|Year : 2011 | Volume
| Issue : 4 | Page : 383-388
Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq
Samer A Al Hilali, Ali M Almohana
Department of Microbiology, College of Medicine, Kufa University. Iraq Najaf Kufa p.o. Box(18), Iraq
|Date of Submission||05-Jun-2011|
|Date of Acceptance||16-Oct-2011|
|Date of Web Publication||24-Nov-2011|
Samer A Al Hilali
Department of Microbiology, College of Medicine, Kufa University. Iraq Najaf Kufa p.o. Box(18)
Source of Support: None, Conflict of Interest: None
Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx1 and stx2 ), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx 1, stx 2, and hlyA genes. Typical EPEC (eaeA +, bfpA +) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.
Keywords: Atypical EPEC, bundle-forming pilus, enteropathogenic Escherichia coli, intimin, typical Enteropathogenic Escherichia coli
|How to cite this article:|
Al Hilali SA, Almohana AM. Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq. Indian J Med Microbiol 2011;29:383-8
|How to cite this URL:|
Al Hilali SA, Almohana AM. Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq. Indian J Med Microbiol [serial online] 2011 [cited 2019 Sep 23];29:383-8. Available from: http://www.ijmm.org/text.asp?2011/29/4/383/90171
| ~ Introduction|| |
Diarrhoeal disease continues to be a health problem worldwide. The causes of diarrhoea include a wide range of viruses, bacteria and parasites. Among the bacterial pathogens, E. coli plays an important role. EPEC is an important category of diarrhoeagenic E. coli which has been linked to infant diarrhoea in the developing world. As with other diarrhoeagenic E. coli strains, transmission of EPEC is faecal-oral, with contaminated hands, contaminated foods or contaminated fomites serving as vehicles. EPEC adhere to the mucosal cells of the small bowel. The result of EPEC infection is watery diarrhoea, which is usually self-limited, but can be chronic.  EPEC strains are noninvasive, infecting their hosts by attaching to intestinal epithelial cells (IEC), effacing the epithelial microvilli and producing pedestal-like structures. The formation of attaching and effacing (A/E) lesions is required for these microbes to cause diarrhoeal disease.  EPEC possess both the bfpA and eae for E. coli-attaching and effacing. It is a well-recognized pathogen in developing countries as class I EPEC or typical EPEC. However, atypical EPEC organisms possessing eae alone have been reported to be more prevalent in both developing and developed countries, and animals can be reservoirs of atypical EPEC, in contrast to typical EPEC, in which humans are the sole reservoir.  In Iraq, till now, little information was available on the prevalence of EPEC infection. However, in Najaf, there is no molecular identification and categorization of EPEC associated with diarrhoea in children younger than 2 years old. The study is aimed to determine the occurrence of EPEC serotypes in children with diarrhoea in Najaf and detecting their virulence properties.
| ~ Materials and Methods|| |
Patients and stool collection
This study was conducted in Najaf. Two hospitals (Alzahraa Teaching Hospital and Alhakeem Teaching Hospital) were included in this study. A total of 656 stool specimens were collected from children younger than 2 years old suffering from watery diarrhoea characterized in Nguyen et al.,  and 54 apparently healthy control. The specimens collected during the period from September to November, 2009.
Isolation and identification of bacterial isolates
All stool specimens were cultured on MacConkey agar (Himedia M081; Himedia, Mumbai, India) and were incubated for 24 h at 37°C. Colonies morphologically resembling E. coli were identified by standard laboratory techniques. 
Escherichia More Details coli serotyping
The E. coli-positive cultures were set for further serological test. Isolates biochemically identified as E. coli were serologically examined by slide agglutination test (remel Escherichia coli Agglutinating Sera; Remel Europe Ltd., UK) according to kit procedures, using polyvalent O antisera for EPEC, (2, 3, and 4), separately. From the colony which showed a positive reaction with polyvalent antisera a subculture was prepared and the isolates were retested by using the following EPEC monovalent O: K antisera: (O26: K60(B6), O55: K59(B5), O111: K58(B4), O119: K69(B14), O126: K71(B16), O86: K61(B7), O114: K90(B), O125: K70(B15), O127: K63(B8), O128: K67(B12), O44: K74(L), O112: K66(B11), O124: K72(B17), O142: K86(B), O18c: K77(B21).
DNA isolation and polymerase chain reaction assay
Enteropathogenic E. coli DNA extraction was done according to the Pospiech and Neumann  method. DNA templates were subjected to PCR using six sets (F and R) of primers targeting virulence properties genes listed in [Table 1].
|Table 1: Virulence properties primers sequences and size of amplified products from selected genes |
Click here to view
The reaction mixture moreover contained GoTaq® Green Master Mix, X2 (Promega M 7122; Promega Corporation, USA), which is premixed ready-to-use solution containing Taq DNA polymerase dNTP, MgCl 2 and according to Promega procedure, the reaction mixtures were prepared in 0.2 ml eppendorf tube with 25 ml reaction volumes. The PCR were performed with PCR system (GeneAmp PCR system 9700; Applied Biosystem, Singapore) at 94°C for 2 min. For 1 cycle followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. For the eaeA gene, 94°C for 2 min for 1 cycle followed by 30 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min for the bfpA gene, 94°C for 5 min. For 1 cycle followed by 30 cycles of 94°C for 1 min, 57°C for 45 s and 72°C for 1 min for the EAF gene, 94°C for 5 min for 1 cycle followed by 35 cycles of 95°C for 40 s, 65°C for 20 s and 72°C for 45 s for stx1 and stx2 genes and 95°C for 5 min for 1 cycle followed by 35 cycles of 95°C for 1 min, 58°C for 1.30 min and 72°C for 1.30 min for the hlyA gene. The amplified PCR products were detected by agarose gel electrophoresis and visualized by staining with ethidium bromide using gel documentation system (BioDocAnalyze Live; Biometra biomedizinische Analytik GmbH, Germany).
The Chi-square test was used to determine the statistical significance of the data. A P value of <0.5 was considered significant.
| ~ Results|| |
It was observed that the number of E. coli isolates from 656 stool specimens were 535 (81.6%) (Basically, 1 E. coli isolate from each patient). A total of 52 (96.3%) E. coli isolates were obtained from 54 healthy individuals.
Of the total of E. coli isolates recovered from 656 children with diarrhoea, only 22 (3.4%) agglutinated with EPEC polyvalent antisera. By contrast, no EPEC isolate belonging to classical EPEC polyvalent serotypes was detected in stool of healthy individuals. However, the highest number of the EPEC isolated belonging to polyvalent 2 sero-group with following serotypes, O55: K59(B5) (22.7%), O111: K58(B4) (18.18%), O26: K60(B6) (13.63%), followed by polyvalent 3 sero-group with the following serotypes, O125: K70(B15) (9.09%), O128: K67(B12) (4.54%), O127: K63(B8) (4.54%), O114: K90(B) (4.45%), followed by polyvalent 4 sero-group with serotype O44: K74(L) (13.63%).
From the total isolates, 6 (27.3%) isolates were PCR-positive for at least one of the targeted virulent genes [Table 2]. Only two (9.1%) isolates gave positive results with the eaeA gene belonging to serotype O125: K70(B15) [Figure 1]. Six (27.3%) isolates gave positive result with bfp, of these, four isolates gave amplified product size equal to 326 bp, belonging to O125: K70(B15) (two isolates), O111: K58(B4) (two isolates), while, two isolates gave amplified product size equal to 200 bp, belonging to O44: K74(L) and O128: K67(B12) [Figure 2]. Only two isolates (9.1%) that belonged to serotype O125: K70(B115) was found to harbour the EAF plasmid gene [Figure 3]. Based on these criteria [Table 2], two isolates (33.3%) were classified as typical EPEC, these isolates belonged to O125: K70(B15) serotype, obtained from infants younger than 6 months. Four (66.7%) isolates were classified as atypical EPEC. The atypical EPEC isolates belonged to O111: K58(B4) (two isolates), O44: K74(L) (one isolate), O128: K67(B12) (one isolate) serotypes, obtained from children with diarrhoea aged from 3 months to 17 months.
|Table 2: Genotypic characterization of typical, atypical and non - EPEC serotypes |
Click here to view
|Figure 1: Ethidium bromide-stained agarose gel of PCR amplified products from extracted E. coli DNA amplified with primers eaeA F and eaeA R. Lane (L), DNA molecular size marker(100-bp ladder). Lane (E3), E. coli O125: K70(B15) show positive results with eaeA gene. Lane (E5), E. coli O125: K70(B15) show positive results with eaeA gene. Lanes (E1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19) EPEC isolates show negative results to eaeA gene Lane (C), negative control|
Click here to view
|Figure 2: Ethidium bromide-stained agarose gel of PCR amplified products from extracted E. coli DNA amplified with primers bfpA F and bfpA R. Lane (L), DNA molecular size marker(100-bp ladder). Lane (E1), E. coli O111: K58(B4)show positive results with bfpA gene 326bp. Lane (E3), E. coli O125: K70(B15) show positive results with bfpA gene326bp. Lane (E5), E. coli O125: K70(B15) show positive results with bfpA gene326bp. Lane (E6), E. coli O128: K67(B12) show positive results with bfpA gene 200bp. Lane (E7), E. coli O127: K63(B8) show positive results with bfpA gene 200bp. Lane (E13), E. coli O111: K58(B4) show positive results with bfpA gene 326bp. Lanes (E2, 4, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18 and 19) EPEC isolates show negative results with bfpA gene Lane (C), negative control|
Click here to view
|Figure 3: Ethidium bromide-stained agarose gel of PCR amplified products from extracted E. coli DNA amplified with primers EAF F and EAF R. Lane (L), DNA molecular size marker(100-bp ladder). Lane (E3), E. coli O125: K70(B15) show positive results withEAF gene. Lane (E5), E. coli O125: K70(B15) show positive results withEAF gene. Lanes (E1, 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19) EPEC isolates show negative results withEAF gene. Lane (C), negative control|
Click here to view
| ~ Discussion|| |
Enteropathogenic E. coli has been identified as important cause of infantile diarrhoea in all the developing countries where it has been looked for, but the incidence has varied greatly in different studies. The present study revealed that EPEC was higher than been demonstrated previously by Almohana  who reported that 37 (2.1%) E. coli isolates recovered from 1798 patients suffering from diarrhoea were assigned as EPEC. The relative frequency has been demonstrated in Tehran, Iran,  that EPEC represent (8.4%) of diarrhoeal causes. A high frequency of EPEC was observed in Korea (56%) and Brazil (34.0%).  In another study, EPEC serogroups were isolated as the sole pathogen from 44.9% of the Iranian children with diarrhoea. 
The WHO has considered isolates in the 12 O serogroups to be EPEC strains;  O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142 and O158. However, the serological investigation revealed 8 E. coli serogroups of the 12 E. coli serogroups described by WHO  as EPEC strains. We found that O55: K59(B5) was the most common EPEC isolates that represented 0.8% of cases of diarrhoea in Najaf. In agreement with this study, the O55 serogroup was the most prevalent (12.6%) in Iran.  While, in Romania the O55 serogroup represent 1.2% diarrhoeal causes in children. 
Although serogrouping proposed by Levine et al.  has been carried out to define the EPEC strains, it is now recognized that the serogrouping is not well correlated with the presence of pathogenic factors. Therefore, at present, the detection of pathogenic genes by PCR may be the best way to identify the diarrhoeagenic E. coil. 
Only 2 (9.1%) isolates belonging to serotype O125: K70(B15) gave positive results with eaeA gene. Since there is no data recorded for the presence of the eaeA gene in the EPEC isolates in Najaf, we cannot identify the difference in the incidence of this virulence factor. However, the isolation frequency of EPEC having the eaeA gene was not as high as expected in Najaf. The isolation frequency having this gene may vary tending on the district, season, child age and so on. Notably, a high incidence of EPEC defined on the basis of the eaeA gene was reported by Nguyen et al.  in Hanoi, Vietnam, who found that all tested EPEC isolates were eaeA PCR positive. In an Iranian study, the eaeA gene was detected in 40.5% of the 111 EPEC examined strains.  A lower incidence of 10.9% has been reported in South Africa.  On the other hand, Nishikawa et al.  reported that all EPEC strains isolated from children with diarrhoea did not react with eaeA-specific primer in Japan. However, the results of this study appeared that EPEC isolates possess the eaeA gene is an uncommon cause of diarrhoea in Najaf.
Only 18.2% isolates gave positive results with bfpA primers that equal to target product size (326 bp). The ethidium bromide-stained agarose gel of PCR-amplified products show other two products (200 bp), which may be considered as positive result depending on a previous study conducted by Carneiro et al.,  who reported the presence of 200 bp amplicons with bfpA primers, suggesting that a deletion on this gene could contribute for the lower efficiency with which these strains attached to cells, producing a localized adherence like type phenotype. The occurrence of bfpA in the present study was analogous to that recorded by other studies such as, Alikhani et al.  who found that 31.5% strains in Iran gave positive results for the bfpA gene. In South Africa, Obi et al.  showed that the bfpA gene coding for EPEC was most frequently detected (22.7%) among E. coli isolates.
The most regular matter in EPEC is the presence of eaeA alone (atypical EPEC) or with bfpA (typical). Unusual in this study was the detection of bfpA without the eaeA gene [Table 2]. This observation was also documented by Kobayashi et al.  who found that two isolates were positive to the bfpA gene but negative to the eaeA gene.
Unusual in this study is the detection of the bfpA gene in four isolates but not the EAF gene [Table 2]. It's questionable whether isolates positive for the bfpA gene but not for EAF plasmid should be classified as EPEC and whether they actually are pathogenic. The reason for these phenomena is currently unknown. However, this may be due to the absence of the EAF probe from the EAF plasmid without any deleterious effects on localized adherence expression.  The EAF probe used to detect EPEC not recognized in some typical EPEC isolates, and the frequency of these organisms in infection may have been underestimated.  There are no report in Iraq that can be compared with the results of the present study and concerning detection of the EAF probe and plasmid in EPEC. However, the absence of the EAF plasmid in most bfpA-positive EPEC isolates in the present investigation was in agreement with the observation of Trabulsi et al.  Based on this study observation, the EAF PCR assay proved to be a nonspecific and inefficient method for the detection of EPEC isolates carrying the EAF plasmid.
In this study, two isolates were classified as typical EPEC (eaeA +, bfpA +). Typical EPEC strains remain an important cause of potentially foetal infant diarrhoea in developing countries.  In Brazil, up to the 1990s, typical EPEC was the main cause of acute diarrhoea in children younger than one year old, of low socio-economic status.  In Iran, Alikhani et al.  reported that the typical EPEC strains continue to be an important cause of diarrhoea in children, mostly encountered among the strains belonging to serogroups O55 and O86. In a meeting on EPEC held in 1995, a consensus definition of atypical EPEC was established, namely that they are EAF- (bfpA- ), eaeA+ strains that promote attaching and effacing lesions.  The present study demonstrated unusual detection of four isolates carrying the bfpA gene without the eaeA gene [Table 2]. Hardegen et al.  detected five isolates which were positive for EAF plasmid only and reported this question whether E. coli strains positive for EAF plasmid but not for eaeA should be classified as EPEC. Alikhani et al.  reported that the presence of only one gene (eaeA or bfpA) used to identify EPEC as atypical. Therefore, these four isolates recovered in the present study were classified as atypical EPEC.
According to the results of the present study, we concluded that the serotyping of E. coli is not likely corresponding to the pathogenic factors. Significant differences were found between the serotyping method and molecular techniques in detection of EPEC (P<0.05). The molecular methods was more specific but slower than the serotyping method. The possession of EPEC-related O and K antigens is no longer deemed an essential characteristic of true pathogenic EPEC strains.
| ~ References|| |
|1.||Dedeiæljuboviæ A, Hukiæ M, Bekiæ D, Zvizdiæ A. Frequency and distribution of diarrhoeagenic Escherichia coli strains isolated from pediatric patients with diarrhoea in Bosnia and Herzegovina. Bosn J Basic Med Sci 2009;9:149-55. |
|2.||Kobayashi RK, Saridakis HO, Dias AM, Vidotto MC. Molecular identification of enteropathogenic Escherichia coli (EPEC) associated with infant diarrohea in Londrina, Parana, Brazil. Braz J Microbiol 2000;31:275-80. |
|3.||Fujihara S, Arikawa K, Aota T, Tanaka H, Nakamura H, Wada T, et al. Prevalence and properties of diarroheagenic Escherichia coli among healthy individuals in Osaka City, Japan. Jpn J Infect Dis 2009;62:318-23. |
|4.||Nguyen TV, Le PV, Le CH, Weintraub A. Antibiotic resistance in diarroheagenic Escherichia coli and Shigella strains isolated from children in Hanoi, Vietnam. Antimicrob Agents Chemother 2005;49:816-9. |
|5.||MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria. 3 rd ed. Philadelphia: Lippincott Williams and Williams; 2000. |
|6.||Pospiech T, Neumann J. Genomic DNA isolation. In: Kieser T, editor. John Innes Center. Norwich NR4 7UH. UK, 1995. |
|7.||Brian MJ, Frosolono M, Murray BE, Miranda A, Lopez EL, Gomez HF, et al. Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome. J Clin Microbiol 1992;30:1801-6. |
|8.||Afset JE, Bergh K, Bevanger L. High prevalence of atypical enteropathogenic Escherichia coli (EPEC) in Norwegian children with diarrohea. J Med Microbiol 2003;52:1015-9. |
|9.||Woodford N, Johnson AP. Molecular Bacteriology, Protocols and Clinical Applications. Totowa, New Jersey : Humana Press Inc.; 1998 |
|10.||Fagan PK, Hornitzky MA, Bettelheim KA, Djordjevic SP. Detection of Shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR. Appl Environ Microbiol 1999;65:868-72. |
|11.||Almohana AM. Prevalence and characterization of verotoxin producing Escherichia coli isolated from patients with diarrhea in Baghdad and Najaf. Ph.D. Thesis. Al-Mustansiryia University; 2004. |
|12.||Jafari F, Shokrzadeh L, Hamidian M, Ahrabi SS, Zali, M. Acute diarrhea due to enteropathogenic bacteria in patients at hospitals in Tehran. Jpn J Infect Dis 2008;61:269-73. |
|13.||Gomes TA, Rassi V, MacDonald KL, Ramos SR, Trabulsi LR, Vieira MA, et al. Enteropathogens associated with acute diarrheal disease in urban infants in Sao Paulo, Brazil. J Infect Dis 1991;164:331-7. |
|14.||Alikhani MY, Mirsalehian A, Aslani MM. Detection of typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhea. J Med Microbiol 2006;55:1159-63. |
|15.||Trabulsi LR, Keller R, Tardelli Gomes TA. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis 2002;8:508-13. |
|16.||Usein C, Chitoiu D, Ciontea S, Condei M, Damian M. Escherichia coli pathotypes associated with diarrhea in Romanian children younger than 5 years of age. Jpn J Infect Dis 2009;62:289-93. |
|17.||Levine MM, Nataro JP, Karch H, Baldini M, Kaper JB, Black RE, et al. The diarroheal response of human to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J Infect Dis 1985;152:550-9. |
|18.||Phantouamath B, Sithivong N, Insisiengmay S, Higa N, Toma C, Nakasone N, et al. The incidence of Escherichia coli having pathogenic genes for diarrohea: A study in the people's Democratic Republic of Lao. Jpn J Infect Dis 2003;56:103-6. |
|19.||Obi CL, Green E, Bessong PO, de Villiers B, Hoosen AA, Igumbor EO, et al. Gene encoding virulence markers among Escherichia coli isolates from diarrhoeic stool samples and river sources in rural Venda communities of South Africa. Water SA 2004;30:243-8. |
|20.||Nishikawa Y, Zhou Z, Hase A, Ogasawara J, Kitase T, Abe N, et al. Diarroheagenic Escherichia coli isolated from stools of sporadic cases of diarroheal illness in Osaka City, Japan between 1997 and 2000: Prevalence of enteroaggregative E. coli heat-stable enterotoxin 1gene-possessing E. coil. Jpn J Infect Dis 2002;55:183-90. |
|21.||Carneiro LA, Lins MC, Garcia FR, Silva AP, Mauller PM, Alves GB, et al. Phenotypic and genotypic characterization of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurized milk. Intern J Food Microbiol 2006;108:15-21. |
|22.||Law D. American society for microbiology adhesion and its role in the virulence of enteropathogenic Escherichia coli. Clin Microbiol Rev 1994;7:152-3. |
|23.||Scotland SM, Smith HR, Said B, Willshaw GA, Cheasty T, Rowe B. Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe. J Med Microbiol 1991;35:278-83. |
|24.||Gomes TA, Irino K, Girão DM, Girão VB, Guth BE, Vaz TM, et al. Emerging enteropathogenic Escherichia coli strains? Emerg Infect Dis 2004;10:127-32. |
|25.||Hardegen C, Messler S, Henrich B, Pfeffer K, Würthner J, MacKenzie CR. A set of novel multiplex Taqman real-time PCRs for the detection of diarrhoeagenic Escherichia coli and its use in determining the prevalence of EPEC and EAEC in a university hospital. Ann Clin Microbiol Antimicrob 2010;9:315-21. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]