Serotypes, Virulence Genes, and Intimin Types ofShiga Toxin (Verotoxin)-Producing Escherichia coli Isolatesfrom Cattle in Spain and Identification of a New Intimin VariantGene(eae-ξ)

ABSTRACT A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx1 genes, 278 (54%) possessed stx2 genes, and 135 (26%) possessed both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new 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 OX177) 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). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx2eae-γ1 ehxA (46 isolates) was the most common, followed by O157:H7 stx1stx2eae-γ1 ehxA (34 isolates), O113:H21 stx2 (25 isolates), O22:H8 stx1stx2ehxA (15 isolates), O26:H11 stx1eae-β1 ehxA (14 isolates), and O77:H41 stx2ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intiminβ 1, 82 O157:H7 isolates possessed intimin γ1, three O111:H- isolates had intimin type γ2, one O49:H- strain showed intimin type δ, 13 (six of serotype O103:H2) isolates had intimin type ε and eight (four of serotype O156:H-) isolates had intimin ζ. We have identified a new variant of the eae intimin gene designated ξ (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.

[1]  S A McEwen,et al.  Distribution and characteristics of verocytotoxigenic Escherichia coli isolated from Ontario dairy cattle , 1992, Epidemiology and Infection.

[2]  V. Gannon,et al.  Immunological Characterization of Escherichia coli O157:H7 Intimin γ1 , 2002, Clinical and Vaccine Immunology.

[3]  J. Paton,et al.  Direct Detection and Characterization of Shiga Toxigenic Escherichia coli by Multiplex PCR for stx1, stx2, eae, ehxA, and saa , 2002, Journal of Clinical Microbiology.

[4]  J. Morris,et al.  Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world. , 1996, Epidemiologic reviews.

[5]  L. Beutin,et al.  Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains , 2002, Journal of Clinical Microbiology.

[6]  H. Flint,et al.  Escherichia coli O157 in Farm Animals , 1999 .

[7]  S. Djordjevic,et al.  Bovine Non-O157 Shiga Toxin 2-Containing Escherichia coli Isolates Commonly Possess stx2-EDL933 and/or stx2vhb Subtypes , 2003, Journal of Clinical Microbiology.

[8]  M. Blanco,et al.  Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina , 2004, European Journal of Epidemiology.

[9]  E. Nielsen,et al.  Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5′ Nuclease PCR Assay , 2003, Journal of Clinical Microbiology.

[10]  F. Scheutz,et al.  Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Cattle, Food, and Children during a One-Year Prospective Study in France , 2000, Journal of Clinical Microbiology.

[11]  G. Dougan,et al.  Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens , 1998, Journal of Clinical Microbiology.

[12]  M. Blanco,et al.  Enterotoxigenic, verotoxigenic, and necrotoxigenic Escherichia coli isolated from cattle in Spain. , 1993, American journal of veterinary research.

[13]  J. Lanser,et al.  Molecular Characterization of a Shiga ToxigenicEscherichia coli O113:H21 Strain Lacking eaeResponsible for a Cluster of Cases of Hemolytic-Uremic Syndrome , 1999, Journal of Clinical Microbiology.

[14]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[15]  J. Blanco,et al.  Distribution and characterization of faecal verotoxin-producing Escherichia coli (VTEC) isolated from healthy cattle. , 1997, Veterinary microbiology.

[16]  W. Wolter,et al.  Shiga‐toxin‐producing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties , 2000, Letters in applied microbiology.

[17]  J. H. Green,et al.  Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle , 1991, Journal of clinical microbiology.

[18]  L. Garber,et al.  Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds , 1995, Applied and environmental microbiology.

[19]  G. Reuter,et al.  Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle , 1990, Journal of clinical microbiology.

[20]  V. Gannon,et al.  Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction , 1993, Journal of clinical microbiology.

[21]  L. Beutin,et al.  Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933 , 1995, Infection and immunity.

[22]  A. Caprioli,et al.  Typing of Intimin Genes in Human and Animal Enterohemorrhagic and Enteropathogenic Escherichia coli: Characterization of a New Intimin Variant , 2000, Infection and Immunity.

[23]  L. Beutin,et al.  Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals , 1993, Journal of clinical microbiology.

[24]  G. Frankel,et al.  Intimin-mediated tissue specificity in enteropathogenic Escherichia coli interaction with human intestinal organ cultures. , 2000, The Journal of infectious diseases.

[25]  J. 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 , 2004, Journal of Clinical Microbiology.

[26]  J. Mainil,et al.  Association between the effacing (eae) gene and the Shiga-like toxin-encoding genes in Escherichia coli isolates from cattle. , 1993, American journal of veterinary research.

[27]  L. Wieler,et al.  Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes , 1996, Journal of clinical microbiology.

[28]  J. Blanco,et al.  Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing Escherichia coli Isolates from Healthy Sheep in Spain , 2003, Journal of Clinical Microbiology.

[29]  J. Blanco,et al.  Prevalence and characteristics of Escherichia coil serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle , 1996, Epidemiology and Infection.

[30]  H. Lior,et al.  Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario , 1996, Epidemiology and Infection.

[31]  M. Karmali,et al.  Infection by verocytotoxin-producing Escherichia coli , 1989, Clinical Microbiology Reviews.

[32]  Hideki Kobayashi,et al.  Prevalence and Characteristics of Shiga Toxin-Producing Escherichia coli from Healthy Cattle in Japan , 2001, Applied and Environmental Microbiology.

[33]  T. Whittam,et al.  Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli. , 1999, Molecular biology and evolution.

[34]  S. McEwen,et al.  Associations between Virulence Factors of Shiga Toxin-ProducingEscherichia coli and Disease in Humans , 1999, Journal of Clinical Microbiology.

[35]  J. Meng,et al.  Virulence genes of Shiga toxin-producing Escherichia coli isolated from food, animals and humans. , 1998, International journal of food microbiology.

[36]  J. Yu,et al.  A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[37]  I. Chinen,et al.  Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil. , 2003, Veterinary microbiology.

[38]  T. Whittam,et al.  Molecular Evolution of the Intimin Gene in O111 Clones of Pathogenic Escherichia coli , 2002, Journal of bacteriology.

[39]  James C. Paton,et al.  Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections , 1998, Clinical Microbiology Reviews.

[40]  R. Sellwood,et al.  Escherichia Coli Associated with Neonatal Diarrhoea in Piglets and Calves , 1981 .

[41]  H. Lior,et al.  Growing Concerns and Recent Outbreaks Involving Non-O157:H7 Serotypes of Verotoxigenic Escherichia coli. , 1996, Journal of food protection.