The Effects Of The Intestinal Environment On Salmonella Pathogenesis And Molecular Identification Of Microbes In The Clinical Laboratory

Abstract

Salmonella causes disease ranging from self-limiting enteritis to septicemia. To cause disease it must first invade the intestinal epithelium. To invade, it employs a type three secretion system and effector proteins encoded on Salmonella Pathogenicity Island 1 (SPI 1). In this work, we investigated the effects of the intestinal environment on Salmonella pathogenesis using an in vivo mouse model of infection as well as in vitro genetic studies. In our in vivo studies, we characterized the intestinal environment in control, streptomycin-treated, Salmonella infected, and streptomycin-treated, Salmonella infected mice. Using 16S rDNA clone libraries we found that the microbiota in the ileum is different from that of the cecum and that streptomycin treatment alters the microbiota in both the ileum and cecum. Upon histopathological examination of the ileum, we also found that pretreatment with streptomycin prior to infection increased Salmonella pathology. We also defined the short chain fatty acids present in both the ileum and cecum using GC-MS and HPLC analysis and found that streptomycin treatment significantly decreased the fatty acid concentrations in the cecum and that this change correlated with an increase in pathology in the cecum. Previous in vitro studies in our lab have shown that the short chain fatty acids propionate and butyrate repress Salmonella SPI 1 invasion genes. In this work, we further confirmed the effects of propionate and butyrate using concentrations of the fatty acids comparable to what we found in the cecum of the mouse. We also found that metabolism of propionate is necessary for the repressive effect on invasion genes and that the metabolic intermediate, propionyl-CoA, is important for this effect. The molecular techniques used to define the microbiota of the intestinal environment are also applicable to the clinical laboratory setting. Use of ribosomal genes for species-level identification has shown promise for both bacteria and yeast. Therefore, we examined the usefulness of these techniques for yeast identification in the clinical veterinary laboratory and found that sequence analysis of the D1/D2 region of the yeast large ribosomal subunit is an effective method of identification.