No abstract

The indigenous microbial community of the gastrointestinal (GI) tract determines susceptibility to Clostridium difficile colonization and disease. Previous studies have demonstrated that antibiotic-treated mice challenged with C. difficile either developed rapidly lethal C. difficile infection or were stably colonized with mild disease. The GI microbial community of animals with mild disease was dominated by members of the bacterial family Lachnospiraceae, while the gut community in moribund animals had a predominance of Escherichia coli. We investigated the roles of murine Lachnospiraceae and E. coli strains in colonization resistance against C. difficile in germfree mice. Murine Lachnospiraceae and E. coli isolates were cultured from wild-type mice. The ability of each of these isolates to interfere with C. difficile colonization was tested by precolonizing germfree mice with these bacteria 4 days prior to experimental C. difficile challenge. Mice precolonized with a murine Lachnospiraceae isolate, but not those colonized with E. coli, had significantly decreased C. difficile colonization, lower intestinal cytotoxin levels and exhibited less severe clinical signs and colonic histopathology. Infection of germfree mice or mice precolonized with E. coli with C. difficile strain VPI 10463 was uniformly fatal by 48 h, but only 20% mortality was seen at 2 days in mice precolonized with the Lachnospiraceae isolate prior to challenge with VPI 10463. These findings confirm that a single component of the GI microbiota, a murine Lachnospiraceae isolate, could partially restore colonization resistance against C. difficile. Further study of the members within the Lachnospiraceae family could lead to a better understanding of mechanisms of colonization resistance against C. difficile and novel therapeutic approaches for the treatment and prevention of C. difficile infection.

The current report presents our initial findings on the immune response to C. difficile infection (CDI) in an acute mouse model. 5-8 week old male C57BL/6 mice were either left untreated or received a cocktail of 5 antibiotics in drinking water for 3 days followed by a single intra-peritoneal dose of clindamycin. The antibiotic-treated mice were then infected with 105 CFUs of the C. difficile strain VPI10463 and sacrificed 42 hours later. Flow-cytometric study of the spleens, mesenteric lymph nodes (MLN), colons and ceca of the infected mice showed a significant increase in the numbers of B cells, CD4 and CD8 T cells, FOXP3+ CD4 cells, NK cells and neutrophils in the colons and ceca, as well as cells of the monocyte/macrophage lineage and dendritic cells in the ceca of the infected mice. The spleens and MLN of the infected mice did not display such an increase. A substantial fraction of the recruited neutrophils in the colons and ceca of the infected mice displayed upregulated levels of CD11b, whereas a similar fraction of CD4 and CD8 cells in the colons and ceca of uninfected and infected mice showed an upregulated level of CD69. Multiplex qPCR analysis of colonic and cecal tissue from the infected mice showed a significant increase in the mRNA levels of numerous neutrophil-attracting chemokines and a number of cytokines produced in an innate immune response, but no indication of T cell polarization, thereby collectively underscoring the innate, local nature of the response.