Stress and survival of clostridium difficile

Abstract

CDI has been implicated in significant morbidity and mortality rates leading to complications such as pseudomembranous colitis, toxic megacolon and sepsis. Several classical symptoms include infectious and profuse diarrhoea, high grade fever, nausea and abdominal cramps.

The aim of this thesis was to insert a functional dnaK heat shock gene in the ClosTron generated C. difficile 630Δerm::dnaK by using a pMTL80000 series plasmid to restore the wildtype phenotype thus correcting the growth rate and temperature sensitivity. The dnaK gene, under the influence of a strong fdx promoter, conjugated successfully into C. difficile 630Δerm::dnaK. For comparison, a novel pyrE- background method established at the University of Nottingham was used to create two further dnaK disruption mutants, C. difficile 630∆erm∆pyrE::dnaK and C. difficile R20291ΔpyrE::dnaK. These pyrE- dnaK disruption mutants were then complemented by the insertion of pMTL-YN series plasmids containing a functional dnaK gene. The process was approached in a logical fashion for construction and complementation of the new dnaK deletion mutants in the pyrE- background. It was verified by PCR that the dnaK gene successfully inserted into the pMTL-YN series plasmids and that these plasmids were successfully conjugated into the required C. difficile isolates. Results achieved from the growth curve experiments and motility assays showed that the dnaK deletion mutants in the pyrE- background and associated complements displayed no difference in their growth in comparison to the wild-type (C. difficile 630Δerm).

Another objective of this thesis was to phenotypically characterise the original dnaK disruption mutant. Three C. difficile isolates (C. difficile 630, C. difficile 630Δerm and C. difficile 630Δerm::dnaK) were subjected to temperature stress ranging from 4 to 45°C and subsequently antibiotic stress using nine various antibiotics with differing gradient concentrations (10-500 μg/mL). Results of these stress tests highlighted differences between all three isolates. The antibiotic stress tests have indicated that C. difficile 630Δerm::dnaK is sensitive to gentamicin compared to C. difficile 630 and C. difficile 630Δerm. This may significantly impact future therapy options giving greater choices of treatment. Data from antibiotic stress tests have also indicated that C. difficile 630 has heteroresistance to metronidazole, one of the first line treatments for CDI. This provided a foundation for further investigations using 108 C. difficile clinical isolates (17 different ribotypes) to identify a possible pattern of heteroresistance to metronidazole among a N. Ireland C. difficile population. Results from the clinical isolates indicated that there was a 15% heteroresistance rate for metronidazole in N. Ireland with C. difficile ribotype 078 being completely heteroresistant. The results from the antibiotic stress studies in particular the heteroresistance to metronidazole highlight the need for new and improved treatment regimes for C. difficile.

Date of Award	Aug 2016
Original language	English
Sponsors	Department for Employment and Learning, Northern Ireland
Supervisor	Nigel Ternan (Supervisor) & Geoffrey Mc Mullan (Supervisor)