Elsevier

Food Control

Volume 121, March 2021, 107640
Food Control

Occurrence, pathotypes, and antimicrobial resistance profiles of diarrheagenic Escherichia coli strains in animal source food products from public markets in Mashhad, Iran

https://doi.org/10.1016/j.foodcont.2020.107640Get rights and content

Highlights

  • The first survey on six pathotypes of Diarrheagenic E. coli in foodstuffs in Iran.

  • Differences in the frequency of strains among months was statistically significant.

  • Shiga-like toxin producing E. coli (STEC) was the most prevalent pathotype.

  • The highest resistance rate was for tetracycline, lowest for gentamicin & cefoxitin.

  • STECs had higher antibiotic resistance rate in comparison with other pathotypes.

Abstract

Diarrheagenic Escherichia coli (DEC) strains are common pathogens that transmitted through the consumption of contaminated foods, and cause acute intestinal diseases in human. The present study was conducted on 300 samples including raw milk, meat and some of their products from November 2016 to October 2017. Microbiological and molecular diagnostic techniques were used to identification of DEC. A total of 69% (207/300) E. coli was isolated and the frequency of DEC was 25.6% (53/207). The pathogenic groups of DEC recovered from the isolates had the following profile: Shiga-like toxin producing E. coli (STEC): 62.3%, enteropathogenic E. coli (EPEC): 24.5%, enteroaggregative E. coli (EAEC): 9.4%, and enterotoxigenic E. coli (ETEC): 3.8%. Neither enteroinvasive E. coli (EIEC) nor diffusely adherent E. coli (DAEC) were found. The highest incidence values were found in hamburgers (46.7%), and the highest rate of DEC positive prevalence was in March. Of the DEC strains, 90.6% (48/53) displayed resistance to at least one antibiotic and the highest level of resistance was found for tetracycline (69%). The obtained results revealed that the studied animal source food products may easily act as a reservoir of DEC with a potential ability to transfer antibiotic resistance and virulence genes to the gastrointestinal microbiota. Therefore, it is of paramount importance to develop effective strategies for improving food safety and updated guidelines for the prudent use of antimicrobial agents in Iran.

Introduction

Diarrheagenic Escherichia coli (DEC) strains are a main etiologic agent of moderate-to-severe diarrhea in humans (Canizalez-Roman, Gonzalez-Nuñez, Vidal, Flores-Villaseñor, & León-Sicairos, 2013). DECs are important foodborne pathogens that have been classified into six pathogenic types on the basis of their specific virulence traits. These types include Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC), Diffusely adherent E. coli (DAEC) and Shiga-like toxin producing E. coli (STEC) (Amézquita-Montes et al., 2015) which constitute the subgroup of Enterohemorrhagic E. coli (EHEC). Outbreaks caused by DEC are closely linked to contaminated food chain worldwide.

EPEC is one of the major pathogens which is responsible for infantile diarrhea in developing countries. This pathotype is determined by the presence of the locus of enterocyte effacement (LEE) region encoding for the intimin (eae gene) and the lack of stx genes. This last trait is also used to distinguish strains of EPEC from STEC. EPEC adherence factor (EAF) plasmid-encoded bundle-forming pilus (bfp) gene is an index for the classification of EPEC. BFP-positive isolates are termed typical EPEC (tEPEC), whereas BFP-negative ones are classified as atypical EPEC (aEPEC) (Canizalez-Roman et al., 2013). ETEC strains are characterized by heat-labile (LT) and/or heat-stable (ST) enterotoxins which are plasmid-encoded. ETEC is recognized as the leading cause of traveler's diarrhea in developing countries and the most common pathotype of E. coli which causes infantile diarrhea. EAEC strains which are known as the cause of acute and persistent diarrhea are distinguished by their aggregative adherence pattern to cultured cells. This phenotype is associated with a plasmid which codes many virulence genes, including an anti-aggregation protein transporter (CVD432), aggregative adherence fimbria (AAF), and the gene aggR that regulates the expression of fimbria (Aslani, Alikhani, Zavari, Yousefi, & Zamani, 2011; Canizalez-Roman et al., 2013). EIEC closely resembles Shigella and causes an invasive and dysenteric form of diarrhea in humans (Castro-Rosas et al., 2012) which is mediated by ipaH and virF genes (Canizalez-Roman et al., 2013). DAEC is a heterogeneous group that is defined by a distinct diffuse pattern of adherence (DA) to HeLa and HEp-2 cells (Bautista-De León, Gómez-Aldapa, Rangel-Vargas, Vázquez-Barrios, & Castro-Rosas, 2013) due to a fimbrial adhesion (F1845) encoded by daaD. STEC strains have been described by a range of symptoms in the human hosts, from mild diarrhea to severe hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). They produce one or two Shiga-like toxins encoded by stx1 and stx2 genes (Canizalez-Roman et al., 2013). The STEC isolates that contain LEE pathogenicity island are frequently termed EHEC (Perna et al., 2001).

Transmission of diarrheagenic pathotypes occurs through the fecal-oral route. Human beings constitute the main reservoir of non-STEC pathotypes, while the intestinal tracts of cattle and other animals are the primary reservoirs of STEC (Winstead, Hunter, & Griffin, 2015). That's why veterinary hygiene and food safety are strictly under control by the European Union to ensure consumer health (Bondoc, 2016a; 2016b). Animal-derived foods which are suspected to be the main source of antibiotic resistance in humans have emerged as a serious global problem (Campos, Gil, Mourão, Peixe, & Antunes, 2015). E. coli also has developed a high level of resistance due to excessive use of antibiotics. In addition, E. coli can transfer mobile resistance elements, such as plasmids, to other enteric pathogens leading to the spread of multi-drug resistance among different enterobacteria (Hannah et al., 2009; Uysal & Durak, 2012). Molecular biology techniques, including multiplex-PCR assays, have been reported in literature in recent decades for the rapid identification of DEC pathotypes. The present study aimed to investigate the animal source foods (raw milk, cheese, meat, and meat products) for the distinction of all six pathogenic groups of DEC and their antibiotic susceptibility profiles. Specific DNA amplification of virulence genes by a panel of PCR reactions has been used for the detection of pathotypes. To our knowledge, this was the first study of the prevalence of six DEC groups in foodstuffs in Iran.

Section snippets

Sampling location

Mashhad is the second most populous city in Iran and the capital of Razavi Khorasan Province. It is located in the northeast of the country with a population of more than 3,000,000. In this city, food products of animal origin are available in eight municipality markets which are located in eight urban regions each entailing several butcheries and retail minimarkets. Moreover, there are 11 milk collecting centers in the suburbs and raw milk is delivered daily from dairy farms to the local milk

Detection of E. coli

The sampling of the current study covered the geographical area of Mashhad in Khorasan Razavi province. Milk collecting centers for raw milk and different municipality markets for meat products and cheese samples were assayed to detect DEC contamination. The isolates were confirmed in molecular analyses by PCR targeting the uidA gene. At the first screening of 300 samples, E. coli was isolated from 207 items (69%), including 104 and 103 of meat and raw milk/cheese samples, respectively (Table 4

Discussion

Foodborne diseases are a significant global public health concern and bacterial pathogens are the most common etiologic agent for this kind of disease (Amézquita-Montes et al., 2015). In this line, European Union has just cracked down the rules for strict control of food safety and public health to cope with developing certain infectious diseases (Bondoc, 2016c; 2016d). The present study investigated the frequencies of six pathogenic groups of DEC in some dairy and meat products in Mashhad,

Conclusion

The obtained results pointed to the high levels of fecal contamination in animal source foods. It is worthy to note that environmental pollution with human wastewater or manipulations during the process by people are the most probable source of pathotypes (Winstead et al., 2015). The obtained results suggested that raw milk and cheese made with unpasteurized milk, as well as undercooked red meat, ground meat and hamburgers, may be involved in the transmission of the foodborne infections caused

CRediT authorship contribution statement

Neda Fallah: Conceptualization, Investigation, Resources, Writing - original draft, Writing - review & editing. Mehran Ghaemi: Methodology, Validation. Kiarash Ghazvini: Validation. Mehrnaz Rad: Validation. Abdollah Jamshidi: Supervision, Writing - review & editing, Funding acquisition.

Declaration of competing interest

The authors have no competing interests to declare.

Acknowledgments

This study was funded by Ferdowsi University of Mashhad. Our deepest appreciation and thanks go to the Department of Molecular Biology of Razavi Khorasan Veterinary Organization National Laboratory team for their invaluable support at different stages of this research.

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