Prevalence of mucus activatable stx2d among eae-negative STEC strains. Association between this Shiga toxin type and severe clinical outcome.

MILIWEBSKY E; DEZA N; GALLI L; CHINEN I; CARBONARI CC; BASCHKIER A; MANFREDI E; RIVAS M

CABA

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli Infections; 2009

Asociación Argentina de Microbiología

Shiga toxin (Stx) is the major virulence factor implicated in the pathogenesis of Shiga toxin producing Escherichia coli (STEC). The Stx1 and Stx2 families were subdivided into Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2-O118, Stx2-OX3a, Stx2d, Stx2e, Stx2f and Stx2g subtypes. The designation of Stx2dactivatable has been derived from its capacity to be activated in its biological activity, demonstrated as a significant increase in its cytotoxicity for cultivated cells, in the presence of intestinal mucus. The presence of activatable Stx in STEC infected humans and the activation of this Stx by intestinal mucus during infection might result in increased virulence of such strains for humans. This toxin has been found among eae-negative STEC strains and significantly associated with the ability to cause severe disease, including bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). The aims of this study were to investigate the prevalence of Stx2d in a collection of human eae-negative STEC strains and to determine the association between Stx2d and clinical outcome. A total of 42 Vero-cells-cytotoxic STEC strains, isolated during 2001-2007 period from patients with BD, non-BD (D) and asymptomatic carriers (AC) were tested for the Stx2dactivatable gene (stx2d) by PCR and PstI restriction analysis. The stx genetic variants of strains included in this study have been previously identified as: stx2 (n=18 strains); stx2vh-b (n=17); stx1/stx2 (n=2); stx2/stx2vh-b (n=2); stx2vh-a (n=1); stx1/stx2vh-b (n=1); stx1c/stx2vh-b (n=1); by PCR-RFLP B-subunit analysis (Tyler, 1991; Zhang, 2002). The stx2d gene was identified in 29 (69%) strains. Twenty out of 21 strains stx2vh-b (alone or in combination with other gene), and 9 out of 20 strains stx2 (alone or with stx1), were positive for stx2d. However, one strain stx2vh-a isolated from D case and other strain stx2vh-b isolated from AC, were negative. The strains harboring stx2d belonged to 13 serotypes, and the most common were O8:H19 (n=3 strains) and O174:H21 (n=10). The analysis of association between stx2d and clinical outcome, including 25 STEC strains that harbored stx2d as the sole stx gene, was performed. In addition, two strains were excluded because the case clinical data were not available. The stx2d gene was detected among strains isolated from 11 out of 18 HUS cases, 7 out of 12 D cases and 5 out of 6 AC. Although, STEC strains that were stx2d account for most of the eae-negative strains isolated from patients with HUS (61.1%), the direct association between stx2d strains with the ability to cause severe disease (HUS) compared with milder disease (D) was not statistically significant (p=0.6). Studies of the stx2d characterization including more strains and the detection of the activation of Stx-cytotoxicity by intestinal mucus in Vero cells assay are necessary to confirm the results. It is important to emphasize that eae-negative STEC stx2d strains were isolated from severe disease and the detection of this stx variant might predict the high pathogenic potential of the isolated STEC.