The objective of this study was to evaluate the biofilm removal efficacy of three root canal treatment systems: ProUltra® PiezoFlow™, traditional needle irrigation, and the GentleWave® system in an ex-vivo benchtop study. Twenty-four extracted maxillary and mandibular molars were selected. Teeth were all instrumented to a master apical file size #25 with 4% taper. Teeth were then randomly divided into four experimental groups and two control groups. The root canals were inoculated with a culture of Enterococcus faecalis and incubated for five weeks to form a biofilm. Each group was then treated with one of the different root canal treatment systems using 6% sodium hypochlorite (NaOCl) as per the respective manufacturer’s recommendation followed by a rinse with water. Following treatment, teeth were decoronated and roots were sectioned longitudinally. Three scanning electron microscope images were taken at the apical level per root half at 5000x magnification. Images were scored by four calibrated examiners blind to group membership using a four-point scoring system (<5% coverage, 5-33%, 34-66%, and >66%). Results were analyzed using mixed model ANOVA. All the experimental groups were significantly better than the positive control group in removing biofilm. Among the experimental groups, the GentleWave® 15/04 group was significantly better than the other groups. There was no significant difference between the GentleWave® and the ProUltra® PiezoFlow™. Traditional needle irrigation scored the worst in reducing E. faecalis biofilm. The GentleWave™ system was as effective at intracanal biofilm removal as the ProUltra® PiezoFlow™ and better than traditional needle irrigation using 6% NaOCl as an irrigant.

Pseudomonas aeruginosa est une bactérie à Gram négatif à caractère ubiquitaire que l’on retrouve dans une grande diversité d’environnements. C’est un pathogène opportuniste qui est responsable chez l’homme d’infections chroniques ou aigües qui peuvent être mortelles pour des patients immuno-déficients. L’établissement d’une infection chronique est généralement associé à la capacité de la bactérie à former un biofilm, qui se définit comme une population bactérienne attachée sur une surface et englobée par une matrice extracellulaire formée entre autre depolysaccharides. La formation du biofilm est un processus bien défini dans le temps et dans l’espace et qui implique la mise en jeu de nombreuses structures de surfaces dont l’assemblage est strictement contrôlé. Une des voies de régulation contrôlant cet assemblage est le système à 2composants Roc1 (« regulation of cup genes »). Les gènes cup codent des composants de la voie « chaperone-usher » qui permet le transport de sous-unités pilines et leur assemblage à la surface bactérienne sous forme de pili. Ces pili Cup sont important dans l’établissement du biofilm. Le système Roc1 est aussi impliqué dans la mise en place du système de sécrétion de type III, qui est communément associé aux infections aigues. De fait le système Roc1 peut être considéré comme un «interrupteur» décidant du mode d’infection associé à P. aeruginosa. Le système Roc1 est constitué d’un senseur non-orthodoxe (RocS1) et de deux régulateurs de réponse, RocA1 et RocR, dont le domaine effecteur est un domaine de liaison à l’ADN ou un domaine EAL à activité phosphodiesterase, respectivement. Il existe également d’autres gènes paralogues de Roc1 qui sont le système Roc2 avec RocS2 et RocA2 très similaire à RocS1 et RocA1, ainsi que RocS3 similaire à RocS1. Le travail réalisé au cours de ma thèse a montré qu’il existe une régulation croisée entre Roc1 etRoc2. Cependant, chacune des branches du réseau de régulation contrôle l’expression d’une série de gènes bien spécifiques. Nous avons montré que la signalisation via RocS2 et RocS1 lorsqu’elle converge sur RocA1 contrôle l’expression des gènes cupC et ce contrôle est totalement indépendantde RocA2. Par contre lorsque la signalisation RocS1 et RocS2 converge vers RocA2 alors ce sont les gènes mexAB-oprM, qui codent une pompe d’efflux impliquée dans la résistance aux antibiotiques, dont l’expression est alors réprimée.En conclusion, nous avons mis en évidence un modèle unique de régulation croisée qui résulte dans un effet antagoniste entre formation du biofilm et résistance aux antibiotiques. Si cela peut paraître inattendu, quelques données cliniques sont en faveur d’une telle balance. En effet, l’analyse de souches de P. aeruginosa, isolées à partir de patients atteints de mucoviscidose, révèle que dans ces isolats la pompe MexAB-OprM est inactive. La raison de cette adaptation n’est pas élucidée, mais l’absence de pompe fonctionnelle pourrait procurer un avantage, une meilleure aptitude à la souche à persister dans cet environnement. Il est également reconnu que dans les poumons de ces patients le mode préféré de développement pour P. aeruginosa est le biofilm. Mises bout à bout ces observations suggèrent donc que le système Roc pourrait être un système de régulation important pour percevoir l’environnement du poumon chez le patient mucoviscidosique et déclencher une réponse adaptée. / The opportunistic pathogen Pseudomonas aeruginosa is responsible for diverse chronic and acute infections in human. Chronic infections are associated with the capacity of P. aeruginosa to form biofilms. One of the pathways controlling biofilm formation is the Roc1 two-component system, involved in the regulation of cup genes allow the assembly of thin fimbriae at the surface of the bacterium. Cup fimbiae are important in biofilm formation. There exist paralogues of the Roc1 system - the Roc2 and Roc3 system. The work in this thesis has shown that cross-regulation occurs between Roc1 and Roc2. However, each branch in this network appears to control the expression of a specific subset of genes whose role and functions are striking in the context of an infection process. We characterized here a unique model of cross-regulation which results in the antagonistic regulation of biofilm formation and antibiotic resistance

This thesis describes the use of different DNA sequencing technologies to investigate the structure and function of microbial communities in two extreme environments, boreal acid sulfate soil and the terrestrial deep biosphere. The first of the two investigated environments was soils containing un-oxidized metal sulfides that are termed ‘potential acid sulfate soil’ (PASS) materials. If these materials are exposed to atmospheric oxygen by either natural phenomena (e.g., land uplift) or human activities (e.g., drainage) then the metal sulfides become oxidized and the PASS becomes acidic and is defined as an ‘acid sulfate soil’ (ASS). The resulting acid and metal release from metal sulfide oxidation can lead to severe environmental damage. Although acidophilic microorganisms capable of catalyzing acid and metal release have been identified from many sulfide mineral containing environments, the microbial community of boreal PASSs/ASSs remains unclear. This study investigated the physicochemical and microbial characteristics of PASSs and ASSs from the Risöfladan experimental field in Vasa, Finland. Sanger sequencing of 16S rRNA gene sequences of microorganisms present in the PASSs and ASSs were mostly assigned to acidophilic species and environmental clones previously identified from acid- and metal-contaminated environments. Enrichment cultures inoculated from the ASS demonstrated that the acidophilic microorganisms were responsible for catalyzing acid and metal release from PASSs/ASSs. Lastly, the study investigated how to mitigate metal sulfide oxidation and the concomitant formation of sulfuric acid by treating ASSs in situ with CaCO3 or Ca(OH)2 suspensions. The DNA sequencing still identified acidophilic microorganisms after the chemical treatments. However, the increased pH during and after treatment suggested that the activity of the acidophiles might be inhibited. This study was the first to identify the microbial community present in boreal PASSs/ASSs and suggested that treatment with basic compounds may inhibit microbial catalysis of metal sulfide dissolution. The second studied environment was the deep, dark terrestrial subsurface that is suggested to be both extremely stable and highly oligotrophic. Despite the scarcity of carbon and energy sources, the deep biosphere is estimated to constitute up to 20% of the total biomass on earth and thus, represents the largest microbial ecosystem. However, due to the difficulties of accessing this environment and our inability to cultivate the indigenous microbial populations, details of the diversity and metabolism of these communities remain largely unexplored. This study was carried out at Äspö Hard Rock Laboratory, Sweden and utilized second-generation sequencing to identify the taxonomic composition and genetic potential of planktonic and biofilm populations. Community DNA sequencing of planktonic cells from three water types at varied age and depth (‘modern marine’, ‘undefined mixed’, and ‘old saline’) showed the existence of ultra-small cells capable of passing through a 0.22 μm filter that were phylogenetically distinct communities from the &gt;0.22 μm fraction. The reduced cell size and/or genome size suggested a potential adaptation to the oligotrophic environment in the terrestrial deep biosphere. The identified planktonic communities were dominated by Proteobacteria, Candidate divisions, unclassified archaea, and unclassified bacteria. Functional analysis of the assembled genomes showed that the planktonic population from the shallow modern marine water demonstrated a predominantly anaerobic and heterotrophic lifestyle. In contrast, the deeper, old saline water was more closely aligned with the hypothesis of a hydrogen-driven deep biosphere. Metagenomic analysis of subsurface biofilms from ‘modern marine’ and ‘old saline’ water types suggested only a subset of populations were involved in initial biofilm formation. The identified biofilm populations from both water types were distinct from the planktonic community and were suggested to be dominated by hydrogen fed, chemolithoautotrophic and diazotrophic populations.

The overall goal of the thesis was to develop and optimize the moving bed biofilm reactor technology under anaerobic conditions. The thesis work was divided into two different series of experiments. Hence, at first, the reactor start-up on synthetic substrate was evaluated and it was proven that the anaerobic moving bed biofilm reactor technology could successfully treat concentrated wastewater. Subsequently, a study on Fort Garry Brewery wastewater was conducted to optimize the process for a typical North American industrial wastewater. The aim was successfully achieved and a potential design to treat Fort Garry Brewery wastewater was developed. The anaerobic moving bed biofilm reactor was found to be capable in treating brewery wastewater with potential savings to the industry paying surcharges for discharging wastewater over the city sewer bylaw limits. / October 2016

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.

Les biofilms sont des communautés structurées de micro-organismes enrobées dans une matrice extracellulaire. Les biofilms sont impliqués dans la persistance de plusieurs maladies infectieuses et la matrice extracellulaire du biofilm protège les bactéries contre les cellules du système immunitaire de l'hôte, les antibiotiques et les désinfectants. Récemment notre laboratoire a démontré que le zinc inhibe la formation de biofilm chez Actinobacillus pleuropneumoniae, une bactérie pathogène du porc. Le but de cette étude est d'évaluer l'effet du zinc sur la croissance et la formation du biofilm chez différentes bactéries pathogènes du porc, telles que Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus et Streptococcus suis. Les bactéries ont été cultivées dans des plaques de 96 puits sous condition optimale de formation de biofilm et les biofilms ont été colorés au cristal violet. La présence du biofilm a été confirmée par microscopie confocale à balayage laser à l’aide du marqueur fluorescent FilmTracerTM FM ® 1-43. À des concentrations micromolaires, le zinc inhibe faiblement la croissance bactérienne et bloque d'une manière dose-dépendante la formation de biofilm d’A. pleuropneumoniae, Salmonella Typhimurium et H. parasuis. De plus, la formation de biofilm de E. coli, S. aureus et S. suis a été faiblement inhibée par le zinc. Nos résultats indiquent que le zinc a un effet inhibiteur sur la formation de biofilm de la plupart des pathogènes bactériens d'origine porcine. Cependant, le mécanisme sous-jacent de l'activité anti-biofilm du zinc reste à être caractérisé. / Biofilms are structured communities of microorganisms enclosed in a self-produced extracellular matrix. Biofilms are responsible for the persistence of most infectious diseases, because the biofilm matrix acts as a form of protection for the bacteria against the host immune system, antibiotic and disinfectants. Recent work in our laboratory demonstrated that zinc could inhibit biofilm formation of Actinobacillus pleuropneumoniae, a swine pathogen. The aim of this study was to evaluate the effect of zinc on growth and biofilm formation of other bacterial swine pathogens, such as Bordetella bronchiseptica, Escherichia coli, Haemophilus parasuis, Salmonella, Staphylococcus aureus, and Streptococcus suis. Bacteria were grown on 96-well plates under optimal biofilm forming conditions and the biofilms were stained with crystal violet. The presence of biofilms was confirmed by confocal laser scanning microscopy with FilmTracerTM FM® 1-43. At micromolar concentrations, zinc weakly inhibited bacterial growth and effectively blocked biofilm-formation by A. pleuropneumoniae, Salmonella Typhimurium, and H. parasuis in a dose-dependent manner. Additionally, biofilm formation of E. coli, S. aureus and S. suis was slightly inhibited by zinc. Our results indicate that zinc has an inhibitory effect on biofilm formation of most bacteria of porcine origin. However, the mechanism behind the antibiofilm activity of zinc has yet to be characterized.

Purpose: The purpose was to examine the maternal influences on the development of infant oral biofim and dominant bacterial strains of at risk populations. Methods: The study used a cross-sectional design to examine factors influencing biofilm colonization and the identification of bacterial strains transmitted from mother to child. Participants were enrolled in Children’s Health Involving Parents of Greater Richmond (CHIP). Plaque and saliva samples were collected from mothers and their children ages 6-36 months. The colonized oral bacteria strains of the mother infant dyads were then compared. Oral bacterial strain identification was completed using the HOMIM Forsythe microbe identification array. Examination for concordant strains was done using the statistical boot strap shuffle in Excel. Results: Forty-one CHIP families were involved in the pilot study. Participants were predominantly non-white , less than high school education 46.3%, and their average age was 29.1 years. Mothers had a caries prevalence of 87.8% and the infant’s caries rate was 26.7%. To date n=14 pairs of the n=41 samples have been processed and analyzed using the HOMIM microarray. Twelve paired samples were not processed due to non-detectable levels of bacterial DNA. Fifteen samples are currently being processed by HOMIM Forsyth. Predominate species transferred from mother to child include S. Oralis, S. parasanguinous, S. mitis, Slakia, and S. anginosis. 425 unique strains of bacteria were analyzed on the array with a maternal concurrence rate of 33%. Conclusion: When comparing total bacterial populations in the oral environment a concurrence of transmission from mother to child was 33%. Higher rates of vertical transmission were observed in S. Oralis, S. sanguinous, and Slakia.

Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We investigated the molecular basis of this phenomenon. Deletion and complementation analysis and thereafter antibody based inhibition assays confirmed a functional role for the surface adhesin clumping factor B as the causative determinant associated with the increased biofilm phenotype. Finally, we investigated the regulation of clumping factor B-mediated biofilm formation and the basis for the strain dependence. Regulation was determined to occur via two novel post-translational networks- one affecting ClfB activity, mediated by Ca2+ binding to the EF-Hand domain, and the other affecting protein stability, mediated by the enzymatic activity of the metalloprotease-aureolysin. Polymorphisms within the aureolysin gene sequence, between strains, was identified as the basis for some strains forming robust biofilms within chelated media versus other than do not exhibit this phenotype.

This thesis focuses on solution antimicrobial effectiveness for copolyoxetanes with quaternary ammonium and PEG-like side chains. Ring opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy) ethoxy) methyl)-3-methyloxetane (ME2Ox) yielded random copolymers with 14-100 (m) mole% BBOx designated P[(BBOx-m)(ME2Ox)]. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m. Mole ratios and molecular weights were obtained from 1H-NMR and end group analysis. Differential scanning calorimetry (DSC) studies showed Tg’s between 69 and -34 °C. Minimum inhibitory concentrations (MIC) against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa showed MIC decreasing with increasing C12 mole% reaching a minimum between C12-43 and C12-60. C12-43 had the lowest MIC for all strains. At 5× MIC (challenge:108 cfu/ml), C12 43 kills ≥ 99% of the tested strains within 1 hr. C12-m copolyoxetane cytotoxicity toward human red blood cells, HFF (Human Foreskin Fibroblast) and HDF (Human Dermal Fibroblast) was low, indicating good prospects for biocompatibility. Cx-m copolyoxetane antimicrobial efficacy, hemolytic activity and cytotoxicity were further explored by changing quaternary alkyl chain length. Copolyoxetanes are represented as Cx-50, where 50 is the mole percent quaternary repeat units and ‘x’ is quaternary alkyl chain length (2 to 16 carbons). Reaction of P[(BBOx-m)(ME2Ox)] with a series of tertiary amines yielded the desired quaternary ammonium segment. DSC studies showed Tg’s between -40 °C and -60 °C and melting endotherms for C14-50 and C16-50. A systematic dependence of alkyl chain length on MIC was found with C8-50 being the most effective antimicrobial. Kill kinetics for C8-50 (5× MIC, challenge: 108 cfu/ml) effected >99% kill in 1 hour for S. aureus (7 log reduction). C8-50 efficacy on biomass and cell viability of P. aeruginosa biofilms was investigated. Crystal violet (CV) staining assays demonstrate that C8-50 had no effect on adhesion of already established P. aeruginosa biofilms, but reduced biofilm formation by killing cells prior to attachment. For anti-adhesion assays, noticeable reduction in biofilm mass occurred at concentrations greater than 2× MIC. Viability studies show a substantial log reduction of 2.1 at MIC. The low cytotoxicity of Cx-m copolyoxetanes coupled with low MICs and favorable biofilm results indicate good prospects for therapeutic applications.

This study compared seven dental unit water line (DUWL) treatments and three in-office bacteria test kits. Sodium hypochlorite (NaOCl) 1:10 in tap water weekly; 3 drops of NaOCl in 1 liter of water; Dentapure® DP 40; ICX™ tablet; Sterilex® Ultra powder; Lines™; and Selective Micro® Dental-Clean. Traditional culture technique was compared to HPC Dental Sampler; Aquasafe™ Dental Unit Water Line Test Kit; and Bacteria in Water Test Kit. Eight dental units in the Virginia Commonwealth University Graduate Endodontic Clinic were randomly assigned treatment regimens. Samples were taken weekly initially and after flushing for 1 minute. In conclusion NaOCl hypochlorite 1:10 in tap water once weekly, Sterilex® Ultra, Lines™, and Selective Micro® Dental-Clean were effective at all sample times while ICX™, 3 drops of NaOCl, and Dentapure® DP 40 were only effective after 1 minute flushing. There was no significant difference between the in-office test kits and traditional culture.