Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2008. / Title from second page of PDF document. Includes bibliographical references (p. 215-246).

Zugl.: Braunschweig, Techn. Univ., Diss., 2008

Würzburg, Univ., Diss., 2002.

Essen, Universiẗat, Diss., 2004--Duisburg.

A água do equipo odontológico pode servir como meio de disseminação de microrganismos, uma vez que é a segunda maior fonte de contaminação na Odontologia. O objetivo desta pesquisa foi avaliar o nível de contaminação por bactérias aeróbias totais em água de equipos odontolóicos (reservatórios, seringas tríplices e alta rotação) e torneiras de 5 Clínicas Odontológicas da Faculdade de Odontologia de Ribeirão Preto - USP, por meio do método convencional (R2A Agar) e o sistema Petrifilm AC (3M St, Paul, MN, USA). Além disso, determinou-se o nível de contaminação por Pseudomonas spp. (Cetrimide Agar Base), coliformes (Endo Agar) e fungos (Petrifilm YM para bolores e leveduras), como também identificou-se as bactérias com série bioquimica na forma de kit comemial (API 20NE), detectou-se bactérias da boca pela técnica de Checkerboard DNA-DNA Hybridization e analisou-se o biofilme formado nas linhas dágua dos equipos (seringas tríplice e alta rotação), com auxilio de microscópio eletrônico de varredura (MEV). Por outro lado, foram sugeridas recomendações para a manutenção da qualidade microbiológica da água de equipos odontológicos. As comparações estatísticas mostraram que os níveis de contaminação bacteriana das águas de torneiras, bem como dos equipos foram mais elevados pelo método R2A Agar do que pelo sistema Petrifilm AC (p<0,001). Embora, as amostras de água das torneiras utilizadas para preencher os reservatórios dos equipos tivessem pequeno número de bactérias (908UFC/ml) - R2A e (2UFC/ml) Petrifilm AC, as dos 25 equipos apresentaram: reservatórios de 0 a 3.900.000 UFC/ml (média de 211.705 UFC/ml) - R2A Agar, e de 0 a 231.000 UFC/ml (média de 14.065 UFC/ml) Petrifilm AC; seringas tríplices de 0 a 5.200.000 UFC/ml (média de 509.068 UFC/ml) - R2A Agar, e de 0 a 610.000 UFC/ml (média de 30.842 UFC/ml) Petrifilm AC; e alta rotação de 0 a 6.300.000 UFC/ml (média de 862.279 UFC/ml) - R2A Agar, e de 0 a 730.000 UFC/ml (média de 61.817 UFC/ml) Petrifilm AC. As placas Petrifllm AC incubadas a 23°C por 7 dias demonstraram um nível maior de contaminação bacteriana do que aquelas incubadas a 35°C por 48h (p<0,001). De acordo com o método de cultura, Escherichia coli, coliformes totais e Pseudomonas spp. estavam ausentes das amostras de água das torneiras, embora 11 (44%) dos equipos tivessem apresentado E. coli e/ou coliformes totais e em 1 (4%) de alta rotação, Pseudomonas aeruginosa. Todavia, segundo o método molecular, 1 (50%) amostra de água de torneira e 36 (48%) de equipos mostraram contaminação por E. coli. Das águas de 10 torneiras e 25 equipos, 1 (10%) e 17 (68%) estavam contaminadas com fungos, respectivamente. As análises com MEV mostraram biofilmes nas linhas d\'água de todos os equipos, constituídos por uma diversidade microbiana embutida em densas e extensas matrizes de substâncias poliméricas extracelulares. As bactérias identificadas por meio do API 20 NE foram Acinetobacter Iwoffii, Brevundimonas vesicularis, Burkholderia cepacia, Moraxella spp., Oligel/a ureo/ytica, Pasteurella spp., P. aeruginosa e Sphingomonas paucimobi/is. Nas águas dos equipos, as bactérias mais prevalentes exclusivas da boca forem Streptococcus gordonii (35/46,7%), Treponema denticola (28/37,3%), e Aggregatibacter actinomycetemcomitans(b) (9/25,6%). Em conclusão, o BIOFILME formado nas linhas d\'água dos equipos funciona como um \"sistema amplificador\" do pequeno número de microrganismos das águas de torneiras, sendo a causa principal dessa alarmante contaminação das águas dos equipos. Assim, recomendações para a manutenção da qualidade microbiológica da água de equipos, bem como a avaliação de fungos deveriam ser acatadas pelos profissionais da Odontologia. / Dental unit water may serve as microorganism dissemination, since it is the second major source of contamination in dentistry. The aim of this research was to assess the contamination level of total aerobic bacteria in water from dental units (reservoirs, air-water syringes and high-speed handpieces) and taps from 5 Dental Clinics at the Faculdade de odontologia de Ribeirão PReto USP using conventional method (R2A Agar) and Petrifilm SYSTEM (3m, St Paul, MN, USA). Moreover, to evaluate the level of contamination by Pseudomonas spp. (Centrimide Agar Base), coliforms (Endo Agar) and fungi (Petrifilm YM for yeasts and molds) as well as to identify the bacteria by means biochemical test in form of kit (API 20NE), to detect mouth microorganisms by Checkerboard DNA-DNA Hybridization technique and to analyze the biofilm formed on dental unit waterlines (air-water syringes and high-speed handpieces) by scanning electron microscopy (SEM). The levels os bacteria in water from tap and dental unit were higher by R2A Agar than Petrifilm AC (p<0.001). Although, the tap water used to supply the dental unit reservoirs had few bacteria (908CFU/ml) R2A and (2CFU/ml) Petrifilm AC, water from 25 dental units showed: reservoir from 0 to 3,900,000CFU/ml (average of 211,705FCU/ml) R2A Agar, and from 0 to 610,000 CFU/ml (average of 30,842CFU/ml) Petrifilm AC; air water syringes from 0 to 5,200,000CFU/ml (average of 509,068CFU/ml) R2A Agar, and from 0 to 610,000CFU/ml (average of 30,842CFU/ml) Petrifilm AC; and high-speed handpieces from0 to 6,000,000CFU/ml (average of 862,279CFU/ml) R2A Agar, and from and0 to 730,000CFU/ml (average of 61,817CFU/ml) Petrifilm AC. The Petrifil AC plates incubated at 23ºC for 7 days demonstrated a level of bacterial contamination higher than those at 35ºC for 48h (p<0.001). According to culture method, Escherichia coli, total coliforms and Pseudomonas spp. Were not detected in water from taps, but E. coli and/or total coliforms were presented in 11 (44%) high-speed handpiece water. However, according to molecular method, 1 (50%) water sample from a tap and 36 (48%) from dental units showed contamination with E. coli. Water from 10 taps and 25 dental units were contamined with fungi in 1 (10%) and 17 (68%) samples, respectively. The analysis by SEM showed in all dental unit waterlines biofilms constituted of a microbial diversity embedded in dense and extensive matrices of extracellular polymeric substances. The bacteria identified by API 20 NE were Acinetobacter iwoffiii, Brevundimonas vesicularis, Burkholderia cepacia, Morexella spp., Oligella ureolytica, Pasteurella spp., P. aeruginosa and Sphingomonas paulcimobilis. The oral bacteria prevalent in dental unit water samples were Streptococcus gordonii (35/46.7%), Treponema denticola (28/37.3%) and Aggregatibacter actiomycetemcomitansb (9/25.6%). In conclusion, BIOFILM formed in dental nit waterlines serve as an amplifier system of few microorganisms from tap water, being the major cause of high contamination of dental unit water. Besides, recommendations to maintain the microbiological quality of dental units as well as the fungal evaluation should be employed for Dentistry professionals

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-29Bitstream added on 2014-06-13T20:24:19Z : No. of bitstreams: 1 lancellotti_m_dr_jabo.pdf: 3240433 bytes, checksum: 4b16953d2baee65e759a77712c2107dc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho objetivou trazer uma contribuição aos estudos relacionados à infecção cruzada, por Staphylococcus spp, dentro do ambiente dos consultórios odontológicos, destacando as principais fontes de contaminação e o provável risco a que os profissionais e pacientes estão expostos. Foram coletadas 160 amostras de água, 300 amostras de fômites e 360 amostras das mãos (direita e esquerda) e da cavidade nasal de dentistas, auxiliares e pacientes em 40 consultórios. A determinação da contagem de Staphylococcus spp na água, pelo método de filtração em Millipore® ,mostrou que 28% não atenderam ao padrão de potabilidade estabelecidos pela American Dental Association. Dentre os consultórios estudados, os de atendimento de convênio apresentaram a maior contaminação da água (62,5%) e os consultórios particulares (36%) e de convênios (35%) apresentaram maior contaminação em relação aos fômites pesquisados. As regiões de fômites mais contaminadas foram: assento (90%), maçaneta (80%), aparelho de Rx (76%) e caneta de alta rotação (70%). A área anatômica mais contaminada foi à cavidade nasal (66%) seguido da mão esquerda (57%) e mão direita (42%). A correlação entre os isolados de Staphylococcus spp nos fômites, água e áreas anatômicas significativa, podendo ser sugerido que houve infecção cruzada nos consultórios odontológicos estudados. As cepas de Staphylococcus spp, isoladas das águas de abastecimento do equipamento odontológico, foram sensíveis aos antibióticos ciprofloxacina (97%) e vancomicina (91%) e resistentes a oxacilina (78%), enquanto, as cepas, isoladas de fômites, das mãos e cavidade nasal foram sensíveis ao antibiótico ciprofloxacina (85%) e resistentes a oxacilina (88%). / This work aimed at making a contribution to studies related to cross infection by Staphylococcus spp in the environment of dental offices, focusing on main contamination sources and possible risk for professionals and patients. There have been collected 160 samples of water, 300 samples of fomites, and 360 samples from hands (right and left) and nasal cavities of dentists, assistants, and patients in 40 dental offices. The count determination of Staphylococcus spp in water, through the Millipore® filtering method, has shown that 28% did not meet the standard of potability established by the American Dental Association. Among studied dental offices, dental care plan offices have presented the highest rate of contamination of water (62,5%), and private offices (36%) and dental care plan offices (35%) have presented the highest rate of contamination as to researched fomites. The most contaminated fomites areas were: chair (90%), door knob (80%), Rx device (76%), and high-speed handpiece (70%). The most contaminated anatomical area was the nasal cavity (66%), followed by left (57%) and right hands (42%). The correlation among isolated Staphylococcus spp in fomites, water, and anatomical areas was significant, therefore, it might be suggested that there has been cross infection in the studied dental offices. Strains of Staphylococcus spp, which had been isolated from dental equipment water, were sensible to antibiotics ciprofloxacin (97%) and vancomycin (91%), and they were resistant to oxacillin (78%); on the other hand, strains isolated from fomites in hands and nasal cavities were sensible to antibiotics ciprofloxacin (85%) and oxacillin (88%).

Les oomycètes phytopathogènes ont co-évolué avec les microbiotes des plantes hôtes. Il en résulte la formation de biofilms et des réseaux complexes d’interactions dont nous commençons juste à comprendre l’incidence sur la biologie et la virulence des oomycètes. Déterminer la nature de ces interactions et leur rôle dans le contexte d’une infection est aujourd’hui un enjeu cognitif qui concerne la caractérisation des mécanismes moléculaires et communautaires sous-jacents. C’est également une opportunité en termes d’innovation pour élaborer des méthodes de lutte alternative à l’usage de fongicides. Dans ce contexte, ce travail de thèse a consisté à étudier, d’une part, la formation de biofilm chez Phytophthora parasitica, un oomycète polyphage et tellurique, ainsi que, d’autre part, les interactions au sein de ce biofilm entre P. parasitica et le microbiote procaryote rhizosphérique de la plante hôte Solanum lycopersicum. L’analyse du génome de P. parasitica et du transcriptome du biofilm a conduit à la caractérisation d’une nouvelle famille de mucines restreinte à la lignée des oomycètes, les protéines MUCL. Ces 315 protéines sécrétées (25-30 kDa) sont réparties en 15 groupes et possèdent deux domaines : un domaine hautement conservé de fonction inconnue ; un domaine caractéristique des mucines, riche en résidus Sérine et Thréonine avec de très nombreux sites putatifs d’O-glycosylation. Chez P. parasitica, les 3 gènes PPMUCL1/2/3 sont exprimés et co-régulés spécifiquement au stade biofilm. / The interactions between a pathogen and the host surface resident microbiota are critical to disease outbreak. These interactions shape the distribution, the density and the genetic diversity of inoculum. However for most plant pathogens how each of these interactions acts on disease as a single one or as a member of a functional network remains to be specified. This issue is addressed here through the analysis of two types of interactions involving the polyphagous oomycete P.parasitica : (i) the intraspecific interaction that leads to monospecific biofilm formation by P. parasitica zoospores on plant surface; (ii) the interspecific interactions that occur between P. parasitica biofilm and the prokaryotic microbiota of Solanum lycopersicum rhizosphere. The biology of monospecific biofilm is investigated through the characterization of MUCL, a new oomycete-specific Mucin-like Protein family. Gene profiling, biochemical and immunohistological analyses define the extent of this family and lead to identify three members, PPMUCL1/2/3, as residing in P. parasitica biofilm. The Phytophthora parasitica-Microbiota interaction is explored using first a metagenomic approach. Two microbial metagenomes derived from a soil of a tomato greenhouse is defined and compared after 16S RNA gene sequencing: M1 which corresponds to the sub-rhizospheric microbiota able to colonize the roots of axenic tomato seedlings; M2, the sub-microbiota able to colonize the tomato seedling roots previously coated with P. parasitica monospecific biofilm. A representative collection of microorganisms from M2 were also obtained through in vitro selection on a medium prepared from P. parasitica extract.

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.

Fluid flow in porous media is important for a number of fields including nuclear waste disposal, oil and gas, fuel cells, water treatment and civil engineering. The aim of this work is to improve the current understanding of how the pore space governs the fluid flow in porous media in the context of nuclear waste disposal. The effects of biofilm formation on flow are also investigated. The thesis begins with a review of the current porous media characterisation techniques and the means for converting the pore space into pore network models and their existing applications. Further, I review the current understanding of biofilm lifecycle in the context of porous media and its interactions with fluid flow. The model porous media used in this project is Hollington sandstone. The pore space of the material is characterised by X-ray CT and the equivalent pore networks from two popular pore network extraction algorithms are compared comprehensively. The results indicate that different pore network extraction algorithms could interpret the same pore space rather differently. Despite these differences, the single-phase flow properties of the extracted networks are in good agreement with the estimates from a direct approach. However, it is recommended that any flow or transport study using pore network modelling should entail a sensitivity study aiming to determine if the model results are extraction method specific. Following these results, a pore merging algorithm is introduced aimed to improve the over segmentation of long throats and hence improve the quality of the extracted statistics. The improved model is used to study quantitatively the pore space evolution of shale rock during pyrolysis. Next, the extracted statistics from one of the algorithms is used to explore the potential of regular pore network models for up-scaling the flow properties of porous materials. Analysis showed that the anisotropic flow properties observed in the irregular models are due to the different number of red (critical) features present along the flow direction. This observation is used to construct large regular models that can mimic that behaviour and to discuss the potential of estimating the flow properties of porous media based on their isotropic and anisotropic properties. Finally, a long-term flow-through column experiment is conducted aiming to understand the effects of bacterial colonisation on flow in Hollington sandstone. The results show that such systems are quite complex and are susceptible to perturbations. The flow properties of the sandstone were reduced significantly during the course of the experiment. The possible mechanisms responsible for the observed reductions in permeability are discussed and the need for developing new imaging techniques that can allow examining biofilm development in-situ is underlined as necessary for drawing more definitive conclusions.

abstract: Contamination by chlorinated ethenes is widespread in groundwater aquifers, sediment, and soils worldwide. The overarching objectives of my research were to understand how the bacterial genus Dehalococcoides function optimally to carry out reductive dechlorination of chlorinated ethenes in a mixed microbial community and then apply this knowledge to manage dechlorinating communities in the hydrogen-based membrane biofilm reactor (MBfR). The MBfR is used for the biological reduction of oxidized contaminants in water using hydrogen supplied as the electron donor by diffusion through gas-transfer fibers. First, I characterized a new anaerobic dechlorinating community developed in our laboratory, named DehaloR^2, in terms of chlorinated ethene turnover rates and assessed its microbial community composition. I then carried out an experiment to correlate performance and community structure for trichloroethene (TCE)-fed microbial consortia. Fill-and-draw reactors inoculated with DehaloR^2 demonstrated a direct correlation between microbial community function and structure as the TCE-pulsing rate was increased. An electron-balance analysis predicted the community structure based on measured concentrations of products and constant net yields for each microorganism. The predictions corresponded to trends in the community structure based on pyrosequencing and quantitative PCR up to the highest TCE pulsing rate, where deviations to the trend resulted from stress by the chlorinated ethenes. Next, I optimized a method for simultaneous detection of chlorinated ethenes and ethene at or below the Environmental Protection Agency maximum contaminant levels for groundwater using solid phase microextraction in a gas chromatograph with a flame ionization detector. This method is ideal for monitoring biological reductive dechlorination in groundwater, where ethene is the ultimate end product. The major advantage of this method is that it uses a small sample volume of 1 mL, making it ideally suited for bench-scale feasibility studies, such as the MBfR. Last, I developed a reliable start-up and operation strategy for TCE reduction in the MBfR. Successful operation relied on controlling the pH-increase effects of methanogenesis and homoacetogenesis, along with creating hydrogen limitation during start-up to allow dechlorinators to compete against other microorgansims. Methanogens were additionally minimized during continuous flow operation by a limitation in bicarbonate resulting from strong homoacetogenic activity. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2012