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This thesis examined the gut microbiota in individuals at risk of rheumatoid arthritis (RA) to investigate the role of the gut microbiome in RA development. The study included participants with pre-clinical RA and healthy controls. Samples were collected at multiple time points over a 15-month period. The results showed that there were changes in the gut microbiome associated with RA progression, specifically affecting the Firmicutes and Proteobacteria phyla. A particular strain of Prevotellaceae was more abundant in at-risk individuals compared to healthy controls. Temporal analysis suggested that RA progression involves the development of a less diverse microbiome characterized by the accumulation of RA-associated pathobionts. These findings confirm and expand on previous research linking bacterial associations, including Prevotellaceae, to the progression of RA. Further investigations will explore the relationship between bacterial specialisation and the risk profile in the development of RA-related autoimmunity.

Cystic fibrosis (CF) is a genetic disease affecting multiple body organs; however, pulmonary complications remain the most common cause of morbidity and mortality. An increasing body of evidence has linked fungi to worsening CF disease, but their role has not been fully explored, partly due to challenges associated with diagnosis. The aim of this thesis was to better understand the role of immune response in the diagnosis of Aspergillus related diseases and to gain a full picture of fungi found in CF airways - the mycobiome, and how it relates to clinical conditions and treatments. Three studies were performed. First, serum samples were analysed from CF patients with Allergic Bronchopulmonary Aspergillosis (ABPA), Aspergillus sensitisation, and Aspergillus bronchitis for their IgE and IgG response to Aspergillus recombinant antigens. Second, 126 CF patients were recruited, and their sputum samples were analysed by conventional culture methods and next generation sequencing comparing two internal transcribed sequence ribosomal DNA regions to assess the mycobiome. In a subset of patients, lung mycobiome was investigated in relation to lung disease severity as well as CFTR modulator therapy. Third, the impact of antifungals on the lung mycobiome was investigated in a longitudinal study where sputum samples from before and during/post treatment were analysed. Asp f1 and f2 antigens were associated with Aspergillus bronchitis, with Asp f1 in particular was significantly linked to culture positivity. Patients with ABPA showed a more heterogeneous response ranging from allergy to infection like presentation. The mycobiome analysis revealed more fungi than conventional methods. Amplification of ITS2 rDNA provided better detection of Aspergillus and Exophiala species. However, fungal burden seems to decline in patients with severe lung disease and in those on CFTR modulators. In patients receiving antifungal therapy, the lung mycobiome remains largely stable.

Clostridioides difficile infection (CDI) is the leading cause of infective antibiotic-associated diarrhoea and is responsible for significant patient morbidity and mortality. Despite appropriate antimicrobial therapy, CDI recurs in approximately 20-30 % of cases, suggesting that C. difficile can occupy a protective niche whereby antimicrobial therapy is ineffective. Biofilms represent such a potential niche. We sought to determine the role of biofilms in recurrent CDI (rCDI) and how the sessile community can affect C. difficile biofilm formation. A triple stage chemostat model of the human colon was used to predict the efficacy of a simulated faecal microbiota transplantation (FMT) and two different dosing regimens of a spore consortium, SER-109, to prevent rCDI and to define a role for biofilms in rCDI. Planktonic and biofilm communities were individually analysed using culture-based techniques and bacterial taxonomic sequencing. Bile acid levels were monitored to investigate potential mechanisms of efficacy. A biofilm batch culture assay was used to investigate the influence of biofilm-associated microbiota on C. difficile biofilm formation. Results show that FMT and a triple dose of SER-109 successfully prevented rCDI, potentially due to reducing the levels of primary bile acids through conversion to secondary bile acids. Despite the ability of microbiome therapies to prevent rCDI, they failed to eradicate C. difficile from the biofilm, suggesting a risk of future CDI. The biofilm-associated C. difficile was able to seed the planktonic phase, resulting in C. difficile germination and proliferation, which proved to be sufficient to induce CDI. Biofilm batch culture experiments indicate that commensal biofilm populations can reduce or increase C. difficile biofilm formation and growth, which required the presence of viable cells. We conclude that biofilms provide a protective niche for C. difficile, which facilitates CDI recurrence, and that patients undergoing microbiome-based therapies potentially remain at risk of CDI with subsequent antibiotic use.

Clostridium difficile infection (CDI) is an infection of the gastrointestinal tract causing symptoms ranging from mild diarrhoea to life-threatening toxic megacolon. Between 10-30% of patients suffer a recurrent episode (rCDI) after an initial episode. Some patients develop multiple recurrent episodes, leading to unpleasant cycles of disease and antimicrobial therapy. This thesis utilises a multidimensional approach to study rCDI. In Chapter 2, previously generated clinical data is used to assess the effect of treatment delay on two outcomes; diarrhoeal duration and risk of recurrence. It was hypothesised that delays initiating treatment result in increased symptom duration and recurrence risk. Logistic regression models highlighted treatment delay has no significant effect on diarrhoeal duration or recurrence risk. The only significant variable associated with risk of recurrence was previous CDI (P < 0.001). These findings suggest clinicians should not be overly concerned by treatment delays in mild/moderate CDI. In Chapter 3, the germination and thermotolerance properties of five strains of C. difficile spores were investigated. In the nosocomial environment spores may be reingested by the patient, germinate and initiate fulminant disease. Additionally, spores can persist in the gastrointestinal tract and germinate in response to stimulatory cues. C. difficile spore recovery was optimised by using variety of media and supplements. The ribotype (RT) 078 strain germinated more efficiently in the absence of additional supplementation. RT 027/078 strains were more thermotolerant. Intrinsic differences in spore germination characteristics between clades could facilitate the increased ability of some strains to cause rCDI. In Chapter 4, an in vitro gut model was used to simulate rCDI. Previous research has characterised changes in the microbiota that occur in response to antibiotics. In this study a metaproteomic approach was utilised to study the overarching metabolic processes occurring during simulated rCDI. Although dysbiosis was evident, the metaproteome remained fairly constant throughout simulated infection.

Clostridium difficile infection (CDI) laboratory diagnostic assays have variable performance, but reasons behind this variability are not well defined. In contrast to previous findings, the PCR ribotype of the organism only appears to be a factor in reduced sensitivity for toxin enzyme immunoassays (EIAs), not glutamate dehydrogenase (GDH) EIAs. Growth curve data demonstrated that GDH is produced during the exponential phase of growth, and the sensitivity of the GDH assay in vivo may be related to the amount of protein produced by the organism, as very high levels of GDH were detected during growth of C. difficile in an in vitro gut model. Indeed the levels of GDH, measured in both gut model and patient samples, correlated with organism bioload. In addition, the median faecal levels of GDH in recurrent CDI cases were significantly higher than in patients with a single infection episode. Interestingly, when patients had sequential faecal samples tested, 27% with an initial GDH-positive/toxin-negative result had a subsequent toxin positive sample, after a median of eight days. Further studies, with supplementary assays for gut inflammation, are required, to determine if these are ‘missed’ infections or insignificant sub-clinical levels of toxin. A laboratory test that could predict risk of recurrence would be an important tool to inform choice of appropriate C. difficile treatment and prevention options. Indeed GDH detection may offer such an opportunity; a cohort of patients has been identified who were consistently GDH positive, even after resolution of symptoms, who subsequently developed recurrent CDI following additional antimicrobial therapy. The cycle threshold (CT) value of PCR assays for the detection of toxin gene may also provide additional information, as low CT (< 25) was significantly associated with toxin positivity, presence of PCR ribotype 027 and mortality. Low CT was also associated with recurrence but was not a significant finding.

Clostridioides difficile is the major cause of infectious antibiotic-associated diarrhoea, imparting a substantial clinical and financial burden on healthcare facilities. Resistance development, particularly to fluoroquinolones, has been implicated in major, international epidemics, predominantly associated with the hyper-virulent ribotype 027. The development of multiple antimicrobial resistance may contribute significantly to the considerable clinical challenges associated with this organism. In this study, optimised germination environments, antimicrobial susceptibility testing and next generation sequencing were utilised in the recovery and characterisation of an historical C. difficile collection (1980-86). Epidemiological comparisons of ribotype distribution and susceptibility patterns with modern surveillance data (2012-2016) sought to reveal antimicrobial resistance variance between two distinct periods. By correlating phenotypic resistance and genetic determinants, the dissemination of resistance genes was evaluated. Contributions of bacterial mutability to resistance propagation were investigated in response to fluoroquinolone exposure amongst seven prevalent, clinical ribotypes; (n=44). Through in vitro batch and continuous co-culture modelling, the impact of resistance-conferring gyrA and gyrB mutations on the fitness of ribotype 027 (n=7) was assessed. The majority of test antimicrobials (n=8/9) were less active against modern vs historical isolates. This is potentially due to increased antimicrobial exposure and subsequent selection/expansion of resistant strains. Moxifloxacin testing demonstrated the largest increase in resistant populations, reinforcing the notion of reduced susceptibility to modern fluoroquinolones as a potential contributory factor in disease. Phylogenetic analyses highlighted the complexity of molecular clock predictions, with 69% of historical genomes correlating with a 95% prediction interval. Elevated mutability was observed amongst ribotype 027, suggesting greater propensity for resistance evolution in this type. Common fluoroquinolone resistance-conferring substitutions revealed advantages to bacterial fitness. Continuous, competitive co-culture modelling of a Thr82 > Ile mutant, 027 strain emphasised the fitness benefits of this polymorphism, retained in the absence of fluoroquinolone pressure. These findings indicate a potential contribution to the success of this ribotype.

William Adam enjoys a reputation as one of the most important trumpet teachers in the twentieth and twenty-first centuries and is known as a pedagogue who develops highly proficient trumpet performers and teachers. Adam earned this reputation primarily as professor of trumpet at Indiana University from 1946-1988.

Clostridium difficile infection (CDI) is associated with significant patient morbidity, mortality and financial burden. Until recently, antimicrobial treatment options were limited to metronidazole and vancomycin, but both agents are associated with recurrence rates of approximately 20%. The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. Sessile organisms are known to cause chronic infection such as cystic fibrosis. Mucosal biofilms exist on surfaces of the gastrointestinal tract, but the existence and role of C. difficile in these structures remains unknown. The present study describes the process undertaken to adapt and validate an in vitro human gut model to study the planktonic and biofilm mode of growth of C. difficile and the indigenous gut microbiota. A triple stage chemostat gut model, primed with a human faecal emulsion was used to induce and treat simulated CDI. A glass rod system was incorporated into the third vessel to facilitate the formation and subsequent analysis of mixed-species biofilms. Sessile and planktonic gut microbiota and C. difficile populations within an in vitro gut model are similar in the absence of antimicrobial intervention. Differences in behaviours of the two modes of growth are evident upon antimicrobial administration, with a delayed response in sessile populations. The sessile mode of growth of C. difficile within mature biofilm structures is complex and variable. Within the redesigned biofilm gut model, sessile C. difficile remained in spore form for the duration of the experiment, despite induction of simulated CDI, treatment of CDI and recurrence of disease evident within planktonic communities. Recalcitrant spores within biofilms may be seeded into the planktonic fluid of the gut model after apparent successful initial treatment and contribute to recurrence of CDI. The role of sessile C. difficile in recurrent CDI should be further investigated.