Immune response to clostridium difficile infection and an investigation of the mechanisms of moxifloxacin resistance in clinical C. difficile isolates

Abstract

Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.