|Year : 2019 | Volume
| Issue : 1 | Page : 87-91
Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran
Ramin Abri1, Afshin Javadi2, Roghayeh Asghari3, Vadood Razavilar4, Taghi Zahraei Salehi5, Firouzeh Safaeeyan6, Mohammad Ahangarzadeh Rezaee7
1 Food & Drug Safety Research Center, Health Management & Safety Promotion Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Food Hygiene, Biotechnology Research Center, Tabriz Branch, Tehran, Iran
3 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
4 Department of Food Hygiene, Science & Research Branch, Islamic Azad University, Tehran, Iran
5 Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
6 Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
7 Infectious & Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Submission||21-Dec-2017|
|Date of Web Publication||30-Sep-2019|
Dr Mohammad Ahangarzadeh Rezaee
Infectious & Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of
E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic
E. coli (EPEC) pathotypes in animal-source foods (ASF).
Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates.
Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples.
Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.
Keywords: Animal source foods - enteropathogenic Escherichia coli - enterotoxigenic Escherichia coli- food borne diseases - food hygiene
|How to cite this article:|
Abri R, Javadi A, Asghari R, Razavilar V, Salehi TZ, Safaeeyan F, Rezaee MA. Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran. Indian J Med Res 2019;150:87-91
|How to cite this URL:|
Abri R, Javadi A, Asghari R, Razavilar V, Salehi TZ, Safaeeyan F, Rezaee MA. Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran. Indian J Med Res [serial online] 2019 [cited 2020 Sep 29];150:87-91. Available from: http://www.ijmr.org.in/text.asp?2019/150/1/87/268217
Diarrhoeal disease is a global problem, particularly in developing countries. Various infectious agents cause diarrhoea, such as rotaviruses, coronaviruses, Campylobacter spp., Clostridium perfringens, Escherichia coli and Salmonella species,. Among the bacterial pathogens, diarrhoeagenic E. coli (DEC) strains are the most common agents of diarrhoea, especially in developing countries. Based on the virulence factors, DEC is classified into six groups: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC),. ETEC is a major cause of childhood diarrhoea in developing countries and in people who travel from industrialized countries to native regions,,. The virulence factors of these bacteria are heat-stable and heat-labile enterotoxins that are encoded by the st and lt genes. Another important pathotype of the species is EPEC, which is the primary cause of diarrhoea in infants, especially in developing countries. EPEC strains exist in two forms: typical EPEC or tEPEC (having eae and bfp genes) and atypical EPEC or aEPEC (lacking bfp gene),.
Because the isolation of E. coli was easier than that of other enteric pathogens, it could be used as an indicator of faecal contamination and other faecal-origin microorganisms in food materials. Ruminants, especially calves, are identified as a major source of pathogenic E. coli. Food products of animal origin, such as fresh meat and raw milk, are considered as common carriers of E. coli. Meat and meat products may be contaminated in several ways, such as through direct contact with faeces or hides during slaughter. Moreover, raw milk and milk products could be rich nutritious medium for many microorganisms. Poor sanitation during collection or storage can also cause contamination. Pathogenic E. coli has been identified as a major cause of foodborne diseases, because animal source foods (ASFs) are constituents of human diet. Hence, it becomes necessary to determine the occurrence of E. coli in ASFs through a reliable and quick test. The aim of this study was to identify ETEC and EPEC as two common pathotypes of E. coli in raw meat and dairy products through duplex-polymerase chain reaction (PCR).
| Material & Methods|| |
This study was conducted from May to September 2016 in the laboratory of Microbiology, Food and Drug Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. A total of 120 samples of meat products (fresh beef, ground beef and hamburger) and 102 dairy products (raw milk, traditional cheese, Doogh and yoghurt) were collected from markets in different localities of Northwest Iran. All specimens were immediately transferred to the laboratory under cold chain and sterile conditions.
Bacterial isolation: For the enrichment of the E. coli strains, all specimens were cultured in lauryl sulphate broth (Merck, Germany) overnight at 37°C and subsequently were streaked onto MacConkey agar (Merck, Germany) and incubated at 37°C for 24 h. The lactose-fermenting colonies were identified as E. coli using Gram staining and conventional biochemical tests as described previously. Finally, E. coli isolates were stored at −80°C in trypticase soy broth supplemented with 20 per cent glycerol for further procedures.
DNA extraction: DNA extraction from overnight cultures was done using the Promega DNA extraction kit (A11125, USA), following the instructions given by the manufacturer.
Polymerase chain reaction assays: The DNA templates were examined by two separate duplex PCRs with specific primers [Table 1]. Duplex-PCR 1 and 2 were for the identification of lt and st genes (for detection ETEC) and eae and bfp genes (for detection EPEC), respectively,,. Both duplex-PCR assays were accomplished in a 25 μl reaction mixture, consisting of 2× PCR master mix [2× concentrated solutions of Taq DNA polymerase, reaction buffer, MgCl2 and dNTPs (CinnaGen Inc., Iran)] with a BioRad T100™ thermal cycler (Bio-Rad Laboratories, Inc., USA). The PCR Master Mix (CinnaGen Inc., Iran) contains all components for PCR, except DNA template and primers. Primers were provided by GeNetBio Inc., Korea. The first duplex-PCR conditions included an initial denaturation at 95°C for five minutes for one cycle followed by 35 cycles of 95°C for 45 sec, 49°C for 45 sec, 72°C for 45 sec and final extension at 72°C for seven minutes. The second duplex-PCR conditions comprised: An initial denaturation at 95°C for three minutes for one cycle followed by 38 cycles of 95°C for one minute, 53°C for one minute, 72°C for one minute and final extension at 72°C for 10 minutes. Amplified PCR products were observed after electrophoresis on one per cent agarose and staining with Safe Dye (CinnaGen Inc., Iran). The PCR products were visualized under ultraviolet UV transilluminator and photographed.
|Table 1: Primers used for the detection of enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes|
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Reference strains: The following standard strains were employed as positive controls: ETEC (H10407) and EPEC (2348/69). These strains were provided by R. Vidal, Microbiology and Mycology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago, Chile.
| Results|| |
Among the 222 samples (120 meat samples, 102 diary samples) examined in this study, 109 (49%) E. coli isolates were obtained [63 (57.7%) from meat products and 46 (42.2%) from dairy products]. The ground beef and hamburger had the highest occurrence of contamination, followed by fresh beef. About dairy products, all of the E. coli isolates belonged to raw milk and traditional cheese samples. No E. coli isolate was identified in fermented dairy products i.e., Doogh, yoghurt.
Occurrence of ETEC and EPEC strains in meat and dairy products: Among E. coli isolates, two ETEC and four EPEC (1 typical EPEC and 3 atypical EPEC) were detected in meat samples while no cases of ETEC and only two EPEC (1 typical EPEC and 1 atypical EPEC) were identified in dairy samples. The distribution of virulence target genes is shown in [Table 1]. Fifty per cent (4 of 8 isolates) of the pathogenic E. coli isolates harboured both eae and bfp genes and were considered as tETEC. Furthermore, no st and lt genes were identified in dairy products [Figure 1] & [Figure 2].
|Table 2: Virulence gene profile of Escherichia coli strains screened by duplex polymerase chain reaction|
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|Figure 1: (A) Ladder 1000 bp, (B) positive control for eae and bfp genes, (C) positive isolate for eae gene, (D) positive isolate for eae and bfp genes, (E) negative control, (F) positive isolate for eae gene.|
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|Figure 2: (A) Ladder 1000 bp, (B) negative control, (C) positive isolate for lt and st genes, (D) positive isolate for st gene.|
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| Discussion|| |
Diarrhoea caused by different intestinal pathogens is one of the major concerns of public health. An important bacterial agent of diarrhoea is E. coli, which causes death among under five children and the elderly. Animal food products, especially raw meat and dairy products that are rich in nutritional value are likely to get contaminated with spoilage and pathogenic bacteria, resulting in the transmission of infections to humans. Therefore, use of rapid diagnostic techniques is important in reducing disease development rate and decreasing the financial burden of the disease on the community health.
In the present study, the occurrence of E. coli was 42.2 and 57.7 per cent in raw milk/raw milk products and raw meat/raw meat products, respectively. In a similar study carried out by Rúgeles et al, E. coli was isolated from 58 per cent of the meat samples. Badri et al reported the prevalence of E. coli in meat samples as 48 per cent. Several other studies have reported the presence of E. coli in raw milk and meat products,,,. It should be noted that in the studied dairy samples, no E. coli isolate was identified in Doogh and yoghurt samples. It could be due to acidic p H and high temperatures created during their fermentation process. Moreover, majority of E. coli identified in meat products were isolated from ground meat and hamburger. It is probable, these products are more contaminated due to poor sanitation of grinder and high manipulation in their production process. According to the results of the duplex-PCR of isolated E. coli, only two EPEC were found in raw milk and raw milk products, whereas two ETEC and four EPEC were identified in raw meat products. In similar studies,, no case of ETEC was found in raw meat products, while EPEC (mostly atypical) was the most isolated pathotype, thereby confirming the results of the present study. Mohammed showed that 15.63 per cent E. coli isolated from meat products were ETEC, which was higher than that of several previous studies. Holko et al, reported 2.1 per cent ETEC and 3.09 per cent EPEC in traditional cheese samples made from sheep milk. A study conducted on raw milk materials, found one ETEC and 13 EPEC. In another study on 206 samples of raw milk, 17 EPEC (8.25%) were identified.
In conclusion, our results showed that the use of unpasteurized milk in traditional dairy products and inadequate sanitation during the preparation, transport and/or storage of ASFs and personal hygiene could be a potential factor in the spread of E. coli pathotypes and other zoonotic pathogens in the community should be considered and as a threat to public health.
Financial support & sponsorship: None.
Conflicts of Interest: None.
| References|| |
Vilchez S, Reyes D, Paniagua M, Bucardo F, Möllby R, Weintraub A. Prevalence of diarrhoeagenic Escherichia coli
in children from León, Nicaragua. J Med Microbiol
Bueris V, Sircili MP, Taddei CR, dos Santos MF, Franzolin MR, Martinez MB, et al.
Detection of diarrhoeagenic Escherichia coli
from children with and without diarrhea in Salvador, Bahia, Brazil. Mem Inst Oswaldo Cruz
Nguyen TV, Le Van P, Le Huy C, Gia KN, Weintraub A. Detection and characterization of diarrhoeagenic Escherichia coli
from young children in Hanoi, Vietnam. J Clin Microbiol
Vendruscolo JW, Waldrich TL, Saikawa GIA, Pelayo JS, Kobayashi RKT, Nakazato G, et al.
Novel multiplex PCR for detection of diarrhoeagenic Escherichia coli
strains isolated from stool and water samples. Genet Mol Res
Canizalez-Roman A, Gonzalez-Nuñez E, Vidal JE, Flores-Villaseñor H, León-Sicairos N. Prevalence and antibiotic resistance profiles of diarrhoeagenic Escherichia coli
strains isolated from food items in Northwestern Mexico. Int J Food Microbiol
Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli
in developing countries: Epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev
Rezaee MA, Rezaee A, Moazzeni SM, Salmanian AH, Yasuda Y, Tochikubo K, et al.
Expression of Escherichia coli
heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae
. J Microbiol
Nataro JP, Kaper JB. Diarrhoeagenic Escherichia coli
. Clin Microbiol Rev
Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL. Epidemiology of Escherichia coli
O157:H7 outbreaks, United States, 1982-2002. Emerg Infect Dis
Osman KM, Mustafa AM, Elhariri M, Abdelhamed GS. The distribution of Escherichia coli
serovars, virulence genes, gene association and combinations and virulence genes encoding serotypes in pathogenic E. coli
recovered from diarrhoeic calves, sheep and goat. Transbound Emerg Dis
Mohammed MAM. Molecular characterization of diarrhoeagenic Escherichia coli
isolated from meat products sold at Mansoura city, Egypt. Food Control
Altalhi AD, Hassan SA. Bacterial quality of raw milk investigated by Escherichia coli
and isolates analysis for specific virulence-gene markers. Food Control
Hall GS. Bailey & Scott's diagnostic microbiology
ed. St. Louis: American Society for Clinical Pathology; 2015.
Alizade H, Ghanbarpour R, Nekoubin M. Phylogenetic of Shiga toxin-producing Escherichia coli
and a typical enteropathogenic Escherichia coli
strains isolated from human and cattle in Kerman, Iran. Int J Enteric Pathog
Aranda KRS, Fagundes-Neto U, Scaletsky ICA, Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli
spp. J Clin Microbiol
Rúgeles LC, Bai J, Martínez AJ, Vanegas MC, Gómez-Duarte OG. Molecular characterization of diarrhoeagenic Escherichia coli
strains from stools samples and food products in Colombia. Int J Food Microbiol
Rajendran P, Ajjampur SSR, Chidambaram D, Chandrabose G, Thangaraj B, Sarkar R, et al.
Pathotypes of diarrhoeagenic Escherichia coli
in children attending a tertiary care hospital in South India. Diagn Microbiol Infect Dis
Badri S, Filliol I, Carle I, Hassar M, Fassouane A, Cohen N. Prevalence of virulence genes in Escherichia coli
isolated from food in Casablanca (Morocco). Food Control
Fadaei A. Bacteriological quality of raw cow milk in Shahrekord, Iran. Vet World
Bonyadian M, Moshtaghi H, Akhavan Taheri M. Molecular characterization and antibiotic resistance of enterotoxigenic and entero-aggregative Escherichia coli
isolated from raw milk and unpasteurized cheeses. Vet Res Forum
Paneto B, Schocken-Iturrino R, Macedo C, Santo E, Marin J. Occurrence of toxigenic Escherichia coli
in raw milk cheese in Brazil. Arquivo Bras Med Vet Zootec
Dehkordi FS, Yazdani F, Mozafari J, Valizadeh Y. Virulence factors, serogroups and antimicrobial resistance properties of Escherichia coli
strains in fermented dairy products. BMC Res Notes
Flores RA, Tamplin ML. Distribution patterns of Escherichia coli
O157:H7 in ground beef produced by a laboratory-scale grinder. J Food Prot
Holko I, Bisova T, Holkova Z, Kmet V. Virulence markers of Escherichia coli
strains isolated from traditional cheeses made from unpasteurised sheep milk in Slovakia. Food Control
[Figure 1], [Figure 2]
[Table 1], [Table 2]