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Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925 / Biomedical Sciences

<p> Glycopeptide Antibiotics (GPAs) such as vancomycin are often used clinically as antibiotics of last resort against infections due to Gram-positive bacteria that are resistant to more commonly used antibiotics such as methicillin. The clinical emergence of vancomycin resistant enterococci (VRE) and vancomycin resistantS. aureus (VRSA) necessitates methods to evade this resistance. </p> <p> GP As consist ofa heptapeptide backbone that is cross-linked to create a pocket that binds the D-Alanyl-D-Alanine terminus of peptidoglycan intermediates, inhibiting strengthening ofthe cell wall and resulting in susceptibility to osmotic stress. Resistance to GPAs occurs when D-Ala-D-Lactate replaces D-Ala-D-Ala and the GPA pocket can no longer bind effectively. In order to create novel binding pockets, we must understand the specificity ofthe P450 monooxygenase enzymes that have been shown to catalyze the cross-links. The 4 P450-encoding genes ofthe GPA A47934 biosynthetic cluster of Streptomyces toyocaensis as well as genes encoding electron transport proteins necessary for P450 function from Streptomyces coelicolor were cloned in Escherichia coli for heterologous expression and characterization. One P450, StaJ was purified and shown to bind CO as expected using spectrophotometric tests. </p> <p> The genes responsible for GP A resistance are regulated by a two component regulatory system consisting ofa sensor kinase (VanS) and a response regulator (V an.R). In order to probe the events leading to VanS autophosphorylation and ultimately resistance activation we utilize a series of GP A derivatives harbouring the photo labile group benzophenone as well as the fluorescent and affinity moieties BODIPY and biotin. Benzophenone permits light controlled covalent binding of the GP A to proteins that bind them while BODIPY allows fluorescence detection and biotin allows enrichment and detection by Western analysis. We report that this system was insufficient to clearly identify vancomycin binding proteins due to background signals despite multiple rounds of troubleshooting. It must be our conclusion that under the conditions tested, there are no proteins that bind the GP A derivative used in this study. </p> / Thesis / Master of Science (MSc)

As the problem of resistance increases in the current health care system, new solutions to this problem are not emerging at a similar rate. The ability to discover novel antibiotics, and modify existing antibiotics, is competing with highly evolving resistance profiles. An alternate solution to this problem may be to search for inhibitors of these resistance mechanisms and pairing them with current antibiotics. Proof of this hypothesis lies in the great success of P-lactamase inhibitors already in the clinic. Inhibitors may be created using synthetic methods, however searching for inhibitors found in the natural environment may lead to a greater success. For example, bacteria in their natural setting must cope with constant exposure to antibiotics secreted by both themselves and by other species. As well, bacteria must be able to handle encounters with other species that are resistant to their own defense mechanisms. With this in consideration, it is possible that these bacteria have already established an ability to challenge resistance encountered in their own environment, such as through the secretion of compounds that inhibit these mechanisms. Screening of such inhibitors can be done against purified resistance elements or via cell-based screens with resistant bacteria. The focus of this research was to develop expression systems which contain inducible antibiotic resistance genes to be used for whole-cell screening for inhibitors of antibiotic resistance. The expression systems studied were pSWEET, for use in the Gram positive bacterium Bacillus subtilis, and pETcoco, for use in the Gram negative bacterium Escherichia coli. It was found that the pSWEET expression system integrated into the B. subtilis chromosome at unspecified locations and was not an ideal system for the proposed screen. pET coco holds promise as a suitable expression system but at this point in time it requires further examination to ensure plasmid stability and reproducibility of results. Therefore further examination of these two systems is needed if they are to be used in a screen for inhibitors, and a search for substitute systems must be undertaken. / Thesis / Master of Science (MSc)