Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient for inducible nitric oxide synthase (iNOS) did not support growth of E. coli by nitrate respiration, suggesting that nitrate generated during inflammation was host-derived. Thus the inflammatory host response selectively enhances growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

SummarySalmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta , the two genes required to produce acetylphosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA . We also found that a D D D D ( ackA-pta ) mutation alone had no effect on virulence but, in combination with D D D D ( barA ), it increased the oral LD 50 24-fold. Thus, the combined loss of the BarA-and acetate-dependent pathways is required to reduce virulence. Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella , whereas those of the large intestine inhibit invasion.

BackgroundChanges in the microbial populations on the skin of animals have traditionally been evaluated using conventional microbiology techniques. The sequencing of bacterial 16S rRNA genes has revealed that the human skin is inhabited by a highly diverse and variable microbiome that had previously not been demonstrated by culture-based methods. The goals of this study were to describe the microbiome inhabiting different areas of the canine skin, and to compare the skin microbiome of healthy and allergic dogs.Methodology/Principal FindingsDNA extracted from superficial skin swabs from healthy (n = 12) and allergic dogs (n = 6) from different regions of haired skin and mucosal surfaces were used for 454-pyrosequencing of the 16S rRNA gene. Principal coordinates analysis revealed clustering for the different skin sites across all dogs, with some mucosal sites and the perianal regions clustering separately from the haired skin sites. The rarefaction analysis revealed high individual variability between samples collected from healthy dogs and between the different skin sites. Higher species richness and microbial diversity were observed in the samples from haired skin when compared to mucosal surfaces or mucocutaneous junctions. In all examined regions, the most abundant phylum and family identified in the different regions of skin and mucosal surfaces were Proteobacteria and Oxalobacteriaceae. The skin of allergic dogs had lower species richness when compared to the healthy dogs. The allergic dogs had lower proportions of the Betaproteobacteria Ralstonia spp. when compared to the healthy dogs.Conclusions/SignificanceThe study demonstrates that the skin of dogs is inhabited by much more rich and diverse microbial communities than previously thought using culture-based methods. Our sequence data reveal high individual variability between samples collected from different patients. Differences in species richness was also seen between healthy and allergic dogs, with allergic dogs having lower species richness when compared to healthy dogs.

The centisome 63 type III secretion system (T3SS-1) encoded by Salmonella pathogenicity island 1 (SPI1) mediates invasion of epithelial cells by Salmonella enterica serotype Typhimurium. Characterization of mutants lacking individual genes has revealed that T3SS-1 secreted proteins (effectors) SopE2 and SopB are required for invasion while the SipA protein accelerates entry into cells. Here we have revisited the question of which T3SS-1 effectors contribute to the invasion of epithelial cells by complementing a strain lacking all of the effector genes that are required to cause diarrhea in a calf (a sipA sopABDE2 mutant). Introduction of either the cloned sipA, the cloned sopB, or the cloned sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for nonpolarized HT-29 cells. However, a contribution of sopA or sopD to invasion was not apparent when invasion assays were performed with the nonpolarized colon carcinoma cell lines T84 and HT-29. In contrast, introduction of either the sopA, the sopB, the sopD, or the sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells. Furthermore, introduction of a plasmid carrying sipA and sopB increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells significantly compared to the introduction of plasmids carrying only sipA or sopB. We conclude that SipA, SopA, SopB, SopD, and SopE2 contribute to S. enterica serotype Typhimurium invasion of epithelial cells in vitro.

Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.