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

摘要

Clostridium difficile is an increasingly common cause of nosocomial infection. C.uddifficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage toudmild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. Itudis not yet fully understood why this spectrum is seen, however, it is believed that theudimmune response mounted by an individual plays an important role in determining theudoutcome of infection.udThis thesis comprises three studies. Firstly, a comparative study of immune celludpopulations within the lamina propria of colonic tissue not exhibiting pathologicaludchanges and taken from individuals with symptomatic CDI (cases); asymptomaticudcarriers; and non-colonised controls. Effector T cells, B cells, plasma cells andudmacrophages were enumerated by means of immunohistochemical staining of tissueudsections. Secondly, a study to establish the prevalence within these three study groupsudof specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8udgenes by PCR genotyping and to determine whether an association existed betweenudthese genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms ofudmoxifloxacin resistance in a collection of clinical isolates. This study also sought touddetermine whether the competitive advantage conferred by resistance to moxifloxacinudinfluenced the fitness of C. difficile isolates, in particular growth and the expression ofudthe virulence factors toxins A and B.udCarriers were found to have fewer of all four immune cell types quantified than bothudcases 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 butudthese differences were not significant. These findings suggest that individuals whoudbecome infected, both symptomatically and asymptomatically, with C. difficile displayudaltered mucosal immune cell populations when compared with those of uninfectedudindividuals.udThe data regarding host polymorphisms are suggestive of an association between theudpresence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2udgenotypes were carried by cases and carriers while the variant TLR5 genotype wasudcarried by cases only. No variant genotypes were present in control subjects.udAll moxifloxacin resistant isolates characterised in this study, with the exception of anudisolate with intermediate resistance and a third-generation mutant with reducedudsusceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Effluxudpumps are known to play a role in multi-drug resistance in many bacterial species. SemiquantitativeudPCR analysis of expression of the putative efflux pumps cme and cdeAudfound no correlation between overexpression and moxifloxacin resistance, suggestingudthat these genes do not play a role. Three novel mutations in the putative promoterudregion of CD3197, a MerR family transcriptional regulator found immediately upstreamudof cme, were identified. No association between the presence of these mutations andudoverexpression of cme or resistance or sensitivity to moxifloxacin was found. Theudcompetitive advantage conferred by resistance to moxifloxacin does not influence theudfitness of C. difficile isolates, as measured in terms of growth and toxin production.