Short Communication
Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain

https://doi.org/10.1016/j.ijfoodmicro.2005.08.025Get rights and content

Abstract

The aim of this study was to determinate the prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli (STEC) strains isolated from different dairy products (DP) in Spain with the purpose of determining whether DP represent a potential source of STEC pathogenic for humans.

A total of 502 DP were examined from 64 different ovine and caprine flocks and 6 dairy plants in Extremadura (Western Spain). Samples were collected monthly between March 2003 and June 2004 and included 360 unpasteurised milk obtained from the bulk tank, 103 fresh cheese curds and 39 cheeses. Samples obtained were examined for STEC using genotypic (PCR) methods.

STEC strains were detected from 39 (10.8%) bulk tank, 4 (3.9%) fresh cheese curds and 2 (5%) cheese, whereas O157:H7 serotype were isolated from one (0.3%) bulk tank. A total of 9 STEC strains (O27:H18, O45:H38, O76:H19, O91:H28, O157:H7, ONT:H7, ONT:H9 and ONT:H21) were identified in this study. One of them, the serotype O27:H18, has not been reported previously as STEC.

PCR showed that 3 strains carried stx1 genes, 5 possessed stx2 genes and 1 both stx1 and stx2. Whereas all STEC caprine isolates showed ehxA genes, only O157:H7 serotype showed eae virulence genes. The strain O157:H7 isolated possessed intimin type γ1 and belonged to phage type 31.

This study confirms that dairy product is an important reservoir of STEC pathogenic for humans.

Introduction

Shiga toxin-producing Escherichia coli (STEC), also called verotoxin-producing E. coli, is the most important recently emerged group of food-borne pathogens, especially the serotype O157:H7 (Paton and Paton, 1998). STEC elaborate two potent phage-encoded cytotoxins called Shiga toxins (Stx1 and Stx2) or verotoxins (VT1 and VT2) (Paton and Paton, 1998). A number of serotypes belonging to STEC can cause serious diseases in humans, including hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombocytopenic purpura (TP), which may prove fatal in immunocompromised patients (Slutsker et al., 1997, Banatvala et al., 2001). It is now generally agreed that the major portion of the histopathological lesions associated with both HC and HUS is a consequence of the interaction of Stx with endothelial cells (Karmali, 1989). In addition to toxin production, another virulence-associated factor expressed by STEC is a protein called intimin, which is responsible for the intimate attachment of STEC to intestinal epithelial cells, causing attaching and effacing lesions in the intestinal mucosa (Jerse et al., 1990). Intimin is encoded by the chromosomal gene eae, which is part of a pathogenicity island termed the locus for enterocyte effacement (LEE) (Donnenberg et al., 1997). A factor that may also affect virulence of STEC is the enterohaemolysin (Ehly), also called enterohaemorrhagic E. coli haemolysin (EHEC-HlyA), which is encoded by ehxA gene (Schmidt et al., 1995).

Since healthy domestic animals, in particular ruminants like cattle, sheep and goats, can harbour STEC and O157:H7 in their faeces, they are regarded as natural reservoirs of these organisms (Beutin et al., 1993, Blanco et al., 2001, Blanco et al., 2004a). Recently, we have demonstrated in our country that a half of the sheep investigated harbour these bacteria (Blanco et al., 2003a, Rey et al., 2003). Transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables, or water contaminated by faeces of carriers. Also, person-to-person transmission has also been documented (Karmali, 1989).

Relatively little information is available about the frequency of isolation of STEC from ill persons in Spain because most laboratories do not routinely culture for this organism (Blanco et al., 2004b).

Given the importance of ovine and caprine livestock in our country, and the fact that many milk products from these species are not treated with any previous bacteriological sterilisation procedures, the aim of the present study was to investigate the prevalence of STEC O157:H7 and STEC non-O157 in ovine and caprine milk and other dairy products in order to determinate the potential risk that the consumption of these products can have from the standpoint of public health and hygiene.

Section snippets

Specimen collection, culture, and STEC screening

Samples were collected monthly in 64 different flocks in Extremadura (Western Spain) between March 2003 and June 2004 and comprise a total of 502 dairy products (DP). The DP included 360 unpasteurised ovine and caprine milk obtained from the bulk tank, 103 fresh cheese curds and 39 cheeses.

According to ISO 16654:2002, a total of 25 g of each sample was added to 225 ml of modified tryptone soya broth (TSB) (CM129, Oxoid, Madrid, Spain) supplemented with novobiocin (20 mg l 1) (N1628, Sigma,

Results and discussion

Shiga-like toxin E. coli (STEC) implicated in hemorrhagic colitis (HC) and hemolytic uraemic syndrome (HUS), has become a serious health problem in various countries. Although many cases are undoubtedly associated with consumption of undercooked meat and meat products, vegetables and drinking water, a number of studies have also reported outbreaks of HC and HUS associated with consumption of milk and dairy products; sources reported to date include pasteurized and unpasteurized cow's milk (

Acknowledgements

This study was supported partially by the Regional Government Junta de Extremadura-Consejería de Educación, Ciencia y Tecnología (2PRO1A049-2PRO2A007) and the Fondo de Investigación Sanitaria (FIS G03/025-COLIRED-O157). The authors wish to thank A. Echeita (National Centre of Microbiology, Majadahonda, Madrid, Spain) for the phage typing of STEC.

References (56)

  • M.P. Vettorato et al.

    Properties of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep in the State of Sao Paulo, Brazil

    Vet. Microbiol.

    (2003)
  • C. Zweifel et al.

    Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland

    Int. J. Food Microbiol.

    (2004)
  • R. Ahmed et al.

    Phage-typing scheme for Escherichia coli O157:H7

    J. Infect. Dis.

    (1987)
  • N. Banatvala et al.

    The United States National prospective hemolytic uremic syndrome study: microbiologic, serologic, clinical, and epidemiologic findings

    J. Infect. Dis.

    (2001)
  • L. Beutin et al.

    Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals

    J. Clin. Microbiol.

    (1993)
  • L. Beutin et al.

    Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period

    J. Clin. Microbiol.

    (2004)
  • M. Bielaszewska et al.

    Human Escherichia coli O157:H7 infection associated with the consumption of unpasteurized goat's milk

    Epidemiol. Infect.

    (1997)
  • J. Blanco et al.

    Epidemiology of verocytotoxigenic Escherichia coli (VTEC) in ruminants

  • M. Blanco et al.

    Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain

    J. Clin. Microbiol.

    (2003)
  • J. Blanco et al.

    Verotoxin producing Escherichia coli (VTEC) in Spain: prevalence, serotypes and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and human infections

    Exp. Biol. Med.

    (2003)
  • M. Blanco et al.

    Serotypes, virulence genes and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain: identification of a new intimin variant gene (eae-ξ)

    J. Clin. Microbiol.

    (2004)
  • J.E. Blanco et al.

    Serotypes, virulence genes and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo (Spain) from 1992 through 1999

    J. Clin. Microbiol.

    (2004)
  • K.N. Brett et al.

    stx1c is the most common Shiga toxin 1 subtype among Shiga toxin-producing Escherichia coli isolates from sheep but not among isolates from cattle

    J. Clin. Microbiol.

    (2003)
  • Outbreak of Escherichia coli O157:H7 infection associated with eating fresh cheese curds—Wisconsin, June 1998

    Jama

    (2000)
  • P.A. Chapman et al.

    A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from cases of bloody diarrhoea, non-bloody diarrhoea and asymptomatic contacts

    J. Med. Microbiol.

    (1996)
  • R.C. Clarke et al.

    Isolation of verocytotoxin-producing Escherichia coli from milk filters in south-western Ontario

    Epidemiol. Infect.

    (1989)
  • A.N. De Araujo et al.

    Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells

    BMC Microbiol.

    (2001)
  • S.P. Djordjevic et al.

    Virulence properties and serotypes of Shiga toxin-producing Escherichia coli from healthy Australian slaughter-age sheep

    J. Clin. Microbiol.

    (2001)
  • Cited by (82)

    • Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style)

      2022, Journal of Dairy Science
      Citation Excerpt :

      For the non-O157 E. coli STEC strain M294aVO, the minimum growth temperature in LTH ewe milk was 6°C, but in raw ewe milk it did not grow at any of the tested conditions. The presence of E. coli O157:H7 in raw ewe milk (Caro et al., 2006; Rey et al., 2006; Otero et al., 2017) raises the possibility of its survival in raw sheep milk cheeses (Caro and García-Armesto, 2007; Baylis, 2009). Cheesemaking involves several different steps such as curd formation, draining, salting, and ripening, although the exact process depends on the cheese variety.

    • Virulence genes and genetic diversity assessment of Shiga toxin-producing Escherichia coli O91 strains from cattle, beef and poultry products

      2018, Microbial Pathogenesis
      Citation Excerpt :

      Shiga toxin-producing Escherichia coli (STEC) O91 has ranked in the top five of the non-O157 serogroups most frequently associated with human cases, and strains belonging to this serogroup are the most common human pathogenic eae-negative STEC strains [1]. They have been isolated from foods of different origins, such as beef, pork, lamb or poultry [2–8] as well as animals [7,9–13]. Clinical cases related to STEC O91 have been regularly reported since the 1990s [1,14–21].

    • Foodborne pathogens in raw milk and cheese of sheep and goat origin: a meta-analysis approach

      2017, Current Opinion in Food Science
      Citation Excerpt :

      After assessing all the information presented in every study, a total of 37 primary studies passed the two quality criteria of having used approved microbiological methods and presenting sufficient data in extractable form. The meta-analyses for goat raw milk and cheese were based on 35 primary studies [3,7–12,13•,14•,15–22,23•,24–26,27•,28–40] while those for sheep raw milk and cheese were based on 16 studies [3,12,18–20,22,24,26,27•,30,32,34,47,40–42]. The occurrence of microbial hazards in goat and sheep raw milk and cheese is a binary trait (i.e., a sample tests either positive or negative for the pathogen), thus the parameter to measure the effect size is the raw proportion p (calculated as the number of successes, that is, positive samples, s, divided by the total sample size, n).

    View all citing articles on Scopus
    View full text