Water and wastewater treatment solutions incorporating biofilm systems are becoming increasingly popular due to more stringent regulations pertaining to drinking water and wastewater effluent discharge in Canada and in other parts of the world. As a major component of biofilm, extracellular polymeric substances (EPS) have been considered as an important factor affecting the physical and chemical properties of biofilm. Further, the selected method of EPS extraction and the methods of detecting the composition of the EPS have shown to affect the results of EPS measurements. In this research, protocols for EPS extraction and EPS composition analysis were investigated and optimized for nitrifying moving bed biofilm reactor (MBBR) biofilm. In addition, the confocal Raman microscopy (CRM) spectra of EPS in nitrifying MBBR biofilm and the protein, polysaccharide and extracellular DNA (eDNA) percent concentrations of the EPS were investigated at various operating temperatures. Further, the CRM spectra and the protein, polysaccharide and eDNA percent concentration of EPS in nitrifying MBBR biofilm along with the biofilm morphology and thickness and the viability of the embedded cells were investigated at various hydraulic retention times (HRTs). The EPS was characterized at various temperatures and HRTs in order to investigate potential correlation between the EPS components of the nitrifying biofilm and the ammonia removal kinetics. The biofilm morphology and thickness along with the bacterial viability of the biofilm were also investigated at various HRTs. Biofilm morphology images and thickness measurements were acquired using a variable pressure scanning electron microscope (VPSEM). The percentages of viable embedded cells in the biofilm were quantified using live/dead staining in combination with confocal laser microscopy (CLSM) imaging. The research demonstrates that an increase in protein content and subsequently a decrease in polysaccharides and eDNA contents in the EPS of nitrifying MBBR biofilm were observed at the lowest operational HRT and the highest temperature in this work. In particular, the EPS protein to polysaccharide (PN/PS) ratio of nitrifying MBBR systems was shown to significantly decrease below a value of 3 when the system was underloaded (observed at the highest operational temperature in this study) or hydraulically overloaded (observed at the lowest HRT in this study). As such, data obtained at lower operational temperatures, with the system no longer underloaded, and at longer HRTs, with the system no longer hydraulically overloaded, all demonstrate an EPS PN/PS ratio of approximately 3. Correlations were observed between the chemically measured EPS PN/PS ratios and the measured Raman spectra intensity ratios; supporting the concept of higher PN/PS ratios of EPS in more optimal nitrifying MBBR operations. Further, the ammonia removal kinetics and EPS response at HRT values of 0.75 and 1.0 h indicate that nitrifying MBBR systems may be optimized to operate at HRTs as low as 0.75 to 1.0 hour as opposed to conventional HRTs of 2.0 to 6.0 h.

Nitrification is currently the most common means of ammonia removal from wastewaters in temperate climates. In conventional suspended growth systems operating in northern climate regions, nitrification completely ceases at temperatures below 8°C. This is a considerable concern in passive treatment systems where wastewater temperatures can reach as low as 1°C for extended periods in the winter months. There is evidence biofilm technologies have the ability to nitrify at low temperatures, however, the literature is missing an understanding of low temperature nitrification and the subsequent impacts during seasonal changes. Additionally, there is an urgent need to gain a fundamental knowledge of the interplay between nitrifying performance optimization, biofilm morphology and the microbiome. This research aims to fill these needs using nitrifying moving bed biofilm reactors (MBBRs) at the lab and pilot scale. This research concluded the most important factor determining MBBR carrier selection is a combination of surface area and pore space size. Although high surface area to volume carriers are attractive, the propensity to clog at high loading rates significantly decreases the removal rates. The viability of the biomass and ammonia oxidizing bacterial communities were not significantly changed, indicating the ammonia removal rates were reduced due to loss of surface area in the clogged carriers. Operation at 1°C demonstrated significant rates of nitrification can be attained and stable for extended periods of operation. This study developed the first kinetic curve at 1°C with a maximum removal rate of 0.35 gN/m2·d. The performance of the post carbon removal nitrifying MBBR systems were shown to be enhanced at 1°C by an increase in the viable embedded biomass as well as thicker biofilm. This effectively increased the number of viable cells present during low temperature operation, which partially compensated for the significant decrease in rate of ammonia removal per nitrifying cell. At all studied loading rates at 1°C, the ammonia oxidizing bacteria were primarily in the family Nitrosomonadaceae (greater than 95 percent abundance of AOB population) and the nitrite oxidizing bacteria were primarily the genus Nitrospira (greater than 99 percent abundance of NOB population). Operation at 20°C demonstrated high rates of removal in high loaded condition and robustness in extreme low loaded conditions. In both high loaded and extreme low loaded conditions the viability of the nitrifying biomass was sustained, with the family Nitrosomonadaceae as the primary ammonia oxidizing bacteria and the genus Nitrospira as the primary nitrite oxidizing bacteria. In extreme low loaded conditions and as well during start-up phases there are high prevalence of bacteria not directly related to the nitrification process. Their presence however indicates a dynamic process with changes in microbial composition within the biofilm matrix in response to varying conditions. Change in microbial composition likely helps stabilize and maintain the biofilm matrix enhancing process robustness in the temperate climates. The new knowledge gained in this research optimizes the operation of nitrifying MBBR systems and elucidates the impacts of operational conditions on the biofilm and microbial community of nitrifying MBBR systems to further our understanding of nitrifying attached growth treatment technologies. The results of this study are anticipated to be used to design the first MBBR treatment system for year round ammonia removal in passive treatment systems located in northern climate regions.

Succinic Acid (SA) was continuously produced using glucose and a Mg2CO3(OH)2 slurry as feed. Glucose feed concentrations of 20 and 40 g ℓ-1 were employed with corresponding Mg2CO3(OH)2 slurry concentrations of 60 and 120 g ℓ-1. The reactor pH was passively maintained between 6,4 and 6,8 by the buffer properties of the slurry in conjunction with the pH adjusted glucose feed. The suspended cell (SC) reactor was operated at 37°C with dilution rates varying between 0,04 h-1and 0,6 h-1. Groperl® particles were used as inert supports in the biofilm reactor; dilution rates of 0,11 h-1 to 1 h-1 were investigated. Two SC fermentations were conducted for the 20 g ℓ-1 glucose feed concentration and one for the 40 g ℓ-1. All SC fermentation runs were operated in excess of 12 days, while the biofilm run lasted 6,5 days. Fermentations were terminated only after contamination by lactic acid bacteria was observed. SC fermentations with the glucose feed concentration of 20 g ℓ-1 achieved a maximum SA productivity of 5,2 g ℓ-1h-1 at 0,6 h-1 with a corresponding SA yield of 0,65 g g-1. SC fermentations with the glucose feed concentration of 40 g ℓ-1 achieved a maximum SA productivity of 3,76 g ℓ-1h-1 at 0,4 h-1 with a SA yield of 0,82 g g-1. The results were comparable to the other continuous studies with Actinobacillus succinogenes, despite the fact that either biofilms or membranes were employed in these studies. The preliminary biofilm study demonstrated the capability of A. succinogenes to produce SA in high productivities and yields. SA productivities and yields for the dilution rates of 0,33 h-1 and 1,0 h-1, were 5,72 g ℓ-1h-1 (0,95 g g-1) and 12 g ℓ-1h-1 (1,0 g g-1), respectively. The biofilm reactor at 0,33 h-1 achieved twice the SA productivity of the SC reactor at 0,3 h-1 with a 42 % increase in SA yield. Copyright / Dissertation (MEng)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

Biofilms are important in nature and in engineered processes. Because of this, a fundamental understanding of their growth and behaviour is required. This work aimed at monitoring biofilm growth using a biological rotating reactor and the Rotoscope biofilm monitor. Both methods worked on the principle of a rotating circular disc that was semi-submerged in water and the light reflected of the area that was outside of the water. Light reflectance on the disc was taken three times a day and the average recorded as the daily reading. It was noticed that in both systems, growth of biofilms on the discs caused a decrease in the amount of light reflected. A decrease in light reflectance indicated an increase in biofilm thickness. The growth of biofilm was confirmed by scanning electron microscopy analysis. The addition of a biocide caused a slight increase in light reflectance indicating partial biofilm removal. The Rotoscope was very sensitive to changes in biofilm characteristics. Rotoscope met the requirements needed for an on-line, real-time and non-destructive biofilm monitoring system. The aged anolyte was effective in killing both suspended and biofilm bacteria at a concentration of 1:10 irrespective of its age and storage conditions. Exposure of aerobic bacteria to different concentrations of sodium nitrite at different time intervals indicated that sodium nitrite had a limited, or no biocidal effect on these bacteria mostly encountered in biofilms. The ready to use chlorine dioxide was also used as the means of controlling biofilms. MIC for RTU ClO2 was found to be 80ppm, which in certain instances killed all bacteria immediately upon exposure while in other cases an exposure time of 1h was required. It was indicated that at this concentration, biofilms were removed. This was confirmed by scanning electron microscopy analysis. Proteins of suspended bacteria treated with 1:10 and 1:100 anolyte dilutions and the control were extracted and compared using SDS-PAGE. Protein bands of bacteria treated with 1:10 NaCl derived anolyte were fewer and fainter as compared to those from untreated cells. More bands were produced in cells treated with 1:100 NaCl derived anolyte as compared to the untreated cells. Cells treated with the non-halide anolyte, both 1:10 and 1:100 dilutions, produced more bands than in the untreated cells. Anolyte destroyed vital proteins for bacterial survival causing cell death or it caused fragmentation of proteins to small peptides, reducing the number of viable cells. NaNO2 was ineffective as biocide while aged anolyte and RTU liquid ClO2 were effective as biocides. SDS-PAGE indicated that anolyte killed bacteria by affecting their proteins. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Continuous fermentations were performed in an external-recycle, biofilm reactor using glucose and CO2 as carbon substrates. The nitrogen source for the auxotrophic Actinobacillus succinogenes was a combination of yeast extract (YE) and corn steep liquor (CSL), and sometimes only YE or CSL was used. In this study, the succinic acid productivity of A. succinogenes decreased by 67% as the amount of YE in the complex nitrogen source mixture decreased from 16 g·L-1 to 0 g·L-1. Succinic acid production increased as the CSL concentration in the nitrogen source increased, and the mass ratio of succinic acid to acetic acid exceeded the theoretical maximum limit of 3,93 g·g-1 when only CSL was used as the nitrogen source. The mass ratio of formic acid to acetic acid was consistently within the theoretical yield limitations (0,77 g·g−1) and decreased as the CSL concentration in the nitrogen source increased. The highest SA concentration in this study was 22,57 g·L-1 when only YE was used as the nitrogen source in the growth medium, and the highest SA productivity obtained in this study was 1,58 g·L-1·h-1 when a combination of YE and CSL was used as a nitrogen source. The highest mass ratio of SA to AA achieved was 8,3 g·g-1 when CSL was the sole nitrogen source. The mass ratio of FA to AA was consistently less than 0,77 g·g-1, approaching 0 g·g-1, as the CSL concentration in the nitrogen source increased. / Dissertation (MSc)--University of Pretoria, 2016. / National Research Foundation (NRF) / Chemical Engineering / MSc / Unrestricted

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-14T18:46:13Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Tacilene Luzia Silva.pdf: 1235794 bytes, checksum: f6e18d8a1d865ebf8536ae8b8a1666f3 (MD5) / Made available in DSpace on 2016-04-14T18:46:13Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Tacilene Luzia Silva.pdf: 1235794 bytes, checksum: f6e18d8a1d865ebf8536ae8b8a1666f3 (MD5) Previous issue date: 2015-02-13 / CNPq / Pseudomonas aeruginosa é uma bactéria Gram negativa, oportunista e ubíqua, frequentemente associada a infecções graves em pacientes imunocomprometidos. Em razão do aumento de resistência dessa bactéria aos múltiplos antimicrobianos, surgem à preocupação e a procura por novas alternativas terapêuticas, com as substâncias bioativas de origem natural representando uma importante fonte para obtenção desses medicamentos. O objetivo do presente estudo foi determinar a atividade sinérgica do timol e agentes antimicrobianos frente a cepas de Pseudomonas aeruginosa multirresistentes e avaliar os efeitos dessa interação sobre a biossíntese de biofilme e de piocianina. Para isso, numa primeira etapa foi determinada a concentração inibitória e bactericida mínima do timol e de antimicrobianos (Polimixina B, ceftazidima, piperacilina/tazobactam, cefepima, ciprofloxacino e meropenem) frente a dez cepas de Pseudomonas aeruginosa. O estudo da interação entre o timol e os agentes antimicrobianos foi realizado pelo método do tabuleiro de xadrez. Os critérios utilizados para avaliar a atividade sinérgica foram definidos pelo Índice da Concentração Inibitória Fracionada (FIC índex). A partir dos melhores valores do FIC índex das associações timol/antimicrobiano foram avaliadas a atividade sobre a produção de biofilme e piocianina. Três cepas (LFBM 01, LFBM 02, LFBM 16) apresentaram um perfil de resistência ao meropenem e cefepima e um efeito sinérgico foi observado entre o timol e meropenem ou cefepima sobre essas cepas. A associação timol/cefepima inibiu a biossíntese do biofilme em até 99,76%, e a associação timol/meropenem mostrou ser mais eficaz na inibição da piocianina cujos valores foram de até 84,33%. O timol associado ao meropenem ou cefepima, age sinergicamente, inibindo cepas de Pseudomonas aeruginosa multirresistentes e interferindo na biossíntese de biofilme e piocianina. / Pseudomonas aeruginosa is a Gram negative bacteria, opportunistic and ubiquitous, often associated with severe infections in immunocompromised patients. Due to the increased resistance of the bacteria to multiple antibiotics, there are the concerns and the search for new therapeutic alternatives, with the bioactive substances of natural origin represents an important source for obtaining these drugs. The aim of this study was to determine the synergistic activity of thymol and antimicrobials agents multiresistant Pseudomonas aeruginosa strains and evaluate the effects of this interaction on the biofilm biosynthesis and pyocyanin. For this, a first step was determined and the minimum inhibitory concentration of thymol and bactericidal antibiotics (polymyxin B, ceftazidime, piperacillin / tazobactam, cefepime, ciprofloxacin and meropenem) compared to ten strains of Pseudomonas aeruginosa. The study of the interaction between the thymol and antimicrobial agents was carried out by the checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Index of Fractional Inhibitory Concentration (FIC index). From the best FIC index values of associations thymol / antimicrobial activity were evaluated on the production of biofilm and pyocyanin. Three strains (LFBM 01, LFBM 02, LFBM 16) showed an meropenem resistance profile and cefepime and a synergistic effect was observed between the thymol and meropenem or cefepime on these strains. The thymol / cefepime combination inhibited biofilm biosynthesis up to 99.76%, and thymol association / meropenem was more effective in inhibiting pyocyanin with values of up to 84.33%. The thymol associated with meropenem or cefepime, acts synergistically by inhibiting multidrug-resistant Pseudomonas aeruginosa strains and interfering in the biosynthesis of biofilm and pyocyanin.

Orientador: Mirna Lucia Gigante / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-30T23:01:21Z (GMT). No. of bitstreams: 1 Ribeiro_MariaCeciliaEnes_D.pdf: 17038739 bytes, checksum: 54c8350faac4b14e773283e6e871aa78 (MD5) Previous issue date: 2015 / Resumo: O objetivo geral deste trabalho foi avaliar o efeito de diferentes matrizes na adesão e formação de biofilme em aço inoxidável por Bacillus cereus, bem como avaliar a eficiência dos procedimentos de higienização no controle de biofilmes de esporos desse micro-organismo. Nas duas primeiras etapas, avaliou-se a capacidade de adesão e formação de biofilme por B. cereus em aço inoxidável, com e sem prévio condicionamento da superfície, utilizando-se água, leite UHT desnatado e integral como matrizes e quatro diferentes tipos de inóculos, pool de células vegetativas de B. cereus isolados da indústria láctea, pool de esporos de B. cereus isolados da indústria láctea, células vegetativas da cepa de B. cereus ATCC 14579 e esporos da cepa de B. cereus ATCC 14579. Na terceira etapa do trabalho avaliou-se a influência da matriz condicionante (água e leite UHT integral), do meio de inoculação do pool de esporos de B. cereus (água e leite UHT integral) e do tempo de exposição (5 min (0,08h), 10, 24, 48 e 72 horas) sobre a adesão e formação de biofilme por B. cereus em aço inoxidável. Na quarta etapa, avaliou-se a eficiência de nove procedimentos de higienização na remoção dos biofilmes formados pelo pool de esporos de B. cereus em aço inoxidável. Todos os experimentos foram repetidos três vezes e os dados estatisticamente avaliados. A hidrofobicidade e o potencial zeta das superfícies dos esporos também foram avaliados. Os resultados das duas primeiras etapas indicaram que o pool de esporos de B. cereus isolados de indústria láctea apresentou a maior capacidade de adesão e formação de biofilme em aço inoxidável quando comparado aos outros tipos de inóculos, em todas as condições avaliadas. O maior grau de adesão de esporos de B. cereus (4,93 log UFC/cm2) foi observado ao se utilizar leite integral como matriz condicionante do aço inoxidável. Entretanto, comparando-se todas as matrizes, a menor adesão (3,01 log UFC/cm2) foi observada quando o pool de esporos de B cereus foi veiculado no leite integral sem prévio condicionamento da superfície. Na terceira etapa do trabalho observou-se que a adesão e formação de biofilme pelo pool de esporos de B. cereus foi maior quando inoculados em água, independente das matrizes de condicionamento. A adesão de B. cereus aumentou 1,02 e 0,3 log UFC/cm2 ao longo do tempo de exposição, quando o pool de esporos de B. cereus foi inoculado em água e leite integral, respectivamente. O biofilme de esporos veiculados na água apresentou maior resistência aos procedimentos de higienização. A sanitização com hipoclorito de sódio foi mais eficiente na remoção dos biofilmes quando comparada ao ácido peracético. O pool de esporos de B. cereus isolados da indústria láctea foi altamente hidrofóbico e apresentou carga negativa em uma ampla faixa de pH, com ponto isoelétrico de aproximadamente 3,0. Os esporos de B. cereus isolados da indústria láctea apresentaram maior capacidade de adesão ao aço inoxidável quando comparados aos outros inóculos avaliados, o que pode estar relacionado à alta hidrofobicidade e a baixa carga de superfície dos esporos / Abstract: The aim of this study was to evaluate the effect of different matrices on the adhesion and biofilm formation by Bacillus cereus on stainless steel, and to evaluate the effectiveness of sanitation procedures for controlling biofilm from spores of this microorganism. The first two parts were carried out in order to evaluate the adhesion and biofilm formation by B. cereus on stainless steel, with and without previous conditioning of the surface, using water, skim and whole UHT milk as matrices and four different types of inocula: a pool of B. cereus vegetative cells isolated from dairy industry, a pool of B. cereus spores isolated from dairy industry, vegetative cells of B. cereus ATCC 14579, and spores of B. cereus ATCC 14579. The third part of the study evaluated the effect of the conditioning matrix (water and whole UHT milk), the inoculation medium of pool of B. cereus spores (water and whole UHT milk) and exposure time (5 min (0.08h), 10, 24, 48 and 72 hours) on the adhesion and biofilm formation by B. cereus on stainless steel. In the fourth part, the effect of nine sanitation procedures on the removal of B. cereus spores biofilm was evaluated. All experiments were repeated three times and data were statistically evaluated. Hydrophobicity and zeta potential from spore¿s surface were also evaluated. Regarding the results to the first and second parts, the pool of B. cereus spores isolated from dairy industry had the highest ability of adhesion on stainless steel when compared to the other inocula, for all tested conditions. After stainless steel surface conditioning with whole milk, B. cereus spores showed the highest adhesion (4.93 log CFU/cm2). However, lower adhesion (3.01 log CFU/cm2) was observed when B. cereus spores were delivered in whole milk as compared to the other matrices, without previous conditioning of the surface. The results of the third part indicated that the adhesion and biofilm formation by the pool of B. cereus spores was higher when they were inoculated in water, regardless of the conditioning matrix. B. cereus spores adhesion increased by 1.02 and 0.3 log CFU/cm2 over exposure time, when the pool of B. cereus spores was inoculated into water and whole milk, respectively. Biofilm of B. cereus spores inoculated in water showed the highest resistance against all tested sanitation procedures. Sodium hypochlorite was the most effective sanitizer for removing all biofilms when compared to the peracetic acid. The pool of B. cereus spores isolated from dairy industry was highly hydrophobic and showed a negative charge at a wide pH range, with an isoelectric point of about 3.0. B. cereus spores isolated from dairy industry showed the highest ability to adhere on stainless steel when compared to the other inocula, which is possibly related to its higher hydrophobicity and lower spore surface charge / Doutorado / Tecnologia de Alimentos / Doutora em Tecnologia de Alimentos

O objetivo do presente estudo foi avaliar a ocorrência de cepas de Staphylococcus aureus no ambiente de ordenha, analisar seu perfil genético e a produção de biofilme, provenientes de 10 propriedades localizadas nas regiões de Franca e Ribeirão Preto, estado de São Paulo, Brasil. Foram analisadas 220 amostras de leite individual de vacas positivas no teste CMT (California Mastitis Test), 120 amostras de leite de tanque de expansão, 389 swabs de utensílios e equipamento relacionados com ordenha e 120 swabs de mãos de manipuladores. As coletas de amostras foram realizadas mensalmente durante o período de agosto/2010 a janeiro/2011. Das 849 amostras analisadas, 56 cepas de S. aureus (6,6%) foram isoladas, sendo 12 (5,4%) de leite individual de vacas, 26 (21,6%) de leite de tanque de expansão, 14 (3,6%) de utensílios e equipamentos e 4 (6,9%) de mãos de manipuladores. Os resultados indicam uma baixa prevalência de S. aureus nas propriedades analisadas, não havendo diferença significativa entre as frequências encontradas nas duas regiões analisadas. A técnica de PFGE (pulsed-field gel electrophoresis) permitiu identificar 31 perfis genéticos (pulsotipos), utilizando-se a enzima de restrição SmaI. Nos ensaios de produção de biofilmes em microplaca, 19 (63,3%) de 30 pulsotipos avaliados foram considerados produtores de biofilme. Nos ensaios conduzidos em aço inox, 13 (43,3%, N=30) foram positivos e, para o silicone, não houve produção de biofilme. O elevado percentual de isolados no leite de tanque de expansão evidencia um problema de saúde pública, considerando que no Brasil muitas vezes o leite é consumido sem pasteurização. A ocorrência de pulsitipos de S. aureus formadores de biofilmes indica a persistência do patógeno em diversos pontos no ambiente de ordenha, bem como provável envolvimento dos mesmos com casos de mastite e sua eliminação no leite, ressaltando a necessidade de práticas higiênicas para prevenir a formação de biofilmes nas propriedades estudadas. / The objective of the present study was to evaluate the occurrence of biofilm-producing strains of Staphylococcus aureus in the milking environment from 10 farms located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Twohundred twenty samples of milk from individual cows previously tested for CMT (California Mastitis Test), 120 samples of bulk tank milk, 389 swabs of equipments and utensils related to milking and 120 swabs of milk\'s handlers hands were analyzed. A total of 56 (6.6%) S. aureus strains were isolated out of 849 samples analyzed, being 12 (5.4%) from milk samples from individual cows, 26 (21.6%) from bulk tank milk, 14 (3.6%) from swabs of equipments and utensils, and 4 (3.3%) from swabs hands of milk\'s handlers. Results indicated a low prevalence of S. aureus in the dairy farms analyzed, and there was no significant difference between the percentages found in the two regions evaluated. On the basis of PFGE results (using SmaI enzyme), 31 profiles (pulsotypes) were found. In the biofilm-production assay using microplates, 19 (63.3%) of 30 pulsotypes tested were considered positive (biofilm producers). For assays conducted in stainless steel, 13 (43.3%) of pulsotypes were biofilm producers, although no pulsotype was able to produce biofilms on the surface of silicon. Results of this trial showed a high percentage of bulk tank milk samples contaminated with S. aureus, hence indicating a public health problem especially in Brazil were milk is often consumed without pasteurization. The occurrence of S. aureus pulsotypes which were also biofilm-producers suggests the persistence of the pathogen in several sites at the milking environment, as well the probable involvement of S. aureus biofilms with mastitis and its excretion in the milk. The need for adoption of hygienic practices to prevent the formation of biofilms of S. aureus in the dairy farms evaluated is stressed.

Staphylococcus aureus é uma bactéria que pode ser encontrada colonizando diversas partes do corpo humano, entretanto os diversos fatores de virulência que a bactéria possui, ancorados a sua superfície ou excretados para o meio extracelular, tornam essa bactéria um potencial patógeno, causando infecções de pele e tecidos moles, osteomielite, infecções respiratórias, infecções relacionadas a cateteres e outros dispositivos e bacteremia. Um dos fatores de virulência da bactéria, é a habilidade em formar biofilmes. Biofilmes são comunidades bacterianas tridimensionais complexas, que vivem organizadas e aderidas a uma superfície biótica ou abiótica, embebidas em uma matriz exopolimérica. Cerca de 80% das bactérias vivem organizadas na forma de biofilme, pois nestas estruturas são menos sensíveis aos antibióticos e à resposta imune do hospedeiro. A habilidade de S. aureus em formar biofilme é importante pois o torna uma das principais bactérias que infecta dispositivos médicos e implantes, aumentando a morbidade e mortalidade dos pacientes que apresentam esse tipo de infecção. Os medicamentos da classe dos β-lactâmicos eram a principal escolha para o tratamento de S. aureus, entretanto nos últimos anos essa bactéria adquiriu resistência a esses antimicrobianos, através da aquisição do gene mecA, tornando escassa as opções terapêuticas. Como se não bastasse, os biofilmes bacterianos são particularmente mais resistentes a tratamentos com antibióticos, não só devido ao aumento da transmissão de mecanismos de resistência dentro da comunidade, mas também por causa das limitações de difusão da droga colocados pela matriz extracelular, inativação de antibióticos pela alta concentração de íons de metal e baixo pH, entre outros fatores. Combinados, esses atributos tornam o biofilme bacteriano em torno de 1000 vezes mais tolerante e/ou resistente aos antimicrobianos comparado às células planctônicas. A investigação de estudos epidemiológicos para prevenção dessas infecções, bom como de novas estratégias para prevenção e tratamento de infecções por biofilmes, especialmente em isolados clínicos sabidamente multirresistentes, é urgentemente necessária. Dentre estas estratégias estão a pesquisa de diferentes mecanismos ou substâncias capazes de provocar a inibição da formação ou a erradicação do biofilme formado. Neste contexto, 8 os sistemas de bombas de efluxo e inibidores de bombas de efluxo representam uma fonte promissora de erradicação do biofilme formado. O principal objetivo deste estudo é investigar características clínico-epidemiológicas em isolados clínicos que estejam associadas a formação de biofilme, bem como investigar o papel de bombas de efluxo, inibidores dessas bombas e novos genes envolvidos na habilidade de isolados clínicos de S. aureus em formar biofilme. O capítulo 1 associa características clínicas e epidemiológicas com a habilidade de formação de biofilme. O capítulo 2 mostra o papel da adição de antimicrobianos na inibição e erradicação de biofilmes, a associação com inibidores de bomba de efluxo para melhor entender os sistemas de bomba de efluxo na capacidade desses isolados em formar biofilme e por último, novos genes que participam desse processo, em isolados clínicos de MRSA. Este estudo permite planejar ações preventivas para essas infecções relacionadas a biofilmes. Além disso, demonstra que os sistemas de bombas de efluxo parecem ser alvos promissores para erradicar infecções associadas a biofilmes bacterianos. / Staphylococcus aureus can be found colonizing the human body, however its virulence factors anchored to its surface or secreted into the extracellular medium, makes this bacteria as a potential pathogenic, causing skin and soft tissue infections, osteomyelitis, respiratory infections, catheter-related and other devices infections and bacteremia. One of the virulence factors that bacteria produce is the ability to form biofilms. Biofilms are complex three-dimensional bacterial communities, living organized and attached on a biotic or abiotic surface, embedded in a matrix exopolimérica. About 80% of live bacteria are organized in the form of biofilms because in these structures are less sensitive to antibiotic and the host immune response. The ability of S. aureus to form biofilms is important because it makes it one of the main bacteria that infects medical devices and implants, increasing patient morbidity and mortality. The class of β-lactam drugs used to be main choice for the treatment of S. aureus infections, however in recent years the bacteria acquired resistance to these antibiotics by acquiring mecA gene, so therapeutic options becoming scarce. Besides that, bacterial biofilms are particularly resistant to antibiotic treatments, not only due to increased transmission resistance mechanisms within the community, but also because limitations in drug diffusion by extracellular matrix, inactivation of antibiotics due to high concentration of metal ions and low pH, and other factors. Combined, these attributes make the bacterial biofilm around 1000 times more tolerant and / or resistant to antimicrobial compared to planktonic cells. Investigation of epidemiological studies to prevent such infections, as well as new strategies for prevention and treatment of biofilm infections, especially in known multidrug-resistant clinical isolates, is urgently needed. Among these strategies we could list the different search engines or substances capable of causing or inhibiting the formation of biofilm eradication. In this context, system efflux pumps and efflux pump inhibitors represent a promising source of biofilm eradication. The aim of this study is to investigate the clinical and epidemiological characteristics in clinical isolates that are associated with biofilm formation and investigate the role of efflux pumps and inhibitors of these pumps in the ability of S. 10 aureus clinical isoltes to form biofilms. The chapter 1 associates clinical and epidemiological characteristics with biofilm formation ability. Chapter 2 shows the role of the addition of antimicrobials in inhibition and eradication of biofilms, the association with efflux pump inhibitors to better understand the efflux pump systems in the ability of these isolates to form biofilm and, finally, new genes important in MRSA clincal isolates biofilm formation. This study allows planning preventive actions for these biofilm-related infections. In addition, it demonstrates that efflux pump systems appear to be promising targets for eradicating infections associated with bacterial biofilms.

Orientador: Marlise Inêz Klein / Resumo: Streptococcus mutans orquestra a formação de biofilmes cariogênicos através da produção da matriz extracelular que contém exopolissacarídeos (EPS), DNA extracelular (eDNA) e ácidos lipoteicóicos (LTA). EPS são um marcador de virulência para cárie, mas não está claro como os genes associados com eDNA (lytS e lytT) e LTA (dltA e dltD) afetam a virulência de S. mutans. Portanto, avaliou-se como os genes lytST, dltAD e gtfB (EPS-insolúveis) afetariam o desenvolvimento de lesões cariosas em ratos e a sobrevivência de larvas (Galleria mellonella) após infecção sistêmica e para esclarecer sua contribuição na patogenicidade dessa bactéria. Ainda, avaliou-se o efeito dos tratamentos tópicos com miricetina (afeta a síntese de EPS), composto 1771 (modula o metabolismo do LTA) e flúor (prevenção da cárie) sobre biofilmes in vitro de S. mutans. Alimentou-se os ratos com dieta cariogênica e inoculou-se com cepa parental UA159 e cepas com deleção de genes ∆lytS, ∆dltD e ΔgtfB (n=14). Após 5 semanas, avaliou-se as populações microbianas (cultivável total e S. mutans) e as lesões de cárie. Injetou-se a cepa parental e as cepas com deleção de genes ΔgtfB, ΔlytS, ΔlytT, ΔdltA e ΔdltD na hemocele de larvas e registrou-se a sobrevivência das larvas longitudinalmente (n=10). Formou-se biofilmes de S. mutans sobre discos de hidroxiapatita revestidos com película salivar e realizou-se tratamentos tópicos duas vezes ao dia: miricetina (Mir), composto 1771, flúor (F) Mir+1771, Mir+F, 1771+F, Mir+1771+... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Streptococcus mutans orchestrates the cariogenic biofilms formation through the production of an extracellular matrix that contains exopolysaccharides (EPS), extracellular DNA (eDNA), and lipoteichoic acids (LTA). EPS is a virulence marker of dental caries, but it is unclear how genes associated with eDNA (lytS and lytT) and LTA (dltA and dltD) affect S. mutans virulence. Therefore, this study evaluated how the genes lytST, dltAD and gtfB (insoluble EPS) affected the development of carious lesions in rats and the survival of larvae (Galleria mellonella) after systemic infection to clarify its contribution to the pathogenicity of S. mutans. Also, it assessed the effect of topical treatments with myricetin (affects EPS synthesis), compound 1771 (modulates LTA metabolism) and fluoride (caries prevention) on in vitro S. mutans biofilms. The rats received a cariogenic diet and were inoculated with the parental strain UA159 and its strains with deletion of genes ΔlytS, ΔdltD, and ΔgtfB (n=14). After five weeks, viable microbial populations (total cultivable and S. mutans) and caries lesions were evaluated. Larvae were inoculated by intra-hemocoel injection with the parental strain and deletion strains ΔgtfB, ΔlytS, ΔlytT, ΔdltA, and ΔdltD, and larval survival was recorded longitudinally (n=10). S. mutans in vitro biofilms were formed on saliva-coated hydroxyapatite discs and topically treated twice-daily: Myricetin (Myr), Compound 1771, Fluoride (F), Myr+1771, Myr+F, 1771+F, Myr+17... (Complete abstract click electronic access below) / Doutor