No description available.

The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA.

No description available.

No description available.

The incidence of antibiotic resistance amongst bacteria has increased steadily due to the over prescription of antibiotics for minor ailments in addition to their overuse in the agricultural industry. As such it is imperative that we identify new therapies to combat the growing threat from antibiotic resistant pathogens. The aim of this study was to investigate the antimicrobial potential of a number of novel alpha helical peptides from diverse sources. These peptides shared common biophysical features of host defense peptides (HDPs) such as size, cationicity and amphipathicity. The nature of these peptides suggests they could function as topical antimicrobial agents and the oral cavity represents an ideal location for their use . This study has shown that truncation of LL-37 to produce shorter mimetics generates peptides with enhanced antimicrobial efficacy against a range of oral pathogens in addition to pathogens from the skin, lung and gut. In one case truncation of LL-37 produced a peptide (KE-18) with increased potential to bind to lipopolysaccharide and lipoteichoic acid. A further mimetic, KR-12 has potent an ti-candidal peptide with decreased haemolytic activity. In addition this study has shown that the truncated mimetics of LL-37 investigated had a similar mechanism of action to that of the parent molecule as all were shown to disrupt the bacterial membrane. Peptides from the African volcano frog (Xenopus amieti) displayed potent antimicrobial efficacy against a range 'of oral pathogens. Then exhibited minimal haemolytic activity suggesting these peptides may be useful templates for the development of possible therapeutics designed for the oral cavity. PGLa-AMl and CPF-AMl both displayed excellent ability to inhibit the growth of Streptococcus mutons suggesting these peptides could have a potential role in the prevention of dental carries. ~. The study identified a number of "cryptic" peptides (the 10 family of peptides) which , displayed antimicrobial activity and some ability to bind to lipopolysaccharide. One peptide (31Q3) merited further investigation as the others showed modest antimicrobial activity against examples of Gram positive, Gram negative and fungal pathogens. Overall, this study has identified a number of alpha helical peptides which have the potential to be investigated further as possible therapeutics for the treatment of a number of oral conditions .

The flagellum of Campy/obaeter jejuni is a major virulence determinant, dependent on about 40 flagellar genes that are co-ordinately expressed. However, the existence of a flagellar regulon in C. jejuni and the genes that may control it are so far undefined. To elucidate this, three putative global regulatory gene homologues, rpoN, fliA and flgR encoding the alternative sigma factors, 0'54, 0'28 and the 0'54 -associated transcriptional activator FlgR respectively, were selected. The genes were cloned and mutated by inverse PCR mutagenesis. Kanamycin resistant mutants were constructed by allelic replacement in two C. jejuni strains, NCTC 11168 and NCTC 11828. Electron microscopic studies showed that the rpoN and flgR mutants were nonflagellate; the fliA mutant had truncated flagella. Immunoblotting confirmed lack of flagellin expression in the rpoN and flgR mutants and partial flagellin expression in the fliA mutant. Thus neither the flaB gene with a 0'54 promoter encoding the minor flagellin, nor the flaA gene with a 0'28 promoter encoding the major flagellin are expressed in the rpoN andflgR mutants. These observations confirm the roles of rpoN,flgR andfliA gene homologues in flagellar expression in C.jejuni. Promoter analysis of all the flagellar genes in C. jejuni shows that rpoN and flgR are essential regulatory genes in the putative flagellar regulon of C. jejuni. Microarray hybridisations and analyses reveal that mutation of the rpoN and/or flgR genes affects regulation of expression of several flagellar genes. These analyses shed light on the distinct difference in flagellar regulon between enteric bacteria and C. jejuni and outline the flagellar regulon of the latter. Studies were also carried out with two putative flagellar regulatory genes, Inr and regX3. However, mutations in these genes did not affect the nature of the flagella.

BACKGROUND. Intensive care units (ICUs) are "hot" areas for antibiotic consumption, infection and antibiotic resistance. Resistance affects patient outcomes, resource utilization and determines whether treatments are adequate. Within Europe, Italy has among the highest rates of antibiotic consumption but resistances - particularly in ICUs - are largely unexplored, both nationally and regionally. OBJECTIVES. To assess variation in antibiotic prescribing, consumption, resistance, and treatment outcomes and to identify critical points for improvement in antimicrobial practice across 5 ICUs in the Veneto region, North-east Italy. RESULTS. From 2002 to 2010, 911 patients were reviewed. Median K'U stay (17 days; IQR, 8-29) and ICU mortality (mean, 24.9%) were similar across sites. Empirical antibiotics were given to 853 patients (83.1%), with penicillin/β-lactamase inhibitor combinations (26%), cephalosporins (20.7%), fluoroquinolones (10.9%), and carbapenems (9.8%) frequently used. Laboratory investigation was often long delayed (median 7 days IQR = 3-14) after treatment initiation, and there were few (37.2%) microbiological-based shifts; 30.9% of empirical regimens were inadequate. Treatment inadequacy (AOR=13.99) and septic shock (AOR=3.29) were the main independent predictors for hospital mortality. Amongst 1908 isolates tested - predominantly, Pseudomonas aeruginosa (22%) and MRSA (14.8%) - 53.7% were multiresistant, with significant inter-hospital differences in resistance rates of Enterobacteriaceae to fluoroquinolones, and for P. aeruginosa to fluoroquinolones and carbapenems) (p < 0.001). The relationship between resistance and use of fluoroquinolones and 3rd_ generation cephalosporins was clear for Enterobacteriaceae (p < 0.001), but weaker for P. aeruginosa. The susceptibility of Escherichia coli to fluoroquinolones decreased over time (X2 = 0.009). Antibiotic use was inflated, especially at one ICU with excess of fluoroquinolone (94.4 DDD/100 bed-days vs. 26.1-35.9 elsewhere) use. CONCLUSIONS. Considerable inter-hospital variation III prescribing affected antibiotic consumption and resistance prevalence. Poor and delayed use of laboratory microbiology was prominent, as was the uncontrolled use of antibiotics. Urgent interventions are needed and improvement strategies are discussed.

Antimicrobial drug resistance is an expanding world-wide problem causing increased mortality and morbidity as well as a considerable financial burden on health care services. Acquired resistant determinants represent themain contributor to drug resistance in bacteria. The IMP-1 metallo-B-lactamase is one of the most important emerging drug resistance mechanisms . IMP is normally encoded on a class 1 integron, and its expression is driven by the Pc integron promoter. The work reported in this thesis aimed to investigate factors that contribute to the expression of horizontally acquired genes using blaIMP-1 as a model system.

Nosocomial antibiotic resistance is an increasing problem world wide; some pathogens, particularly Pseudomonas aeruginosa and Acinetobacter baumannii, in some countries have become resistant most useful antibiotics. These bacteria can sequester antibiotic resistance genes and incorporate them into their genome, subsequently spreading them to other bacterial species via a variety of mechanisms. The distribution of resistance genes among both gram-positive and gram-negative pathogens often as not involves mobile genetic elements such as plasmids, transposons, integron and insertion sequence.

In this thesis, I present my published works in the broad field of medical microbiology. Over a period of thirty years I have worked in a range of settings,. including hospital and public health laboratories, major reference laboratories and academic science departments. I have studied significant public health issues associated with each position, and therefore, while there is no single theme in my work, the emphasis of my research has been very much on the development and validation of methods and their application to public health microbiology. My early work was on obligately anaerobic bacteria. I began to study Clostridium difficile during my doctoral research and continued for several years after I completed my PhD. My expertise in anaerobic microbiology led to several book chapters and ultimately to an undergraduate textbook and an edited volume on anaerobic microbiology methods. My work on anaerobic bacteria forms one of three themes in my commentary. During my tenure at the University of Ulster; I began to broaden my research to encompass pathogens in the environment. Areas of interest included the survival of pathogenic bacteria during anaerobic digestion of cattle slurry and the anaerobic bacterial flora of flax retting. I moved to the University of West Indies in Barbados, where for 13 years I taught microbiology to medical students and provided clinical microbiology support to the hospital and public health laboratories. This venue provided me with the opportunity to follow a diverse research agenda, with a strong concentration on diseases of local and regional public health importance. I have included some of this work in my third commentary theme, clinical and public health microbiology. My major contributions to the field have undoubtedly been in the diagnosis, taxonomy and epidemiology of leptospirosis. I began working on leptospirosis in 1992 in the former Medical Research Council laboratory in Barbados. My work here included both serological and molecular diagnostic studies, and epidemiological research on reservoir animals and other zoonotic diseases. After moving to the Centers for Disease Control and Prevention (CDC), my work was focussed on developing tools for molecular diagnosis and molecular typing of leptospires and on taxonomy of leptospires. At the CDC, I was also the Acting Chief of the Special Bacteriology Reference Laboratory. In this capacity, I had the opportunity to lead a group focussed on the identification and characterization of previously unrecognised bacterial pathogens. Some of this work is included in my third commentary theme. I moved to Saskatchewan in 2003, to my current position in the provincial public health laboratory. My research in Saskatchewan has been directed towards the use of molecular techniques for diagnosis and epidemiological typing of significant public health pathogens, while mentoring younger scientists. Community-acquired methicillin-resistant Staphylococcus aureus is a public health challenge in our communities and I have made significant contributions to a collaborative effort to understand and control these infections in Saskatchewan. In recent years I also have assumed a leadership role in biosafety and biosecurity regulatory issues and in quality assurance and proficiency testing. An abbreviated copy of my curriculum vitae is included to provide an indication both of the range of my research work and my other contributions to the field. In the interests of brevity, papers presented at conferences and published abstracts are not included.