Characterization of Shiga-toxin producing E.coli (STEC) and enteropathogenic E.coli (EPEC) using multiplex Real-Time PCR assays for stx1 , stx2 , eaeA
-
Pejman Abbasi
,
Mohammad Kargar
,
Abbas Doosti
,
Jalal Mardaneh
,
Sadegh Ghorbani-Dalini
,
Mohammad Ali Dehyadegari
Abstract
Background and Objective: Diarrheal disease is still a major health problem, especially in developing countries, where it is considered as one of the leading causes of morbidity and mortality especially in children. Studies showed that Diarrheagenic E. coli (DEC) such as STES and EPEC strains are among the most prevalent causative agents in acute diarrhea, particularly in children. Aim of the present study was to investigate the presence and the frequency of STEC and EPEC as etiologic agent of diarrhea in children less than 2 years of age with diarrhea in Shiraz.
Materials and Methods: A total of 285 stool samples were collected from patients with diarrhea in Shiraz, in 2012. Diarrheagenic E. coli (DEC) strains were isolated by standard biochemical analysis. In this study, we used multiplex Real time PCR and single PCR to detect the presence of indicator genes stx1 , stx2 and eaeA for STEC and EPEC strains, respectively.
Results: A total of 285 stool samples were tested in which 49 (17%) were identified as contaminated with E. coli by biochemical tests. Out of total samples, 15 STEC (31%) and 13 EPEC (27%) were identified using multiplex Real-Time PCR assay. Among STEC isolates, 2 strains were stx1 (+), 8 isolates stx2 (+), 3 isolates were stx1 (+) , stx2 (+) and 2 isolates were stx1 (+) , stx2 (+), eaeA (+).
Conclusion: In this study, we found rather high occurrence of STEC and EPEC virulence genes in children with diarrhea. The results of this study showed that, real time PCR can be used as a replacement for conventional PCR assay in the detecting virulence genes of STEC and EPEC strains. Real-time PCR offers the advantage of being a faster, more robust assay, because it does not require post-PCR procedures to detect amplification products.
Asadi Karam M, Bouzari S, Oloomi M, Aslani M, Jafari A. Phenotypic and genotypic characterization of Enteropathogenic Escherichia coli (EPEC) strains in Tehran, Iran. Iran J Microbiol 2010; 2: 3-7.
Moyo SJ, Maselle SY, Matee MI, Langeland N,1. Mylvaganam H. Identification of diarrheagenic Escherichia coli isolated from infants and children in Dar el Salaam, Tanzania. BMC Infect Dis 2007; 7: 1-7.
Blanco M, Schumacher S, Tasara T, Zweifel C, Blanco 2. JE, Dahbi G, et al. Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae- η2). BMC Microbiol 2005; 5: 1-11.
Lai Y, Rosenshine I, Leong JM, Frankel G. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli. Cell Microbiol 2013; 15:1796-808.
Jafari A, Aslani MM, Bouzari S. Escherichia coli: a brief review of diarrheagenic pathotypes and their role in diarrheal diseases in Iran. Iran J Microbiol 2012;4:102-117.
Kaper JB, Nataro JP, Molby HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004; 2: 123-140.
Blanco M, Blanco JE, Dahbi G, Alonso MP, Mora 4.A, Coira MA, et al. Identification of two new intimin types in atypical enteropathogenic Escherichia coli. Int Microbiol 2006; 9: 103-110.
Lozer DM, Souza TB, Monfardini MV, Vicentini F, Kitagawa SS, Scaletsky IC, Spano LC. Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities. BMC Infect Dis 2013; 13: 418.
Souza MR, Klassen G, Toni FD, Rigo LU, Henkes C, Pigatto CP, et al. Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains. Mem Inst Oswaldo Cruz 2010; 105: 318-321.
Donnenberg MS, Whittam TS. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J Clin Invest 2001; 107: 539-548.
Preußel K, Höhle M, Stark K, Werber D. Shiga Toxin- producing Escherichia coli O157 is more likely to lead to hospitalization and death than non-O157 serogroups - except O104. PLoS One 2013; 8: e78180.
Tesh VL. Virulence of enterohemorrhagic Escherichia coli: role of molecular crosstalk. Trends Microbiol 1998; 6: 228-233.
Vidal M, Kruger E, Durán C, Lagos R, Levine M, Prado V, et al. Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections. J Clin Microbiol 2005; 43: 5362-5365.
Boerlin P, McEwen SA, Boerlin-Petzold F, Wilson JB, Johnson RP, Gyles CL. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J Clin Microbiol 1999; 37: 497-503.
Beutin L, Krause G, Zimmermann S, Kaulfuss S, Gleier K. Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period. J Clin Microbiol 2004;42: 1099-108.
Kaper JB, Nataro JP, Molby HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004; 2: 123-140.
Bueris V, Sircili MP, Taddei CR, dos Santos MF, Franzolin MR, Martinez MB, et al. LR. Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil. Mem Inst Oswaldo Cruz 2007; 102: 839-844.
Alikhani MY, Hashemi SH, Aslani MM, 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: 42-47.
Hartland EL, Leong JM. Enteropathogenic and enterohemorrhagic E. coli: ecology, pathogenesis, and evolution. Front Cell Infect Microbiol 2013; 3:15.
Pourakbari B, Heydari H, Mahmoudi S, Sabouni F, Teymuri M, Ferdosian F, et al. Diarrhoeagenic E. coli pathotypes in children with and without diarrhoea in an Iranian referral pediatrics centre. EMHJ 2013; 19: 7.
Jafari F, Garcia-Gil LJ, Salmanzadeh-Ahrabi S, Shokrzadeh L, Aslani MM, Pourhoseingholi MA, et al. Diagnosis and prevalence of enteropathogenic bacteria in children less than 5 years of age with acute diarrhea in Tehran childrens hospitals. J infect 2009; 58: 21-27.
Alikhani MY, Mirsalehian A, Aslani MM. Detection of 20 Typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhoea. J Med Microbiol 2006; 55: 1159-1163.
López-Saucedo C, Cerna JF, Villegas-Sepulveda N, Thompson R, Velazquez FR, Torres J, et al. Single multiplex polymerase chain reaction to detect diverse loci associated with diarrheagenic Escherichia coli. Emerg Infect Dis 2003; 9: 127-131.
Wang G, Clark CG, Rodgers FG. Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157: H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J Clin Microbiol 2002; 40:3613-3619.
Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerg Infect Dis 2005; 11: 603-609.
Zumbrun SD, Melton-Celsa AR, Smith MA, Gilbreath JJ, Merrell DS, O’Brien AD. Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157: H7 colonization and disease. Proc Natl Acad Sci U S A2013;110: E2126-33.
Scallan E, Griffin PM, Angulo FJ, Tauxe RV, Hoekstra RM. Foodborne illness acquired in the United States-- unspecified agents. Emerg Infect Dis 2011; 17: 16-22.
Salmanzadeh-Ahrabi S, Habibi E, Jafari F, Zali MR.Molecular epidemiology of Escherichia coli diarrhoea in children in Tehran. Annals Tropic Paediatrics 2005;25: 35-39.
Presterl E, Zwick RH, Reichmann S, Aichelburg A, Winkler S, Kremsner PG, et al. Frequency and virulence properties of diarrheagenic Escherichia coli in children with diarrhea in Gabon. Am J Trop Med Hyg 2003; 69: 406-10.
Nweze EI. Aetiology of diarrhoea and virulence properties of diarrhoeagenic Escherichia coli among patients and healthy subjects in southeast Nigeria. J Health Popul Nutr 2010; 28: 245-252.
Sharifi Yazdi MK, Akbari A, Soltan Dallal MM.Multiplex polymerase chain reaction (PCR) assay for simultaneous detection of shiga-like toxin (stx and stx ), intimin (eae) and invasive plasmid antigen H(ipaH) genes in diarrheagenic Escherichia coli. African Biotechnal 2011; 10: 1522-1526.
Chen HD, Frankel G. Enteropathogenic Escherichia coli: unravelling pathogenesis. FEMS Microbiol Rev 2005; 29: 83-98.
Ishii S, Meyer KP, Sadowsky MJ. Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources. App Environ Microbiol 2007; 73: 5703-5710.
Echeverria P, Orskov F, Orskov I, Knutton S, Scheutz F, Brown JE. Attaching and effacing Enteropathogenic Escherichia coli as a cause of infantile diarrhea in Bangkok. J Infect Dis 1991; 164: 550-554.
Gomez TA, Rassi V, Macdonald KL, Ramos SR, 30.Trabulsi LR, Vieira MA. Enteropathogens associated with acute diarrheal in urban infants in Sao Paulo, Brazil. J Infect Dis 1991; 164: 331-337.
Pabst WL, Altwegg M, Kind C, Mirjanic S, Hardegger D, Nadal D. Prevalence of enteroaggregative Escherichia coli among children with and without diarrhea in Switzerland. J Clin Microbiol 2003; 41: 2289-2293.
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| Issue | Vol 6 No 3 (2014) | |
| Section | Articles | |
| Keywords | ||
| Children EPEC Multiplex Real-Time PCR STEC Shiraz | ||
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