Search

Cutibacterium acnes (C. acnes) (formerly known as Propionibacterium acnes) forms part of the normal resident host microflora of the skin, large intestine and oral cavity. Despite increasing evidence for the opportunistic pathogenic role of this species within intervertebral disc (IVD) herniation and associated Modic changes, C. acnes is considered a low virulence microorganism. This thesis aimed to investigate the virulence characteristics of 67 clinical C. acnes isolates belonging to phylotypes IA1, IB, II and III obtained from human herniated IVD tissue material and eight acne lesion isolates. Phenotypic isolate characterisation was undertaken using crystal violet biofilm formation assays, Galleria mellonella (G. mellonella) waxworm infection assays, exocellular enzyme production agar assays and Western blot protein analysis. Associations between phylotype grouping and site of isolation of the isolates were then investigated. Biofilm formation crystal violet assays showed that phylotype III disc tissue isolates failed to form significant biofilms. Semi-quantitative biofilm formation classification criteria identified the majority of disc tissue and acne lesion isolates as weak biofilm formers over 7-days, indicative of the slow growth rate of this species. Phylotype-specific exocellular enzyme production demonstrated that phylotype IA1and IB isolates commonly produced β-haemolysins (84% and 67%, respectively). Gelatinaseproduction was phylotype and site of isolation-specific. The highest rates of non-specific protease enzyme production were recorded for C. acnes phylotypes IA1 and III, with 100% of these isolates showing protease activity. Ubiquitous lipase production was recorded amongst all C. acnes disc tissue and acne lesion isolates tested. Comparison of waxworm larvae mortality rates between differing C. acnes phylotypes and culture preparation techniques (cell-free, heat-killed and cell-containing inoculum preparations) showed no significant effects on G. mellonella survival rates, suggesting that C. acnes does not induce significant mortality within this infection model. However, all phylotypes of C. acnespromoted G. mellonella melanisation, indicative of insect humoral immune response activation, with cell-containing cultures of C. acnes isolates 82 (IB) and 80 (III) leading to the greatest degrees of melanisation. Therefore, the G. mellonella infection model issensitive and can discriminate between the pathogenicity of C. acnes belonging to a range of phylotypes. Additionally, following Western blotting, disc tissue C. acnes samples produced CAMP factor 1 protein. Overall, site of C. acnes isolation (IVD or acne lesion) and phylotype appeared to influence phenotypic characteristics. This suggests that the role of C. acnes within lumbar disc herniation etiology should not be readily dismissed and highlights the need for further research into the applicability of anti-CAMP factor vaccines within C. acnes disc disease management.

With the steady increase in global antimicrobial resistance rates, concomitant with a recent paucity of novel antibiotic discovery, it is essential that new antimicrobial compounds be discovered to prevent a return to the pre-antibiotic era over coming decades. Knowledge of the medicinal properties of traditional plants has given rise to many of the modern day medicines. Consequently, the aim of this work was to assess the antimicrobial and antibiofilm activities of a number of traditional African herbal remedies against a range of clinically important bacterial species. After a pilot study was performed, Prosopis africana and Uvaria chamae were selected from a range of medicinal plants due to their greater antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli than the other remedies tested. Crude extracts from P. africana (PAM, PAS and PAU) and U. chamae (UCM, UCS and UCU) were prepared using maceration, Soxhlet and ultrasound extraction method respectively. Cultures of both aerobic and anaerobic bacterial species were exposed to the crude extracts in agar-well diffusion and broth micro-dilution assays. In addition, semi-quantitative and quantitative antibiofilm assays were performed using PAS, PAU, UCS and UCU extracts against preformed biofilms of P. aeruginosa, S. aureus, S. epidermidis, Propionibacterium acnes and Clostridium difficile strains. Fractionation of the crude extracts was then performed using column chromatography; with antimicrobial, simple phytochemical and spectrophotometric analyses being performed on the resultant fractions. It was found that all extracts had both bacteriostatic and bactericidal activity against all of the bacterial species tested, although the Soxhlet and ultrasound extracts demonstrated a significantly greater activity than the maceration extracts. The extracts demonstrated antibiofilm activity against preformed biofilm of P. aeruginosa, S. aureus, S. epidermidis, P. acnes and C. difficile strains by reducing the density of biofilm biomass significantly by at least 11.5 % compared to the control. In the antimicrobial activity test, the fractions that were the most effective against P. aeruginosa and S. aureus with MIC and MBC value ranges of 0.195 % - 2.60 % and 0.391 % - 6.25 % respectively, contained the following phytochemical groups; saponins, quinones, flavonoids, and alkaloids which have been previously shown to be antimicrobial by other workers. Absorption spectrum analysis further suggests that the fractions of U. chamae and P. africana with the greatest antimicrobial activity might contain flavonoid-related compounds as their major active components. This study demonstrated that P. africana and U. chamae extracts are effective antimicrobial and antibiofilm agents and therefore the isolation and evaluation of their active ingredients could yield potentially new leads to fight antimicrobial resisance.