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Title: Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

item AMEZQUITA-LOPEZ, BLANCA - Center For Research In Food And Development (CIAD)
item Quiñones, Beatriz
item Lee, Bertram
item CHAIDEZ, CRISTOBAL - Center For Research In Food And Development (CIAD)

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2014
Publication Date: 1/30/2014
Publication URL:
Citation: Amezquita-Lopez, B.A., Quinones, B., Lee, B.G., Chaidez, C. 2014. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells. Frontiers in Cellular and Infection Microbiology. DOI: 10.3389/fcimb.2014.00007.

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) are considered to be a major cause of food-borne disease. STEC can cause a wide variety of disease symptoms in humans, ranging from diarrhea, watery and bloody diarrhea, to life-threating diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS). Cattle are considered to be the major carrier of STEC strains. However, recent studies have indicated that small domestic ruminants, including sheep and goats, have been implicated as carriers of STEC. The severity of the disease in humans has been associated with more than 100 serotypes of STEC. A lot of cases of HC and HUS have been accredited to STEC serotype O157:H7 strains, which is the most commonly-associated serotype with HUS in North America. Recent epidemiological studies have documented additional non-O157 serotypes, including O26, O45, O91, O103, O104, O111, O113, O121, and O145, among STEC strains that were associated with severe disease symptoms in the United States, Europe and Latin America. The pathogenesis of STEC in humans requires several bacterial virulence factors. In particular, the production of Shiga toxins (Stx) is thought to contribute to the development of severe symptoms in humans. Stxs have been classified in two major types, Stx1 and Stx2, and several variants of the stx1 and stx2 genes have been identified. Of which, the Stx1 has three variants (stx1a, stx1c and stx1d), while the Stx2 group contains seven variants, corresponding to stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g. Epidemiological studies suggest that STEC strains positive for stx2a may be more virulent than strains positive for only stx1a or both stx1a and stx2a. However, additional studies documented that infection by STEC that have the stx1a subtype can cause HUS, whilst STEC harboring stx1c appear to be associated with mild disease or asymptomatic carriage. Additional molecular typing studies of STEC have demonstrated that there is a strong correlation between strains with stx2 subtypes and severe illness, such as bloody diarrhea and HUS. For example, among stx2 positive STEC strains, those harboring both subtypes stx2a and stx2d were found to be associated with higher virulence and severe disease symptoms (HUS). Many STEC strains that produce Shiga toxin do not cause HUS, demonstrating that additional virulence factors are required to cause illness. For example, virulence factors present on pathogenicity islands (PAIs), such as the locus of enterocyte effacement (LEE) and the non-LEE. The LEE contains the eae gene (intimin) that is an important virulence factor responsible for intimate attachment to intestinal epithelial cells. The non-LEE PAI contains genes such as iha, an iron-regulated gene A homolog adhesin. The non-LEE effector (Nle) molecules are proposed to be involved in STEC infection. Additionally, other chromosomal and plasmid virulence genes, encoding proteases, cytotoxins, adhesins, and receptor proteins, are suggested to contribute to STEC pathogenesis by permitting these bacterial pathogens to attach and colonize the human epithelium. The detection of these virulence genes in STEC strains would provide key information on the identification of those risk factors that may contribute to the development of human disease. In the present study, STEC strains were isolated from feces of domestic animals that were raised in small farms located in rural communities. The small rural farms were located in rural communities where the primary purpose of raising livestock is for local consumption. To determine the relative virulence of STEC strains isolated from domestic farm animals in rural areas of Mexico, the present study identified the several virulence factors and Shiga toxin variants, and examined the relative cytotoxicity in mammalian of stx subtypes expressed by STEC strains

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammalian cells by STEC strains isolated from domestic farm animals in rural farms in Mexico. The STEC strains were analyzed by using PCR with sequence-specific primers to determine the presence of genes encoding intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system protein (etpD), subtilase cytotoxin (subA), autoagglutinating adhesion (saa), type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2), Ol-71 (nleH1-2 and nleA) and the distinct variants of Shiga toxin (stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g). Also, we evaluated the cytotoxicity the STEC strains by use of a fluorescent Vero cell-based method. Most of the STEC strains (93%; 27/29) carried a gene coding for at least one of the stx2 variants. In particular, the stx2a subtype, which is associated with severe illness, was present in 10% (3/29) of the STEC strains. Other stx subtypes identified were stx1a, stx1c, stx2b, stx2c and stx2d. A total of 45% (13/29) of the STEC strains were PCR-positive for more than one stx subtype, and among them the virulence gene profile that was most frequently-observed was stx1a, stx1c, stx2b. The virulence gene more commonly detected was ehxA in 80% (26/29) of the STEC strains. Other virulence genes such as espP, katP and etpD were more associated with the serotype O157:H7. The cytotoxicity in mammalian cells was higher in the non-O157 STEC strains with serotypes O8:H19, O75:H8, O146:H8 and O146:H21. Moreover, the stx subtypes associated with higher cytotoxicity were stx1a/stx1c/stx2b and stx1a/stx2a/stx2c, identified in 83% (24/27) and 16.7% (4/29) of the tested STEC strains, respectively. In conclusion, the STEC strains analyzed were found to be diverse in their genetic composition, and the presence of more than one stx gene variant in the same isolate may contribute to a higher cytotoxicity in mammalian cells.