Relevance of Biofilms in the Pathogenesis of Shiga-Toxin-ProducingEscherichia coliInfection

Natalia Angel Villegas,José Baronetti,Analía Etcheverría,Inés Albesa,Maria Becerra,Rosana Polifroni,Nora Padola,Alberto Parma,Maria Paraje

doi:10.1155/2013/607258

Abstract

The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

Highlights

Hemorrhagic colitis, which occasionally progresses to hemolytic uremic syndrome (HUS) in children and other susceptible groups of individuals, is a hallmark of human infection with Shiga toxin-producing Escherichia coli (STEC)
A quantitative analysis of biofilm formation indicated that the three STEC strains showed “weak biofilm producer” biofilm formation in tryptic soy broth (TSB) (Figure 1(a))
When assays were performed with thioglycollate medium in aerobiosis with the addition of glucose, an increase in biofilm formation was seen in strain N∘ 1 (BBU = 1.87 ± 0.05), N∘ 2 (BBU = 2.16 ± 0.07), and N∘ 3 (BBU = 1.97 ± 0.07) too ( #P versus thioglycollate medium < 0.01)

Summary

Introduction

Hemorrhagic colitis, which occasionally progresses to hemolytic uremic syndrome (HUS) in children and other susceptible groups of individuals, is a hallmark of human infection with Shiga toxin-producing Escherichia coli (STEC). Escherichia coli O157 : H7 is the serotype most commonly associated with clinical disease and foodassociated outbreaks. Vero cells have a high sensitivity to Stx, and the cytotoxicity assay using this cell line is often used as the gold standard to evaluate diagnostic immunoassays. These cells have on their plasma membrane receptors a high concentration of Gb3 and Gb4, allowing detection of Stx, Stx, and its variants. STEC strains released toxins into the culture medium and the specific cytotoxicity on Vero cells is used to determine the ability of bacterial isolates from clinical specimens and Stx producing food. Stx toxin is produced and released into the medium continuously during the exponential growth phase, while Stx toxin accumulates in the periplasmic space of the bacteria and it released at the end of the exponential phase [6, 7]

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