Molecular analysis of virulence mechanisms associated with adherent-invasive Escherichia coli (AIEC)

Crohn's Disease (CD) is a chronic inflammatory bowel disease of unknown etiology. Recent work has shown that a new pathotype of Escherichia coli, Adherent Invasive E. coli (AIEC) may be associated with CD. AIEC has been shown to adhere to and invade epithelial cells and to replicate within macrophages (together this is called the AIEC phenotype). In this thesis, the AIEC phenotype of 84 E. coli strains were determined in order to identify the prevalence of this phenotype within the E. coli genus. This study showed that a significant proportion of E. coli strains (approx. 5%) are capable of adhering to and invading epithelial cells and undergoing intramacrophage replication. Moreover, the results presented in this study indicate a correlation between survival in macrophage and resistance to grazing by amoeba supporting the coincidental evolution hypothesis that resistance to amoebae could be a driving force in the evolution of pathogenicity in some bacteria, such as AIEC. In addition, this study has identified an important regulatory role for the CpxA/R two component system (TCS) in the invasive abilities of AIEC HM605, a colonic mucosa-associated CD isolate. A mutation in cpxR was shown to be defective in the invasion of epithelial cells and this defect was shown to be independent of motility or the expression of Type 1 fimbriae, factors that have been shown to be involved in the invasion of another strain of AIEC, isolated from a patient with ileal CD, called LF82. The CpxA/R TCS responds to disturbances in the cell envelope and has been implicated in the virulence of a number of Gram negative pathogens. In this study it is shown that the CpxA/R TCS regulates the expression of a potentially novel invasin called SinH. SinH is found in a number of invasive strains of E. coli and Salmonella. Moreover work presented here shows that a critical mechanism underpinning AIEC persistence in macrophages is the repair of DNA bases damaged by macrophage oxidants. Together these findings provide evidence to suggest that AIEC are a diverse group of E. coli and possess diverse molecular mechanisms and virulence factors that contribute to the AIEC phenotype. In addition, AIEC may have gone through different evolutionary histories acquiring various molecular mechanisms ultimately culminating in the AIEC phenotype. The gastrointestinal (GI) tract harbors a diverse microbiota; most are symbiotic or commensal however some bacteria have the potential to cause disease (pathobiont). The work presented here provides evidence to support the model that AIEC are pathobionts. AIEC strains can be carried as commensals in healthy guts however, when the intestinal homeostasis is disrupted, such as in the compromised gut of CD patients, AIEC may behave as opportunistic pathogens and cause and/or contribute to disease by driving intestinal inflammation.

Supporting therapist engagement in evidence based practice: Developing a continuing education programme through participatory action research

Therapists find it challenging to integrate research evidence into their clinical decision-making because it may involve modifying their existing practices. Although continuing education (CE) programmes for evidence-based practice (EBP) have employed various approaches to increase individual practitioner’s knowledge and skills, these have been shown to have little impact in changing customary behaviours. To date, there has been little attempt to actively engage therapists as collaborators in developing educational processes concerning EBP. The researcher collaborated with seven clinical therapists (one occupational therapist, four physiotherapists and two speech and language therapists) enrolled in a new post-qualification Implementing Evidence in Therapy Practice (IETP) MSc module to monitor and adapt the learning programme over ten weeks. The participating therapists actively engaged in participatory action research (PAR) iterative cycles of reflecting→ planning→ acting→ observing→ reflecting with the researcher. Mixed methods were used to evaluate the IETP module and its influence on therapists’ subsequent engagement in EBP activities. Data were gathered immediately on completion of the module and five months later. Immediate post-module findings revealed four components as being important to the therapists: 1) characteristics of the learning environment; 2) acquisition of relevant EBP skills; 3) nature of the learning process; and 4) acquiring confidence. The two themes and sub-themes which emerged from individual interviews conducted five months post-module expanded on the four components already identified. Theme 1: Experiencing the learning (sub-themes: module organisation; learning is relational; improving the module); and theme 2: Enacting the learning through a new way of being (sub-themes: criticality and reflection; self agency; modelling EBP behaviours; positioning self in an EB work culture). The therapists’ perspectives had by then shifted from that of a learner to that of a clinician constructing a new sense of self as an evidence-based practitioner. Findings from this study underline the importance of the process of socially constructed knowledge and of empowering learners through collaboratively designed continuing education programmes. In the student-driven learning environment, therapists chose repetitive skill-building and authentic problem-solving activities which reflected the complexity of the environments to which they were expected to transfer their learning. These findings have implications for educators designing EBP continuing education programmes, during which students develop professional ways of being.