Molecular detection of Enterotoxigenic Escherichia coli Toxins and Colonization Factors from Diarrheic Children in Pediatric Teaching Hospital, Sulaymaniyah, Iraq


  • Hezhan Faeq Rasul Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq
  • Sirwan Muhsin Muhammed Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq
  • Hunar Hiwa Arif Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq
  • Paywast Jamal Jalal Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq



Escherichia coli, Enterotoxigenic E. coli, Stable toxin, Labile toxin


Enterotoxigenic Escherichia coli (ETEC) is one well-established causative agent of diarrhea in the developing countries among young children. This prospective study was performed at Laboratories of University of Sulaimani (in Sulaymaniyah City/Iraq) from September to October 2021which aimed to determine the prevalence of ETEC among children and the most prevalence colonization factor (CFA/I) among ETEC. One hundred and twenty-five fresh stool samples were collected from hospitalized – children with diarrhea at Dr. Jamal Ahmed Rashid’s Pediatric Teaching Hospital. The collected samples were cultured on MacConkey and eosin methylene blue agar as selective and differential media for Gram- negative bacteria. Colonies were identified through Gram staining and biochemical tests including: Indole, methyl red, and catalase reaction test. Vitek-2 machine was depended to test some obtained isolates. Most of isolates (60%) showed positive results for E. coli – out of this percentage, 14 (18.66%) were positive for ETEC using polymerase chain reaction assay identifying stable and labile toxins (LTs). It was noticed that all of the ETEC isolates were stable toxin producer isolates whereas LT producer isolates were not identified. Colonization factor 5 (CS5) has been detected among three ETEC isolates (21.42%), meanwhile, 11 isolates (78.57%) have not expressed colonization factors at all.

Author Biographies

Sirwan Muhsin Muhammed, Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq

Asstantan professor at Universty of Sulaimani , College of Science , Department of Biology 

Head of biology department 

Hunar Hiwa Arif, Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq

Lecturer of Universty of Sulaimani , College of Science , Biology Department 


Paywast Jamal Jalal, Department of Biology, College of Science, University of Sulaymaniyah, Sulaymaniyah, Iraq

Lecturer At Universty of Sulaimani , College Science , Biology Department 


A. D. Mare, C.N. Ciurea, A. Man, B. Tudor, V. Moldovan, L. Decean, et al. Enteropathogenic Escherichia coli-A summary of the literature. Gastroenterology Insights, vol. 12, pp. 28-40, 2021.

S. Ramos, V. Silva, M. L. E. Dapkevicius, M. Caniça, M. T. Tejedor-Junco, Igrejas G, et al., Escherichia coli as commensal and pathogenic bacteria among food-producing animals: Health implications of extended spectrum β-lactamase (ESBL) production. Animals (Basel), vol. 10, p. 2239, 2020.

J. P. Nataro and J. B. Kaper. Diarrheagenic Escherichia coli. Clinical Microbiology Reviews, vol. 11, pp. 142-201, 1998.

G. D. Christensen, W A Simpson, J. J. Younger, L. M Baddour, F. F Barrett, D. M. Melton, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology, vol. 22, pp. 996-1006, 1985.

Y. Zhou, X. Zhu, H. Hou, Y. Lu, L. Yu, L. Mao, et al. Characteristics of diarrheagenic Escherichia coli among children under 5 years of age with acute diarrhea: A hospital based study. BMC Infectious Diseases, vol. 18, p. 63, 2018.

S. M. Turner, A. Scott-Tucker, L. M. Cooper and I. R. Henderson. Weapons of mass destruction: Virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiology Letters, vol. 263, pp. 10-20, 2006.

J. M. Fleckenstein, K. Roy, J. F. Fischer and M. J. I. Burkitt. Identification of a two-partner secretion locus of enterotoxigenic Escherichia coli. Infection and Immunity, vol. 74, pp. 2245-2258, 2006.

C. K. Porter, M. S. Riddle, D.R. Tribble, A. Louis Bougeois, R. McKenzie, S. D. Isidean, et al., A systematic review of experimental infections with enterotoxigenic Escherichia coli (ETEC). Vaccine, vol. 29, pp. 5869-5885, 2011.

F. Qadri, A. M. Svennerholm, A. S. G. Faruque and R. B. Sack. Enterotoxigenic Escherichia coli in developing countries: Epidemiology, microbiology, clinical features, treatment, and prevention. Clinical Microbiology Reviews, vol. 18, pp. 465-483, 2005.

T. P. Madhavan and H. Sakellaris. Colonization factors of enterotoxigenic Escherichia coli. Advances in Applied Microbiology, vol. 90, pp. 155-97, 2015.

Y. Zhang, P. Tan, Y. Zhao and X. Ma. Enterotoxigenic Escherichia coli: Intestinal pathogenesis mechanisms and colonization resistance by gut microbiota. Gut Microbiota, vol. 14, p. 2055943, 2022.

I. Bölin, G. Wiklund, F. Qadri, O. Torres, A. L. Bourgeois, S. Savarino, et al. Enterotoxigenic Escherichia coli with STh and STp genotypes is associated with diarrhea both in children in areas of endemicity and in travelers. Journal of Clinical Microbiology, vol. 44, pp. 3872-3877, 2006.

M. A. Lasaro, J. F. Rodrigues, C. Mathias-Santos, B. E. C. Guth, A. Balan, M. E. Sbrogio-Almeida, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. Journal of Bacteriology, vol. 190, pp. 2400- 10, Apr 2008.

A. Sjoling, G. Wiklund, S. Savarino, D. Cohen and A. M. Svennerholm. Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. Journal of Clinical Microbiology, vol. 45, pp. 3295-3301, 2007.

C. M. Müller, A. Aberg, J. Straseviçiene, L. Emody, B. E. Uhlin and C. Balsalobre. Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP. PLoS Pathogens, vol. 5, p. e1000303, 2009.

O. Puiprom, S. Chantaroj, W. Gangnonngiw, K. Okada, T. Honda, T. Taniguchi, et al. Identification of colonization factors of enterotoxigenic Escherichia coli with PCR-based technique. Epidemiology and Infection, vol. 138, pp. 519-524, 2010.

T. Dadheech, R. Vyas and V. Rastogi. Prevalence, bacteriology, pathogenesis and isolation of E. coli in sick layer chickens in Ajmer region of Rajasthan, India. International Journal of Current Microbiology and Applied Sciences, vol. 5, pp. 129-136, 2016.

G. Y. Lee, H. I. Jang, I. G. Hwang and M. S. Rhee. Prevalence and classification of pathogenic Escherichia coli isolated from fresh beef, poultry, and pork in Korea. International Journal of Food Microbiology, vol. 134, pp. 196-200, 2009.

R. A. Pollack, L. Findlay, W. Mondschein and R. R. Modesto. Laboratory Exercises in Microbiology. John Wiley and Sons, Hoboken, 2018.

M. P. MacWilliams. Indole Test Protocol. American Society for Microbiology, Washington, D.C, 2012.

S. McDevitt. Methyl Red and Voges-Proskauer Test Protocols. vol. 8, American Society for Microbiology, Washington, D.C, 2009.

K. Reiner. Catalse Test Protocol. American Society For Microbiology, Washington, D.C, 2014.

M. Ligozzi, C. Bernini, M. G. Bonora, M. De Fatima, J. Zuliani and R. Fontana. Evaluation of the VITEK 2 system for identification and antimicrobial susceptibility testing of medically relevant gram-positive cocci. Journal of clinical microbiology, vol. 40, pp. 1681- 1686, 2002.

I. Espinosa, M. Báez, M. I. Percedo and S. Martínez. Evaluation of simplified DNA extraction methods for Streptococcus suis typing. Revista de Salud Animal, vol. 35, pp. 59-63, 2013.

L. Lin, B. D. Ling and X. Z. Li. Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-resistant clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex. International Journal of Antimicrobial Agents, vol. 33, pp. 27-32, 2009.

M. Yavzori, N. Porath, O. Ochana, R. Dagan, R. Orni-Wasserlauf and D. Cohen. Detection of enterotoxigenic Escherichia coli in stool specimens by polymerase chain reaction. Diagnostic Microbiology and Infectious Disease, vol. 31, pp. 503-509, 1998.

C. Rodas, V. Iniguez, F. Qadri, G. Wiklund, A. M. Svennerholm and A. Sjöling. Development of multiplex PCR assays for detection of enterotoxigenic Escherichia coli colonization factors and toxins. Journal of Clinical Microbiology, vol. 47, pp. 1218-1220, 2009.

S. C. Pawar, A, T. A. P. Devi, C. Setti, R. Gajula, S. Srikanth, S. Kalyan. Molecular evolution of pathogenic bacteria based on rrsA gene. Journal of Biotechnology and Biomaterials, vol.2, pp.12-18, 2018.

R. Mulchandani, F. Massebo, F. Bocho, C. L. Jeffries, T. Walker and L. A. Messenger. A community-level investigation following a yellow fever virus outbreak in South Omo Zone, South-West Ethiopia. Peer Journal, vol. 7, p. e6466, 2019.

H. K. Hasan, N. A. Yassin and S. H. Eassa. Bacteriological and molecular characterization of diarrheagenic Escherichia coli pathotypes from children in Duhok City, Iraq. Science Journal of University of Zakho, vol. 8, pp. 52-57, 2020.

T. W. Shatub, N. A. H. Jafar and A. K. Krekor Melconian. Detection of diarrheagenic E.coli among children under 5’s age in Tikrit city of Iraq by using single multiplex PCR technique. Plant Archives, vol. 21, pp.1230-1237.16-118, 2021.

Z. K. Khalil. Isolation and identification of different diarrheagenic (DEC) Escherichia coli pathotypes from children under five years old in Baghdad. Iraqi Journal of Medical Sciences, vol. 28, pp. 126- 132, 2015.

Z. K. Abdul-hussein, R. H. Raheema and A. I. Inssaf. Microbiology. Molecular diagnosis of diarrheagenic E. coli infections among the pediatric patients in Wasit Province, Iraq. Journal of Pure and Applied Microbiology, vol. 12, pp. 2229-2241, 2018.

E. Abbasi, M. Mondanizadeh, A. van Belkum and E. J. I. Ghaznavi- Rad. Multi-drug-resistant diarrheagenic Escherichia coli pathotypes in pediatric patients with gastroenteritis from central Iran. Infection and Drug Resistance, vol. 13, p. 1387, 2020.

A. Emami, N. Pirbonyeh, F. Javanmardi, A. Bazargani, A.Moattari, A. Keshavarzi, et al. Molecular diversity survey on diarrheagenic Escherichia coli isolates among children with gastroenteritis in Fars, Iran. Future Microbiology, vol. 16, pp. 1309-1318, 2021.

H. K. Saka, N. T. Dabo, B. Muhammad, S. García-Soto, M. Ugarte- Ruiz and J. Alvarez. Diarrheagenic Escherichia coli pathotypes from children younger than 5 years in Kano State, Nigeria. Frontiers in Public Health, vol. 7, p. 348, 2019.

G. Shahbazi, M. A. Rezaee, F. Nikkhahi, S. Ebrahimzadeh and F. Hemmati. Characteristics of diarrheagenic Escherichia coli pathotypes among children under the age of 10 years with acute diarrhea. Asian J Med Sci, vol. 25, p. 101318, 2021.

G. Ochien and L. Atieno. Prevalence of Enterotoxigenic Escherichia coli Among Children Under Five Years in Siaya County, Western Kenya. Maseno University, Kenya, 2021.

O. Lukjancenko, T. M. Wassenaar and D. W. Ussery. Comparison of 61 sequenced Escherichia coli Genomes. Microbial Ecology, vol. 60, pp. 708-720, 2010.

S. K. Arif and L. I. F. Salih. Identification of different categories of diarrheagenic Escherichia coli in stool samples by using multiplex PCR technique. Asian J Med Sci, vol. 2, pp. 237-243, 2010.

C. I. C. Ifeanyi, N. F. Ikeneche, B. E. Bassey, N. Al-Gallas, A. A. Casmir and I. R. Nnennaya. Characterization of toxins and colonization factors of enterotoxigenic Escherichia coli isolates from children with acute Diarrhea in Abuja, Nigeria. Jundishapur Journal Of Microbiology, vol. 11, p. e64269, 2018.

S. Eybpoosh, S. Mostaan, M. M. Gouya, H. Masoumi-Asl, P. Owlia, B. Eshrati, et al. Frequency of five Escherichia coli pathotypes in Iranian adults and children with acute diarrhea. PLoS One, vol. 16, p. e0245470, 2021.

S. Zheng, F. Yu, X. Chen, D. Cui, Y. Cheng, G. Xie, et al. Enteropathogens in children less than 5 years of age with acute diarrhea: A 5-year surveillance study in the Southeast Coast of China. BMC infectious Diseases, vol. 16, pp. 434-434, 2016.

E. Kipkirui, M. Koech, A. Ombogo, R. Kirera, J. Ndonye, N. Kipkemoi, et al. Molecular characterization of enterotoxigenic Escherichia coli toxins and colonization factors in children under five years with acute diarrhea attending Kisii Teaching and Referral Hospital, Kenya. Tropical Diseases Travel Medicine and Vaccines, vol. 7, pp. 1-7, 2021.

A. M. Svennerholm. From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development. Indian Journal of Medical Research, vol. 133, p. 188, 2011.

S. Nazarian, S. L. M. Gargari, I. Rasooli, M. Alerasol, S. Bagheri and S. D. Alipoor. Prevalent phenotypic and genotypic profile of enterotoxigenic Escherichia coli among Iranian children. Japanese Journal of Infectious Diseases, vol. 67, pp. 78-85, 2014.

H. Zeighami, F. Haghi, F. Hajiahmadi, M. Kashefiyeh and M. Memariani. Multi-drug-resistant enterotoxigenic and enterohemorrhagic Escherichia coli isolated from children with diarrhea. Journal of Chemotherapy, vol. 27, pp. 152-155, 2015.

S. Gupta, J. Keck, P. K. Ram, J. A. Crump, M. A. Miller and E. D. Mintz. Part III. Analysis of data gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human development index countries, 1984-2005. Epidemiology and Infection, vol. 136, pp. 721-738, 2008.

H. Alizade, R. Ghanbarpour and M. R. Aflatoonian. Molecular study on diarrheagenic Escherichia coli pathotypes isolated from under 5 years old children in southeast of Iran. Asian Pacific Journal of Tropical Disease, vol. 4, pp. S813-S817, 2014.

E. Joffré, A. von Mentzer, A. M. Svennerholm and Å. Sjöling. Identification of new heat-stable (STa) enterotoxin allele variants produced by human enterotoxigenic Escherichia coli (ETEC). International Journal of Medical Microbiology, vol. 306, pp. 586- 594, 2016.

J. M. Fleckenstein, P. R. Hardwidge, G. P. Munson, D. A. Rasko, H. Sommerfelt and H. Steinsland. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes and Infection, vol. 12, pp. 89-98, 2010.

O. Torres, W. González, O. Lemus, R. A. Pratdesaba, J. A. Matute, G. Wiklund, et al. Toxins and virulence factors of enterotoxigenic Escherichia coli associated with strains isolated from indigenous children and international visitors to a rural community in Guatemala. Epidemiology and Infection, vol. 143, pp. 1662-1671, 2015.

S. X. Zhou, L. P. Wang, M. Y. Liu, H. Y. Zhang, Q. B. Lu, L. S. Shi, et al. Characteristics of diarrheagenic Escherichia coli among patients with acute diarrhea in China, 2009-2018. Journal of Infection, vol. 83, pp. 424-432, 2021.

M. Paredes-Paredes, P. C. Okhuysen, J. Flores, J. A Mohamed, R. S Padda, A. Gonzalez-Estrada, et al. Seasonality of diarrheagenic Escherichia coli pathotypes in the US students acquiring diarrhea in Mexico. Journal of Travel Medicine, vol. 18, pp. 121-125, 2011.

L. F. Peruski Jr., B. A. Kay, R. A. El-Yazeed, S. H. El-Etr, A. Cravioto, T. F. Wierzba, et al. Phenotypic diversity of enterotoxigenic Escherichia coli strains from a community-based study of pediatric diarrhea in periurban Egypt. Journal of Clinical Microbiology, vol. 37, pp. 2974-2978, 1999.

H. I. Shaheen, S. B. Khalil, M. R. Rao, R. A. Elyazeed, T. F. Wierzba, L. F. Peruski Jr, et al. Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt. Journal of Clinical Microbiology, vol. 42, pp. 5588-5595, 2004.

D. G. Evans, D. J. Evans Jr and W. J. I. Tjoa. Hemagglutination of human group A erythrocytes by enterotoxigenic Escherichia coli isolated from adults with diarrhea: Correlation with colonization factor. Infection and Immunity, vol. 18, pp. 330-337, 1977.

M. G. Jobling and R. K. Holmes. Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encode. PLoS One. vol. 7, p. e29898, 2012.

F. Qadri, S. K. Das, A. S. Faruque, G. J. Fuchs, M. J. Albert, R. B. Sack, et al. Prevalence of toxin types and colonization factors in enterotoxigenic Escherichia coli isolated during a 2-year period from diarrheal patients in Banglades. Journal of Clinical Microbiology, vol. 38, pp. 27-31, 2000.

B. Juma, P. Waiyaki, W. Bulimo, E. Wurapa, M. Mutugi and S. Kariuki. Molecular detection of Enterotoxigenic Escherichia coli surface antigens from patients in Machakos District Hospital, Kenya. ECAMJ, vol. 1, p. 62-68, 2014.

M. R. C. Nunes, F. Penna, R. Franco, E. Mendes and P. Magalhaes. Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes. Journal of Applied Micorbiology, vol. 111, pp. 224-232, 2011.