Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil

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Abstract

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H, O119:H and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H, and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type θ/γ2 was the most frequent among STEC, whereas the intimin β1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin μB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H, O113:H21, O118:H16, and O174:H21 were isolated.

Introduction

Shiga toxin-producing Escherichia coli (STEC), also called verotoxin-producing E. coli (VTEC), is the most important recently emerged group of foodborne pathogens. It is a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (HC) or the hemolytic uremic syndrome (HUS), which is the main cause of acute renal failure in children (Karmali, 1989, Paton and Paton, 1998, Beutin et al., 2004, Blanco et al., 2004a). Since its identification as a pathogen in 1982, STEC O157:H7 has been the cause of a series of outbreaks that happened especially in Canada, Japan, U.K. and U.S. (Karmali, 1989, Mora et al., 2004). Domestic ruminants, mainly cattle, have been implicated as the principal reservoir (Gansheroff and O'Brien, 2000, Blanco et al., 2001, Blanco et al., 2003, Blanco et al., 2004b, Meichtri et al., 2004). Transmission occurs through consumption of undercooked meat, unpasteurized dairy products and vegetables or water contaminated by feces of carriers, because STEC strains are found as part of the normal intestinal flora of the animals. Person-to-person transmission has also been documented (Blanco et al., 2001, Mora et al., in press).

Most outbreaks of HC and HUS have been attributed to strains of enterohemorrhagic serotype O157:H7 (Karmali, 1989, Mora et al., 2004). However, as non-O157 STEC strains are more prevalent in animals and as contaminants in foods, humans are probably more exposed to these strains (Beutin et al., 2004, Blanco et al., 2004a). STEC strains that cause human infections belong to a large number of O:H serotypes (a total of 472 serotypes are listed in the authors' website, http://www.lugo.usc/ecoli). Infections with some non-O157 STEC types, such as O26:H11 or H, O91:H21 or H, O103:H2, O111:H, O113:H21, O117:H7, O118:H16, O121:H19, O128:H2 or H, O145:H28 or H and O146:H21 are frequently associated with severe illness in humans, but the role of other STEC non-O157 types in human disease needs further examination (Karmali, 1989, Pradel et al., 2000, Beutin et al., 2004, Blanco et al., 2004a).

Human and bovine STEC elaborate two potent phage-encoded cytotoxins called Shiga toxins (Stx1 and Stx2) or verotoxins (VT1 and VT2) (Karmali, 1989, Paton and Paton, 1998). In addition to toxin production, another virulence-associated factor expressed by STEC is a protein called intimin which is responsible for intimate attachment of STEC to the intestinal epithelial cells, causing attaching and effacing (A/E) 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) (Kaper et al., 1998). Severe diarrhea (especially HC) and HUS were closely associated with STEC types carrying the eae gene for intimin (Karmali, 1989, Paton and Paton, 1998). Differentiation of intimin alleles represents an important tool for STEC typing in routine diagnostics as well as in epidemiological and clonal studies. The C-terminal end of intimin is responsible for receptor binding, and it has been suggested that different intimins may be responsible for different host tissue cell tropism (Zhang et al., 2002, Fitzhenry et al., 2002, Torres et al., 2005). Intimin type-specific PCR assays identified 19 variants of the eae gene that encode 19 different intimin types and subtypes (α1, α2, β1, ξR/β2B, δ/κ/β2O, γ1, θ/γ2, ε1, νR/ε2, ζ, η1, η2, ι1, μR/ι2, λ, μB, νB, ξB, ο) (Adu-Bobie et al., 1998, Oswald et al., 2000, Tarr and Whittam, 2002, Zhang et al., 2002, Jores et al., 2003, Blanco et al., 2003, Blanco et al., 2004b, Blanco et al., 2005, Ramachandran et al., 2003, Garrido et al., 2006). Apart from the capability to produce Shiga toxins and intimins, STEC may possess accessory putative virulence factors such as the enterohemolysin (Ehly), also called enterohemorrhagic E. coli hemolysin (EHEC-HlyA), which is encoded by ehxA gene (Schmidt et al., 1995) and the STEC autoagglutinating adhesin (Saa) encoded by saa gene (Paton and Paton, 2002).

Enteropathogenic E. coli (EPEC) strains are defined as intimin-containing diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes (Kaper, 1996). Most human classic EPEC strains belong to a series of O antigenic groups known as EPEC serogroups: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158. EPEC are further classified as typical, when possessing the EAF (for EPEC adherence factor) plasmid that encodes localized adherence (LA) on cultured epithelial cells mediated by the Bundle Forming Pilus (BFP); whereas atypical EPEC strains do not possess the EAF plasmid (Trabulsi et al., 2002). Typical EPEC, a major cause of infant diarrhea in developing countries, are rare in industrialized countries, where atypical EPEC seem to be a more important cause of diarrhea (Trabulsi et al., 2002). Typical EPEC strains are isolated mainly from humans, whereas atypical EPEC strains have been isolated from different animal species, including cattle, sheep and goats (Trabulsi et al., 2002, Orden et al., 2003, Blanco et al., 2005, Cortés et al., 2005).

In Brazil, only a few studies have reported the isolation and the characteristics of STEC in cattle (Cerqueira et al., 1999, Leomil et al., 2003, Irino et al., 2005). The only two studies on bovine STEC isolation in São Paulo State were from beef cattle (Leomil et al., 2003) and dairy cattle (Irino et al., 2005). However, both studies did not include the detection of the eae gene and the intimin subtyping. Moreover, the occurrence of EPEC strains was not examined. In the present study we describe the isolation of STEC and EPEC strains among diarrheic and healthy cattle from different farms in São Paulo State (Brazil) and also compare their phenotypic and genotypic characteristics.

Section snippets

Fecal samples

Fecal samples were obtained from beef cattle from 18 different farms in São Paulo (Brazil). Farms were located in different counties of São Paulo State. Two hundred and sixty-four diarrheic and 282 non-diarrheic samples from calves (1 to 6 months old) were processed. The feces were collected with swabs and inoculated in Stuart medium tubes (MEUS, Piove di Sacco, Italy), transported to the laboratory, and stored in the refrigerator for 24–48 h.

E. coli strains

The fecal samples were streaked on MacConkey (Difco,

Prevalence of STEC and EPEC strains in diarrheic and healthy cattle

Two hundred and sixty-four diarrheic and 282 non-diarrheic samples from calves (1 to 6 months old) were processed. Among diarrheic calves, 31 (12%) STEC and 7 (2.6%) EPEC strains were isolated. Among healthy cattle, 24 (8.5%) STEC strains and 8 (2.8%) EPEC strains were isolated.

Virulence genes of STEC strains

A total of 55 STEC strains were characterized in this study: 31 from diarrheic cattle and 14 from healthy cattle. PCR showed that 27 (49%) isolates carried stx1 genes, nine (16%) possessed stx2 genes and 19 (34%) both

Discussion

In the last two decades, STEC O157:H7 has become a prominent pathogen involved in sporadic and large foodborne outbreaks of bloody diarrhea and HUS particularly in the United States, Canada, United Kingdom, and Japan. On the other hand, infections caused by non-O157 STEC strains have also been increasingly reported in many countries (Karmali, 1989, Paton and Paton, 1998, Beutin et al., 2004, Blanco et al., 2004a). Thus, STEC strains that cause human infections belong to a large number of O:H

Conclusion

The isolation and diversity of STEC serotypes presently described point the beef cattle as important reservoir of STEC in Brazil. The isolation of serotypes previously described and associated with severe diseases in humans (O111:H, O113:H21, O118:H16, and O174:H21) in Brazil and other countries (Guth et al., 2002, Vaz et al., 2004, Beutin et al., 2004, Blanco et al., 2004a) may represent a risk for the population of acquiring severe infections in the future. Moreover, cattle can be considered

Acknowledgements

We thank Monserrat Lamela for skillful technical assistance. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Spanish Fondo de Investigación Sanitaria (FIS G03-025-COLIRED-O157) and the Xunta de Galicia (grants PGIDIT02BTF26101PR, PGIDIT04RAG261014PR, PGIDIT05BTF26101PR). A. Mora and G. Dahbi acknowledge the Xunta de Galicia and the Agencia Española de Cooperación

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