Intestinal mucus-derived metabolites modulate virulence of a clade 8 enterohemorrhagic Escherichia coli O157:H7.

The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetyl-galactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease.

Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfbO157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfAO113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.

Cytotoxic effects of Shiga toxin-2 on human extravillous trophoblast cell lines.

Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may have on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound-healing assay and invasion was determined by the 'transwell chamber' assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branch formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose-dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.

Experimental combination therapy using low doses of benznidazole and allopurinol in mouse models of Trypanosoma cruzi chronic infection.

This study evaluated the effectiveness of low doses of benznidazole (BNZ) on continuous administration (BNZc), combined with allopurinol (ALO), in C57BL/6J and C3H/HeN mice infected with Trypanosoma cruzi Nicaragua strain and T. cruzi Sylvio-X10/4 clone. TcN-C57BL/6J was also treated with intermittent doses of BNZ (BNZit). The drug therapy started 3 months post infection (pi) in the chronic phase of mice with heart disease progression, followed-up at 6 months pi. TcN-C57BL/6J treated with BNZc was also monitored up to 12 months pi by serology and electrocardiogram. These mice showed severe electrical abnormalities, which were not observed after BNZc or BNZit. ALO only showed positive interaction with the lowest dose of BNZ. A clear parasitic effect, with significant reductions in antibody titres and parasitic loads, was achieved in all models with low doses of BNZ, and a 25% reduction of the conventional dose showed more efficacy to inhibit the development of the pathology. However, BNZ 75 showed partial efficacy in the TcSylvio-X10/4-C3H/HeN model. In our experimental designs, C57BL/6J allowed to clearly define a chronic phase, and through reproducible efficacy indicators, it can be considered a good preclinical model.

Shiga Toxin-Producing Escherichia coli Infections during Pregnancy.

Gastrointestinal infection with Shiga toxin-producing Escherichia coli (STEC) causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS), characterized by hemolytic anemia, thrombocytopenia and acute renal failure. The main virulence factor of STEC is Shiga toxin (Stx), which is responsible for HUS development. STEC can produce Stx type 1 and/or 2 (Stx1, Stx2) and their variants, Stx2 being more frequently associated with severe cases of HUS. This pathology occurs in 5⁻15% of cases with STEC infection when Stx gain access to the bloodstream and causes damage in the target organs such as the kidney and brain. STEC infections affect mainly young children, although the large HUS outbreak with a new Stx2-producing STEC O104:H4 in Europe in 2011 involved more adults than children, and women were over-represented. Maternal infections during pregnancy are associated with adverse pregnancy outcomes. Studies in rats showed that Stx2 binds to the utero-placental unit and causes adverse pregnancy outcomes. In this article, we provide a brief overview of Stx2 action on placental tissues and discuss whether they might cause pregnancy loss or preterm birth.