Background: Introduction: Enterotoxigenic Escherichia coli (ETEC) is the most important bacterial cause of watery  travelers' diarrhea  in  developing countries. Watery diarrhea is can cause serious life-threatening dehydration. ETEC was caused diarrhea by the secretion of two heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs) which increase intestinal secretion. Routine laboratory methods are not appropriate to detect ETEC and other diarrheagenic E. coli pathotypes. The molecular techniques such as PCR are rapid and accurate methods that have been developed for detection of ETEC. We were recognized ETEC by PCR on lt and st genes from E. coli isolates from patients with diarrhea collected from selected Tehran educational hospitals.Materials and Methods: The E. coli isolates were collected from total 140 patients with diarrhea and 110 patients without diarrhea using culture and IMViC test. DNA was extracted by boiling method and the presence of the uidA, lt and st genes was detected by PCR.Results: Among 140 E. coli isolates from diarrheal stools 5 (3.6%) isolates were positive for, just lt gene, 3 (2.1%) co-amplified for both lt/st and 1 (0.7%) was positive for just the st gene which were considered as ETEC. In the E. coli isolates from non-diarrheal control samples just one (0.9%) isolate was positive for both lt and st genes.Conclusion: The results showed that the ETEC as a significant cause of diarrhea, usually ignored by laboratories using traditional methods. Sometimes the ETEC causes severe diarrhea and can threaten for patient's life. Thus a rapid diagnostic test such as PCR can be very helpful in the treatment of patients

Dabiri, Hossein

Fallah, Fatemeh

Goudarzi, Hossein

Mohammadzadeh, Mohammad

English

Background: Introduction: Enterotoxigenic Escherichia coli (ETEC) is the most important bacterial cause of watery  travelers' diarrhea  in  developing countries. Watery diarrhea is can cause serious life-threatening dehydration. ETEC was caused diarrhea by the secretion of two heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs) which increase intestinal secretion. Routine laboratory methods are not appropriate to detect ETEC and other diarrheagenic E. coli pathotypes. The molecular techniques such as PCR are rapid and accurate methods that have been developed for detection of ETEC. We were recognized ETEC by PCR on lt and st genes from E. coli isolates from patients with diarrhea collected from selected Tehran educational hospitals.Materials and Methods: The E. coli isolates were collected from total 140 patients with diarrhea and 110 patients without diarrhea using culture and IMViC test. DNA was extracted by boiling method and the presence of the uidA, lt and st genes was detected by PCR.Results: Among 140 E. coli isolates from diarrheal stools 5 (3.6%) isolates were positive for, just lt gene, 3 (2.1%) co-amplified for both lt/st and 1 (0.7%) was positive for just the st gene which were considered as ETEC. In the E. coli isolates from non-diarrheal control samples just one (0.9%) isolate was positive for both lt and st genes.Conclusion: The results showed that the ETEC as a significant cause of diarrhea, usually ignored by laboratories using traditional methods. Sometimes the ETEC causes severe diarrhea and can threaten for patient's life. Thus a rapid diagnostic test such as PCR can be very helpful in the treatment of patients.</p

Mohammad Mohammadzadeh

Hossein Goudarzi

Hossein Dabiri

Fatemeh Fallah

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  Novelty in Biomedicine NBM 144   Novelty in Biomedicine 2015, 3, 144-7 Original Article   Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples referred to educational hospitals in Tehran-Iran   Mohammad Mohammadzadeh, Hossein Goudarzi*, Hossein Dabiri, Fatemeh Fallah  Department of Medical Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Abstract Background: Introduction: Enterotoxigenic Escherichia coli (ETEC) is the most important bacterial cause of watery  travelers' diarrhea  in  developing countries. Watery diarrhea is can cause serious life-threatening dehydration. ETEC was caused diarrhea by the secretion of two heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs) which increase intestinal secretion. Routine laboratory methods are not appropriate to detect ETEC and other diarrheagenic E. coli pathotypes. The molecular techniques such as PCR are rapid and accurate methods that have been developed for detection of ETEC. We were recognized ETEC by PCR on lt and st genes from E. coli isolates from patients with diarrhea collected from selected Tehran educational hospitals. Materials and Methods: The E. coli isolates were collected from total 140 patients with diarrhea and 110 patients without diarrhea using culture and IMViC test. DNA was extracted by boiling method and the presence of the uidA, lt and st genes was detected by PCR. Results: Among 140 E. coli isolates from diarrheal stools 5 (3.6%) isolates were positive for, just lt gene, 3 (2.1%) co-amplified for both lt/st and 1 (0.7%) was positive for just the st gene which were considered as ETEC. In the E. coli isolates from non-diarrheal control samples just one (0.9%) isolate was positive for both lt and st genes. Conclusion: The results showed that the ETEC as a significant cause of diarrhea, usually ignored by laboratories using traditional methods. Sometimes the ETEC causes severe diarrhea and can threaten for patient's life. Thus a rapid diagnostic test such as PCR can be very helpful in the treatment of patients. Keywords: Enterotoxigenic Escherichia coli, Diarrhea, lt, st  *Corresponding Author: Hossein Goudarzi, Department of Medical Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Email: hgod100@yahoo.com  Please cite this article as: Mohammadzadeh M, Goudarzi H, Dabiri H, Fallah F. Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples referred to educational hospitals in Tehran-Iran. Novel Biomed. 2015;3(3):144-7.    Introduction Enterotoxigenic Escherichia coli (ETEC) is the most important bacterial cause of watery travelers'diarrhea in all age groups in areas with poor sanitation especially developing countries1,2. ETEC is the most common bacteria which are detected in 30-70% of patients with traveler’s diarrhea3.Watery diarrheais usually self-limiting, but can cause serious life-threatening dehydration.There are more than 650 million cases of ETEC infection, which result in 800,000 deaths in each year4. There are Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples…             Mohammadzadeh et al. NBM 145 Novelty in Biomedicine 2015, 3, 144-7 virulentplasmids in ETEC strains that encode colonization factors (CFs), toxins, and other adhesins.The ETEC colonizes the surface of the small bowel  via a group of surface structures such colonizationfactor antigen I (CFA/I) and produces enterotoxins, that raise intestinal secretion5,6. ETEC enterotoxins is classified to heat-labile enterotoxins (LTs) and the heat-stable enterotoxins (STs)7,8. ETEC may contain one of  LT, ST, or both Simultaneously (9). LT is closely related in structure and function to vibrio cholera enterotoxin (CT)9.These toxinsbe made of a single A subunit and five identical B subunits which B subunits mediate binding of the cell surface gangliosides such GM1 and GD1b, and the A subunit has ADP-ribosyl transferase activity that upregulates adenylate cyclase. This function leads to increased levelsof intracellular cAMP and result to increase intestinal secretion10,11. STs are small, single-peptide toxins that include two classes STa and STb that are different in structure and mechanism. Only the STa is related to diarrhea. The STa toxin is a small peptide, with guanylate cyclase activitywhich increase intracellular cGMP and lead to intestinal secretion12. Traditionally, thediagnosis of  ETEC is based on the production of LT or ST in animal models and serological and immunological assays13,14. These tests are difficult and time consuming with high rate false results.Also routine medical laboratories lack essential Facilities to perform the mentioned assays and culture methods are not able to distinguish E. coli pathotypes such as ETEC. Thus arapid and accurate methodsare necessary to detect this critical pathogen. Recently, molecular techniques such aspolymerase chain reaction (PCR) has been developedfor detection ofETEC, whichis based on amplification of lt and st genes. According to the importance of ETEC infections and weakness of traditional methods, further molecular studies are necessary to evaluate the ETEC in patients with diarrhea. The current study aims to detect the ETEC in patients with watery diarrhea who was referred to selected Tehran educational hospitals during 2013-2014 using PCR.  Methods Sampling and culture In this descriptive study, a totalof 140stool samples from patients with diarrhea (Cases) and 110 stool samples from patients without diarrhea (Controls) collected from three Tehran educational hospitals (Emam Hossein, Loghman and Taleghani hospitals). Patients which have three or more watery stools were evaluated in this study. Sampling  was performed during April 2013 to September 2013. Stool samples were cultured on Macconkey and Hekton enteric agar (Merck, Germany)  and were incubated in 37°C overnight. Lactose positive colonies were selected and examined with IMViC tests to identify E. coli isolates. All stool samples were examined microscopically for WBC and RBC. DNA extraction DNA was extracted with boiling method. The E. coli isolates was cultured in 5ml Luria Bertani broth (Merck, Germany) and incubated in 37°C overnight. All isolates was poured in 1.5 ml microtubes and centrifuged at 3000 rpm for 3 minutes. The supernatant was discarded and was added 100 µl distilled water on pellets and the microtubes boiled for 10 minutes. The microtubes was centrifuged at 12000 rpm for 2 minutes and The DNA containing supernatant was collected and stored in -20°C for molecular process. PCR assay All E. coli isolates were tested for beta D-glucuronidase encoding uidA gene as a E. coli housekeeping gene. E. coli K12 was used as positive control. The uidA positive isolates were evaluated for lt and st genes by PCR using specific primers (Table 1). ETEC H10407 was used as positive control. PCR amplification was performed in a 25µl reaction mixture containing 2μl of DNA template, 12μl ready to use Mastermix (Fermentase. Germany), 9μl of distilled water and 1μL of 20pmols forward and reverses primers.  DNA amplification was carried out the following PCR cycling program: initial denaturation at 95°C for 5 min was followed by 30 cycles of amplification (denaturation at 95°C for 30 Sec, annealing for 30 Sec at 67°C for uidA, 58°C for lt and 61°C for stand extension at 72°C for 30sec) and final extension at Mohammadzadeh et al.              Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples… NBM                                                                            146                                   Novelty in Biomedicine 2015, 3, 140-7 72°C for 5 min. Results In total, 140 E. coli isolates from patients with diarrhea and 110 E. coli isolates from patients without diarrhea were collected for this study. The mean age of the patients was 38 years and in controls was 34. All E. coli isolates first were confirmed with IMViC tests and then isolates were tested genotypically by amplification of uidA gene that all isolates were positive PCR results. Among 140 E. coli isolates from diarrheal stools 5 (3.6%) isolates were positive for justlt gene, 3 (2.1%) co-amplified for both lt/stand 1 (0.7%) was positive for just the st gene which were considered as ETEC. In the E. coli isolates from non-diarrheal control samples just one (0.9%) isolate was positive for both lt and st genes. The calculated p value was 0.046 that was confirmed a significant association of ETEC with diarrhea among the studied patients. The mean age of patients with ETEC was 26 years. Also, all stool samples with ETEC isolates were negative for WBC and RBC. Discussion ETEC pathotypes are a major cause of diarrheal disease in childhood in developing countries and a frequent cause of traveler’s diarrhea among visitors to these countries 16. About 20-60% of the peoples who travel to countries with poor sanitations, will suffer from travelers’ diarrhea, Which ETECis the most common agent 2,17. The diarrhea caused by two heat-labile (LT) and the heat-stable (ST)  enterotoxins. ETEC may contain only an LT, an ST, or both LT and ST. About 35% of ETECs express ST, 35% express ST and LT, and the remnants express only LT18. Like other diarrheagenic E. coli pathotypes, The ETEC did not identify by routine laboratory culture methods, thus molecular methods such PCR are necessary to distingush these pathotypes. Many studies use the PCR for detecting ETEC from diarrheal samples by amplifying lt and st genes. In a recent study, we detect ETEC in 6.4% of patients with diarrhea and 0.9% of control cases. Also lt present only in 5 (55.6%) isolates, both lt and st in 3 (33.3%), and st only in one (11.1%) isolate. Aslani et al used lt and st gene to detect ETEC from 193 E. coli isolates from diarrheal samples which were collected from different urban and rural area of Iranian 2005. They showed that 14 (7.2%) of E. coli isolates were ETEC. The lt and st distribution in their ETEC isolates was 7 (50%) isolate only st, 6 (42.9%) only lt and one (7.1%) both st and lt19.  Alikhani et al. in 2009 detect ETEC in 3.7% of 187 adult and adolescent patients with diarrhea in western Iran, using PCR for lt and st gene. They explain that the 5 (71.4%) isolates produced ST, 1 (14.3 %) produced LT, and 1 (14.3%) of the isolates produced both ST and LT, respectively20. In another study in 2011 in Turkey Iseri et al reported ETEC in 4.9% of 245 patients with diarrhea by using passive latex agglutination and enzyme immunoassay methods. Of these, 5 (41.7 %) were ST and 7 (58.3%) were LT21. Pourakbari et al in a study on children with and without diarrhea in an Iranian referral pediatric center reported ETEC in 26% of cases and 10% of controls22. The ETEC was reported by Oscar et al. among Escherichia coli isolates associated with childhood diarrhea in Colombia. A case-control study was conducted on 815 samples from children younger than Table 1: Base sequences and product sizes of the oligonucleotide primers to amplify specific, fragments of the uidA, lt and st genes.  Primer Sequence (5'–3') Product size bp Reference  uidA 5-GCGTCTGTTGACTGGCAGGTGGTGG-3 5- GTTGCCCGCTTCGAAACCAATGCCT-3 503 bp 15 lt 5-GCACACGGAGCTCCTCAGTC-3 5-TCCTTCATCCTTTCAATGGCTTT-3 218 bp 15 st 5-GCTAAACCAGTAGAGCTCTTCAAAA-3 5-CCCGGTACAGAGCAGGATTACAACA-3 149 bp 15  Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples…             Mohammadzadeh et al. NBM 147 Novelty in Biomedicine 2015, 3, 144-7 five years of age. The ETEC was detected in 17 (4.87%) in cases and 8 (2.29%) in controls. Based on the presence of enterotoxins, 11 (44%) ETEC isolates were LT positives, 10 (40%) were ST positive and only 4 (16%), were LT/ST positive23. Because of differences in time, place and the study conditions seem to be reasonable for different reported results in reviewing studies. The reviewed results showed that the ETEC can be seen as a significant cause of diarrhea cases, which usually ignored by microbiology laboratories using traditional culture methods. Sometimes the ETEC causes severe watery diarrhea that can threaten the patient's life, thus a rapid diagnostic test such as PCR can be very helpful in the treatment process. Conclusion Sometimes the ETEC causes severe watery diarrhea that can threaten the patient's life, thus a rapid diagnostic test such as PCR can be very helpful in the treatment process. Acknowledgements Special thanks to Research Vice-Chancellor of Shahid Beheshti University of Medical sciences, school of medicine, Tehran, Iran for supporting this study. References 1. Nataro J P, andKaper J B. Diarrheagenic Escherichia coli .Clin Microbiol  Rev 1998; 11: 142–201. 2. Hill D R, Beeching N J. Travelers’ diarrhea. Curr Op Infect Dis 2010; 23: 481-487. 3. Wiedermann U, Kollaritsch H. Vaccines against traveler’s diarrhea and rotavirus disease – a review. Wein Klin Wochenscher. 2006;118:2-8. 4. Qadri  F, SvennerholmA M, Faruque AS, and Sack B. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin. Microbiol. Rev. 2005;18:465–83. 5. Croxen  MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. 2010;8:26-38. 6.  Wolf MK. Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin. Microbiol. Rev. 1997;10:569–84. 7. Sahl JW, Sistrunk JR, Fraser CM,  Hine E, Baby N,  Begum Y, et al. Examination of the EnterotoxigenicEscherichia coli Population Structure during Human Infection. M Bio. 2015;6(3):1-6. 8. Johnson TJ, Nolan LK. Pathogenomics of the virulence plasmids of Escherichia coli. Microbiol Mol Biol Rev. 2009;73:750-74. 9. Frost L, Leplae R, Summers O, Toussaint A. Mobile genetic elements: the agents of open source evolution. Nat. Rev. Microbiol. 2005;3:722–32. 10. Beddoe T, Paton AW, Le Nours J, Rossjohn J, Paton JC. Structure, biological functions of the AB5 toxins. Cell. 2010;35:411-8. 11. Tobias J, Vutukuru S R. Simple and rapid multiplex PCR for identification of the main human diarrheagenic Escherichia coli. Microbiological Research. 2012;167:564-70. 12. Turner S M, Scott-Tucker A, Cooper L M, Henderson R. Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 2006;263:10–20. 13. Shepard M, Danzeisen L, Isaacson E, Seemann T,  Achtman M, Johnsona J. Genome Sequences and Phylogenetic Analysis of K88- and F18-Positive Porcine Enterotoxigenic Escherichia coli. Journal of Bacteriology. 2012;395-405. 14. Honda T, Akhtar Q, Glass RI, Kibriya AK. A simple assay to detect Escherichia coli producing heat labile enterotoxin: results of a field study of the Biken tests in Bangladesh. Lancet .1981; ii:609-10. 15. Oscar G, Gomez D, Octavio A, Delfina U, Jing B, Julio G, et al. Detection of  Escherichia coli Enteropathogens by Multiplex Polymerase Chain Reaction from Children’s Diarrheal Stools in Two Caribbean–Colombian Cities. Foodborn pathogens and diseases. 2010; 7: N2. 16.  Nassif X, Pujol C, Morand P, and Eugene E. Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle? Mol. Microbiol .1999;32:1124–3112. 17. Wiedermann U, Kollaritsch H. Vaccines against traveler’s diarrhea and rotavirus disease – a review.Wein Klin Wochenscher. 2006;118:2-8. 18. Gaastra W, and Svennerholm A M. Colonization factors of human enterotoxigenicEscherichia coli (ETEC). Trends Microbiol 1996;4:444–52. 19.  Aslani M, Ahrabi S, Alikhani M, Jafari F, Zali R, Mani M. Molecular detection and antimicrobial resistance of diarrheagenic Escherichia coli strains isolated from diarrheal cases. Saudi med J. 2008;29(3):388-92. 20. Alikhani Y, Hashemi H, Aslani M, Farajnia S. Prevalence and antibiotic resistance patterns of diarrheagenic Escherichia coli isolated from adolescents and adults in Hamedan, Western Iran. Iran. J. Microbiol. 2013;5(1):42-7. 21. Iseri L, Apan Z, Aksoy A, Koc F, Gocmen S, Nuristani D. 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Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples referred to educational hospitals in Tehran-Iran

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Distribution of Enterotoxigenic Escherichia coli among E. coli isolates from diarrheal samples referred to educational hospitals in Tehran-Iran

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