Doctor en Ciencias con mención en Microbiología

Resíduos de Serviços de Saúde (RSS), ainda que tratados e dispostos em aterros sanitários, podem ser causas de impactos ambientais por apresentarem algum indicador de periculosidade. Segundo o apêndice IV da Resolução da Agência Nacional de Vigilância Sanitária (Anvisa) RDC nº 306/2004, para as tecnologias de tratamento desses resíduos é necessário que se atinja pelo menos o Nível III de inativação microbiana. Perante a ausência de dados na literatura que revelassem o tempo de exposição, temperatura e pressão ideais para a inativação microbiana no tratamento de RSS por autoclavagem e se a fração ocupada dos resíduos na autoclave poderia interferir na eficiência da desinfecção definiu-se o objetivo do trabalho para otimização do processo. Para os testes foram utilizados endósporos de Geobacillus stearothermophilus como bioindicadores e instalados cinco termopares na autoclave para aferição da temperatura dentro de todo o espaço da câmara. O RSS foi sintetizado diante das caracterizações como composição gravimétrica, distribuição granulométrica, densidade específica aparente, massa específica aparente e teor de umidade. Apresentou-se uma proposta metodológica para a avaliação do tratamento de RSS frente às dificuldades encontradas. Realizou-se ensaios com 116ºC, 125ºC e 134ºC, observando a fração de inativação em seis tempos de exposição diferentes. Sabendo-se que é padronizada em estabelecimentos de saúde a temperatura ideal para esterilização de materiais de 121ºC, apesar de não terem sido encontrados trabalhos científicos que comprovem a eficiência da esterilização nessa circunstância, foram realizados testes a 50 e 60 minutos de exposição para avaliar essa condição. Inoculou-se 106 endósporos nas amostras e a recuperação foi feita com filtração após a lavagem dos resíduos, sendo realizada a técnica de pour plate para contagem das unidades formadoras de colônias (UFC). O mesmo procedimento foi feito com a amostra retirada da autoclave. A fração de inativação dos endósporos atingiu 100% no tempo de 30 minutos de exposição à temperatura máxima de 134ºC e pressão absoluta de 2,3 kgf/cm2, resultado obtido através da relação do número de micro-organismos recuperados considerados como inoculados e o número de micro-organismos sobreviventes ao tratamento. A 121ºC houve recuperação de UFC após tratamento. A fração de ocupação não foi um fator delimitante para a inativação de endósporos, pois a temperatura se manteve a mesma independentemente da quantidade de resíduo submetido à autoclavagem. Um outro objetivo era avaliar experimentalmente a reprodução dos micro-organismos frente às condições operacionais de autoclavagem e do tempo de permanência do rejeito em condições ambientais. Esse estudo comprovou que a inativação nessas condições otimizadas esteriliza o resíduo, sendo que nenhum micro-organismo voltou a se reproduzir após dias expostos em temperatura ambiente, ou seja, não haveria riscos de contaminação em aterro sanitário quando depositados os rejeitos. Ter encontrado essas condições ideais e avaliado o processo de autoclavagem pode significar um grande avanço nas próprias unidades de tratamento, que terão um parâmetro estabelecido para trabalho. / Healthcare waste, even when treated and disposed of in landfills, can be causes of environmental impacts because they present some hazard indicator. According to appendix IV of Resolution of the National Health Surveillance Agency of Brazil (Anvisa) RDC nº 306/2004, for technologies of healthcare waste treatment, it is necessary to achieve at least Level III of microbial inactivation. The purpose of this research to optimize the process was defined taking into account the absence of data in the literature that revealed the optimal exposure time, temperature and pressure for microbial inactivation through the healthcare treatment by autoclaving and whether the fraction occupied by waste in the autoclave could interfere with the effectiveness of the disinfection. For the tests, endospores of Geobacillus stearothermophilus were used as bioindicators and five thermocouples were installed in the autoclave for temperature measurement throughout the chamber space. The healthcare waste was synthesized by characterizations such as gravimetric composition, granulometric distribution, apparent specific density, apparent specific mass and moisture content. A methodological proposal for the evaluation of waste treatment was introduced taking into consideration the difficulties encountered. Tests were performed at 116ºC, 125ºC and 134ºC, observing the fraction of inactivation at six different exposure times. Since the sterilization of materials at 121ºC is standardized in health establishments, even though no scientific studies were found to prove the sterilization efficiency in these conditions, tests were performed at 50 and 60 minutes of exposure to evaluate this condition. The concentration of 106 endospores were inoculated in the samples and the recovery was done with filtration after washing the waste, and the \"pour plate\" technique was used to count the colony forming units. The same procedure was done with the sample removed from the autoclave. The inactivation fraction of the endospores reached 100% in the time of 30 minutes of exposure to the maximum temperature of 134ºC and absolute pressure of 2.3 kgf/cm2, a result obtained by the ratio of the number of recovered microorganisms considered as inoculated and the number of microorganisms surviving the treatment. At 121ºC there was recovery of colony forming units after treatment. The occupation fraction was not a limiting factor for the inactivation of endospores, since the temperature remained the same regardless of the amount of waste submitted to autoclaving. Another objective was to experimentally evaluate the reproduction of the microorganisms taking into consideration the operational conditions of autoclaving and the amount of time that the waste remains under environmental conditions. This study proved that the inactivation under these optimized conditions sterilizes the waste, and that no microorganism would reproduce again after days exposed at room temperature, therefore, there would be no risk of contamination in a landfill when the waste is deposited. Having found these ideal conditions and evaluated the autoclaving process can mean a major advance in the treatment units themselves, which will have an established parameter for work.

Resíduos de Serviços de Saúde (RSS), ainda que tratados e dispostos em aterros sanitários, podem ser causas de impactos ambientais por apresentarem algum indicador de periculosidade. Segundo o apêndice IV da Resolução da Agência Nacional de Vigilância Sanitária (Anvisa) RDC nº 306/2004, para as tecnologias de tratamento desses resíduos é necessário que se atinja pelo menos o Nível III de inativação microbiana. Perante a ausência de dados na literatura que revelassem o tempo de exposição, temperatura e pressão ideais para a inativação microbiana no tratamento de RSS por autoclavagem e se a fração ocupada dos resíduos na autoclave poderia interferir na eficiência da desinfecção definiu-se o objetivo do trabalho para otimização do processo. Para os testes foram utilizados endósporos de Geobacillus stearothermophilus como bioindicadores e instalados cinco termopares na autoclave para aferição da temperatura dentro de todo o espaço da câmara. O RSS foi sintetizado diante das caracterizações como composição gravimétrica, distribuição granulométrica, densidade específica aparente, massa específica aparente e teor de umidade. Apresentou-se uma proposta metodológica para a avaliação do tratamento de RSS frente às dificuldades encontradas. Realizou-se ensaios com 116ºC, 125ºC e 134ºC, observando a fração de inativação em seis tempos de exposição diferentes. Sabendo-se que é padronizada em estabelecimentos de saúde a temperatura ideal para esterilização de materiais de 121ºC, apesar de não terem sido encontrados trabalhos científicos que comprovem a eficiência da esterilização nessa circunstância, foram realizados testes a 50 e 60 minutos de exposição para avaliar essa condição. Inoculou-se 106 endósporos nas amostras e a recuperação foi feita com filtração após a lavagem dos resíduos, sendo realizada a técnica de pour plate para contagem das unidades formadoras de colônias (UFC). O mesmo procedimento foi feito com a amostra retirada da autoclave. A fração de inativação dos endósporos atingiu 100% no tempo de 30 minutos de exposição à temperatura máxima de 134ºC e pressão absoluta de 2,3 kgf/cm2, resultado obtido através da relação do número de micro-organismos recuperados considerados como inoculados e o número de micro-organismos sobreviventes ao tratamento. A 121ºC houve recuperação de UFC após tratamento. A fração de ocupação não foi um fator delimitante para a inativação de endósporos, pois a temperatura se manteve a mesma independentemente da quantidade de resíduo submetido à autoclavagem. Um outro objetivo era avaliar experimentalmente a reprodução dos micro-organismos frente às condições operacionais de autoclavagem e do tempo de permanência do rejeito em condições ambientais. Esse estudo comprovou que a inativação nessas condições otimizadas esteriliza o resíduo, sendo que nenhum micro-organismo voltou a se reproduzir após dias expostos em temperatura ambiente, ou seja, não haveria riscos de contaminação em aterro sanitário quando depositados os rejeitos. Ter encontrado essas condições ideais e avaliado o processo de autoclavagem pode significar um grande avanço nas próprias unidades de tratamento, que terão um parâmetro estabelecido para trabalho. / Healthcare waste, even when treated and disposed of in landfills, can be causes of environmental impacts because they present some hazard indicator. According to appendix IV of Resolution of the National Health Surveillance Agency of Brazil (Anvisa) RDC nº 306/2004, for technologies of healthcare waste treatment, it is necessary to achieve at least Level III of microbial inactivation. The purpose of this research to optimize the process was defined taking into account the absence of data in the literature that revealed the optimal exposure time, temperature and pressure for microbial inactivation through the healthcare treatment by autoclaving and whether the fraction occupied by waste in the autoclave could interfere with the effectiveness of the disinfection. For the tests, endospores of Geobacillus stearothermophilus were used as bioindicators and five thermocouples were installed in the autoclave for temperature measurement throughout the chamber space. The healthcare waste was synthesized by characterizations such as gravimetric composition, granulometric distribution, apparent specific density, apparent specific mass and moisture content. A methodological proposal for the evaluation of waste treatment was introduced taking into consideration the difficulties encountered. Tests were performed at 116ºC, 125ºC and 134ºC, observing the fraction of inactivation at six different exposure times. Since the sterilization of materials at 121ºC is standardized in health establishments, even though no scientific studies were found to prove the sterilization efficiency in these conditions, tests were performed at 50 and 60 minutes of exposure to evaluate this condition. The concentration of 106 endospores were inoculated in the samples and the recovery was done with filtration after washing the waste, and the \"pour plate\" technique was used to count the colony forming units. The same procedure was done with the sample removed from the autoclave. The inactivation fraction of the endospores reached 100% in the time of 30 minutes of exposure to the maximum temperature of 134ºC and absolute pressure of 2.3 kgf/cm2, a result obtained by the ratio of the number of recovered microorganisms considered as inoculated and the number of microorganisms surviving the treatment. At 121ºC there was recovery of colony forming units after treatment. The occupation fraction was not a limiting factor for the inactivation of endospores, since the temperature remained the same regardless of the amount of waste submitted to autoclaving. Another objective was to experimentally evaluate the reproduction of the microorganisms taking into consideration the operational conditions of autoclaving and the amount of time that the waste remains under environmental conditions. This study proved that the inactivation under these optimized conditions sterilizes the waste, and that no microorganism would reproduce again after days exposed at room temperature, therefore, there would be no risk of contamination in a landfill when the waste is deposited. Having found these ideal conditions and evaluated the autoclaving process can mean a major advance in the treatment units themselves, which will have an established parameter for work.

Predictable, robust genetic parts including constitutive promoters are one of the defining attributes of synthetic biology. Ideally, candidate promoters should cover a broad range of expression strengths and yield homogeneous output, whilst also being orthogonal to endogenous regulatory pathways. However, such libraries are not always readily available in non-model organisms, such as the industrially relevant genus Geobacillus. A multitude of different approaches are available for the identification and de novo design of prokaryotic promoters, although it may be unclear which methodology is most practical in an industrial context. Endogenous promoters may be individually isolated from upstream of well-understood genes, or bioinformatically identified en masse. Alternatively, pre-existing promoters may be mutagenised, or mathematical abstraction can be used to model promoter strength and design de novo synthetic regulatory sequences. In this investigation, bioinformatic, mathematic and mutagenic approaches to promoter discovery were directly compared. Hundreds of previously uncharacterised putative promoters were bioinformatically identified from the core genome of four Geobacillus species, and a rational sampling method was used to select sequences for in vivo characterisation. A library of 95 promoters covered a 2-log range of expression strengths when characterised in vivo using fluorescent reporter proteins. Data derived from this experimental characterisation were used to train Artificial Neural Network, Partial Least Squares and Random Forest statistical models, which quantifiably inferred the relationship between DNA sequence and function. The resulting models showed limited predictive- but good descriptive-power. In particular, the models highlighted the importance of sequences upstream of the canonical -35 and -10 motifs for determining promoter function in Geobacillus. Additionally, two commonly used mutagenic techniques for promoter production, Saturation Mutagenesis of Flanking Regions and error-prone PCR, were applied. The resulting sequence libraries showed limited promoter activity, underlining the difficulty of deriving synthetic promoters in species where understanding of transcription regulation is limited. As such, bioinformatic identification and deep-characterisation of endogenous promoter elements was posited as the most practical approach for the derivation of promoter libraries in non-model organisms of industrial interest.

The effects of thermosonication (high intensity ultrasound coupled with thermal treatment), on the reduction of thermophilic spore-forming microorganisms and its effects on the solubility index in reconstituted skim milk powder (RSMP) were evaluated. Thermosonication was applied to RSMP at various solids concentrations, temperatures, and lengths of time based on commercial milk powder processing conditions. Microbial counts were determined prior to and after treatments to determine the log reduction of Geobacillus stearothermophilusvegetative cells and spores. Log reductions were recorded, and data were analyzed by response surface analysis. The log reductions induced by temperature and time without high intensity ultrasound (HIU) were compared to reductions observed with HIU. Thermosonication was also applied to RSMP to determine effects on solubility using a continuous flow cell system. Thermosonication yielded a significantly higher level of microbial destruction for both vegetative cells and spores than heat treatment alone. For experiments involving vegetative cells, the interaction of treatment time and temperature proved to have the greatest influence on microbial inactivation. In comparison, the interaction of total solids content and length of HIU treatment demonstrated the greatest effect on the increased log reductions for spores. The solubility of RSMP treated with HIU did not significantly differ from the solubility of RSMP not treated with HIU. Further data showed the implementation of HIU, or thermosonication, during milk powder processing would be most effective before and after the evaporation stage when the total solids content of product is 9.2% and 50% at 75°C and 60°C, respectively. Based on preliminary data, it is assumed HIU applied for 10 s at these two locations would produce an additive effect, thereby reducing overall microbial counts by 5.76 log and 0.51 log for G. stearothermophilus vegetative cells and spores, respectively, in the product prior to entering the drying stage. All research findings and observations suggest HIU, or thermosonication, to be a successful method for reducing microbial populations during milk powder processing without sacrificing skim milk powder solubility

Masters of Science / Thermophilic bacteriophages provide simple model systems for understanding biochemical and biological adaptation mechanisms at elevated temperatures. The essential objectives of this study were to characterise the physicochemical properties of select Geobacillus bacteriophages and to sequence their complete genomes. The later objective is believed to be an essential prerequisite to the engineering of a sitespecific integration vector for the stable cloning of exogenous genes into host bacteria. Bacteriophages were assayed at 55oC by the agar overlay technique using dry Karoo soils as source material. A pure strain of bacteriophage called GV1 (for Geobacillus stearothermophilus virus 1) was isolated with the strain Geobacillus stearothermophilus TAU3A1. Plaques were medium sized (2 to 4 mm diameters), with regular contour, clear, and without resistant cells. Host range specificity study showed that GV1 was lytic on thirteen thermophilic Bacillus-like strains tested, including strains of Geobacillus stearothermophilus, G. thermoglucosidasius, B. licheniformis, Anoxybacillus idirlerensis, and A. kuwalawohkensis. However, GV1 failed to infect a mesophilic strain of Bacillus megaterium. TEM analysis of semipurified particles revealed that the phage belongs to the family of Siphoviridae. Morphological characteristics included a long tail of approximately 100 nm and a hexagonal head of approximately 50 nm diameter. Viability and stability studies showed that the phage was best maintained at -80oC in PMN buffer supplemented with 20% glycerol. It was stable at a pH range of 5.5 to 7.5 and MgCl2 and CaCl2 concentration of 0.001 M. hermostability experiments, conducted over short periods of time, showed that GV1 was stable over the temperature range 50 to 75oC, with optimum at 55oC. The study of phage-host interactions showed that phage articles inhibited the initial growth of infected cultures in the first six hours post-infection, presumably while mature phages were released. This was followed by a steady recovery of the growth rate. Atempts to obtain pure particles and to extract and sequence phage DNA were unsuccessful due to the low titer nature of the phage.

Objectives: Dental instrument sterilization with steam autoclaves is critical to maintaining infection control standards in dental practice, and preventing patient-to-patient transmission of pathogenic bacteria and viruses. The American Dental Association and the United States Centers for Disease Control and Prevention recommend, and many state dental laws require, weekly use of biological spore tests to verify dental instrument sterilization outcomes. However, the most widely used biological spore test needs microbial culture incubation for 2 days after autoclave exposure, which limits swift identification of sterilization failure. To address this issue, this study evaluated the reliability of a new rapid biological spore test for determining the sterilization efficacy of dental steam autoclaves within 20 minutes. Methods: Two commercial biological spore tests were evaluated in Temple University dental school steam autoclaves, 1.) the Steris Celerity 20 Steam Biologic Indicator with a 20-minute outcome time requirement, and 2.) the 3M Attest 1262 Biological Indicator with a 48-hour outcome time requirement. Both biological spore tests employed live thermoresistant Geobacillus stearothermophilus spores as an indicator of whether sterilization conditions in steam autoclaves were met or not. To compare their efficacy, a total of 157 pairs of the two biological spore tests were placed into dental steam autoclaves with dental instrument cassettes, and subjected to manufacturer-recommended steam autoclave temperature and air pressure operating conditions for an adequate sterilization time of 15 minutes. Two additional groups of 10 pairs each of the two biological indicators were subjected to appropriate steam autoclave temperature and air pressure settings, but only for aborted non-sterilizing time periods of 10 and 5 minutes, respectively. Subsequent aseptic processing and laboratory incubation of both biological indicators was initiated within 2-24 hours, and followed manufacturer recommendations. After autoclave exposure, Steris Celerity 20 Steam Biologic Indicator test ampoules were incubated in a specialized instrument for 20 minutes at 57 °C, which also spectrophotometrically evaluated the microbial culture medium for fluorescent α- glucosidase enzyme signal changes. No change in fluorescent intensity represented successful sterilization, whereas increased fluorescence indicated survival of viable G. stearothermophilus spores germinating into vegetative bacterial cells after failed sterilization. 3M Attest 1262 Biological Indicator ampoules were incubated for 48 hours in a laboratory heating block at 57 °C, after which a pH-based color change in the microbial culture broth was visually assessed. No change in the color of the culture broth (purple color remains) indicated successful sterilization, whereas development of a yellow color in the culture broth, as a result of viable G. stearothermophilus spore germination into vegetative bacterial cells, denoted failed sterilization. Results: A total of 354 biological indicators were exposed to dental steam autoclaves sterilization cycles, incubated for either 20 minutes or 48 hours, and evaluated for G. stearothermophilus spore growth. The Steris Celerity and 3M Attest biological spore tests both uniformly detected successful sterilization, with no G. stearothermophilus spore growth, after 15 minutes of steam autoclave exposure at manufacturer recommended steam autoclave temperature and air pressure operating conditions. This provided 100% agreement, and no statistically significant difference in the prevalence of successful sterilization outcomes, between 157 pairs of both biological indicator types after 15 minutes of steam autoclave exposure. Similarly, both biological spore test systems were also in complete agreement after only 5 minutes of steam autoclave exposure, with 100% of both biological indicators positive for G. stearothermophilus spore growth, indicating failed sterilization. In contrast, after 10 minutes of steam autoclave exposure, there was a complete lack of agreement between the two types of biological indicators. All 10 Steris Celerity spore tests were positive, whereas all 10 3M Attest ampoules were negative, for G. stearothermophilus spore growth after 10 minutes of steam autoclave exposure. Relative to this disagreement, a non-biological chemical indicator strip that was part of the Steris biological indicator test system failed to have a darkened bar develop and extend into the “Accept (OK)” portion of the strip for all Steris Celerity spore tests exposed to either 5 minutes or 10 minutes of steam autoclave exposure, indicating that adequate autoclave steam, temperature and/or time parameters had not been attained for sterilization. Conclusions: The Steris Celerity biological spore test was successful in rapidly determining the sterilization efficacy of dental steam autoclaves within only a 20-minute incubation time period, as compared to 48 hours of incubation required by the widely-used 3M Attest biological spore test. As a result, this rapid assay offers earlier detection of steam autoclave sterilization failure before potentially contaminated dental instruments are used in clinical patient care. The alarming failure of 3M Attest biological spores to grow after a non-sterilizing 10-minute steam autoclave exposure time warrants further product evaluation. / Oral Biology

Group II Introns are small segments of DNA that reside in the chromosome of bacteria or the organelles of primitive eukaryotes. These elements have some very interesting properties. First, they are retrotransposons that can move from one location to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-mRNA. A Group II Intron type protein with similarity to reverse transcriptase was discovered in the thermophilic bacterium Geobacillus stearothermophilus strain 10 (Vellore et al., 2004, Appl. Environ. Microbiol. 70: 7140-7147). Numerous copies of the intron, designated Gs. Int1, are present in the chromosome of strain 10 but absent from a related strain ATCC 12980. Experiments to detect the in vivo splicing of intron Gs.Int1 from G. stearothermophilus cells did not work. Plasmids to that will over-express the Gs. Int1 intron to detest splicing in vivo in Escherichia coli have been constructed.

The crystal structures of two thermostable carboxylesterase Est30 and Est55 from Geobacillus stearothermophilus were determined to help understand their functions and applications in industry or medicine. The crystal structure of Est30 was determined at 1.63 Å resolution by the multiple anomalous dispersion method. The two-domain Est30 structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. A 100 Da tetrahedral ligand, propyl acetate, was observed to be covalently bound to the side chain of Ser94 in the catalytic triad. This ligand complex represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily. Est55 is a bacterial homologue of the mammalian carboxylesterases involved in hydrolysis and detoxification of numerous peptides and drugs and in prodrug activation. Est55 crystals were grown at pH 6.2 and pH 6.8 and the structures were determined at resolutions of 2.0 and 1.58 Å respectively. Est55 folds into three domains, a catalytic domain, an α/β domain and a regulatory domain. This structure is in an inactive form; the side chain of His409, one of the catalytic triad residues, is pointing away from the active site. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the entry of substrate to its binding site. This structure suggested a self-inactivation mechanism, however, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side chains at this position were favorable, while polar serine was unfavorable for enzyme activity. Both Est30 and Est55 were shown to hydrolyze the prodrug CPT-11 into the active form SN-38. Therefore, Est30 and Est55 are potential candidates for use with irinotecan in cancer therapy. The catalytic efficiency (kcat/Km) of Est30 is about 10-fold lower than that of Est55. The effects of the Cys408 substitutions on Est55 activity differed for the two substrates, p-NP butyrate and CPT-11. Mutant C408V may provide a more stable form of Est55.

This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.