RESEARCH ARTICLE
Miguel Blanco1 Nora L. Padola2 Alejandra Krüger2 Marcelo E. Sanz2 Jesús E. Blanco1 Enrique A. González1 Ghizlane Dahbi1 Azucena Mora1 María Isabel Bernárdez1 Analía I. Etcheverría2 Guillermo H. Arroyo2 Paula M.A. Lucchesi2 Alberto E. Parma2 Jorge Blanco1* 1E. coli Reference Laboratory (LREC), Department of Microbiology and Parasitology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain 2Laboratory of Immunochemistry and Biotechnology, Faculty of Veterinary Sciences, University Nacional del Centro PBA, Tandil, Argentina	Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina   Summary. A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type ε1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type θ/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC. [Int Microbiol 2004; 7(4):269-276] Key words: Escherichia coli O157:H7 · intimin · serotypes of STEC · Shiga- toxin-producing E. coli · virulence genes
Received 10 February 2004 Accepted 21 October 2004   *Corresponding author: J. Blanco Laboratorio de Referencia de E. coli (LREC) Departamento de Microbiología y Parasitología Facultad de Veterinaria Universidad de Santiago de Compostela 27002 Lugo, Spain Tel. & Fax +34-982285936 E-mail: jba@lugo.usc.es

 

 

Genes de virulencia y tipos de intiminas de Escherichia coli productoras de toxinas Shiga aisladas de ganado bovino y carne de vacuno en Argentina Resumen. En este estudio hemos caracterizado un total de 153 Escherichia coli productores de toxinas Shiga (STEC) aisladas de las heces de ganado bovino y de carne picada y hamburguesas de vacuno en Argentina. Los ensayos de PCR mostraron que 22 (14%) aislamientos llevaban el gen stx1, 113 (74%) presentaban el gen stx2 y que 18 (12%) tenían ambos genes. Los genes de virulencia para la intimina (eae), la enterohemolisina (ehxA) y la adhesina autoaglutinante de STEC (saa) fueron detectados en 36 (24%), 70 (46%) y 34 (22%) de los aislamientos, respectivamente. Ninguno de los 34 aislamientos saa-positivos llevaba el gen eae, pero 31 eran ehxA-positivos. Catorce aislamientos (7 del serotipo O26:H11 y 4 del serotipo O5:H-) tenían la intimina b1, 16 poseían la intimina g1 (11 del serotipo O145:H- y 5 del serotipo O157:H7), 5 aislamientos eran positivos para la intimina tipo ε1 (4 de los serotipos O103:H- y O103:H2), y un aislamiento O111:H- mostró la intimina tipo θ/g2. Aunque los 153 aislamientos de STEC pertenecían a 63 seropatotipos, sólo 12 constituían el 58% de los aislamientos. El seropatotipo ONT:H- stx2 (18 aislamientos) fue el más común, seguido por el O171:H2 stx2 (12 aislamientos), etc. La mayoría de los aislamientos (84%) de STEC pertenecían a serotipos encontrados previamente en seres humanos y el 56% a serotipos asociados con STEC aislados de pacientes con el síndrome urémico hemolítico (HUS). Por tanto, este estudio confirma que el ganado bovino es un importante reservorio de STEC patógenos para humanos. Según nuestra información, este es el primer estudio que describe la presencia del gen saa en STEC de los serotipos O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, y ONT:H21. Los serotipos O120:H19 y O185:H7 tampoco habían sido descritos previamente en STEC de origen bovino. [Int Microbiol 2004; 7(4):269-276] Palabras clave: Escherichia coli O157:H7 · intimina · serotipos de STEC · E. coli productoras de toxinas Shiga · genes de virulencia

Genes de virulencia e tipos de intiminas de Escherichia coli produtoras de toxinas Shiga isolados de gado bovino e carne bovina na Argentina Resumo. Neste estudo, um total de 153 Escherichia coli produtoras de toxinas Shiga (STEC) isoladas de fezes de gado bovino e de carne moida e hamburgues de carne bovina na Argentina foram caracterizados. Os ensaios de PCR mostraram que 22 (14%) dos isolados possuiam o gene stx1, 113 (74%) o gene stx2 e que 18 (12%) tinham ambos os genes. Os genes de virulência para a intimina (eae), a enterohemolisina (ehxA) e a adesina autoaglutinante de STEC (saa) foram detectados em 36 (24%), 70 (46%) e 34 (22%) dos isolados, respectivamente. Nenhum dos 34 isolados saa-positivos tinham o gene eae, no entanto 31 eram ehxA-positivos. Catorze isolados (7 do serótipo O 26:H11 e 4 do serótipo O5:H-) tinham a intimina b1, 16 possuiam a intimina g1 (11 do serótipo O145:H- e 5 do serótipo O157:H7), 5 isolados foram positivos para a intimina tipo ε1 (4 dos serótipos O103:H- e O103:H2), e um isolado O111:H- apresentou a intimina tipo θ/g2. Enquanto os 153 isolados de STEC pertenciam a 63 seropatótipos, somente 12 representaram 58% dos isolados. O seropatótipo ONT:H- stx2 (18 isolados) foi o mais comum, seguido pelo O171:H2 stx2 (12), etc. A maioria dos isolados (84%) de STEC pertenciam à serótipos encontrados previamente em seres humanos e cêrca de 56% à serótipos associados com STEC isolados de pacientes com a síndrome urémica hemolítica (HUS). Esses dados confirmam que o gado bovino é um importante reservatório de STEC patogênicos para humanos. Segundo nossa informação, este é o primeiro estudo que descreve a presença do gene saa em STEC dos serótipos O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, e ONT:H21. Os serótipos O120:H19 e O185:H7 também não haviam sido descritos previamente em STEC de origem bovina. [Int Microbiol 2004; 7(4):269-276] Palavras chave: Escherichia coli O157:H7· intimina · serótipos de STEC · E. coli productoras de toxinas Shiga · genes de virulência

 

Introduction

Shiga-toxin-producing Escherichia coli (STEC), also called verotoxin-producing E. coli (VTEC), is the most important recently emerged group of food-borne pathogens. These bacteria can cause severe disease in humans, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) [3,5,6,17,30]. Cattle, especially young animals, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of food-borne outbreaks [2,4,5,7,9,18,20,25-27,34,41]. Argentina has one of the highest recorded frequencies of HUS in the world (300-400 cases/year). It also registers the highest consumption of bovine meat (60 kg/person year) [19].

Human and bovine STEC elaborate two potent phage-encoded cytotoxins, called Shiga-toxins (Stx1 and Stx2) or verotoxins (VT1 and VT2) [16,30]. 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 [16]. Intimin is encoded by the chromosomal gene eae, which is part of a pathogenicity island termed the locus for enterocyte effacement (LEE) [16]. Severe diarrhea (especially in HC) and HUS are closely associated with STEC types carrying eae [17,30]. 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 [42]. Intimin type-specific PCR assays identified 17 variants of eae encoding 17 different intimin types and subtypes (a1, a2, b1, ξR/b2B, δ/κ/b2O, g1, θ/g2, ε1, νR/ε2, ζ, η, ι1, μR/ι2, λ, μB, νB, ξB) [1,6,8,12,24,36,38,42]. Apart from the capability to produce Shiga toxins and intimins, STEC may have accessory putative virulence factors, such as the enterohemolysin (Ehly), also called enterohemorrhagic E. coli hemolysin (EHEC-HlyA), which is encoded by ehxA [35], and the STEC autoagglutinating adhesin (Saa), encoded by saa [14,29].

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.es/ecoli). Most outbreaks of HC and HUS have been attributed to strains of the enterohemorrhagic serotype O157:H7 [4,17,23]. However, as STEC non-O157 are more prevalent in animals and as contaminant in foods, humans are probably more exposed to these strains. 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 non-O157 STEC types in human disease needs further examination [2-4,6,10,17,37]. Although more than 400 different O:H serotypes of STEC have been isolated from cattle (a total of 435 serotypes are listed in the authors' website, http://www.lugo.usc.es/ecoli), there is a lack of information regarding associations between serotype, intimin types, and virulence factor profiles among bovine STEC isolates [9,11,21,33,40].

Thus, the aim of this study was to establish the serotypes, virulence genes and intimin types of STEC isolated from cattle and beef products in Argentina in order to establish whether bovine STEC have the same serotypes and virulence-factor profiles as STEC strains that cause human infections.

Data from this study have been partly presented as a poster communication at the 5^th Int. Symp. on "Shiga toxin (verocytotoxin)-producing Escherichia coli infections", Edinburgh, UK [Blanco JE, et al. (2003) Serotypes and virulence genes (vt1, vt2, eae, ehxA, saa) of VTEC isolated from cattle, food and humans in Argentina. Abstr. P196, p 177].

Materials and methods

E. coli isolates and control strains. A total of 153 STEC isolated as indicated previously [26,27,34] from feces of diarrheic and healthy cattle (n = 131) and beef products (hamburgers and ground beef) (n = 22) in Argentina were characterized in this study. Only one isolate for each animal and food sample was included. E. coli strains used as controls were: EPEC-2348 (human, O127:H6, eae-a1), AEEC-IH2498a (human, O125:H6, eae-a2), EPEC-337 (human, O111:H2, eae-b1), EPEC-359 (human, O119:H6, eae-ξR/b2B), AEEC-6044/95 (human, O118:H5, eae-δ/κ/b2O), STEC-EDL933 (human, O157:H7, stx₁, stx₂, eae-g1, ehxA), STEC-TW07926 (human, O111:H8, stx₁, stx₂, eae- θ/g2), STEC-VTB-286 (bovine, O103:H2, stx₁, eae-ε1), AEEC-IH3205a (human, O123:H19, eae-νR/ε2), STEC-VTO-50 (ovine, O156:H-, stx₁, eae-ζ), AEEC-CF11201 (human, O125:H-, eae-η), AEEC-7476/96 (human, O145:H4, eae-ι1), AEEC-217-2 (human, O101:H-, eae-μR/ι2), AEEC-68-4 (human, O34:H-, eae-λ), EPEC-373 (human, O55:H51, eae-μB), AEEC-IH1229a (human, O10:H-, eae-νB), STEC-B49 (bovine, O80:H-, stx₁, eae-ξB) and K12-185 (negative for stx₁, stx₂, eae, ehxA, and saa). Strains were stored at room temperature in nutrient broth with 0.75% agar.

Detection of virulence genes by PCR and serotyping. The methodology used for the detection of virulence genes and for serotyping has been described elsewhere [8,9,13]. Base sequences and predicted sizes of amplified products for specific oligonucleotide primers used in this study are shown in Table 1.

Results

Virulence genes. A total of 153 STEC isolates from cattle and beef products in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx₁ genes, 113 (74%) possessed stx₂ genes and 18 (12%) both stx₁ and stx₂. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried eae, and 31 were ehxA positive (Table 2).

Serotypes and seropathotypes. STEC isolates belonging to 33 O serogroups and 47 O:H serotypes were identified. However, 57% were of one of the following 12 serogroups (O5, O20, O26, O39, O91, O103, O113, O117, O145, O157, O171, and O174) and 69% of the isolates belonged to only 13 serotypes (O5:H-, O20:H19, O26:H11, O39:H49, O91:H21, O113:H21, O117:H7, O145:H-, O157:H7, O171:H2, O174:H21, ONT:H- and ONT:H21). The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.

Although the 153 STEC isolates belonged to 63 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx₂ (18 isolates) was the most common, followed by O171:H2 stx₂ (12 isolates), ONT:H21 stx₂ (9 isolates), O145:H- stx₂ eae-g1 ehxA (9 isolates), O174:H21 stx₂ (8 isolates), O117:H7 stx₂ (7 isolates), O26:H11 stx₁ eae-b1 ehxA (5 isolates) and O157:H7 stx₂ eae-g1 ehxA (5 isolates).

Gene saa was detected in eae-negative strains of serotypes O8:H16, O20:H19 (5 isolates), O22:H8, O39:H49 (5 isolates), O74:H28, O79:H19, O91:H21 (4 isolates), O113:H21, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, ONT:H8, ONT:H16, ONT:H21 and ONT:HNT.

Typing of eae (intimin) genes. Fourteen isolates (7 of serotype O26:H11 and 4 of serotype O5:H-) had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type ε1 (4 of serotypes O103:H- and O103:H2), and one isolate, O111:H-, showed intimin type θ/g2 (Table 2).

Discussion

In view of the increasing importance of STEC as emerging food-borne pathogens and reports on O157:H7 and non-O157 STEC causing severe illness, evaluation of associated virulence factors of such isolates is required. In the present work, we established the serotypes, virulence genes and intimin types of STEC isolated from cattle and beef products in Argentina in order to determine whether bovine STEC possess the same serotypes and virulence-factor profiles as STEC isolates that cause human infection. To our knowledge, this study is the first to document the detection of saa and to have carried out intimin typing in STEC isolated in Argentina.

It should be noted that the majority (84%) of the 153 bovine STEC isolates characterized in this study belonged to serotypes previously found in human STEC, and 56% to serotypes associated with STEC isolated from patients with HUS. Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. Similarly, Meichtri et al. [20] found in Argentina 86 bovine STEC isolates belonging to 34 serotypes, 17 of which had been previously associated with human disease. The typeable bovine STEC isolated by Meichtri et al. [20] belonged to 17 O serogroups, and, in order of frequency, were: O8 (16 isolates), O113 (14), O103 (5), O91 (4), O171 (3), O174 (3), O25 (2), O112 (2), O145 (2), O2 (1), O11 (1), O104 (1), O121 (1), O128 (1), O143 (1), O146 (1) and O157 (1). The serotype O8:H19 was the most prevalent (13%) [20].

In Spain, Blanco et al. [9] found that 514 STEC isolates belonged to 66 O serogroups and 113 O:H serotypes. However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and O177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19), including 10 serotypes also found among STEC that cause human infections and six serotypes associated with HUS (O20:H19, O22:H8, O26:H11, O105:H18, O113:H21, and O157:H7). Likewise, among the 20 serotypes most frequently isolated in cattle or cattle products in Canada by Johnson et al. [15], 18 were isolated from humans, and 11 of those were serotypes associated with bloody diarrhea/hemorrhagic colitis and/or HUS. Pradel et al. [31] in France, Beutin et al. [2], and Montenegro et al. [22] in Germany, and Wells et al. [39] in the USA also found that many STEC recovered from cattle belonged to serotypes previously associated with human disease. Along with other authors [11,15,21,40], we observed that bovine and human STEC of the same serotype have similar known virulence-associated properties. Of the 153 STEC isolates serotyped in the present study, 35 (23%) were considered O non-typeable (ONT) with all available O antisera (O1 to O185). This highlights the need to extend the serotyping scheme to include new and emerging STEC serogroups. Our study also identified two new serotypes (O120:H19 and O185:H7) not previously reported in bovine STEC strains.

Gene eae, which has been shown to be necessary for attaching and effacing activity, encodes a 94- to 97-kDa outer-membrane protein (OMP), which is termed intimin [16]. Many investigators have underlined the strong association between carrying eae and the capacity of STEC to cause severe human disease, especially HUS [1,3,6,24,30]. This important virulence gene was detected in 100% of STEC O157:H7 and in 21% of non-O157 bovine isolates assayed in the present study. A similar prevalence of the intimin gene has also been found in other studies [4,9]. Nevertheless, the production of intimin is not essential for pathogenesis, because a number of sporadic cases of HUS have been caused by eae-negative non-O157 STEC isolates. Thus, STEC O104:H21 and O113:H21 isolates lacking eae were responsible for an outbreak and a cluster of three HUS cases in the USA and Australia, respectively [28,30]. Paton and Paton [29] recently described a novel autoagglutinating adhesin, designated Saa, in an eae-negative O113:H21 STEC isolate responsible for an outbreak of HUS. In our study, the saa-positive STEC isolates (22% of all isolates) were all eae-negative and often ehxA-positive. However, a recent study showed that there is no significant association between STEC isolated from patients with HUS and saa [14]. Nevertheless, the possible role of saa in human infection remains controversial. Further studies are required to elucidate the contribution of this gene to milder human gastrointestinal conditions, such as diarrhea. However, in the present and in previous studies [14,43], saa was frequently found in bovine and ovine STEC, suggesting that Saa may have a role in the attachment to bovine and ovine gut. To our knowledge, this is the first study to describe the association of saa with serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21.

Analysis of the nucleotide sequences of the intimin genes from different STEC and enteropathogenic E. coli (EPEC) strains has shown a high degree of homology in the 5´ two-thirds of the genes and a significant degree of heterogeneity in the 3´ one-third of the genes [1,12]. Seventeen variants of eae were identified by intimin type-specific PCR assays using oligonucleotide primers complementary to the 3´-end of the specific intimin genes that encode for the intimin types and subtypes a1, a2, b1, ξR/b2B, δ/κ/b2O, g1, θ/g2, ε1, νR/ε2, ζ, η, ι1, μR/ι2, λ, μB, νB, ξB [1,6,8,24,32,36,38,42]. As in human strains, the intimin types b1 and g1 are the most frequently found among bovine STEC isolates. Intimin b1 was mainly found among strains belonging to serotypes O5:H- and O26:H11, whereas intimin type g1 was detected in all STEC isolates of serotypes O145:H- and O157:H7 assayed.

The highly virulent seropathotypes O26:H11 stx₁ or stx₂ eae-b1 ehxA (7 isolates), O103:H2/ H- stx₁ and/or stx₂ eae-ε1 ehxA (4 strains), O118:H16 stx1 eae-b1 ehxA (1 isolate), O145:H- stx₁ or stx₂ eae-g1 ehxA (11 isolates), and O157:H7 stx₂ eae-g1 ehxA (5 isolates) were detected in 28 bovine STEC characterized in the present study. Although our results and those of other authors indicate that STEC strains of human and animal origin with the same serotype are similar with respect to the presence of known virulence-associated factors [2,10,11,21,33], the results of Boerlin et al. [10] suggest that STEC isolates from humans form a different population from those found in the bovine reservoir or that they are only a subpopulation of the latter. Thus, future studies are necessary to establish whether animal and human strains represent the same clones or are only related subpopulations.

Acknowledgements. We thank Monserrat Lamela and María R. Ortiz for skillful technical assistance and Alejandro L. Soraci, Javier Margueritte and Pablo Molina for their collaborations. This work was supported by grants from the Fondo de Investigación Sanitaria (grants FIS G03-025-COLIRED-O157 and FIS 98/1158), the Xunta de Galicia (grants PGIDIT02BTF26101PR and PGIDIT04RAG261014PR), from the Comisión Interministerial de Ciencia y Tecnología (grants CICYT-ALI98-0616 and CICYT-FEDER-1FD1997-2181-C02-01), and European Commission (FAIR programme grants CT98-4093 and CT98-3935), FONCYT (PICT9-5115 BID1201 OC/AR), SECYT-UNCPBA, and Scientific Researches Commission Prov. Buenos Aires (CIC). A. Mora and G. Dahbi acknowledge the Xunta de Galicia and the Agencia Española de Cooperación Internacional (AECI) for research fellowships. A.E. Parma and A.I. Etcheverría are members of CIC; A. Krüger and G.H. Arroyo are members of CONICET.

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