Dissertations / Theses on the topic 'Anti-biofilm agents'

Carbon dots (CD) have emerged as the new eye-catching theranostic nanomaterials due to their distinctive features, including tunable emission, facile surface modification, high biocompatibility and low cytotoxicity. These distinguishing features allow full customizations of CD according to the needs of various studies. Of note, they have been widely employed as nano-vehicles with live-tracking systems in many biological applications to deliver medicine with low bioavailability to targeted sites. Candida albicans, a commonly seen commensal fungus accounts for life-threatening infections in humans, is the leading cause of oral candidiasis. Yet, the efficacy of the "gold standard" Amphotericin B (AmB) has been limited due to poor water solubility and dose-dependent cytotoxicity. In addition, the interactions of CD with Candida cells/biofilms and human epithelial tissues have not been fully investigated, and very limited studies have been done on CD-based antifungal drugs delivery for topical administration. Herein, AmB-conjugated guanylated CD (CD-Gu + -AmB) tackling oral fungal infections were synthesized and possessed potent antifungal/anti-biofilm effects against C. albicans. Moreover, CD-Gu + -AmB exhibit low cytotoxicity to primary human oral keratinocytes and can selectively accumulate in the cell nuclei. Above all, the first evidence of studying the penetration and exfoliation profiles of CD in a three- dimensional organotypic human oral epithelial tissue model was provided, and the accumulation of CD-Gu + -AmB in the epithelial tissue can form a 'shielding' layer on oral epithelia against C. albicans. This study demonstrates that CD-Gu + -AmB may serve as a promising antifungal agent for tackling C. albicans and Candida-induced oral candidiasis through fast epithelial penetration, extra-/intra-cellular embedding and gradual exfoliation

Bacterial biofilms are a leading cause of life-threatening and device-related infections worldwide. Biofilm related infections are notoriously difficult to treat as they are highly tolerant to conventional antibiotics. This project has designed and synthesised a new class of antibiotics to circumvent biofilm tolerance and shown that the prepared compounds could eradicate several medically important pathogens (P. aeruginosa, E. coli, and S. aureus). Importantly, as these compounds are hybrids of drugs that are already used clinically as stand-alone therapies, they demonstrate great potential to be translated into therapies in the near future.

The emergent resistance of bacteria against the conventional antibiotics has motivated the search for novel antimicrobial agents. Nature abounds with a number of antimicrobial peptides that are a part of our innate immune system and protect us against a variety of pathogenic bacteria. While they are broad-spectrum in their activity and show less drug-resistance induction, their intrinsic metabolic stability limits their potential therapeutic applications. Herein we describe the development of novel broad-spectrum bioactive antimicrobial peptidomimetics AA-peptides. AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides are discussed in this dissertation "[aacute]-AApeptides" and " ã-AApeptides" The therapeutic potential of these AApeptides were accessed by conducting antibacterial assays against a series of both gram-positive, gram-negative bacteria and fungi. These oligomers were characterized using MALDI-TOF and Circular Dichroism spectroscopy (CD). Their invitro toxicity was evaluated against human erythrocytes .We attempted to study their mechanism of action via membrane depolarization assay. We have successfully identified them as antimicrobial agents, pro-inflammatory immune response suppressing agents and as anti-biofilm agents.

Foram avaliados dentifrícios experimentais à base de Ricinus communis (DR), Triclosan (DT) e Cloramina-T (DC) para higiene de próteses totais, tendo como controle dentifrício sem agente antimicrobiano (DB) e água. Para análise in vitro foram realizados ensaios físico-químicos (medida da densidade, pH, consistência e características reológicas); ensaio de abrasividade, avaliada em 30 espécimes de resina acrílica antes e após a escovação artificial e análise microbiológica com a formação de biofilme multiespécies (S. mutans, C. albicans e C. glabrata) sobre espécimes em resina acrílica. Estes, após contaminação, foram escovados por 60s com DR, DT, DC e DB e água (n=10). Foram empregados controles positivo (contaminado e não escovado) e negativo (sem contaminação). Para análise in vivo, seguiu-se o modelo \"crossover\" com \"washout\" de 7 dias. Os voluntários escovaram suas próteses superiores 3 vezes ao dia por 07 dias. A capacidade de remoção do biofilme foi avaliada empregando evidenciação, fotografia e quantificação com software Image Tool 3.0. Na avaliação antimicrobiana, o biofilme foi desprendido da prótese por escovação com solução salina e a suspensão resultante, semeada em meios de cultura específicos para Candida spp, S. mutans, S. aureus e bactérias Gram-negativas. As espécies de Candida foram identificadas pelo meio de cultura Chromagar e pela PCR (Polymerase Chain Reaction). Na avaliação dos dentifrícios pelos participantes foi aplicado questionário. Os resultados das características organolépticas e físico-químicas foram informados em tabelas autoexplicativas. Os dados de rugosidade foram analisados por ANOVA e os dados da ação antimicrobiana in vitro, pelo teste de Kruskal-Wallis. Para os dados das variáveis clínicas (in vivo), empregou-se teste de Friedman e o teste de Cochran. Os testes estatísticos foram realizados com p<0,05. Os dentifrícios não apresentaram diferença quanto à abrasividade (DB=0,264±0,098; DR=0,236±0,236; DT=0,265±0,116; DC=0,203±0,105), porém promoveram aumento da rugosidade comparando à água (0,027±0,004). Frente às espécies de Candida, in vitro, o DT foi o mais eficaz (p=0,00; m=1,30) seguido do DC (m=2,6), DB (m=3,26) e DR (m=3,59). Para o S. mutans houve diferença entre a água (m=3,86) e os dentifrícios (p=0,001), porém não entre estes (DB: m=0; DR: m=2,3 e DC: m=0). DT inibiu o crescimento de S. mutans. Quanto à capacidade de remoção do biofilme, não houve diferença entre os dentifrícios (p=0,055; DB: m=7,39; DR: m=7,94; DT: m=10,16; DC: m=8,14), porém houve redução do biofilme comparando ao Baseline (m=16,53). Os dentifrícios não apresentaram diferença antimicrobiana, in vivo, contra Candida spp. (p=0,495), S. mutans (p=0,497), S. aureus (p=0,845) e bactérias Gram-negativas (p=0,425). Na identificação das espécies de Candida pelo Chromagar não houve diferença quanto ao seu aparecimento independente do dentifrício (p=0,466). O resultado pela PCR foi semelhante à identificação convencional e as espécies de C. albicans, C. tropicalis e C. glabrata foram mais prevalentes, respectivamente. Na avaliação dos dentifrícios pelos participantes não houve diferença (p>0,05) entre eles para nenhuma questão. Os dentifrícios apresentaram resultados satisfatórios, apresentando potencial para uso clínico e controle do biofilme de próteses totais.
This study evaluated experimental dentifrices based on Ricinus communis (DR), Triclosan (DT) and Cloramina-T (DC) for complete denture cleaning. To in vitro analysis were performed physicochemical tests (measurement of density, pH, consistency and rheological characteristics); abrasiveness test evaluated in 30 acrylic resin specimens before and after artificial brushing and microbiological analysis with multi-species biofilm formation (Streptococcus mutans, C. albicans and C. glabrata) on the specimens of acrylic resin. This specimens were manually brushed for 60 seconds with DR, DT, DC and DB and water (n = 10). Positive controls were used (contaminated and not brushed) and negative (no contamination). To in vivo analysis the study followed the crossover model with washout of 7 days. The volunteers brushed their upper dentures 3 times daily for 07 days. The removal of biofilm capacity was evaluated employing evidenciation, photography and quantification with Image Tool 3.0 software. For the evaluation of antimicrobial activity, the biofilm was removed from the denture by brushing with saline solution and the suspension was seeded in culture media specific for Candida spp, S. mutans, S. aureus and Gram-negative bacteria. The Candida species were identified by culture medium Chromagar and PCR method (Polymerase Chain Reaction). One questionnaire was used for the dentifrices evaluation by the participants. The results of physicochemical characteristics were informed in self-explanatory tables. The roughness data were analyzed by ANOVA and antimicrobial activity in vitro data by the Kruskal-Wallis test. To the data of the clinical variables (in vivo) was used Friedman test and Cochran test. Statistical tests were performed with p<0.05. The dentifrices showed no difference in the abrasiveness (DB=0.264 ± 0.098, DR=0.236 ± 0.236, DT=0.265 ± 0.116, DC=0.203 ± 0.105), but promoted increased roughness when compared to water (0.027 ± 0.004). To Candida species, in vitro, the DT was the most effective (p=0.00, m=1.30) followed by DC (m=2.6), DB (m=3.26) and DR (m=3.59). To S. mutans there was difference between the water (m=3.86) and dentifrices (p=0.001), but these did not showed difference from each other (DB: m=0; DR: m=2.3 and DC: m=0). DT inhibited the growth of S. mutans. There was no difference among the dentifrices for biofilm removal (p=0.055; DB: m=7.39; DR: m=7.94; DT: m=10.16; DC: m=8.14), but the biofilm decreased when compared to Baseline (m=16.53). The dentifrices showed no difference antimicrobial, in vivo, against Candida spp. (p=0.495), S. mutans (p=0.497), S. aureus (p=0.845) and Gram-negative bacteria (p=0.425). In the identification of Candida species by Chromagar there was no difference in the appearance of their independent dentifrices (p=0.466). The result by PCR was similar conventional identification, and the species of C. albicans, C. tropicalis and C. glabrata were more prevalent, respectively. In the evaluation of dentifrices by the participants there was no difference (p>0.05) among them to any question. The dentifrices showed satisfactory results with potential for its specificity.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Terapias biológicas tem buscado novas substâncias/protocolos que promovam a eliminação microbiana e induzam ou estimulem a regeneração pulpar e o desenvolvimento completo radicular de dentes permanentes jovens com processos patológicos pulpares. Os objetivos do estudo foram avaliar a a tividade antimicrobiana/antibiofilme de algumas combinações de antibióticos sobre microrganismos de interesse endodôntico e analisar o efeito da combinação de antibióticos com melhor ação antimicrobiana associada à sinvastatina na expressão de marcadores odontoblásticos em células da polpa dental humana (CPDH). A atividade antimicrobiana dos seguintes antibióticos : Metronidazol (ME), Ciprofloxacina (CI), Minociclina (MI), Doxicilina (DO) e Fosfomicina (FO), isolados ou em combinação dupla (ME+CI, ME+MI, ME+DO, ME+FO, CI+MI, CI+DO, CI+FO, DO+FO, MI+FO) ou tripla (ME+CI+MI, ME+CI+FO, ME+MI+FO, ME+CI+DO, ME+DO+FO, CI+DO+FO, CI+MI+FO) foram testados contra Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Candida albicans em condições planctônicas. Biofilmes mono-espécie de E. faecalis e biofilmes em dual-espécies de E. faecalis and C. albicans foram preparados em blocos de dentina para testar a atividade antibiofilme das combinações de antibióticos com os melhores resultados microbiológicos. O efeito antibiofilme das combinações antibióticas sobre biofilme de E. faecalis dentro dos túbulos dentinários foi também avaliada por microscopia confocal. Culturas de CPDH foram expostas à combinação antibiótica com melhor resultado microbiológico e sinvastatina e determinada a viabilidade celular, atividade da fosfatase alcalina (ALP), deposição de nódulos de mineralização e expressão de DSPP (sialofosfoproteína dentinária), importante marcador odontoblástico de mineralização denti nária. Os dados foram 9 analisados estatisticamente, considerando p<0,05 . Todas as combinações de antibióticos reduziram o crescimento bacteriano, exceto por CI+DO e DO+FO para A. Israelii. ME+CI+MI e ME+MI+FO inibiram significantemente o crescimento de A. israelii e E. faecalis, e ME+MI+FO eliminou S. mutans. ME+MI+FO e ME+CI+FO tiveram o melhor efeito contra biofilme de E. faecalis, em mono ou dual-espécies e dentro dos túbulos dentinários. CI e ME+CI+FO afetaram a viabilidade das células pulpares, em 1 e 7 dias. A atividade de ALP aumentou com a presença de sinvastatina para todos os grupos, exceto para CI e ME+CI+FO. Grupos contendo sinvastatina mostram maior deposição de nódulos de mineralização e expressão de DSPP que os grupos sem sinvastatina. Pode-se concluir que a combinação de antibióticos tripla ME+CI+FO teve efeito marcante contra os microrganismos endodônticos, em condições planctônicas e em biofilme. A sinvastatina estimulou a expressão de marcadores odontoblásti cos de mineralização dentinária pelas HDPC; entretanto, seu efeito foi reduzido pela presença da CI.
Biological therapies have searching for substances/protocols, which promote microbial elimination and induce or stimulate pulp regeneration and completion of apical root development in young permanent teeth with pulp pathological processes . The objectives of this study were to evaluate the antimicrobial /anti-biofilm activity of some antibiotics combinations on endodontic microorganisms and the effect of the combination of antibiotics with the best antimicrobial action associated with simvastatin on expression of odontoblast markers by human dental pulp cells (HDPC). The antimicrobial activity of the following antibiotics : Metronidazole (ME), Ciprofloxacin (CI), Minocycline (MI), Doxycycline (DO) and Fosfomycin (FO), either alone or in double (ME+CI, ME+MI, ME+DO, ME+FO, CI+MI, CI+DO, CI+FO, DO+FO, MI+FO) or triple combinations (ME+CI+MI, ME+CI+FO, ME+MI+FO, ME+CI+DO, ME+DO+FO, CI+DO+FO, CI+MI+FO) were tested against Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii and Candida albicans in planktonic conditions. Mono-species biofilm of E. faecalis and dual-species biofilms of E. faecalis and C. albicans were prepared in dentin blocks to test the anti -biofilm activity of antibiotic combinations with the best microbiological results. Antibiofilm effect of antibiotic combination on E. faecalis biofilm inside dentin tubules was also evaluated by confocal microscopy. Cultures of HDPC were exposed to the antibiotic combination with the best antimicrobial effect and simvastatin and determined cell viability, alkaline phosphatase activity, deposition of mineralization nodules and expression of Dspp (dentin sialophosphoprotein), important odontoblast markers of dentin mineralization. Data were analyzed statistically, considering p<0.05. All antibiotic combinations reduced statistically the growth of bacteria tested, except by CI+DO and DO+FO for A. israelii. ME+CI+MI and ME+MI+FO inhibited significantly growth of A. 11 israelii and E. faecalis, and ME+MI+FO eliminated S. mutans. ME+MI+FO and ME+CI+FO had the best effect against E. faecalis biofilm, in mono and dual -species biofilms and inside dentin tubules, similar to CHX. CI and ME+CI+FO affected HDPC viability, 1 and 7 days. ALP activity increased with the presence of simvastatin for all groups, except by CI and ME+CI+FO. Groups containing simvastatin had higher mineralized nodule deposition and higher DSPP expression than groups without simvastatin. It can be concluded that triple antibiotic combination of ME+CI+FO ha d remarkable effect against endodontic microorganisms, in planktonic and biofilm conditions. Simvastatin stimulated the expression of odontoblast markers of dentin mineralization by HDPC; however, its effect was reduced i n the presence of CI.
FAPESP: 2014/00589-7

New strategies are needed to counter the growing problem of bacterial resistance to antibiotics. One such strategy is to design compounds that target bacterial virulence, which could work separately or in concert with conventional bacteriostatic or bactericidal antibiotics. Pilicides are a class of compounds based on a ring-fused 2-pyridone scaffold that target bacterial virulence by blocking the chaperone/usher pathway in E. coli and thereby inhibit the assembly of pili. This thesis describes the design, synthesis, and biological evaluation of compounds based on the pilicide scaffold with the goal of improving the pilicides and expanding their utility. Synthetic pathways have been developed to enable the introduction of substituents at the C-2 position of the pilicide scaffold. Biological evaluation of these compounds demonstrated that some C-2 substituents give rise to significant increases in potency. X-ray crystallography was used to elucidate the structural basis of this improved biological activity. Furthermore, improved methods for the preparation of oxygen-analogues and C-7 substituted derivatives of the pilicide scaffold have been developed. These new methods were used in combination with existing strategies to decorate the pilicide scaffold as part of a multivariate design approach to improve the pilicides and generate structure activity relationships (SARs). Fluorescent pilicides were prepared using a strategy where selected substituents were replaced with fluorophores having similar physicochemical properties as the original substituents. Many of the synthesized fluorescent compounds displayed potent pilicide activities and can thus be used to study the complex interactions between pilicide and bacteria. For example, when E. coli was treated with fluorescent pilicides, it was found that the compounds were not uniformly distributed throughout the bacterial population, suggesting that the compounds are primarily associated to bacteria with specific properties. Finally, by studying compounds designed to inhibit the aggregation of Aβ, it was found that some compounds based on the pilicide scaffold inhibit the formation of the functional bacterial amyloid fibers known as curli; these compounds are referred to as 'curlicides'. Some of the curlicides also prevent the formation of pili and thus exhibit dual pilicide-curlicide activity. The potential utility of such 'dual-action' compounds was highlighted by a study of one of the more potent dual pilicide-curlicides in a murine UTI model were the compound was found to significantly attenuate virulence in vivo.

L’adhésion de bactéries et par suite la formation de biofilms sur la surface de matériaux sont des problèmes récurrents qui peuvent avoir de graves conséquences aussi bien dans le domaine de la santé qu’au niveau industriel. L’éradication des biofilms demeure aujourd’hui un véritable défi et une stratégie proposée consiste en la vectorisation d’agents biocides à l’aide de nanoparticules polymères. La présente étude rapporte l’élaboration de nanoparticules (NPs) biodégradables et biocompatibles de poly(acide lactique-co-glycolique) (PLGA) par le procédé de nanoprécipitation. Les NPs ont aussi été pégylées afin de moduler leur interaction avec les milieux biologiques. Les particules ont été chargées en ciprofloxacine (CIP) avec un taux de charge d’environ 5 %, ce qui permet une libération progressive de la CIP sur 5-6 jours. Des essais microbiologiques ont été réalisés sur la bactérie Gram positif Staphylococcus aureus. Cette bactérie est l’une des principales causes d’infections chroniques et nosocomiales, impliquant le plus souvent des biofilms. Les NPs de PLGA nues et pégylées ont montré une activité antibactérienne en mode planctonique sur deux souches de S. Aureus (ATCC 29213 et 610520), sans doute liée à un effet de taille (échelle nanométrique). Ces NPs n’ont pas montré de cytotoxicité in vitro sur des cellules neuronales. Enfin, des études microbiologiques sur les NPs chargées en CIP ont été menées sur une souche de S. Aureus (ATCC 29213) en modes planctonique et biofilm. La CIP encapsulée reste efficace après séquestration et s’avère plus active pour éradiquer le biofilm que la CIP libre (diminution de la concentration minimale d’éradication du biofilm)
The adhesion of bacteria and hence formation of biofilms on the surface of materials are recurring problems that can have serious consequences at both public health and industrial level. The eradication of biofilms today remains a challenge and a proposed strategy is the vectorization of biocidal agents by using polymeric nanoparticles. This study reports the elaboration of biocompatible and biodegradable nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA) by the nanoprecipitation method. The nanoparticles were also pegylated in order to modulate their interaction with the biological media. The particles were loaded with ciprofloxacin (CIP) with a drug content of about 5 %, which allows a gradual release of the CIP during 5-6 days. Microbiological tests were made against gram positive bacteria Staphylococcus aureus. This bacteria is one of the major causes of chronic and nosocomial infections, most often involving biofilms. The naked NPs and the pegylated ones exhibited an antibacterial activity on planktonic cells against two strains of S. Aureus (ATCC 29213 and 610520), probably related to a size effect (nanoscale). These NPs showed no cytotoxicity in vitro on neuronal cells. Finally, the antibacterial activity studies of NPs loaded with CIP were conducted against a S. Aureus (ATCC 29213) on planktonic cells and biofilm. Encapsulated CIP remains effective after sequestration and is more active to eradicate biofilm than free CIP (decrease of the minimum biofilm eradication concentration)

The prevalence of oral diseases such as dental caries and periodontitis and the universal need for effective control of oral health has stimulated a great deal of interest in oral hygienic formulations both scientifically and commercially driven. Such formulations are normally deployed as complex formulations commonly containing antimicrobial actives together with excipients, where both classes of ingredients may contribute to the bacteriological effect of the oral hygienic product. However, the mode of action and/or the bacteriological and microecological effects of exposure of microorganisms to oral hygiene products are poorly understood. In this context, this doctoral dissertation represents a series of investigations to contribute to knowledge in the area. The impact of selected oral antimicrobial actives (triclosan, sodium lauryl sulphate, stannous fluoride and zinc lactate) on a key aspect of bacterial cellular membrane function was investigated. This involved measuring major cellular respiratory pathways during exposure to the test agents using two types of tetrazolium dyes possessing different redox potentials as respiration pathway indicators. Spectrophotometric analyses indicated that sub-lethal levels of triclosan and sodium lauryl sulphate act as uncoupling agents, an observation not previously been reported. Sub-lethal concentrations of stannous fluoride and zinc lactate however, blocked cellular respiration with resulting shifts towards glycolytic/fermentative pathways. The contribution of a variety of test agents to the overall antimicrobial effect of a complex formulation (Listerine®) was investigated in order to understand the relative efficacy of the actives. This was achieved by testing the essential oils present in the formulation singly and in combination utilising in vitro models. The use of the hydroxyapatite disc model (HDM) to grow salivary microcosms to test the efficacy of the ingredients revealed hitherto unreported synergistic activity between the active ingredients thymol and menthol. Proprietary dentifrices (Colgate Total® and Crest ProHealth®) containing the antimicrobial agents triclosan or stannous fluoride/zinc lactate, respectively, were comparatively evaluated. This was performed by simultaneously establishing salivary microcosms in Sorbarod Biofilm Devices (SBDs). Following the establishment of dynamic steady-states, paired devices were dosed with each of the two proprietary dentifrices. Bacteriological data generated after multiple dosing indicated that both dentifrices were comparably effective in the reduction of all tested bacterial functional groups in the plaque models. However, data generated using HDM models indicated greater reductions in Gram-negative anaerobes after exposure to Colgate total®. The observations presented in this thesis may contribute to the development of oral formulations with optimised antimicrobial efficacies against adventitious pathogens present in the oral cavity and help in reducing the incidence of oral diseases and potentially related systemic interface.

Najčešća komplikacija nakon ugradnje silikonskih implantata za dojku je kontraktura fibrozne kapsule (KK), koja se normalno stvara oko implantata u sklopu reakcije oko stranog tela. Najozbiljnija komplikacija nakon ugradnje silikonskih implantata za dojku je anaplastični krupnoćelijski limfom koji se javlja isključivo kod pacijentkinja koje imaju ugraĎene implantate (eng. Breast-implant associated anaplastic large cell lymphoma – BIA ALCL). Uzrok nastanka ovih komplikacija ostaje nepoznat. Ustanovljeno je da se KK manje javlja kod implantata koji imaju makroteksturisanu površinu i kod onih koji su presvučeni poliuretanskom penom. S druge strane, BIA-ALCL se češće dijagnostikuje kod pacijentkinja kojima su ugraĎeni upravo makroteksturisani implantati. Subklinička infekcija koja predstavlja odgovor organizma na postojanje biofilma na ugraĎenim implantatima, predstavlja jedan od najznačajnijih etioloških faktora za nastanak KK i BIA-ALCL. Biofilm je konglomerat mirkoorganizama uronjenih u matriks koji ih štiti od dejstva antibiotika i antiseptika. Kako je nemoguće delovati medikamentozno na eradikaciju biofilma, brojni autori daju razne preporuke u cilju izbegavanja kontaminacije implantata tokom operativnog zahvata, a time i formiranja biofilma. Pored brojnih mera, savetuje se i ispiranje džepa u koji će se plasirati proteza kao i same proteze, nekim od antiseptičkih ili antibiotskih rastvora. Do sada ne postoje prihvaćene jasne preporuke o načinu ispiranja različitih implantata, objavljena su samo lična iskustva raznih autora. Ciljevi ovog istraživanja su bili da se ustanovi mogućnost formiranja biofilma četiri različite bakterije (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa i Ralstonia pickettii) na tri različito teksturisana silikonska implantata za dojku (sa porama veličine 70-150 μm, 50–900 μm i 13 μm) u in vitro uslovima; da se ispita da li ispiranje antisepticima (oktenidindihidrohloridom i povidon jodom), ili antibiotikom (cefuroksimom) ili istovremeno mešavinom povidon joda i antibiotika pre bakterijske kontaminacije sa četiri različite bakterije ima uticaja na formiranje biofilma na tri različito teksturisana implantata za dojke u in vitro uslovima; i da se ispita efekat antiseptika u odnosu na efekat antibiotika na formiranje bakterijskog biofilma na tri različito teksturisana silikonska implantata za dojku. Istraživanje je koncipirano kao prospektivna studija u vidu eksperimenta koji je izveden u Laboratoriji za mikrobiologiju, Instituta za javno zdravlje Vojvodine u Novom Sadu. Za izvoĎenje eksperimenta korišćeni su uzorci tri vrste silikonskih implantata za dojku sa različito teksturisanom površinom, odnosno porama različite veličine: 70-150 μm, 50–900 μm, i 13 μm. Od svakog od navedenih implantata su pravljeni uzorci, sečenjem kapsula implantata na komadiće veličine 1x1 cm. Ukupno je bilo 1440 uzoraka. Na osnovu teksture uzorci su podeljeni u tri grupe: Grupa 1 (pore veličine 70-150 μm), Grupa 2 (pore veličine 50–900 μm) i Grupa 3 (pore veličine 13 μm). Svaka od ovih grupa je dalje podeljena u jednu kontrolnu grupu i po četiri ispitivane grupe. Nakon sterilizacije uzoraka svaka kontrolna grupa je kontaminirana sa po 100μl bakterijskog bujona Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) i Ralstonia pickettii (n=30). Ispitivane grupe se bile podeljene prema načinima ispiranja na one u kojima su uzorci prvo ispirani: oktenidin – dihidrohloridom ili povidon jodom ili cefuroksimom ili kombinacijom povidon joda i dva antibiotika, pa potom kontaminirani sa po 100μl bakterijskog bujona Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) i Ralstonia pickettii (n=30). Po završenoj kontaminaciji, uzorci su se inkubirali na temperaturi od 37°C u trajanju od 96h, čime su stvoreni uslovi za formiranje biofilma. Nakon inkubacije, svaki pojedinačni uzorak je uronjen u sterilan tripton soja bujon, izlagan soničnoj energiji u trajanju od 1minuta i zatim vorteksiran 1 minut, čime je omogućeno odvajanje nastalog biofilma od implantata. Za ispitivanje sposobnosti formiranja biofilma korišćena je modifikovana tehnika sa mikrotitar pločom po Stepanoviću. Rezultati su pokazali da sve četiri ispitivane bakterije S. epidermidis, S. aureus, P. aeruginosa i Ralstonia pickettii statistički značajno više stvaraju biofilm na implantatima sa porama veličine 50–900 μm u odnosu na pore 70-150 μm i u odnosu na pore veličine 13 μm. Biofilm se statistički značajno više stvara na porama veličine 70-150 μm u odnosu na pore 13 μm. Jedini izuzetak je Pseudomonas aeruginosa kod kojeg ne postoji statistični značajna razlika u produkciji biofilma na teksturisanim implantatima sa porama veličine 70-150 μm u odnosu na one sa porama 13 μm. TakoĎe, sve četiri ispitivane bakterije statistički značajano manje stvaraju biofilm nakon ispiranja povidon jodom, oktenidin-dihidrohloridom ili rastvorom antibiotika u sve tri grupe implantata, u odnosu na površine koje nisu ispirane. Izuzetak je S. epidermidis u Grupi 3 kod kojeg nije utvrĎeno statistički značajno manje formiranje biofilma nakon ispiranja oktenidin dihidrohloridom u odnosu na neispiranje. Cefuroksim je bio efikasniji u sprečavanju formiranja biofilma sve četiri ispitivane bakterije u odnosu na neispiranje u Grupi 1, kao i za S. epidermidis i Ralstoniu Pickettii u Grupi 2. Cefuroksim se nije pokazao statistički značajno efikasnim u sprečavanju formiranja biofilma S. aureus i P. aeruginosa u Grupi 2, kao ni kod jedne bakterije u Grupi 3. Dalje je dokazano da su antiseptici (oktenidin-dihirohlorid i povidon jod) kao i mešavina povidon joda i dva antibiotika (cefuroksim i gentamicin), statistički značajno efikasnji od ispiranja samo antibiotikomcefuroksimom u smanjenju formiranja biofilma sve četiri ispitivane bakterije kod sva tri ispitivana, različito teksturisana silikonska implantata. Rezultati su pokazali da je ispiranje povidon jodom statistički značajno efikasnije u prevenciji stvaranja biofilma kod skoro svih ispitivanih bakterija od ispiranja oktenidin- dihidrohloridom u sve tri grupe implantata. Statistički značajna razlika nije utvrĎena u prevenciji stvaranja biofilma Staphylococcus aureusa kod sve tri grupe implantata prilikom ispiranja povidon jodom u odnosu na oktenidin- dihidrohlorid, kao i kod Ralsotnia pickettii u Grupi 2. Na osnovu rezultata ove studije, preporuka je da se koriste mikroteksturisani implantati kao i da se oni, pre ugradnje isperu povidon jodom ili mešavinom povidon jod i dva antibiotika (cefuroksim i gentamicin), u cilju prevencije stvaranja biofilma, a time i postoperativnih komplikacija koje mogu nastati nakon ugradnje implantata.
The most common complication after breast implant surgery is contracture of capsule, which is normally formed around implants as part of foreign body reaction. The most sincere complication after this kind of surgery is breast implant associated anaplastic large cell lymphoma (BIA-ALCL). The cause of these complications is still unknown. It is evident that capsular contracture (CC) is seen less frequently in patients with macro-textured implants and in those with implants covered with polyurethane foam. On the other hand, BIA-ALCL is diagnosed more frequently in patients with those, macro-textured implants. Subclinical infection, defined as an response of organism on presence of biofilm on the implant, is considered to be one of the most important etiologic factors for CC and BIA-ALCL. Biofilm is a conglomerate of microorganisms immersed into matrix, which protects them from influence of antibiotics and antiseptics. As it is impossible to eradicate biofilms with medicaments, many authors suggest different steps in order to avoid contamination of the implant during the operation and therefore, prevent the formation of biofilm. Among many tips, it is recommended to irrigate the pocket for breast implant and the implant itself, with some antiseptic or antibiotic solution. Up till now, there is no agreed consensus on the type of irrigation for different implants. Only personal experiences of a few authors have been published. Aims of this research were: to establish the possibility of biofilm formation of four different bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa and Ralstonia pickettii) on three differently textured breast implants (with pore diameter of 70-150 μm, 50–900 μm and 13 μm) in vitro; to examine whether the irrigation of implant with antiseptics (povidone iodine and octenidine dihydrochloride), antibiotics (cefuroxime) or mixture of povidone iodine and two antibiotics, before the contamination with bacteria, has an influence on the incidence on biofilm formation on three differently textured implants; and to examine the effect of antiseptics in contrast to the effect of antibiotics on biofilm formation on three differently textured breast implants. The study was conducted as a prospective research that took place at the Laboratory for microbiology, at the Institute of public health of Vojvodina in Novi Sad. For the experiment, three types of silicone breast implants were used with different pore sizes: 70-150 μm, 50–900 μm and 13 μm. Samples were made by cutting each of these types of implants into pieces sized 1x1cm. There were 1440 samples in total. According to texture, samples were divided it three groups: Group 1 (pore size 70-150 μm), Group 2 (pore size 50–900 μm) and Group 3 (pore size 13 μm). Furthermore, each of these groups was divided in one control and four test groups. After sterilisation of samples, every control group was contaminated with 100μl of bacterial broth of Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) and Ralstonia pickettii (n=30). Tested groups were divided according to type of irrigation into those where samples were firstly irrigated with either: octenidine dihydrochloride of povidone iodine or cefuroxime of mixture of povidone iodine with two antibiotics, and after the irrigation, contaminated with 100μl bacterial broth of Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) and Ralstonia pickettii (n=30). After contamination, samples were incubated on 37°C for 96h, which created excellent conditions for biofilm formation. After incubation, each sample was dipped into sterile tripton soy broth, and then exposed to sonic energy for 1 minute and vortexed for 1 minute, which made biofilm separate from the implant. For testing the capability of biofilm formation, modified technique with microtitar plates described by Stepanović was used. Results show that all four examined bacteria S. epidermidis, S. aureus, P. aeruginosa and Ralstonia pickettii form more biofilm on implants with pore sizes 50–900 μm compared to implants with pore size 70-150 μm and those with 13 μm. Statistical significance was found in biofilm formation on implants with pores 70-150 μm compared to implants with pores 13 μm. Furthermore, all four examined bacteria form statistically less biofilm after the irrigation with any of used solutions: povidone iodine, octenidine dihydrochloride, antibiotic solution of mixture of povidone iodine and two antibiotics, in all three groups of implants compared to surfaces that were not irrigated. The exception is S. epidermidis in Group 3, where no statistical significance was found on biofilm formation after the irrigation with octenidine dihydrochloride compared to non-irrigation. Cefuroxime was more efficient in biofilm prevention for all four tested bacteria compared to non-irrigation in Group 1 and for S. epidermidis and Ralstonia pickettii in Group 2. There was no statistical significance found in prevention of S. aureus i P. aeruginosa biofilms when irrigating with cefuroxime in Group 2, as well as for all tested bacteria in Group 3. Furthermore, it was verified that antiseptics (octenidin dihydrochloride and povidone iodine) and mixture of povidone iodine and two antibiotics (cefuroxime and gentamycin), were statistically more efficient in biofilm prevention of all four examined bacteria in all groups of implants, compared to irrigation with antibiotic-cefuroxime alone. Results show that irrigation with povidone iodine is statistically more efficient in biofilm prevention of almost all examined bacteria compared to irrigation with octenidine dihydrochloride in all groups of implants. There was not found any statistical significance in prevention of Staphylococcus aureus biofilm when irrigating with povidone iodine compared to octenidine dihydrochloride in all groups of implants, and also in biofilm prevention of Ralsotnia pickettii in Group 2. According to results of this research, it is recommended to use micro-textured implants and to irrigate them with povidone iodine or mixture of povidone iodine and two antibiotics (cefuroxime and gentamycin) prior the implementation, in order to prevent biofilm formation which is most probable cause of postoperative complications after implant surgery.

Les infeccions bacterianes han esdevingut un greu problema a escala mundial per culpa del mal ús que hom ha fet dels antibiòtics. L’adquisició de resistències als antimicrobians s’ha accelerat i cada cop hi ha més bacteris resistents a tots els antibiòtics coneguts. Aquest problema es veu agreujat quan els bacteris formen un biofilm i les infeccions es tornen cròniques. Els bacteris creixents en forma de biofilm produeixen una matriu extracel·lular protectora i adquireixen així un mecanisme extra de protecció vers els antibiòtics. A més, en l’actualitat no hi ha cap antibiòtic desenvolupat que sigui capaç d’actuar específicament contra els biofilms bacterians madurs i, per tant, es necessiten nous procediments i fàrmacs per a poder tractar-los. En aquest treball s’estudien noves metodologies per al tractament de bacteris creixent en forma de biofilm des de tres punts de vista diferents. En primer lloc, s’intenta buscar noves teràpies i, per això, s’analitza l’acció antimicrobiana i antibiofilm de noves molècules, que han sigut modificades químicament per a millorar el seu potencial contra els biofilms de Staphylococcus aureus. Una part d’aquestes molècules són derivades del triclocarban i unes altres són derivades de l’àcid oleanòlic i l’àcid maslínic. En ambdós casos s’han aconseguit molècules menys tòxiques i més actives que els compostos dels quals deriven, fins i tot contra la soca de S. aureus resistent a meticil·lina (MRSA). En segon lloc, es pretén desenvolupar una tecnologia que permeti determinar, de mantera senzilla i amb precisió, la sensibilitat antibiòtica dels biofilms, ja que els mètodes que es fan servir actualment es basen en el creixement microbià en forma planctònica i no són fiables en bacteris creixent en forma de biofilm. Seguint aquest fil, s’ha desenvolupat el dispositiu “BiofilmChip”, una estructura microfluídica que permet el creixement de biofilms de manera senzilla i l’anàlisi de la seva sensibilitat sense la necessitat d’usar un microscopi confocal. En tercer i últim lloc, es volen desenvolupar noves tecnologies per al tractament de biofilms utilitzant enzims disgregadors del biofilm i fent servir mètodes alternatius d’alliberament de fàrmacs (drug delivery), com són les nanopartícules. Així, s’ha treballat amb els enzims alginat liasa i desoxiribonucleasa I (DNasa I), que degraden components de la matriu extracel·lular dels biofilms i en trenquen l’estructura, fent possible que els antibiòtics travessin aquesta barrera i puguin actuar. Per una banda, s’ha analitzat l’especificitat, l’activitat disgregadora i la sinergia amb antibiòtics de cinc alginat liases contra els biofilms de Pseudomonas aeruginosa. Per altra banda, s’ha treballat amb unes nanopartícules que milloren l’activitat de l’antibiòtic tobramicina, ja que contenen l’enzim DNasa I que permet la penetració d’aquest antibiòtic en els biofilms de S. aureus i P. aeruginosa. Aquestes nanopartícules, a més, estan marcades amb un fluoròfor, la qual cosa ha permès visualitzar la seva interacció amb la matriu extracel·lular del biofilm.

L’émergence et la multiplication des infections impliquant des bactéries résistantes et multi-résistantes aux traitements par voie antibiotique sont actuellement un défi majeur dans le domaine de la santé. En effet, la résistance des microorganismes aux molécules antibiotiques est devenue un phénomène de plus en plus préoccupant notamment en milieu hospitalier d’où la nécessité de faire appel à de nouvelles thérapies et à de nouveaux agents antimicrobiens plus efficaces. De nombreux agents antibiotiques classiques ont été développés ces dernières années, mais beaucoup d’entre eux présentent encore des risques d'effets secondaires plus ou moins toxiques sur les cellules eucaryotes, et en dépit de leur efficacité importante contre des microorganismes multi-résistants. Ainsi, les peptides antimicrobiens sont considéré comme de bons candidats dans la lutte contre multi-résistantes microorganismes, principalement en raison de leur faible toxicité sur les cellules eucaryotes et de leurs différents modes d'action par rapport aux antibiotiques classiques. En effet, ces derniers sont généralement non spécifiques et sont moins susceptibles de mener aux phénomènes de résistance observés pour les antibiotiques classiques. L'objectif des travaux menés dans ce mémoire était d'étudier les modes d'action des deux agents antimicrobiens différents; i) la colistine, un polypeptide cyclique déjà utilisé pour traiter les infections causées par des bactéries multi-résistantes et ii) la catestatine bovine (CAT), un peptide linéaire récemment découvert faisant partie de la famille des HDP (Host Defense Peptides), c’est-à-dire produite par le système endocrinien et immunitaire des mammifères. Cette étude a été réalisée principalement à l’aide de différentes méthodes de caractérisation physico-chimique telles que la microscopie à force atomique (AFM) et la spectroscopie infrarouge à transformée de Fourier en réflexion totale atténuée (ATR-FTIR). L'activité de la colistine sur des membranes phospholipidiques pures et mixtes (à base de DPPC, DOPC et DPPE) a été suivie en temps réel et in-situ par ces deux techniques. Les modifications de l'empreinte biochimique des membranes, en particulier au niveau de la bande Amide II et du rapport d'intensité intégré Amide II/C=O nous a permis de renforcer l'hypothèse selon laquelle l'activité du peptide était plus intense sur les membranes mixtes que sur les membranes purs. Des modifications similaires dans l'empreinte biochimique de ces membranes ont été observées quand elles avaient été exposées à la catestatine. En outre, la spectroscopie infrarouge a également mis en évidence des changements conformationnels dans la structure de la catestatine, notamment par le passage d’une structure en pelote dite « random coil » à une structure en hélice alpha, et ce uniquement au contact avec la membrane. De tels changements conformationnels pourraient être impliqués dans l'activité antimicrobienne et le mode d'action de ce peptide. En outre, nous nous sommes également intéressé à l’action des deux peptides sur des membranes phospholipidiques plus complexes puisque constituées principalement d’extraits naturels de lipopolysaccharides bactériens (lipide A, LPS-s et le LPS-re). Nos résultats ont mis en évidence que les deux agents antimicrobiens étaient à l’origine d’une réorganisation de la structure des membranes et dans certains cas, le peptide était à l’origine de la formation des pores de différentes tailles. L'influence de l'élasticité de la membrane a également été étudiée à l’aide de la spectroscopie de force (AFM). Cette étude a mis en évidence un impact considérable des peptides sur les propriétés mécaniques des membranes et en particulier sur leur élasticité. Afin de se rapprocher des conditions réelles d'un traitement antimicrobien, nous avons exposé des biofilms bactériens de E. coli différentes de doses de deux peptides antimicrobiens. [...]
The emergence and multiplication of infections involving resistant and multi-resistant antibiotic-resistant bacteria is currently a major challenge in the field of health. Indeed, the resistance of microorganisms to antibiotic molecules has become an increasingly worrying phenomenon, particularly in hospitals, hence the need for new therapies and new antimicrobial agents that are more effective. Many conventional antibiotic agents have been developed in recent years, but many of them still present risks of more or less toxic side effects on eukaryotic cells, and despite their high effectiveness against multi-resistant microorganisms. Thus, antimicrobial peptides are considered good candidates in the fight against multi-resistant microorganisms, mainly because of their low toxicity to eukaryotic cells and their different modes of action compared to conventional antibiotics. Indeed, the latter are generally non-specific and are less likely to lead to the observed resistance phenomena for conventional antibiotics.The aim of the work carried out in this thesis was to study the modes of action of the two different antimicrobial agents; (I) colistin, a cyclic polypeptide already used to treat infections caused by multi-resistant bacteria; and (ii) bovine catestatin (CAT), a recently discovered linear peptide belonging to the Host Defense Peptides Ie produced by the endocrine and immune system of mammals. This study was carried out mainly using different physico-chemical characterization methods such as Atomic Force Microscopy (AFM) and Fourier Transform Infrared Spectroscopy (ATR-FTIR). The activity of colistin on pure and mixed phospholipid membranes (based on DPPC, DOPC and DPPE) was monitored in real time and in situ by these two techniques. The changes in the biochemical fingerprint of the membranes, in particular in the Amide II band and in the Amide II / C = O integrated intensity ratio allowed us to reinforce the hypothesis that the activity of the peptide was more intense On mixed membranes than on pure membranes. Similar changes in the biochemical footprint of these membranes were observed when they were exposed to catestatin. In addition, infrared spectroscopy has also demonstrated conformational changes in the structure of catestatin, in particular by the passage from a so-called random coil structure to an alpha-helix structure, and only in contact with the structure membrane. Such conformational changes could be implicated in the antimicrobial activity and mode of action of this peptide. In addition, we have also been interested in the action of the two peptides on more complex phospholipid membranes since they consist mainly of natural extracts of bacterial lipopolysaccharides (lipid A, LPS-s and LPS-re). Our results showed that the two antimicrobial agents were responsible for a reorganization of the structure of the membranes and in some cases the peptide was at the origin of the formation of the pores of different sizes. The influence of the elasticity of the membrane has also been studied using force spectroscopy (AFM). This study revealed a considerable impact of the peptides on the mechanical properties of the membranes and in particular on their elasticity. In order to approximate the actual conditions of antimicrobial treatment, we exhibited different bacterial E. coli biofilms from doses of two antimicrobial peptides. This latter study was carried out in real time and in situ using infrared spectroscopy and atomic force microscopy. Infrared spectroscopy allowed us to follow the modifications of the biochemical fingerprint of the biofilm on the course of the treatment. Also provided information on possible changes in bacterial metabolism. In parallel with these measurements, the AFM allowed us to observe the changes in the morphology and mechanical properties of the bacterial biofilm as a function of the antimicrobial treatment applied. [...]

Chapter 1 of this thesis reviews the current literature on Chronic Rhinosinusitis (CRS) including its aetiology and the role of bacterial biofilms in CRS. It also presents an updated review on all current and emerging topical anti-biofilm options in the management of CRS. Chapter 2 describes the optimisation of an in vivo model using Chitogel (CG) as a drug delivery vehicle for anti-biofilm agents to treat Staphylococcus aureus biofilms. Budesonide and Mupirocin were incorporated as they have some efficacy in the management of recalcitrant CRS in the form of topical irrigations. This study showed that Chitogel- Budesonide-Mupirocin gel significantly reduces S. aureus biofilms in vivo and is the first study to suggest an alternative mode of topical drug delivery to the sinuses. Chapter 3 furthers the study of topical anti-biofilm gel by incorporating novel antimicrobial agents Deferiprone and Gallium Protoporphyrin (DG) into Chitogel. DG has a synergistic anti-biofilm effect against S. aureus and Methicillin Resistant S. aureus (MRSA) biofilms by targeting the iron metabolism pathway that is crucial for bacterial growth and survival. This study has shown that Chitogel- Deferiprone- Gallium Protoporphyrin is safe and effective in eradicating S. aureus biofilms in vivo. Chapter 4 explores the potential wound healing properties of Deferiprone in vitro. In this study Deferiprone was shown to delay primary nasal fibroblast migration without affecting epithelial migration, decrease collagen and reactive oxygen species (ROS) production and reduce interleukin 6 (IL-6) production. This anti-inflammatory potential and ability to limit scar tissue formation has wide prospective applications in the field of sinus surgery. Chapter 5 explores the long-term safety of S. aureus bacteriophage (NOV012) in vivo as a novel treatment option in CRS. Bacteriophage (Phage) therapy was first proposed as an antibacterial treatment in the 1910s and has been recently revived in the face of a global antibiotic resistance crisis. In this study we have shown that S. aureus phage are safe as a topical sinus irrigation in vivo for up to 3 weeks. Chapter 6 furthers the study of Pseudomonas aeruginosa phage (CT-PA) in vivo. Topical sinus irrigation of CT-PA was safe and effective in reducing P. aeruginosa biofilms in vivo over 3 weeks. Chapter 7 explores the translation of our in vivo studies into the first phase-1 clinical trial investigating the safety and preliminary efficacy of phage in S. aureus CRS. It was safe and well tolerated up to 3×109 PFU twice daily for 14 days with no dose-limiting side effects. Preliminary efficacy indicated favourable outcomes across all cohorts with 2 out of 9 patients achieving eradication of infection. Chapter 8 presents a pilot study investigating the evidence behind alternative treatments like colloidal silver (CS) in recalcitrant CRS. This is the first clinical study looking at the safety and efficacy of CS as a topical sinus irrigation in the management of CRS despite it being widely available for purchase over-the-counter. Twice daily CS sinonasal rinses for 10 days is safe but not superior to culture-directed oral antibiotics. We believe that a larger study and further optimisation of treatment duration could further the potential of this therapy. Chapter 9 investigates the role of manuka honey (MH) sinus irrigations with augmented methylglyoxal (MGO) in recalcitrant CRS. Twice daily MH rinses for 14 days have not been shown to be superior to culture-directed oral antibiotics and saline rinses in bacterial eradication but have shown significant improvement in endoscopic scores comparable to control. In the face of a global antibiotic resistance crisis, now more than ever, clinicians are practising more judicious use of systemic oral antibiotics. With an eye to the future, we hope that this thesis on novel topical anti-biofilm therapies would spur further research and someday offer clinicians viable alternatives to systemic oral antibiotics in the management of recalcitrant CRS.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Introduction: Recent evidence has demonstrated the presence of bacterial biofilms on the mucosa of patients with Chronic Rhinosinusitis (CRS), suggesting their role in the pathogenesis of the condition. This thesis contains two separate studies. The studies investigated novel topical therapies by using previously established in vitro and in vivo biofilm growth and detection methods. In the first study, several different proposed anti-biofilm agents were evaluated in a sheep biofilm model, each with varying degrees of immediate and short-term success against Staphylococcus aureus biofilms. A second study was conducted to determine the in vitro anti-bacterial and anti-biofilm properties of Chitosan/Dextran (CD) gel, a novel chitosan-based product with remarkable mucosal healing and haemostatic properties. Methods: Three alternative anti-biofilm treatments: Mupirocin, CAZS (Citric Acid Zwitterionic Acid) and Gallium Nitrate were evaluated in a prospective randomized controlled single-blinded trial using a previously established sheep biofilm model of CRS. The sheep mucosal samples were analyzed for presence of S. aureus biofilms using BacLight staining and CLSM, and the degree of biofilm involvement was determined using FISH (Fluorescence In-Situ Hybridization). The MIC/MBC values for CD gel and its constituents were determined by macro-dilution methods described by Jorgensen et al.[1]. Established in vitro biofilms grown from common CRS pathogens (ATCC strains and clinical isolates) were subjected to treatment by CD gel and its components (chitosan and dextran). A 96-well micro-titre crystal-violet staining method described by O’Toole and Kolter [2] was used to determine the anti-biofilm profile of CD gel against several bacterial strains with known biofilm-forming capacity. Results: Following 8 days of inoculation with S. aureus, all treatment groups in the sheep biofilm model showed a statistically significant reduction in biofilm surface coverage compared to no treatment. Importantly, sheep frontal sinuses treated with twice-daily mupirocin flushes for 5 days showed almost negligible biofilm growth after the follow-up period of 8 days (0.84% ± 1.25% surface area coverage per visual field). The overall data from the in vitro studies suggest that CD gel has marked anti-microbial activity against planktonic and biofilm-forming bacteria. It was inhibitory and bacteriocidal at sub-clinical concentrations (25mg/mL) for all bacteria tested except for E. coli. When tested against a nutrient-free environment as well as a positive growth control, bacteria were essentially unable to grow in its presence. Conclusion: Recalcitrant CRS is a difficult condition to manage and its pathogenesis has been closely linked to the presence of bacterial biofilms. Using a standardized biofilm sheep model of CRS, regular treatment with mupirocin flushes over a 5 day period showed an almost complete eradication of biofilms as assessed by mucosal surface coverage, with sustained effects over the 8 day period of follow-up. Equally as efficacious in the in vitro setting, CD gel demonstrated potent anti-bacterial and anti-biofilm activity against a number of pathogenic organisms suspected of being involved in acute and chronic rhinosinusitis. CD gel’s favourable haemostatic and mucosal healing profile posits it as an ideal post-ESS packing material. These two topical agents therefore hold promise as effective treatment options in the management of CRS.
Thesis (M.S.) -- University of Adelaide, School of Medicine, 2010

Indiana University-Purdue University Indianapolis (IUPUI)
Introduction: One of the main objectives of endodontic therapy is to reduce microbes and remove inflamed pulpal tissue within the root canal system (RCS). This is accomplished through chemomechanical debridement of the RCS using hand and rotary instrumentation along with an antimicrobial irrigant. Today, the most commonly used irrigant is sodium hypochlorite (NaOCl), often at concentrations toxic to human cells. The use of ozone as an endodontic irrigant is a novel technique that has been proven to be antimicrobial against several microorganisms. However, independent research is lacking on ozone’s efficacy against an established endodontic biofilm. If ozone’s efficacy against biofilms is confirmed, the use of toxic and potentially dangerous sodium hypochlorite could be replaced in some clinical situations (i.e., regeneration, immature teeth, resorption) with a safer and effective alternative. Objective: The aim of the current study was to evaluate the anti-biofilm activity of different concentrations of ozonated water compared to various concentrations of NaOCl against an established endodontic biofilm of Enterococcus faecalis in root canal treated teeth in vitro. Materials and Methods: The crowns of similarly sized, maxillary anterior teeth were removed, and the roots cut to a standard length (12 mm). All root canals were instrumented to a standard size. Specimens were sterilized and then inoculated with E. faecalis, which were allowed to grow for two weeks to form an established biofilm. There were six treatment groups: 1) 6% NaOCl; 2) 1.5% NaOCl; 3) 16µg/mL ozonated water; 4) 25µg/mL ozonated water; 5) 50µg/mL ozonated water, and 6) saline. Following treatment, samples were collected, plated, and incubated for two days. The number of CFU/mL were determined, and samples visualized using confocal imaging. The effect of treatment group on bacterial counts was made using one-way ANOVA followed by pair-wise comparisons. Null Hypothesis: Endodontically treated teeth irrigated with ozonated water will not demonstrate a statistically significant decrease in the E. faecalis biofilm compared to those treated with sodium hypochlorite Results: CFUs were converted to log10 and compared using Fisher’s Exact tests or one-way ANOVA followed by pair-wise tests. In all observations utilizing NaOCl irrigation, no colonies formed following treatment. The two NaOCl groups, with 0 CFU/mL, were significantly different than the other four groups (p=0.009). Saline showed a trend towards higher CFU/mL than 50 µg/ml O3 (p=0.068). None of the other comparisons approached statistical significance (p=0.453 25 µg/ml O3, p=0.606 16 µg/ml O3, p=0.999 25 µg/ml O3 vs 50 µg/ml O3, p=0.990 16 µg/ml O3 vs 50 µg/ml O3, p=1.000 16 µg/ml O3 vs 25 µg/ml O3). Confocal imaging helped illustrate effects of irrigation and confirm CFU findings. Conclusion: The results of this study failed to reject the null hypothesis. There was a statistically significant difference in the E. faecalis biofilm remaining in the groups treated with ozonated water compared to those treated with NaOCl. However, there was a trend towards higher CFU/mL in the saline group compared to the 50µg/mL ozonated water group. According to this finding, future studies should evaluate the effects of higher concentrations of ozonated water against an established E. faecalis biofilm. In addition, other follow-up studies might include ozonated water’s effect on human cells, such as the stem cells of the apical papilla that are so critical to the success of regenerative endodontic procedures. Due to university and laboratory closures caused by the COVID-19 pandemic, this project was stopped short and an insufficient sample size did not allow for proper statistical power. Additional occasions should be run upon the university’s re-opening to allow for proper statistical power.

Indiana University-Purdue University Indianapolis (IUPUI)
The primary goal of root canal treatment is to eliminate microbes from the root canal system, which is the cause of pulpal and periapical infections. Research shows that after a single visit of chemomechanical debridement microbes continue to remain within the canal system. An interappointment medication step has been advocated to maximize potential elimination of microbes within the root canal system. Previous studies have shown propolis to be antibacterial against common endodontic microbes. Studies have shown trends in different microbes being present in primary verus secondary endodontic infections. The majority of literature has focused on the efficacy of propolis against Enterococcus faecalis, a microbe commonly implicated in secondary endodontic 95 infections. The aim of this study was to demonstrate the efficacy of propolis against Fusobacterium nucleatum, a microbe commonly found in primary endodontic infections. This study aims to demonstrate the efficacy of propolis against a bacterium of primary endodontic infections (F. nucleatum) as well as against microbial biofilm to further support its potential use as a novel intracanal medicament. Dilutions of propolis were added to cultures of F. nucleatum in microtiter plates in a range from 390 μg/ml to 50,000 μg/ml. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the minimum biofilm inhibitory concentration (MBIC) were determined. The MIC was determined of the total solution (biofilm+planktonic), planktonic, and biofilm (MBIC) after a 48-hour incubation period. The MBIC was determined by fixing biofilm to the wells and using crystal violet staining with spectrophotometry. The MBC was examined by plating solution from each concentration test well and reading the plates after 48 hours of incubation. The results show that the MIC of the total (biofilm+planktonic) appears to occur at a concentration of 6250 μg/ml. The MBIC appears to occur at the concentration of 1562.5 μg/ml. The planktonic results exhibit no significant difference in test and control wells. There was no MBC at any of the test concentrations. The propolis appears to inhibit bacterial growth and biofilm formation but does not appear to be bactericidal at any of the tested concentrations. The results of this study indicate that propolis has an MIC and MBIC when tested in vitro against F. nucleatum, although it does not show an MBC. There appears to be potentially significant interaction of propolis with biofilm as displayed by the lower concentration needed to exibit inhibitory effects on biofilm formation. This information 96 may contribute to the ability to develop a proper concentration of propolis to use in vivo when treating endodontic infections.

We investigated the antibacterial effect of low concentrations of double antibiotic paste (DAP) loaded into a methylcellulose system against bacterial biofilms obtained from mature and immature teeth with necrotic pulps. Standardized radicular dentin specimens were randomly divided into six experimental groups (n = 20). Group 1: 5mg/mL DAP treatment. Group 2: 1mg/mL DAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose. Group 5: No treatment. Group 6: No bacteria or treatment. Clinical bacterial isolates were obtained from mature and immature teeth with necrotic pulps indicated for endodontic regeneration or routine endodontic treatment, respectively. Specimens in each group were inoculated with either bacterial isolates (n = 10) and incubated anaerobically for 3 weeks. Specimens were then treated for one week with the assigned group treatment. Treatments were rinsed with sterile saline and biofilms were detached and spiral plated using biofilm disruption assays. Wilcoxon Rank Sum tests followed by pair-wise comparisons were used for statistical analyses. Treatment of infected dentin with 1 mg/ml of DAP, 5 mg/mL of DAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste. Furthermore, 1 mg/mL of DAP caused complete eradication of biofilm obtained from mature tooth with necrotic pulp. However, the same concentration was not able to completely eradicate biofilm obtained from the immature tooth with necrotic pulp. Low concentrations of DAP (1-5 mg/mL) loaded into a biocompatible methylcellulose system demonstrated significant antibacterial effects against biofilm obtained from both mature and immature teeth with necrotic pulps.

M. Tech. Pharmaceutical Sciences
Bacteria are present in natural environments and can develop into biofilms. Mucus-like extracellular matrix produced by biofilms provides protection to biofilm formers by inhibiting antimicrobial penetration and de-activating antimicrobial molecules, while allowing strong attachment onto surfaces. Biofilm development is associated with otitis media and cystic fibrosis. In this study, selected biofilm-formers implicated in otitis media and cystic fibrosis, Pseudomonas aeruginosa and Moraxella catarrhalis, were used to evaluate the effect of combinations of mucoactive substances and antibiotics against their biofilms. Microtiter-plate assay and optical density measurements were used to evaluate antimicrobial and antibiofilm activities. Confocal scanning laser microscopy was used to visualise the effect of selected treatments against biofilms.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Dentistry Indiana University Endodontic Department
The residual antibacterial effects of antimicrobials used in endodontic regeneration against biofilm bacteria obtained from immature and mature teeth Jordon C. Jacobs DDS, Richard L Gregory PhD, Ygal Ehrlich DMD, Kenneth Spolnik DDS, MS, Josef S. Bringas DMD, DDS, MS, and Ghaeth Yassen BDS, MSD, PhD We explored the residual antibacterial properties of dentin pretreated with low concentrations of double antibiotic paste (DAP) against biofilm bacteria obtained from different clinical sources. Dentin blocks were sterilized and randomized into 4 treatment groups and 2 control groups (n=20). Blocks from treatment groups were pretreated with DAP (1 or 5 mg/ml) loaded into methylcellulose, calcium hydroxide (Ca(OH)2), or methylcellulose paste. After one week, the treatment pastes were removed and all blocks were immersed in PBS. The dentin blocks from treatment groups and one of the control groups were then inoculated with bacterial isolates obtained from immature or mature teeth with pulpal necrosis(n=10). The remaining control group received no bacteria and was used as a sterile control. Blocks were then incubated anaerobically for 3 weeks. Biofilm disruption assays were conducted for all samples. Two-way ANOVA and pair-wise comparisons were used for statistical analyses. The residual antibacterial effect of dentin pretreated with 5 mg/ml of DAP was significantly higher than all other groups regardless of the source of biofilm. Dentin pretreated with 1 mg/ml of DAP demonstrated significantly higher residual antibacterial effects in comparison to dentin pretreated with placebo paste and Ca(OH)2 only in bacterial isolates obtained from mature teeth with pulpal necrosis. Dentin pretreated with Ca(OH)2 did not demonstrate any residual antibacterial effects. Dentin pretreated with 1 or 5 mg/ml of DAP demonstrated significantly better residual antibacterial effects against biofilm bacteria obtained from mature teeth with pulpal necrosis in comparison to bacterial isolates obtained from immature teeth with pulpal necrosis.