No description available.

Strains of pathogens are typically described as virulent or non-virulent. However, in the majority of pathogens, strains often vary continuously and quantitatively in their virulence and pathogencity. Biofilm formation is one of the recently recognized virulence factors in many human pathogens and little is known about the variation and evolution of biofilms among natural strains. In this study, I examined quantitative variation of biofilms among natural strains of the human pathogenic yeast Candida albicans. A total of 115 natural strains of C. albicans from three sources (vaginal, oral and environmental) were quantified by two mebods: (i) the XTT tetrazolium reduction assay, and (ii) optical density following staining by crystal violet dye. Mature biofilm was confirmed by observation using confocal laser scanning microscopy. My analyses indicated that strains from each of the three scurces varied widely in biofilm formation abilities and that biofilm formation ability was positively correlated to cell surface hydrophobicity (CSH). For each strain, multilocus genotypes were determined by PCR-RFLP, my comparative genotype and biofilm analyses denonstrated that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variation for biofilm formation. I also examined potential interactions among strains within C. albicans and between different Candida species. My preliminary results suggest significant variation and complex patterns of strains or species interaction during Candida biofilm development. / Thesis / Master of Science (MS)

Yes / Bacterial quorum sensing has been implicated in a number of pathogenic bacterial processes, such as biofilm formation, making it a crucial target for developing materials with a novel antibiotic mode of action. This paper describes poly(N-isopropyl acrylamide) that has been covalently linked, at multiple chain ends, to homoserine lactone to give a highly branched polymer functionalized with a key messenger molecule implicated in QS. This novel functional material has shown promising anti-QS activity in a Chromobacterium violaceum assay.

Bacteria can actively communicate and interact with each other to establish multicellular communities. Many of these processes involve functional differentiation of cells into specialised subpopulations by expression of varying genetic programmes. This leads to division of labour between the arising subpopulations of cells in the community. One type of such community is the biofilm, which is composed of microbial cells enclosed in a biopolymeric matrix. Such biofilms can be formed in a large range of environments from sea beds to animal tissues. Bacillus subtilis is a soil dwelling Gram-positive rod that was shown to closely interact with plants and establish a protective symbiosis by formation of biofilms on the roots. The biofilm matrix synthesised by B. subtilis is composed of the exopolysaccharide, for which the chemical structure is not yet established, and a protein TasA that forms amyloid-like fibres spanning between the cells and anchored to the cells by an accessory protein TapA. A third protein of unknown function, YuaB, has also been shown to be necessary for establishment of a biofilm. However, the mechanism of function for YuaB has not been elucidated. The data presented in this report focus on the role played by YuaB during formation of the biofilm. By analysis of cell differentiation patterns YuaB was shown to be required for maturation of the biofilm. The localisation of YuaB is identified in two “subtypes” of biofilm, a biofilm pellicle floating on the air-liquid interface and complex colonies formed on solid surfaces. This is achieved using a combination of biofilm fractionation combined with Western blotting and a newly developed method for immuno-fluorescent labelling of biofilm proteins. YuaB acts in synergy with the exopolysaccharide and TasA, as both components of the biofilm matrix are synthesised in the absence of YuaB but the biofilm is not made. The initial structural characterisation of YuaB is presented based on in silico predictions and physiological and biophysical analysis of the mutations introduced into the sequence of YuaB. The experimental data is concluded with a hypothesis that YuaB forms a hydrophobic protective layer necessary for support of the structure of the matured biofilm.

Listeria monocytogenes has been found to withstand harsh environmental conditions including desiccation. The pathogen is also known to form biofilm when in co-culture with other bacteria found in food products. This study investigated the desiccation survival of L. monocytogenes in mixed biofilms with Pseudomonas fluorescens, Serratia liquefaciens and Shewanella putrefaciens. To this end, mono- or binary species biofilms were formed and desiccated (43% relative humidity, 21 days at 15°C) on stainless steel coupons and the double Weibull model was fitted to the resulting survivor curves. The presence of the competitor Gram-negative food spoilage bacteria with the exception of Sh. putrefaciens suppressed (p<0.05) L. monocytogenes during biofilm formation (100% relative humidity, 15°C and 48 h) and subsequently decreased (P<0.05) the desiccation survival in L. monocytogenes without affecting the resistance of individual cells. Microscopic approaches revealed different biofilm forming capabilities in the mono- and binary bacterial combinations.

Biofilms are formed in aquatic environments by the attachment of bacteria to submerged surfaces, to the air/liquid interface, and to each other. Although biofilms are associated with disease and biofouling, the robust nature of biofilms; for example, their ability to tolerate chemical and physical stresses, makes them attractive for beneficial biotechnology applications such as bioremediation and biofuels. Based on an understanding of diverse signals and regulatory networks during biofilm development, biofilms can be engineered for these applications by manipulating extracellular/intercellular signals and regulators. Here, we rewired the global regulator H-NS of Escherichia coli to control biofilm formation using random protein engineering. H-NS variant K57N was obtained that reduces biofilm formation 10-fold compared with wild-type H-NS (wild-type H-NS increases biofilm formation whereas H-NS K57N reduces it) via its interaction with the nucleoid-associated proteins Cnu and StpA. H-NS K57N leads to enhanced excision of the defective prophage Rac and results in cell lysis through the activation of a host killing toxin HokD. We also engineered another global regulator, Hha, which interacts with H-NS, to disperse biofilms. Hha variant Hha13D6 was obtained that causes nearly complete biofilm dispersal by increasing cell death by the activation of proteases. Bacterial quorum sensing (QS) systems are important components of a wide variety of engineered biological devices, since autoinducers are useful as input signals because they are small, diffuse freely in aqueous media, and are easily taken up by cells. To demonstrate that biofilms may be controlled for biotechnological applications such as biorefineries, we constructed a synthetic biofilm engineering circuit to manipulate biofilm formation. By using a population-driven QS switch based on the LasI/LasR system and biofilm dispersal proteins Hha13D6 and BdcAE50Q (disperses biofilms by titrating cyclic diguanylate), we displaced an existing biofilm and then removed the second biofilm. Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Here, we engineered toxin MqsR with greater toxicity and demonstrated that the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

Dentre as oito espécies do gênero Listeria, a única transmitida por alimentos, patogênica para humanos, é a Listeria monocytogenes. De grande preocupação para a indústria alimentícia, este microrganismo pode ser encontrado em diversas superfícies de plantas processadoras de alimentos, sendo capaz de se aderir e formar persistentes biofilmes. No presente trabalho, foi estudada a influência da concentração de glicose e do tempo de incubação na formação de biofilme por uma cepa de L. monocytogenes, sorotipo 1/2a, cultivada em três diferentes temperaturas, 7°C, 25°C e 37°C, através de planejamento experimental utilizando a Metodologia de Superfície de Resposta. As duas variáveis testadas, para os intervalos estudados, foram significativas (p<0,05) na formação de biofilme somente na temperatura de 37°C e, tanto concentrações de glicose tendendo a zero combinadas com tempos de incubação próximos a 26 h, como maiores concentrações de glicose combinadas com menores tempos de incubação, dentro da faixa de estudo aplicada, representaram boas condições para a formação de biofilme. Foi também realizada a análise transcricional de genes relacionados à formação de biofilme e virulência em duas cepas de L. monocytogenes, sorotipos 1/2a e 4b, cultivadas a 7°C e 37°C (condição controle), pela técnica de PCR quantitativo em tempo real (RT-qPCR). Para ambas as cepas, os genes sigB, prfA, luxS, sufS, sufU, ltrC e flaA apresentaram transcrição significativamente aumentada (p<0,05) a 7°C, em comparação aos resultados para a condição de cultivo a 37°C, enquanto que o gene hly não variou significativamente a sua transcrição entre as duas temperaturas. O gene actA foi mais transcrito a 7°C para a cepa denominada 55, do sorotipo 1/2a, enquanto que não variou significativamente a sua transcrição entre as duas condições de crescimento para a cepa denominada 47, do sorotipo 4b. No entanto, para a cepa 55, o gene degU não demonstrou diferença estatisticamente significativa entre seus níveis de transcrição nas duas temperaturas estudadas, mas para a cepa 47, apresentou transcrição significativamente aumentada a 7°C. Interessantemente, a presença do gene agrA não foi detectada na cepa 47, e os seus níveis de transcrição foram menores a 7°C para a cepa 55. Estes resultados demonstram que os dois sorotipos estudados, responsáveis por muitos dos casos humanos de listeriose, apresentam mecanismos moleculares diferentes, nas temperaturas de 7°C e 37°C. / Among the eight species of genus Listeria, the only transmitted by food, pathogenic for humans, is Listeria monocytogenes. Of great concern to the food industry, this microorganism can be found in various areas of food processing plants, being able to adhere and form persistent biofilms. In the present work, the influence of glucose concentration and incubation time on biofilm formation by a strain of L. monocytogenes, serotype 1/2a, grown in three different temperatures, 7°C, 25°C and 37°C, have been studied, through an experimental design using the Response Surface Methodology. The two variables tested, for the studied intervals, were significant (p<0,05) in the biofilm formation only at a temperature of 37°C and, both glucose concentrations tending to zero combined with incubation times near to 26 h, and higher glucose concentrations combined with lower incubation times, within the applied range study, represented good conditions for biofilm formation. Transcriptional analysis of genes related to biofilm formation and virulence in two strains of L. monocytogenes, serotypes 1/2a and 4b, grown at 7°C and 37°C (control condition), was also performed, by real-time quantitative PCR (RT-qPCR). For both strains, sigB, prfA, luxS, sufS, sufU, ltrC and flaA genes showed significantly increased transcription (p<0,05) at 7°C, in comparison with results for the growth condition at 37°C, whereas hly gene did not varied significantly its transcription between the two temperatures. The actA gene was more transcribed at 7°C for the 55 strain, serotype 1/2a, while did not varied significantly its transcription between the two growth conditions for the 47 strain, serotype 4b. However, for 55 strain, the degU gene did not showed statistically significant difference for its transcription levels between the two studied temperatures, but for 47 strain, presented significantly increased transcription at 7°C. Interestingly, the presence of agrA gene was not detected in 47 strain, and its transcription levels were lower at 7°C for 55 strain. These results demonstrate that the two studied serotypes, responsible for many of the human listeriosis cases, have different molecular mechanisms, at temperatures of 7 and 37°C.

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