recorded in fifth age group. The prevalence of Enteropathogenic E. coli infection infections was high in first and second years , also all Enteroaggregative E.coli infections were detected under 2 years, while Enteroinvasive E. coli were high between 2-3 years also cause infection in first age group, in time all Enterotoxigenic E.coli infections were all above 1 year as shown in figure (1).
E. coli pathotypes, in our study were identified and isolated successfully by using Multiplex PCR. PCR products visualized to measured product size results from amplification the primers in compared with (100 bp) ladder as shown in figures (2,3,4,5,6,7,8,9).

DISCUSSION
The distribution of DEC in our study was 42 (20%) among 210 diarrheal cases. Our result concur to other study in Iraq reported by Hamada et al 12 in Kirkuk (36%) and globally with other studies in Iran Heidary et al 19 (28 %), while our result contrast with other studies were showed less prevalence to Diarrheageic E. coli Konateet al 20 revealed (7.4%) in Burkina Faso, Salmaniet al 21 in Iran who showed (88%). These differences reflecting the difference in distribution of geographical areas , quality of sanitation .
Among all the Diarrheagenic E.coli pathotypes , Enteropathogenic E.coli (EPEC) were found to be the most common pathotypes for children with (45.3%), our result compatible with localstudy by Sakhi 22 who showed EPEC as most than other pathotypes (63%), and in contrast with Khalil 11 and Al-Dulaimi 23 where they show it came second after EAEC . Our finding was , however , similar to globally studies with Zhou et al 1 , Thakur et al 7 and Chellapandietal 6 that also reported a high frequency of EPEC pathotypes associated with pediatric diarrhea.
EPEC are sub-grouped into typical (tEPEC, eae+ bfpA+) and atypical (aEPEC, eae+ bfpA-) strains that differ in several respects Naji and Nasser 24 . From 19 isolates detected as EPEC which was watery diarrhea 10 ( 52.6%) isolates of them are atypical EPEC showed eaeA gene found without bfpB gene, and 9 (47.4 %) were typical EPEC which showed eaeA gene together with bfpB gene.    All 19 isolates in our study don't produce nether stx1 or stx2, in addition one of aEPEC showed astA gene. Our study is close to study by Arif and Salih 23 in Sulaimani, Iraq, and global reports by Malvi 25 in India, that showed the distribution of atypical EPEC was higher than typical EPEC. Ochoa and Contreras 26 report that atypical EPEC (aEPEC) are more prevalent than typical-EPEC (tEPEC).       . Antibiotic susceptibility testing of isolates showed high resistance rate to Cephalothin (85.7%). The emergence of multidrug resistance especially in E. coli has become a critical public concern, which was designated as resistance to one agent in three or more antibiotic classes. Kamwati 47 ; Alizadi 48 . Many factors responsible for an increase in rates of antimicrobial resistance include misuse/over use of antibiotic by healthcare professionals and general public Magiorakos et al 49  Imipenem with no resistant agreement with 0% resistant from Al-Hilali 38 and Shamkhi 39 . The Impenim was the most effective antibiotic against DEC followed by Amikacin, Ciprofloxacin and Chloramphenicol. Impinem has been highly effective against gram negative bacteria Mohammed et al 53  The statistical analysis to susceptibility test results in this study showed high resistance among Diarrheagenic E. coli isolates which were collected from hospitalized children than isolates collected from private pediatric clinics (without history of hospital admitted) as shown in table (3).
This result goes with report of Kamwati 47 who showed isolates from children who had been hospitalized were more resistance than those isolated from children not previously hospitalized , and he conclude that recent history of antimicrobial use and hospitalization is a serious predisposing factor to carriage of Multi Drug Resistant strains. Fox-Lewis 55 also mention that hospital-acquired Escherichia coli isolates were multidrug resistant than isolates were community-acquired. Multi drug resistance MDR may be acquired from other patients who have received antibiotics. Infections caused by Multi drug resistance gram negative bacteria are difficult to treat and so may cause more prolonged symptoms in the site of infection Hawkey et al 56 .

CONCLUSION
E n t e r o p a t h o g e n i c E . c o l i a n d Enteroaggregative E. coli was the most common types of Diarrheagenic E. coli among children less than 2 years of age presented with diarrhea in Wasit province. Enterotoxigenic E. coli and Enteroinvasive E. coli were more common in children more than 2 years of age in Wasit province.
This study highlights the Using of multiplex PCR in identifying and successful isolation of Diarrheagenic E.coli from normal flora and can be used as a rapid and accurate method for the isolation of pathogenic strains of E. coli, this will greatly help pediatricians to decrease the use of antibiotic in treatment of diarrhea in children and decreasing the problem of increasing antibiotic resistance. The results of antibiotic sensitivity test revealed that the most active compound against Diarrheagenic E. coli isolates was Imipenem followed by Amikacin, Ciprofloxacin and Chloramphenicol .