[ES] Las biopelículas bacterianas y fúngicas contribuyen enormemente a la persistencia de muchas infecciones graves y potencialmente mortales, las cuales anualmente provocan millones de defunciones. Además, estas bacterias y hongos que crecen adheridas formando biopelículas son hasta 1.000 veces más resistentes a los tratamientos antimicrobianos convencionales, generando una carga económica significativa y dificultando su diagnóstico y tratamiento. Por tanto, es necesario buscar nuevas herramientas fiables para estudiar la dinámica de formación de biopelículas con el fin de mejorar las estrategias de tratamiento.El objetivo general de la tesis doctoral es la puesta a punto de un sistema basado en medidas de impedancia eléctrica para el estudio de la formación y dinámica de crecimiento de las biopelículas bacterianas (gram-positivas y gram-negativas) y fúngicas, así como de biopelículas complejas multi-especie como las de la placa dental subgingival de muestras periodontales humanas. Tras la puesta a punto del sistema, los objetivos específicos de la tesis doctoral son su aplicación como herramienta en la identificación de tratamientos efectivos contra biopelículas persistentes, la búsqueda de nuevos compuestos antimicrobianos con actividad anti-biofilm, así como la evaluación de novedosas nanopartículas autopropulsadas para la erradicación de biofilms multirresistentes. Finalmente, se ha evaluado su aplicación clínica directa en la selección de la terapia antibiótica para el tratamiento personalizado de pacientes con enfermedad periodontal. / [CA] Les biopelícules bacterianes i fúngiques contribueixen en gran manera a la persistència de moltes infecciones greus i potencialment mortals les quals provoquen anualment milions de morts. A més, estes bactèries i fongs que creixen adherides en forma de biopelícules son fins a 1000 vegades més resistents als tractaments antimicrobians convencionals, generant una càrrega econòmica significativa i dificultant el diagnòstic i tractament. Per això, es necessari trobar noves eines fiables per a estudiar la dinàmica de formació de biopelícules amb l'objectiu de millorar les estratègies de tractament. El objectiu general de la tesis doctoral es la posta a punt de un sistema basat en mesures d'impedància elèctrica per al estudi de la formació i dinàmica de creixement de les biopelícules bacterianes (gram-positives i gram-negatives) i fúngiques, així com de biopelícules complexes mutiespècie com les de la placa dental subgingival de mostres periodontals humanes. Una vegada posat a punt el sistema, els objectius específics de la tesis doctoral son la aplicació com a eina de la identificació de tractaments efectius contra biopelícules persistents, la recerca de nous compostos antimicrobians amb activitat antibiopelícula, així com la avaluació de noves nanopartícules autopropulsades per a l'eliminació de biofilms multiresistents. Finalment, s'ha avaluat l'aplicació clínica directa en la selecció de la teràpia antibiòtica per al tractament personalitzat de pacients amb periodontitis. / [EN] Bacterial and fungal biofilms contribute enormously to the persistence of many life-threatening infections, causing millions of deaths annually. In addition, bacteria and fungi growing as biofilms are up to 1.000 times more resistant to conventional antimicrobial treatments, resulting in a significant economic burden and challenging diagnosis and treatment. Therefore, there is a need to search for new reliable tools to study biofilm formation dynamics to improve treatment strategies. This doctoral thesis aims to set up an impedance-based system to study biofilm formation and dynamics of bacterial (gram-positive and gram-negative) and fungal species, as well as complex multi-species biofilms such as subgingival plaque collected from patients with chronic periodontitis. After the impedance system is set up, the specific objectives of the doctoral thesis are its application as a tool in the identification of effective treatment against persistent biofilms, testing new antimicrobial and anti-biofilm compounds, and the evaluation of novel self-propelled nanoparticles on the eradication of multi-resistant S. aureus biofilms. Finally, a clinical application of the impedance system is proposed, aiming at determining the best individual antibiotic therapy in dental clinics (personalized use of antibiotics). / Work performed at Genomics & Health Department at FISABIO Foundation and described in this doctoral thesis was supported by the Spanish Ministry of Science, Innovation and Universities scholarship FPU17/01302 to Miglė Žiemytė and a grant RTI2018-102032-B-I00 to Alex Mira Obrador. / Ziemyte, M. (2023). Towards Personalized Medicine in Antibiotic Treatment: Development of a Real-Time Cell Analysis System for Biofilm Studies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195434

One of the most concerning characteristics of microbial biofilms is that of increased resistance to antimicrobial agents such as the commonly used biocide chlorhexidine (CHX). This can have huge impact on clinical, household and environmental settings. This is particularly alarming when it involves opportunistic pathogenic environmental organisms such as Delftia acidovorans as routine mitigation practices may fail to be effective. This thesis examines tolerance mechanisms of D. acidovorans biofilms exposed to CHX at inhibitory and sub-inhibitory concentrations. To achieve the study goals and objectives, a CHX-tolerant D. acidovorans strain (WT15), (Minimum Inhibitory Concentration; MIC-15 μg ml-1) was compared to a CHX-sensitive strain (MT51, MIC-1 μg ml-1) that was obtained by mutating the wild type strain using transposon mutagenesis. Specific morphological, structural and chemical compositional differences between the CHX-treated and untreated biofilms of wild type and mutant strains were documented using microscopic techniques including confocal laser scanning microscopy (CLSM), scanning transmission x-ray microscopy (STXM), transmission electron microscopy (TEM) and infrared (IR) spectroscopy. Molecular level changes between biofilms formed by these two strains due to CHX treatment were compared using whole-cell proteomic analysis (determined using differential in-gel electrophoresis, or DIGE) along with fatty acid methyl ester (FAME) analysis. The gene disrupted by transposon insertion that led to increased susceptibility to CHX in the mutant strain was identified as tolQ. CLSM revealed differences in biofilm architecture and thickness between the biofilms formed by strains WT15 and MT51. STXM analyses showed that WT15 biofilms contained two morpho-chemical cell variants; whereas, only one type was detected in MT51 biofilms. STXM and IR spectral analyses revealed that CHX-susceptible MT51 cells accumulated the highest levels of CHX, an observation supported by TEM wherein prominent changes in the cell envelope of CHX-susceptible MT51 cells were observed. DIGE analysis demonstrated that numerous changes in protein abundance occurred in biofilm cells following CHX exposure and that most of these proteins were associated with amino acid and lipid biosynthesis, protein translation, energy metabolism and stress-related functions. Overall, these studies indicate the probable role of the cell membrane and TolQ protein in CHX tolerance in D. acidovorans biofilms, in association with various proteins that are differentially-expressed.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Biofouling of water systems is a problem extensively experienced in industry. Although this subject is the focus of many studies, the ability of microorganisms to survive exposure to biocides is still poorly understood. This study aimed to assess the biocidal effect of ozone on planktonic cells and biofilm communities, to evaluate different ozone generation techniques, and to follow population shifts within the biofilm community. Specific objectives included determining the effect of different ozone concentrations, the effect of different exposure times, and an assessment of microbial responses after exposure to sub-lethal ozone concentrations. Typically, 300 ml of an ovemight bacterial culture was exposed to ozone that was generated by anodic oxidation (0.3% wt or 18- 20% wt, respectively) or silent electric discharge (3.5% wt 03). The ozone was purged into the culture for 5-, 7-, 10- and 15 min., respectively. Enumeration of cells following ~10 min. exposure to 18-20% wt ozone showed a significant reduction in viable cell numbers. In contrast, when exposed to the two lower 03 concentrations, there was little change in the viable cell numbers even after prolonged exposure (30- and 60 min.). To evaluate biofilms, ozone was bubbled into the irrigation that was pumped through replicate flow cell channels. Response to ozone exposure was evaluated after staining the biofilms with the Baclight Viability probe, observation with fluorescence microscopy, and image analysis. The higher ozone concentration (18-20% wt 03) more effectively disrupted the biofilm structure of denser biofilms than the lower concentration, especially after 90 min. exposure. When compared to the controls, the 90 min. exposure resulted in a notable reduction in viable cells from 69% to 38% and a corresponding increase in nonviable cells from 29% to 62%. The lower concentration ozone (3.5% wt 03) was effective against the less dense, thinner biofilms evaluated, but not effective against the thicker biofilm. An analysis of the differences between continuous culture biofilms and batch culture biofilms showed that the biofilms in the batch system were less rigid. To evaluate microbial response to biocides, techniques such as Biolog whole-community metabolic profiles and terminal restriction fragment length polymorphisms (T-RFLP) were used. Biolog analysis of planktonic cells revealed changes following exposure to sub-lethal biocide concentrations, however carbon utilisation profiles resembled that of the controls after 24-48 hours. For biofilm communities, no carbon utilization differences could be detected under these conditions. There was, however differences in T-RFLP patterns between treated and untreated biofilm communities. / AFRIKAANSE OPSOMMING: Biobevuiling van watersisteme is 'n probleem wat algemeen in industriëe ervaar word. Alhoewel hierdie onderwerp die fokus van vele studies is, word die vermoëns van mikroorganismes om blootstelling aan biosiede te weerstaan steeds swak verstaan. Die doel van hierdie studie was om die biosidiese effek van osoon op planktoniese selle en biofilm gemeenskappe waar te neem, om die verskillende osoon generasie tegnieke te evalueer, asook om verskuiwings in die samestelling van die biofilm gemeenskap waar te neem. Spesifieke doelwitte sluit in die bepaling van die effek van verskillende osoon konsentrasies, die blootstellingtye, en 'n waarneming van mikrobiese reaksies na blootstelling aan sub-dodings osoon konsentrasies. Drie honderd ml van 'n oornag bakteriese kultuur was aan osoon, wat deur anodiese oksidasie (0.3% wt of 18% - 20% wt) of geluidlose elektriese ontlading (3.5% wt), gegenereer is, blootgestel. Tye van blootstelling was 5-, 7-, 10-, of 15 min., onderskeidelik. Bepaling van selgetalle na :2:10 min. blootstelling aan 18 - 20% wt osoon, het 'n betekenisvolle verlaging in die getal lewensvatbare mikrobeselle getoon. In teenstelling hiermee, het blootstelling aan twee laer osoon konsentrasies min verskil in die lewensvatbare selgetalle, selfs na verlengde blootstellingstye (30- en 60 min.), getoon. Om biofilms te evalueer is osoon in die medium geborrel wat deur replikaat vloeisel kanale gepomp is. Na osoon blootstelling, was die vloeisel onderwerp aan beeld analise deur gebruik te maak van die Baclight lewensvatbare peiler en fluoressensie mikroskopie. Die hoër osoon konsentrasie (18 - 20% wt 03) het die struktuur van dikker biofilms meer effektiefuiteengeskeur as die laer konsentrasie, veral na 90 min. blootstelling. In vergelyking met die onderskeie kontroles, het die getalle van lewensvatbare selle na 90 min. blootstelling drasties verlaag vanaf 69% tot 38% en 'n ooreenstemmende toename in die nie-lewensvatbare selgetalle vanaf 29% tot 62%. Die laer osoon konsentrasie (3.5% wt 03) was meer effektief teenoor die minder digte en dunner biofilms wat ge-evalueer was, maar nie so effektief teenoor die dikker biofilms nie. 'n Analise van die verskille tussen kontinue-kultuur biofilms en lotkultuur biofilms het getoon dat die lot-kultuur biofilms minder rigied is. Vir die evaluering van mikrobiese reaksies na biosied blootstelling, is tegnieke soos Biolog gemeenskap metaboliese profiele en eind-restriksie-fragment-lengte polimorfisme (TRFLP) gebruik. Biolog analise van planktoniese selle het verskille getoon na blootstelling aan sub-dodelike biosied konsentrasies. Koolstof benutting het wel na 24 - 48 ure met dit van die kontrole ooreengestem. Vir biofilm gemeenskappe was daar geen noemenswaardige verskille in koolstof benutting nie. Daar was wel verskille in T-RFLP patrone tussen die onbehandelde en biosied-behandelde biofilm gemeenskappe.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Microorganisms show a tendency to accumulate on surfaces in aqueous environments to form biofilms. Microbial biofilms represent a significant problem in public health microbiology as the development of these microbial communities, especially in water distribution systems, may lead to (i) the enhanced growth of opportunistic pathogens, (ii) the development of organoleptic problems, (iii) the reduction in the flow rate and (iv) the regrowth of microorganisms. In this project, biofilm monitors were installed in a large water distribution system to study biofilm phenomena in drinking water systems, and to deduce the biological stability and quality of the potable water. Measurements of biofilm formation potential showed that biofilms did not reach a steady state after 100 to 150 days. The microbial cells in these biofilms were mostly non-culturable. The contribution of the heterotrophic colony count to active biomass, as determined with cell numbers based on ATP measurements were often < 1%, while the ratio of heterotrophic plate counts and direct acridine orange counts were also <1%. The ratio between cell numbers based on ATP measurements and direct acridine orange counts were often < 100%. Results also showed that under certain conditions, such as those investigated in the present study, 1 pg of ATP may not be equal to approximately 104 active bacteria/cells, as stipulated by previous investigations, and that the average ATP content per active bacterial cell is indeed less than 10-16 - 10-15 g. It was calculated that threshold values for assimilable, and dissolved organic carbon below -5 IJg Gil and -0.5 mg Gil, respectively, should be target values for the control of biofilm formation in this system. It was shown that polyethylene, polyvinylchloride, teflon, plexiglass, copper, zinc-coated steel and aluminium provide favourable attachment surfaces that allowed primary colonisation and subsequent biofilm formation. Significant (p < 0.05) differences in surface colonisation on the materials were observed, indicating that the composition of the material has a direct influence on microbial colonisation. The two grades of stainless steel evaluated in this study were the least favourable materials for biofilm formation. It was further demonstrated that the nature of the surface of these materials, flow conditions and water type all had a direct influence on biofilm formation. While modification of the attachment surface did not result in significant differences (p > 0.05) in disinfection efficiency of two commonly used biocides, the concentration of the biocide, as well as the material to which the biofilm is attached, greatly influenced biocidal efficiency. The results show that biofilm monitoring needs to be implemented at the water treatment plants in addition to common biostability measurements. / AFRIKAANSE OPSOMMING: Mikro-organismes neig om te akkumuleer aan oppervlaktes in akwatiese omgewings om biofilms te vorm. Mikrobiese biofilms verteenwoordig In betekenisvolle probleem in publieke gesondheidsmikrobiologie omdat die ontwikkeling van hierdie mikrobiese gemeenskappe in waterverspreidingsisteme mag lei tot (i) die verhoogde groei van opportunistiese patogene, (ii) ontwikkeling van organoleptiese probleme, (iii) die vermindering in die vloeitempo en (iv) die hergroei van mikro-organismes. In hierdie projek was biofilm monitors geïnstalleer in In groot waterverspreidingsisteem om biofilm fenomene in drinkwatersisteme to bestudeer, en om die biologiese stabiliteit en kwaliteit van drinkwater af te lei. Bepalings van biofilmvormingspotensiaal het aangetoon dat biofilms nie In stabiele stadium na 100 tot 150 dae bereik nie. Die mikrobiese selle in hierdie biofilms was meestal niekweekbaar. Die bydrae van die heterotrofiese kolonie tellings tot aktiewe biomassa, soos bepaal deur seltellings gebaseer op ATP metings was dikwels < 1%, terwyl die verhouding van die heterotrofiese plaatteIIings en direkte akridien oranje tellings ook < 1% was. Die verhouding tussen seltellings, gebaseer op ATP metings en direkte akridien oranje tellings was dikwels < 100%. Resultate het ook aangetoon dat onder sekere omstandighede, soos dié wat ondersoek was in die huidige studie, 1 pg ATP nie gelyk is aan min of meer 104 aktiewe bakterieë/selle soos gestipuleer deur vorige ondersoeke nie, en dat die gemiddelde ATP inhoud per aktiewe bakteriële sel inderdaad minder as 10-16 tot 10-15 g is. Dit was bereken dat die drempelwaardes vir assimileerbare en opgeloste organiese koolstof onder -51-1g C/l en -0.5 mg C/l, onderskeidelik, teikens moet wees vir die beheer van biofilmvorming in hierdie sisteem. Dit was aangetoon dat polyetileen, polyvinielchlroried, teflon, plexiglas, koper, sink-bedekte staal en aluminium gunstige aanhegtings oppervlaktes voorsien wat primêre kolonisering en daaropvolgende biofilmvorming toelaat. Betekinisvolle (p <0.05) verskille in oppervlak kolinisering op die materiale was waargeneem, wat aandui dat die samestelling van die materiaal In direkte invloed op mikrobiese kolonisering het. Die twee tipes vlekvryestaal wat geëvalueer was in hierdie studie, was die minder gunstige materiale vir biofilmvorming. Dit was verder gedemonstreer dat die aard van die oppervlak van hierdie materiale, vloeitoestande, en water tipe almal In direkte invloed het op biofilmvorming. Terwyl die aanpassing van aanhegtingsoppervlak nie die ontsrnettinqsdoeltreffendheid resultaat van die twee algemeen-gebruikte biosiede betekinisvol (p > 0.05) beïnvloed het nie, het die konsentrasie van die biosiede doeltreffendheid grootliks beïnvloed. asook die aanhegtings-materiaal, biosied Die resultate het aangetoon dat biofilm monitering geïmplementeer moet word by waterbehandelingsaanlegte as In alternatief vir algemene biostabiliteit metings.

Biofilms are communities of microorganisms associated with surfaces encased in a protective extracellular matrix. These communities often pose clinical and industrial challenges due to their ability to tolerate biocidal treatments and removal strategies. Understanding the ecological interactions that take place during biofilm establishment is a key element for designing future treatment strategies. In this work, I utilized unique methods for studying factors contributing to cooperative antibiotic detoxification in a polymicrobial biofilm model. Subsequently, I tested a novel compound mixture that exhibited promising antibiofilm properties. Escapin is an L-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called Escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. To test the combination of EIP and H2O2 on bacterial biofilms, Pseudomonas aeruginosa was selected as a model, due to its role as an important opportunistic pathogen. Specifically, I examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to untreated controls or to EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to controls. Area layer analysis of biofilms post-treatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, which are significantly lower concentrations than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to either EIP or H2O2 alone. Collectively, these results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.

Une grande partie de la flore microbienne des réseaux de distribution d’eau potable est située au niveau des parois des canalisations. Ce biofilm, génère un certain nombre de problèmes pour la gestion des réseaux tels que la consommation du désinfectant, la croissance bactérienne ou encore l’incorporation de pathogènes qui peuvent conduire au non-respect des critères de qualité des eaux par le relargage d’une partie de sa biomasse dans la masse d’eau. Différents paramètres, parmi lesquels le régime hydraulique, permettent de contrôler la prolifération du biofilm. Nous avons cherché à savoir quel était le paramètre clé liant l’hydrodynamique et les biofilms. En utilisant des réacteurs de Couette-Poiseuille dont la géométrie permet de faire varier indépendamment le gradient de vitesse pariétal (?) et le débit, nous avons montré que le gradient de vitesse pariétal est bien le paramètre important pour le contrôle des biofilms. En utilisant ce résultat, nous avons mis au point un modèle de diffusion-convective permettant de décrire le transport et l’accumulation des bactéries sur la surface. Nous avons ainsi mis en évidence que l’accumulation du biofilm est fonction de ?1/3*t. L’hydrodynamique affectant l’organisation de la biomasse, nous avons quantifié par analyse d’images, l’impact du gradient de vitesse pariétal sur la taille, la forme et l’orientation des éléments du biofilms. Les phénomènes de relargage ont été étudiés par le biais d’inoculation de particules de polystyrène et E. coli dans des biofilms formés en chambres d’écoulement ainsi que par la réalisation d’à-coups hydrauliques dans un écoulement en conduite. / Most of the microbial flora present in the drinking water distribution network is located on the pipe walls. This biofilm is a major concern for drinking water providers as it increases disinfectant decay, provides a shelter for pathogens and is the main site of bacterial growth, thus leading, through bacterial detachment, to the non-respect of drinking water quality criteria. Hydraulic regime is one among several parameters that can be used to control biofilm proliferation. Our first objective was to determine which was the key parameter in the hydraulic regime that was controlling biofilm accumulation. Using Couette-Poiseuille reactors which geometry allow to vary independently wall shear rate (?) an d flow rate, we showed that the wall shear rate was indeed the important parameter for biofilm control. From this result, we built a convective-diffusion model in order to describe bacterial transport and accumulation to the pipe surface. It appeared that biofilm accumulation was a function of ?1/3*t. As hydraulic regime was known to affect biofilm organization, we quantified through image analysis, the effect of wall shear rate, on size, shape and orientation of the biofilm elements. Biofilm stability was studied by inoculations of polystyrene microspheres and E. coli in flow chambers colonized by biofilms. The consequences of the rapid variation of the flow rate on a biofilm formed in a pipe flow were also studied.

As dificuldades do reprocessamento dos endoscópicos gastrointestinais, o risco de contaminação e os casos de infecções representam desafios amplamente conhecidos na comunidade cientifica. Neste sentido, investigou-se o reprocessamento de endoscópios gastrointestinais subsidiado nos níveis de sujidade, contaminação microbiana e presença de biofilme. Trata-se de um experimento laboratorial realizado em três fases por meio da bioluminescência com adenosina trifosfato (ATP) para avaliação do nível de sujidade; polimerase chain reaction (PCR) para carga bacteriana e cultura microbiana para determinação do nível de contaminação. Ainda, avaliou-se as superfícies internas de canais de endoscópios por microscopia eletrônica de varredura (MEV) quanto à presença de biofilme. A análise estatística dos dados foi subsidiada em medidas de tendência central, testes de Man-Whitney, Wilcoxon e Spearman, p<0,05, por meio do software IBM SPSS Statistics versão 23.0. A avaliação de 99 endoscópios antes e após limpeza manual demonstrou a eficácia do processo na redução de sujidade (p<0,001) e contaminação microbiana (p=0,03), inclusive com baixo percentual da amostra com micro-organismos viáveis. Dos 75 endoscópios avaliados após o reprocessamento evidenciou-se uma redução do nível de sujidade (todos com <50URL, interna e externamente); entretanto a presença de carga bacteriana foi de 3log de bactéria/mL e 10,6% de positividade das culturas. Os micro-organismos isolados dos lavados de endoscópios após o reprocessamento foram Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Roseomonas gilardii e Micrococcus luteus. Na avaliação dos canais de biópsia de endoscópios provenientes do Brasil observou-se maior contaminação do que os da Austrália (p<0,001). Todos os canais apresentaram biofilme e danos em suas superfícies, entretanto as amostras brasileiras apresentaram particularidades, como, presença de hemácias, neutrófilos e fungos. Assim, observou-se que apesar da baixa carga microbiana nos endoscópios há o risco potencial de infecção cruzada associado ao biofilme, ameaçando a qualidade e segurança do reprocessamento. Adiciona-se a necessidade de avanços tecnológicos e científicos contra o biofilme na prática do reprocessamento de endoscópios / The difficulties on gastrointestinal endoscope reprocessing, contamination risk and outbreaks are a recognized challenge in science. It was investigated gastrointestinal endoscope reprocessing for dirtiness, contamination level and presence of biofilm. Laboratorial experiment performed in three phases using adenosine triphosphate (ATP) bioluminescence for dirtiness evaluation; polymerase chain reaction (PCR) for bacterial load and microbial culture for contamination level evaluation. In addition, it was evaluated biofilm on endoscope channels internal surfaces by scanning electron microscopy (SEM). Statistics analysis was performed by descriptive analysis, ManWhitney, Wilcoxon and Spearman tests, p<0,05, using IBM SPSS Statistics versão 23.0. Before and after cleaning analysis of 99 endoscopes showed it efficacy on reducing dirtiness (p<0,001) and microbial contamination level (p=0,03), including a small percentage of culturable microorganism. From 75 endoscopes tested after reprocessing demonstrated dirtiness level reduction (all samples <50RUL, from internal and external area); but, also, presence of bacterial load of 3 log of bacteria/mL and 10,6% of culture positive. The microorganisms isolated from endoscope flush after reprocessing were Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Roseomonas gilardii and Micrococcus luteus. Endoscope biopsy channel analysis showed that samples from Brazil had higher contamination level than the ones from Australia (p<0,001). All channels analyzed presented biofilm and damage on their surfaces, however Brazilian samples showed particularities, like, blood cells, neutrophils and fungus. So, ever with low microbial load on endoscopes there is a potential risk of crossinfection associated with biofilm, compromising reprocessing quality and safety. Additionally, there´s the need of scientific and technologic improvement on endoscope reprocessing against biofilm

Master of Science - Molecular and Cell Biology / The bacterial contamination profile of a typical commercial yeast factory was assessed by three replicate microbiological surveys. In order to detect low-level contamination in samples, this study made use of a preliminary incubation technique (24h at 37°C), which boosted bacterial counts for the identification of sources of contamination. Numbers of bacteria were quantified by standard pour- and spread-plate techniques and various selective media. Raw materials were negligible in contributing to the bacterial contamination of commercial yeast, with the exception of soda ash, used to control the pH of fermentations, which contained 2 log CFU/ ml Enterococcus and aerobic bacteria. It was found that the scale up of seed yeast biomass was the primary site for contamination with Enterococcus, which progressively increased in number as the product passed down the production line. Coliforms were present at low levels, with significant increases (P < 0.05) observed during the storage of yeast cream; extrusion of compressed yeast; and packaging of dry yeast. The environment surrounding the compressed yeast production line was identified as a potential source of airborne contamination. Although Salmonella spp. and S. aureus were not detected, L. monocytogenes was isolated from compressed and dry yeast products. In addition, Bacillus spp. commonly associated with the rope-spoilage of bread, were isolated from 67% of all dry yeast product samples. Shelf-life investigations, showed that cream and compressed yeast samples were spoiled with lengthened storage periods, and especially at higher temperatures (>10°C), whilst vacuum-packaged dry yeast remained bacteriologically stable. During shelf-life studies, isolates from spoiled cream and compressed yeast samples were predominantly Lactobacillus (up to 78%), while populations of Enterococcaceae predominated in vacuum-packaged dry yeast samples (up to 68%). The use of stainless steel surfaces, attached to processing equipment used in the manufacturing of Baker’s compressed yeast, in conjunction with SEM illustrated the accumulation of yeast and bacterial cells with early stages of biofilm formation, with time. Where populations of Gram-positive members of the lactic acid bacteria family, Lactobacillus and Enterococcaceae, were isolated in the highest proportion from processing equipment surfaces used in the manufacturing of Baker’s compressed yeast (81-100%).

De acordo com a Food and Agriculture Organization of the United Nations (FAO, 2013), o consumo mundial de carne de frango tem aumentado de maneira significativa nas últimas décadas. Por outro lado, a preocupação dos produtores de alimentos, com a inocuidade de seus produtos também tem aumentado na mesma proporção. Durante as etapas da operação de abate de maneira geral, as contaminações cruzadas são as principais causas de disseminação de microrganismos patogênicos nos produtos obtidos e causadores de gastroenterites no consumidor. Espécies da enterobactéria Salmonella se enquadram como um dos maiores riscos desse tipo de doença devido a sua associação com os inúmeros surtos ocorridos a nível mundial, após o consumo desses produtos. Algumas enterobactérias possuem um gene blaKPC, que codifica a enzima carbapenemase, que confere resistência a antibióticos carbapenêmicos, agravando mais a situação. Alguns fatores de virulência encontrados nesse gênero de bactéria podem ainda estar associados a capacidade de adesão e formação de biofilmes em superfícies inertes, dificultando operações de higienização nas linhas de processamento. Assim sendo, a presente pesquisa objetivou a verificação da capacidade de estirpes de Salmonella spp isoladas de aviário e linha de abate de frangos de um frigorífico no estado do Rio Grande do Sul produzirem biofilmes e apresentarem resistência a antibióticos carbapenêmicos. Na avaliação da produção de biofilme, foi empregada a técnica de microplacas de polietileno e produção de cápsula segundo Stepanovic et al. (2004) e Rodrigues et al. (2006), respectivamente.Foram pesquisados os fatores de virulência de salmonelas, representados pelos genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH,e spvC, utilizando o método de Reação de cadeia de Polimerase (PCR), descrita por Borges et al. (2013). As estirpes foram submetidas ao teste de resistência a antibióticos carbapenêmicos pelo método de disco difusão com carbapenêmicos segundo Clinical and Laboratory Standards Institute-CLSI (2010) e pesquisa do gene de resistência a carbapenêmicos blaKCP, pela técnica de PCR, segundo NAAS et al. (2007). Obteve-se quatro perfis genéticos das estirpes de Salmonella spp.: perfil 1: genes ifpA, agfA, invA e avrA; perfil2: genes ifpA, agfA, sefA, invA e avrA; perfil 3: genes invA e avrA; perfil4: genes ifpA,agfA e invA. Observou-se a resistência das estirpes somente ao antibiótico imipenen. Entre as 36 estirpes de Salmonella spp. isoladas, todas foram consideradas produtoras de biofilme in vitro, sendo que 69% destas, apresentaram-se como fortes produtoras, 25% como moderadas, e apenas 6% como fracas produtoras. O método Agar Vermelho Congo não se mostrou eficiente para teste presuntivo de produção de biofilme para estirpes de Salmonella spp. Não foi evidenciado o gene blaKPC nas estirpes de Salmonella spp. isoladas na presente pesquisa. / According to Food and Agriculture Organization of the United Nations (FAO, 2013), the world consumption of meat of chicken has been higher significantly in the last decades. On the other hand, the concern of the food producers with the safety of their products also has increased in the same proportion. During the steps of the slaughter operation in general, cross contamination are the main causes of pathogenic microorganisms dissemination in the obtained products and the causes of gastroenteritis in the consumer. Species of the Enterobacter Salmonella fall as the highest risks of this kind of disease, due to their association with countless outbreaks which occurred worldwide, after the consumption of these products. Some enterobacter have a blaKPC gene, which codes for the carbapenemase enzyme that confers resistance to carbapenems antibiotics, aggravating the situation. Some virulence factors found in this bacteria genes can also be associated to the ability of adherence and the formation of biofilms in inert surfaces, making it difficult the operations of sanitation in the processing lines. Thus, the present research aimed to verify the capacity of Salmonella spp. strains isolated from aviary and slaughter line of chicken in a fridge of Rio Grande do Sul state to produce biofilms and be resistant to carbapenems antibiotics. In the evaluation of biofilm production, it was used the polyethylene microplates and capsule production technique according to Stepanovic et al. (2004) and Rodrigues et al. (2006), respectively. The virulence factors of salmonella were researched, represented by the genes IpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, e spvC, using the Polymerase Chain Reaction (PCR) method described by Borges et al. (2013). The lineages were submitted to the carbapenems antibiotic resistence test by the disk diffusion method with carbapenems according to the Clinical and Laboratory Standards Institute-CLSI (2010), and the research of the resistance to carbapenems gene blaKCP, by the strains Salmonella spp.: profile 1: genes ifpA, agfA, invA and avrA.; profile 2: genes ifpA, agfA, sefA, invA and avrA; profile 3: genes invA and avrA; profile 4: genes ifpA, agfA and invA. It was observed the resistance of the strains only to the imipenen antibiotic. Among the other 36 cultures of Salmonella spp. isolated, all were considered to produce biofilm in vitro, of which 69% were strong producers, 25% moderate, and only 6% were low producers. The method Congo Red Agar was not efficient to the presumptive test of biofilm production for the Salmonella spp. strains. It was not evidenced the gene blaKPC in Salmonella spp. strains isolates in this research.

Considerando que a obtenção de uma formulação farmacêutica pode ser melhor planejada se algumas condições biológicas inerentes à cavidade oral forem contempladas e que os sistemas de liberação modificada podem ser ferramentas para o controle de doenças orais, o objetivo do trabalho é obter uma formulação contendo clorexidina, avaliando seu efeito sobre os mesmos. Este trabalho foi dividido em duas etapas, sendo que a primeira envolveu a obtenção da formulação mais adequada aos experimentos biológicos e a segunda avaliou seus efeitos sobre biofilmes patogênicos orais. Assim, formulações com diferentes concentrações de clorexidina foram preparadas e avaliadas visualmente em relação à sua integridade física nas condições de crescimento dos biofilmes, seguido de ensaio de liberação em meio estático utilizando tampão como meio de dissolução (meio de dissolução convencional). A formulação selecionada foi submetida a ensaios de liberação em meio estático contendo os diferentes caldos de cultura bacterianos como meio de dissolução. Na segunda etapa do trabalho, o efeito da formulação selecionada foi avaliado em biofilmes cariogênicos de Streptococcus mutans ou biofilmes periodontopatogênicos de Porphyromonas gingivalis. Biofilmes de S. mutans (n=6) ou P. gingivalis (n=3) foram formados em caldos de cultura sob lâminas de vidro por 6 e 3 dias, respectivamente, sendo expostos a um dos seguintes tratamentos: 1) Formulação contendo 92% de quitosana e 8% de hidroxipropil metilcelulose (CV, como controle do veículo) ou 2) Formulação contendo 82% de quitosana, 8% de hidroxipropil metilcelulose e 10% de clorexidina (CHX, grupo experimental). Um grupo sem exposição à formulação foi incluído como controle negativo (CN). Após o período experimental, a viabilidade bacteriana, o pH dos biofilmes e a quantificação de clorexidina liberada para os caldos de cultura foram determinados. Os dados foram analisados estatisticamente por teste de Tukey-Kramer e Tukey, sendo o nível de significância estabelecido em 5%. Os resultados sugerem que ainda que a liberação de clorexidina da formulação nos caldos de cultura de S. mutans e P. gingivalis tenha sido menor se comparada ao meio de dissolução convencional (p<0,05), o efeito biológico promovido foi observado para ambos os biofilmes. Para o pH dos biofilmes de S. mutans, os grupos CN e CV não apresentaram diferença entre si (p>0,05), mas apresentaram quedas de pH maiores se comparados à CHX (p<0,05). CHX também resultou em menor viabilidade bacteriana dos biofilmes se comparada aos controles (p<0,05), que não apresentaram diferença entre si (p>0,05). Para P. gingivalis também houve mais morte celular nos biofilmes expostos à CHX (p<0,05) mas CV também apresentou perda de viabilidade em comparação à CN (p<0,05). Apesar da liberação de clorexidina da formulação ter sido dificultada pela presença dos microrganismos, os resultados sugerem que o sistema de liberação obtido foi capaz de diminuir a patogenicidade dos biofimes de Streptococcus mutans e de Porphyromonas gingivalis. Assim, o presente estudo sugere a importância de aliar os estudos de diferentes áreas de conhecimento de forma a contribuir no planejamento das formulações, vislumbrando futuros benefícios clínicos para o controle das doenças orais. / Considering that obtaining a pharmaceutical formulation can be better planned if some biological conditions inherent to the oral cavity are contemplated and that the modified release systems may be tools for the control of oral diseases, the aim of the work is to obtain a formulation containing chlorhexidine and evaluate its effect in pathogenic biofilms. This work was divided in two stages, the first involved obtaining the most appropriate formulation for biological experiments and the second evaluate its effect on oral pathogenic biofilms. Thus, formulations with different concentrations of chlorhexidine were prepared and evaluated visually for their physical integrity under the biofilm growth conditions, followed by the static media release assay using buffer as the dissolution medium (conventional dissolution medium). The selected formulation was subjected to static release tests containing the different bacterial culture broths as the dissolution medium. In the second stage of the work, the effect of the selected formulation was evaluated in cariogenic biofilms of Streptococcus mutans or periodontopathogenic biofilms of Porphyromonas gingivalis. Biofilms of S. mutans (n = 6) or P. gingivalis (n = 3) were formed in culture broths under glass slides for 6 and 3 days respectively, being exposed to one of the following treatments: 1) Formulation containing 92 % of chitosan and 8% of hydroxypropyl methylcellulose (CV, vehicle control) or 2) Formulation containing 82% of chitosan, 8% of hydroxypropyl methylcellulose and 10% of chlorhexidine (CHX, experimental group). A group without exposure to the formulation was included as negative control (CN). After the experimental period, bacterial viability, biofilms pH and quantification of chlorhexidine released into the culture broths were determined. Data were statistically analyzed by Tukey-Kramer and Tukey test with a level of significance of 5%. Results suggest that although chlorhexidine release from formulation in the culture broths of S. mutans and P. gingivalis was lower compared to the conventional dissolution medium (p <0.05), the treatment promoted biological effect for both biofilms. Regarding S. mutans biofilms pH, CN and CV groups showed no difference (p> 0.05), but showed higher pH drops when compared to CHX (p <0.05). CHX also resulted in lower bacterial viability of biofilms compared to controls (p <0.05), which did not show any difference (p> 0.05). For P. gingivalis, there was higher cell death in the biofilms exposed to CHX (p <0.05) and CV presented loss of viability compared to CN (p <0.05). Although the release of chlorhexidine from the formulation has been hampered by the presence of microorganisms, results suggest that the release system was able to reduce the pathogenicity of Streptococcus mutans and Porphyromonas gingivalis biofilms. Thus, the present study suggests the importance of combining studies from different areas of knowledge in order to contribute to the design of formulations aiming future clinical benefits for the oral diseases control.