Immune response to clostridium difficile infection and an investigation of the mechanisms of moxifloxacin resistance in clinical C. difficile isolates
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Date
2010Author
Wroe, Allison J.
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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.