Nosocomial, or hospital acquired, infections, are ubiquitous within the modern clinical setting leading to over $5 billion annually of related healthcare costs in North America. All indwelling devices are highly susceptible to bacterial colonization where physico-chemical interactions between bacteria and biomaterial surfaces have been implicated as determinant factors in the fate of the initial adhesion processes. It has been proposed that by exploiting interference strategies within this critical step of infection the ability to create 'non-infective' biomaterials may be developed. / This thesis demonstrates the effectivity of North American cranberry (Vaccinium macrocarpon) derived proanthocyanidins in preventing the adhesion of pathogenic bacteria to biomaterial surfaces. Specifically, using a model of catheter associated urinary tract infection, significant reductions in initial adhesion of uropathogenic Escherichia coli and Enterococcus faecalis to PVC and PTFE were observed. With the application of colloidal theory, a mechanism of steric interference was determined as responsible for these effects. / The evidence presented implicates PAC as a molecule of interest for the development of novel biomaterials with increased resistance to bacteria colonization.

A Bacteroides gingivalis adhesin mediating attachment to red blood cells and buccal epithelial cells was isolated, cloned and characterized. The isolation procedure involved gentle stirring of the cells followed by ammonium sulphate precipitation, ion-exchange and gel chromatography. The native molecule had a Mr in excess of 10⁶ kDa and was made up of subunits with an Mr of 43 kDa. Antisera raised to the adhesin and its subunits reacted with antigens on the surface of B. gingivalis cells. No reaction with fimbriae was seen. The IgG fractions from these antisera inhibited the adherence of B. gingivalis to host tissue. Proteolytic enzymes destroyed binding capability of whole cells and of the purified adhesin but the molecular weight of the haemagglutinin was not altered. A genomic library of B. gingivalis DNA was created in E. coli JM83. 5500 colonies were screened by a colony immunoassay with anti-S. gingivalis serum and by a direct haemagglutinating assay. 337 clones tested positive by the immunoassay and two clones, 1-3,and 1-49 tested positive for haemagglutinating activity. Both haemagglutinating positive recombinants had inserts of 3.2 kb. One clone, 1-49 was chosen for further characterization. E. coli 1-49 expressed a protein of 43 kDa that was not present in E. coli JM 83 control as seen by SDS-PAGE and Western blot analysis. Anti-1-49 serum inhibited the haemagglutinating activity of B. gingivalis and E. coli 1-49. This serum reacted with surface molecules on B. gingivalis and E. coli 1-49 as seen by immunogold electron microscopy and immunofluorescence, and to the purified haemagglutinin by Western blot analysis. Like the haemagglutinin on B. gingivalis, the haemagglutinating activity of E. coli 1-49 was destroyed by heating and proteolytic enzymes but the apparent size of the molecule as determined by SDS-PAGE was not affected. A bacterial coaggregating adhesin from B. gingivalis was isolated and characterized. The isolation procedure involved adsorption of the solubilized adhesin on S. mitis followed by elution with glycine buffer. SDS-PAGE of the boiled adhesin revealed a protein with an Mr of 46 kDa. Proteolytic digestion destroyed all bacterial aggregating activity and hydrolysed the 46 kd protein. Antisera raised to the 46 kDa protein reacted with surface molecules on all strains of B. gingivalis tested. This antiserum inhibited the coaggregation reaction between B. gingivalis and other bacteria. Vesicles produced by B. gingivalis were found to enhance the binding of S. sanguis to serum coated hydroxy apatite (SeHA). Maximum vesicle mediated binding took place at 37°C and was destroyed by heating. The lipopolysaccharide from several black pigmented bacteroides were isolated and characterized physically, chemically and immunologically. All of the LPS were of the smooth type and contained the sugars rhamnose, glucose, galactose, glucosamine and galactosamine; no KDO or heptose were found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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The effectiveness of selected sanitizers against bacteria attached to synthetic meat processing surfaces was determined. In initial experiments, in vitro suspensions of Shw. putrefaciens, P. fragi, S. typhimurium, and L. monocytogenes were challenged with sanitizers according to the AOAC Germicidal and Detergent Sanitizer Test (GDST). Germicides employed were chlorine (200 ppm), iodophor (25 ppm), quaternary ammonium compound and phosphoric acid (200 ppm), and peracetic acid (185 ppm). In subsequent studies, the same sanitizers were tested against bacteria attached to polyvinyl chloride, polyurethane, and high density polyethylene surfaces. Test surfaces in sterile poultry slurries were inoculated with bacterial cultures, agitated (100 rpm) for 2 hr at 18°C, and then incubated for 22-28 hr at 26°C (Shw. putrefaciens and P. fragi) or 37°C (S. typhimurium and L. monocytogenes). Scanning electron microscopy (SEM) was employed to determine microbial-surface interactions. Attached organisms remaining on surfaces after vortexing were challenged with germicides. In some cases, surfaces were treated with detergents before applying Sanitizers. Impedance microbiology was used to estimate surviving bacterial populations remaining after chemical treatments. All test sanitizers reduced levels of suspended bacterial cultures >5 logs after 30 sec, and thus, were deemed acceptable according to GDST guidelines. From SEM micrographs, the 22-28 hr surface biofilms of Shw. putrefaciens, S. typhimurium, and L. monocytogenes could be characterized as single adherent cells or cell monolayers. Conversely, the P. fragi biofilm occurred as cell aggregates or microcolonies. Although sanitizers were effective according to GDST results, in many instances, attachment to surfaces increased bacterial resistance to germicidal agents (a 5 log reduction was not observed even after 1 min of chemical exposure). Peracetic acid, overall, was the most effective sanitizer in reducing levels of attached bacteria. Where resistance to other germicides could be observed for up to 20 min, peracetic acid typically eliminated biofilm populations after 1 min. Treatment of surfaces with detergents and then sanitizers led to more effective reductions of attached bacteria. The extensive fibril production by attached S. typhimurium (versus other attached organisms in this research) may explain the greater overall sanitizer resistance of this organism as compared to other test bacteria. However, higher initial numbers of surface bacteria (time 0) may have been the reason for greater sanitizer resistance (higher survivor levels) in some experiments. It is hoped that results of this study can aid processors in developing sanitizer schemes to minimize processing surface contamination with organisms of quality and safety concern. / Ph. D.

The efficacy of six sanitizers, [chloline (200 ppm), iodophor (2S: ppm), acid anionic (200 ppm), peracetic acid (200 ppm), and fatty acid sanitizer (200 ppm)], was evaluated against bacteria attached to gasket materials. Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Listeria monocvtogenes were capable of significant attachment to both buna-N nlbber and Teflon® gasket surfaces in either pure or mixed cultures. Differences in initial attachment rates were evident in a mixed culture of P. fluorescens, Y. enterocolitica, and Listeria monocytogenes in vitro. Sanitizer effectiveness depended upon the bacterium being enumerated, the type of surface, if the bacterium was attached in pure culture or as part of a mixed culture, and the system of evaluation, (i.e. whether or not sanitizer was used alone or as part of a cleaning system). Peracetic acid was the most effective. Removal of bacteria was more pronounced on the Te'f1on® surface with all sanitizers used. The cleaning system, which consisted of a pre-rinse with warm water, application of the cleaning solution, post-rinse with warm water, and application of the sanitizing solution, allowed microorganisms to remain, when the bacteria were present as a pure culture, but resulted in the complete removal of bacteria in mixed culture. / Ph. D.

Les modifications de surface par du chitosane ont été effectuées dans cette thèse de deux façons : physisorption de chitosane de différents paramètres moléculaires (« spin coating » de solutions acides homogènes) et greffage de chaînes de chitooligosaccharides dérivées propargyl (« grafting to ») sur les surfaces de silicium. Le premier cas d'étude vise à apporter une compréhension sur le lien existant entre les propriétés physicochimiques des films et les paramètres moléculaires du chitosane et l'impact de ses propriétés physicochimiques et paramètres moléculaires sur les réponses biologiques. A cette fin, les propriétés de mouillabilité, morphologiques, structurales des films d'une part et d'autre part les réponses d'adhésion et de prolifération de bactéries sur les films de chitosane ont été caractérisées. Dans cette étude, nous avons également pris en compte l'effet du type d'acide utilisé lors de la préparation des solutions ainsi que le vieillissement des films sur leurs propriétés physicochimiques. A l'issue de cette étude, les tests biologiques, notamment l'adhésion et la prolifération de bactéries sur les films de chitosane, ont montré des résultats plutôt liés aux paramètres moléculaires des films qu'à leurs propriétés physicochimiques. Le deuxième cas de modification de surface vise à fonctionnaliser chimiquement les surfaces de silicium par des chitooligosaccharides par la méthode « grafting to ». Cette modification a été réalisée en trois étapes : silanisation, azidation et greffage des chitooligosaccharides. Toutes ces étapes ont été validées par différentes caractérisations de la surface après chaque étape de greffage par ellipsométrie, tensiométrie, AFM, spectroscopie infrarouge et spectrométrie TOF-SIMS / The surface modifications have been carried out in this thesis by two different routes: by physisorption of chitosan chains with different molecular parameters onto silicon surfaces (spin coating of homogeneous acidic solutions) and grafting of propargyl- terminated chito-oligosaccharides chains onto silicon surfaces (grafting to). The first study case was to understand the relationship between the physicochemical properties of chitosan films and the molecular parameters, and to find the impact of these physicochemical properties and molecular parameters on the biological responses. To this end, the physicochemical properties such as the wettability, morphology, chemical and physical structure of chitosan films on the one hand, and the bacteria adhesion and spreading on chitosan films on the other hand, have been characterized. In this study, we have also considered the effect of the type of acid used when solubilizing the chitosan on the films neutralization as well as the film ageing effects on the physicochemical properties. At the end of the study, the biological response of the chitosan films showed more sensitivity towards the chitosan molecular parameters than towards the physicochemical properties of the films. The aim in the second case of surface modification was to functionalize the silicon surfaces with chito-oligosaccharides by the “grafting to” method. It was conducted in three steps: silanisation, azidation and grafting of chito-oligosaccharides. All these steps were validated one by one by carrying out various characterizations using ellipsometry, tensiometry, AFM, infrared spectroscopy and TOF-SIMS spectrometry