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Kinsmore, Natasha Louise (2019) Understanding certain Clostridium difficile virulence and antibiotic resistance factors and how these relate to patient clinical outcome. PhD thesis, University of Nottingham.

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Abstract

Clostridium difficile is known for causing the nosocomial diarrhoeal disease, C. difficile infection (CDI). The aim of this study was to investigate the molecular and phenotypic basis of certain C. difficile virulence factors, such as C. difficile transferase (CDT) and sporulation, as well as rifaximin antibiotic resistance, and how these relate to patient clinical outcome.

The molecular basis of CDT was studied by creating in-frame deletion mutants (R20291ΔcdtR, R20291ΔcdtAΔcdtB, R20291ΔcdtRΔcdtAΔcdtB, R20291ΔPaLoc, R20291ΔPaLocΔcdtR, R20291ΔPaLocΔcdtAΔcdtB and R20291ΔPaLocΔcdtRΔcdtAΔcdtB) via allelic exchange gene knock-out. Transcriptional linkage of the binary toxin gene locus by reverse transcriptase (RT) PCR, and in silico promoter analysis was completed. The investigation identified promoters upstream of the cdtR and cdtA genes, however, the locus was also discovered to be transcriptionally linked. This knowledge was used to create complementations of the CDT in-frame deletion mutant strains (R20291∆cdtR*cdtR and R20291ΔcdtRΔcdtAΔcdtB*cdtR*cdtA*cdtB) using a two-step method. All generated strains were subsequently characterised for differences in growth, cytotoxicity and biofilm formation. A key outcome from this characterisation was that the Vero cell cytotoxic effect in all CDT knock-out strains were significantly decreased when compared to the R20291 wildtype (WT) strain, despite the presence of the PaLoc.

Part of the research was completed in conjunction with the Nottingham Digestive Diseases Biomedical Research Unit (NDDBRU) on the Rifaximin for Preventing Relapse of Clostridium Associated Diarrhoea (RAPID) clinical trial. C. difficile was isolated from patient stool samples and ribotyped. A CDT PCR was established to amplify and sequence the CDT locus from patient isolates and then compared to known strains. This analysis revealed that the cdtR in ribotype 078 strains possess a premature stop codon, and in clinical strains containing the ghost CDT gene locus, the cdtR is well conserved. Further work is necessary to determine the significance of these findings to patient outcome. One of the most interesting outcomes of the RAPID clinical trial was the development of rifaximin resistance in five patients. Four single nucleotide polymorphisms (SNPs) (Gln489Lys, Arg505Lys, Ser507Leu and Ile750Met) were determined in eight isolates. Lastly, sporulation was investigated in the clinical samples with the colony-forming units per ml (cfu/ml) shown to be increased at later time points. Concurrently, a ClosTron mutant was created to knock out the C. difficile 630 gene 26350, which encodes for a putative spore envelope protein. A sporulation assay demonstrated that the cfu/ml was reduced when compared to the control strain, indicating a potential important role in sporulation. However, the exact mechanism and identification of numerous genes which influence sporulation, remain unknown.

The precise role of certain C. difficile virulence and antibiotic resistance factors, and how these relate to patient outcome, is yet to be completely alluded to. However, the work presented in this study has demonstrated that the CDT gene locus, and indeed the ghost locus, are significant. It also indicates in certain individuals that exposure to rifaximin increases the risk of developing resistance. Nonetheless, many questions remain about the binary toxin and the synergistic effect with toxin A and B, the effect on colonisation and adherence, and the impact on the immune response. It is also necessary to research strain and host specific interactions before recommending rifaximin as appropriate treatment for the prevention of C. difficile relapse. Furthering knowledge on certain virulence factors will ultimately lead to improved diagnostics, therapeutic care and prevention/eradication measures.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Minton, Nigel Kuehne, Sarah
Keywords:	C. difficile virulence factors; C. difficile transferase; Sporulation; Rifaximin antibiotic resistance
Subjects:	Q Science > QR Microbiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	57076
Depositing User:	Kinsmore, Natasha
Date Deposited:	27 Mar 2020 15:48
Last Modified:	15 Oct 2021 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/57076

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