O tetracloroeteno (PCE), um dos principais contaminantes de águas subterrâneas, é uma molécula recalcitrante, com toxicidade elevada. Processos de biorremediação de água ou solo contaminados com PCE são normalmente limitados pela baixa eficiência de microrganismos sabidamente envolvidos em sua degradação. No entanto, a prospecção de novos microrganismos, mais eficientes na degradação do PCE é uma alternativa para otimizar esses processos. Os objetivos deste estudo foram desenvolver uma técnica de biorremediação utilizando um reator horizontal de leito fixo (RHLF) contendo consórcios de microrganismos eficientes na degradação do PCE, e caracterizar a via de degradação do PCE utilizada pelo consórcio selecionado. Para tanto, amostras de sedimento de dois poços de monitoramento de água foram coletadas de uma metalúrgica com histórico de contaminação com PCE. Os sedimentos foram imobilizados, acondicionados em RHLFs específicos e submetidos a cultivo de enriquecimento em meio mínimo suplementado com PCE. A estrutura das comunidades e a diversidade bacteriana dos RHLFs foram avaliadas e comparadas com as amostras do PM1 e PM2, por PCR-DGGE e sequenciamento de bibliotecas de clones do gene rRNA 16S. Os resultados evidenciaram a seleção de populações bacterianas no RHLF contendo o inóculo do PM1 (In1) após o cultivo de enriquecimento, enquanto no RHLF contendo o inóculo do PM2 (In2), a estrutura da comunidade bacteriana não diferiu daquela observada no PM2. Ensaios de degradação do PCE nos RHLFs, usando cromatografia gasosa associada à espectrometria de massas (CG/EM), mostraram, após 12 horas, uma eficiência de 87 % na degradação do PCE no reator com In1 e 96 % no reator com In2. Foi feito o isolamento e identificação, por sequenciamento do gene rRNA 16S, das bactérias dos RHLFs, sendo identificados 4 isolados do In1, similares a Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans e Stenotrophomonas maltophilia e 7 isolados do In2, similares a Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii e Comamonas sp. Compostos orgânicos voláteis nos reatores com In1 e In2 foram analisados por CG/EM, identificando a produção de clorofórmio (TCM) e 1,1,1-tricloroetano (TCA) como produtos da degradação do PCE. Consórcios formados por bactérias isoladas dos reatores In1 (IIn1) e In2 (IIn2) foram imobilizados e acondicionados em RHLFs distintos para avaliar o potencial dos mesmos na degradação do PCE. Após 12 horas, 92 % do PCE foi degradado nos reatores com IIn1 e IIn2, com produção de TCM e TCA. Testes de degradação usando células em suspensão foram conduzidos para avaliar a eficiência de cada isolado na degradação do PCE. O isolado I8 do In2 (I8In2), identificado como Comamonas sp., teve 68 % de eficiência na degradação do PCE. Ensaios com inibidor de monoxigenases do citocromo P-450 (1-aminobenzotriazole) mostraram que a degradação do PCE nos RHLFs, contendo IIn1, IIn2 e I8In2, foram dependentes dessa enzima. Como conclusão, nós identificamos uma nova via de degradação do PCE altamente eficiente, aeróbia e mediada por monoxigenases e isolamos cepas bacterianas que podem ser usadas como consórcios imobilizados nos RHLFs como uma alternativa eficiente na remediação de áreas contaminadas com PCE. / Tetrachloroethene (PCE), one of the main contaminants of groundwater, is a recalcitrant molecule with high toxicity. Bioremediation processes of water or soil contaminated with PCE are usually limited by the low efficiency of microorganisms known to be involved in its degradation. However, the exploration of new and more efficient microorganisms in the degradation of PCE is an alternative to optimize these processes. The objectives of these studies were to develop a bioremediation technique using horizontal fixed bed reactor (HFBR) containing microbial consortia effective in the PCE degradation, and to characterize the PCE degradation pathway used by the selected consortium. For that, sediment samples of two groundwater monitoring wells were collected from a metallurgical plant with historical of PCE contamination. The sediments were immobilized, packed in specific HFBRs and subjected to enrichment in minimal medium supplemented with PCE. The bacterial community structure and diversity in the HFBRs were evaluated and compared to samples from the MW1 and MW2, by PCR-DGGE and sequencing of 16S rRNA gene clone libraries. The results revealed the selection of bacterial populations in the HFBR containing inoculum from MW1 (In1) after enrichment, while in the HFBRs containing inoculum from MW2 (In2), the bacterial community structure did not differ from that observed in MW2. Tests of PCE degradation in HFBRs using gas chromatography-mass spectrometry (GC/MS) showed, after 12 hours, an efficiency of 87 % in the PCE degradation in the In1 reactor and 96 % in the In2 reactor. Bacteria from HFBR were isolated and identified by sequencing of 16S rRNA gene, and 4 isolates from In1, similar to Burkholderia sp., Pseudomonas stutzeri, P. oryzihabitans and Stenotrophomonas maltophilia, and 7 isolates from In2, similar to Microbacterium trichotecenenolyticum, S. maltophilia, Klebsiella sp., Exiguobacterium acetylicum, P. oryzihabitans, Acinetobacter junii and Comamonas sp. were identified. Volatile organic compounds in the reactors with In1 and In2 were analyzed by GC/MS, showing the production of chloroform (TCM) and 1,1,1-trichloroethane (TCA) as PCE degradation products. Consortia composed of bacteria isolated from the In1 (IIn1) and In2 (IIn2) reactors were immobilized and packed in distinct HFBRs to evaluate the potential of specific consortia in PCE degradation. After 12 hours, 92 % of PCE was degraded in reactors with IIn1 and IIn2, with the production of TCM and TCA. Degradation tests using cells suspension were conducted to evaluate the efficiency of each isolate in PCE degradation. Isolate I8 from In2 (I8In2), identified as Comamonas sp., showed 68 % efficiency in the PCE degradation. Assays using inhibitor of monooxygenases cytochrome P-450 (1-aminobenzotriazole) showed that the PCE degradation in HFBRs containing IIn1, IIn2 and I8In2 were dependent of this enzyme. In conclusion, we have identified a new highly efficient PCE degradation pathway, aerobic and mediated by monooxygenases, and isolated bacterial strains that may be used as consortia which immobilized in HFBRs as an efficient alternative in the remediation of PCE contaminated areas.

Microcistinas são potentes hepatotoxinas e promotoras de tumores encontradas em águas doces que causam riscos à saúde pública e, portanto, representam um sério problema para as estações de tratamento de água. O gênero de cianobactéria Microcystis é o mais conhecido produtor dessas toxinas e também o mais comumente encontrado formando florações em reservatórios de água usados para abastecimento público. Entretanto, algumas bactérias são capazes de utilizar essas toxinas como fonte de carbono o que pode contribuir para a sua remoção da água. Neste estudo foi avaliado o potencial de degradação da microcistina-XR (MCYST-XR) por bactérias heterotróficas isoladas de um sistema de abastecimento público de água da cidade de Piracicaba-SP. A cianotoxina MCYST-XR avaliada foi isolada da linhagem Microcystis aeruginosa NPLJ-4 e purificada. Culturas puras de 35 bactérias isoladas do sistema de abastecimento de água foram testadas. Para isso, cada bactéria foi inoculada em meio mínimo de sais contendo 60 g mL-1 de MCYST-XR purificada e após 144 horas a degradação da toxina foi avaliada por análise em LC-MS/MS. Os picos indicando a massa da microcistina-XR (m/z 1037) não foram detectados no meio de cultura de seis bactérias, as quais foram identificadas pelo sequenciamento quase completo do gene de RNAr 16S como pertencentes aos gêneros Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. e Acinetobacter sp. Este é o primeiro relato de uma linhagem de Acinetobacter capaz de degradar MCYST. A cinética de biodegradação da MCYST-XR mostrou redução de 80% em 72 horas, período em que as bactérias apresentaram o máximo crescimento. A presença do gene mlrA codificante da enzima microcistinase envolvida no metabolismo da microcistina foi avaliada nos genomas dos seis isolados bacterianos usando iniciadores de PCR específicos. Fragmentos de aproximadamente 800 pb foram amplificados em dois isolados, Bacillus sp.e Microbacterium sp. Ensaios de toxicidade utilizando o cládocero Daphnia similis foram realizados para verificar a efetiva remoção da MCYST-XR e a não formação de subprodutos tóxicos na via de biodegradacão. Os resultados negativos dos bioensaios após 48 horas indicaram a ausência de subprodutos tóxicos na via da degradação. / Microcystins are potent hepatotoxins and tumor promoters found in freshwaters that cause public health risks and thus represent a serious problem for water treatment plants. The cyanobacterium genus Microcystis is the most known toxin-producer and the most common bloom-forming in water reservoirs used for public supply. However, some bacteria are able to use these toxins as carbon source, which can contribute to its removal from water. This study assessed the potential for degradation of microcystin-XR (MCYST-XR) by heterotrophic bacteria isolated from a public water supply system of the city of Piracicaba-SP. The cyanotoxin MCYSTXR evaluated was isolated from Microcystis aeruginosa strain NPLJ-4 and purified. Pure cultures of 35 bacteria isolated from the water supply system were tested. For this, each bacterium was inoculated in minimal medium salts containing 60 g mL-1 of purified MCYST-XR and after 144 hours the toxin degradation was evaluated by LC-MS/MS analysis. The peaks indicating the mass of microcystin-XR (m/z 1037) were not detected in the culture medium of six bacteria, which were identified by almost complete sequencing of the 16S rRNA gene as belonging to the genera Pseudomonas sp., Sphingomonas sp., Microbacterium sp., Agromyces sp., Bacillus sp. and Acinetobacter sp. This is the first report of an Acinetobacter strain able to degrade MCYST. The kinetic of the MCYST-XR biodegration showed 80% reduction in 72 hours, period in which the bacteria showed the maximum growth. The presence of the mlrA gene encoding the microcystinase enzyme involved in the metabolism of microcystin was evaluated in the genomes of the six bacterial isolates using specific PCR primers. Fragments of approximately 800 bp were amplified in two isolates, Bacillus sp. and Microbacterium sp. Toxicity assays using the cladoceran Daphnia similis were conducted to verify the effective removal of MCYST-XR and the non formation of toxic by-products in the biodegradation pathway. The negative results of the bioassays after 48 hours indicated absence of toxic by-products in the biodegradation pathway.

Terra Preta Antropogênica (TPA) é um dos tipos de solos mais \"férteis\" do mundo. A TPA recebe esta denominação por ser encontrada em sítios arqueológicos onde viveram grupos pré-históricos. São pequenas faixas de solos que apresentam altas concentrações de nutrientes, matéria orgânica e encontram-se distribuídas aleatoriamente pela região Amazônica. A verdadeira origem destes solos ainda não está bem esclarecida. Devido à falta de informações sobre sua diversidade bacteriana, este trabalho estudou a diversidade bacteriana em amostras de solos TPA coletadas em duas regiões: Lagoa Balbina - sítio Terra preta (Amazônia Central- Amazonas) e Floresta Nacional de Caxiuanã - sítio arqueológico Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O DNA genômico total das amostras de solo foi extraído e usado como molde em uma reação de PCR utilizando oligonucleotídeos específicos do gene 16S rRNA para o Domínio Bactéria. O produto de PCR amplificado foi clonado no vetor pGEM-T e 980 clones foram selecionados e comparados com o banco de dados de 16S rRNA do RDP II e GenBank (NCBI-EUA). Os resultados apresentaram predominância com microrganismos não-conhecidos representando 41,6 % das seqüências de solo TPA- Balbina, 68,3 % das seqüências de ADJ-Balbina, 84,8% das seqüências de solo TPAMina e 47,7 % das seqüências de ADJ-Mina. O filo mais predominante nas amostras TPABalbina foi Firmicutes, representando 37,1% do total de seqüências analisadas. Os filos em destaque foram Proteobacteria (9,6%), seguidos de Verrucomicrobia (5,6%), Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) e Nitrospira (0,5%). Por outro lado, em ADJ-Balbina destacaram-se os filos Proteobacteria 15,1%, Acidobacteria (12,5%), Firmicutes (2,3%), Nitrospira (1,1%) e Verrucomicrobia (0,8%). Em TPA-Mina, os filos apresentados foram Proteobacteria (6,5%), Acidobacteria (4,7%), Firmicutes (1,4%), Nitrospira (1,1%), Planctomycetes (1,1%) e Verrucomicrobia (0,4%). Contudo, na biblioteca ADJ-Mina verificou a presença dos filos Acidobacteria (27,2%), Proteobacteria (14,2%), Firmicutes (3,8%), Verrucomicrobia (3,8%), Nitrospira (1,3%), Planctomycetes (1,3%), Actinobacteria (0,4%) e Gemmatimonadetes (0,4%). O pH do solo pode ser um dos atributos do solo que pode ter influência direta na diversidade bacteriana dos solos estudados, assim como pode ter efeito uma floresta natural sobre as populações microbianas em seu solo, fato observado na adjacência do solo Terra Preta em Caxiuanã - PA. A estimativa da riqueza de UTOs pelo Bootstrap corroborou diretamente os valores de diversidade obtidos pelos índices de Simpson e Shannon. De um modo geral, uma maior probabilidade de ocorrência de UTOs únicas empregadas pelo estimador Jackknife se correlacionou com uma maior percentagem de baixas frequências de filotipos nas quatro bibliotecas. Os métodos não-paramétricos ACE e Chao1 para a estimativa da riqueza de UTOs também corroboraram com os valores obtidos com o estimador Jackknife. / Anthropogenic Dark Earth (ADE) is one of the most fertile soils in the world. ADE soils have received this nomination due to the pre-historical origin of these archaeological sites, established by pre-colombian populations. ADEs are small areas of soil which present high nutrient and organic matter contents and are randomly distributed throughout the Amazonian region. The true origin of these soils is not known yet. Due to the lack of information concerning the bacterial diversity, this work studied the bacterial diversity in ADE soils collected from two regions: Lagoa Balbina - site Terra Preta (Central Amazonia- Amazonas state) and National Forest of Caxiuanã - archaeological site Mina I (Oriental Amazonia - Pará state), using culture-independent molecular techniques. The total genomic DNAs extracted from the soil samples were used as templates in the PCR reactions using the universal primers for the 16S rRNA bacterial gene. The PCR-products were cloned into the pGEM-T vector and 980 clones were selected and searched using the GenBank (NCBI-USA) and the RDP II program. Data analyses indicated predominance of unknown microorganisms, representing 41.6 % among the sequences from ADE-Balbina, 68.3 % from Adjacent-Balbina, 84.8% from ADE-Mina and 47.7 % from Adjacent-Mina. The predominant phylum in ADEBalbina was Firmicutes, representing 37.1% of the total sequences from that site, followed by Proteobacteria (9.6%), Verrucomicrobia (5.6%) Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) and Nitrospira (0.5%). On the other hand, in the adjacent soil ADJ-Balbina, the predominant phylla were Proteobacteria (15.1%), Acidobacteria (12.5%), Firmicutes (2.3%), Nitrospira (1.1%) and Verrucomicrobia (0.8%). In the Oriental Amazon, the prevalent phylla from the ADE-Mina soil were Proteobacteria (6.5%), Acidobacteria (4.7%), Firmicutes (1.4%), Nitrospira (1.1%), Planctomycetes (1.1%) and Verrucomicrobia (0.4%). In the ADJ-Mina verificou a presença dos filos Acidobacteria 27.2%, Proteobacteria 14.2%, Firmicutes 3.8%, Verrucomicrobia 3.8%, Nitrospira 1.3%, Planctomycetes 1.3%, Actinobacteria 0.4% e Gemmatimonadetes 0.4%. The soil pH may be of the soil attributes which may have directly influenced the bacterial diversity in those soils, as well as the above-ground vegetation from the natural forest in Caxiuanã-Pará. The estimates of the Operational Taxonomic Units (OTUs) richness using Bootstrap directly corroborated the diversity values obtained from the Simpson and Shannon indexes. Unique UTOs using Jackknife estimator were correlated with a higher percentage of the low frequencies of phylla in all the four clone libraries. The nonparametric ACE and Chao1 methods to estimate the OTUs richness also corroborated the Jackknife values.

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

This thesis deals with the question of whether planktonic protits of the genus Dinophysis have permanent plastids (=chloroplasts) or practice kleptoplasty, i.e. acquire plastids via predation on other microorganisms. Sequencing the plastid 16S rDNA of Dinophysis spp. collected from 4 different geographical regions unveiled two different plastid genotypes within this genera: one that was found at all locations investigated, identical to that of the free-living cryptophyte Teleaulax amphioxeia, and another found only in the Greenland Sea, closely related to that of the cryptophyte Geminigera cryophila. Both types were found within the species D. acuminata. These findings imply that the plastids in Dinophysis spp. were not inherited from a common ancestor, but acquired from feeding. By using flow cytometry in combination with an acidotrophic probe, it was shown that 71 % of the cells in a D. norvegica population in the aphotic zone of the Baltic Sea had food-vacuoles. Dinophysis used to be regarded as a primarily phototrophic organism, and this was a higher proportion of cells with food-vacuoles than reported earlier. To further study if Dinophysis needs constant refill of new plastids from the environment, a new method combining flow-cytometry and quantitative real-time PCR was developed to compare the levels of nuclear and plastid DNA in different phases of the cell-cycle. Results showed that plastid acquisition in Dinophysis was uncoupled with the cell-cycle, which is different than the pattern seen in microalgal species with permanent plastids. Furthermore, when quantitative real-time PCR combined with flow-cytometry was used to follow D. caudata cultures during a 65 days starvation/feeding experiment, the cells first went through a steady decrease in plastid DNA during starvation. In contrast, after feeding on the ciliate Myrionecta rubra, plastid DNA in starved cells increased 7-fold, thereby directly revealing the kleptoplastic behavior. The main conclusion from this thesis is that Dinophysis cells are actively taking up kleptoplastids from the ciliates on which they feed, and that kleptoplasty is an important key to understand Dinophysis ecology. Part of this thesis work has also been dedicated to the application and optimization of new methods, and it shows how quantitative real-time PCR, flow cytometry and molecular methods in different combinations can be used as powerful tools for the study of plankton ecology.

Hospital infections are the most frequent complications occurring in hospitalized patients, especially in trauma hospitals, where the seriousness of the injury is predictive factor for the emergence of infections. This study evaluated the profile of infection in the Emergency Unit of Agreste (UEA) through data from CCIH and analysis of clinical samples. The antimicrobial resistance profile was determined too. Isolates have been subjected to 16S rRNA sequencing and analysis in GenBank and RDP. Among 271 patients analyzed, 154 (57%) exhibited positivity in culture (PC). Automobile accidents were the main causes of hospitalization (47,2%). The median of hospitalization was 26 days. PC patients were 10 days more than the median. The use of urinary catheter was 15±13 days, being more 9,5 days in patients PC, on average. Of the 252 PC, 43 were isolated over a microorganism, totaling 269 bacteria and 26 yeasts. Pseudomonas aeruginosa (16,9%) and Acinetobacter baumannii (12,9%) were more frequent. Antimicrobial empirical therapy was made by 76,6% of patients, being 58,1% by cephalosporins. Vancomycin (100%) and chloramphenicol (82,4%) were the most sensitive between the antimicrobial Gram positive cocci, and carbapenems (90,1%) among Gram negative bacilli. The results of UEA compared to international monitoring programmes exhibited greater resistance. However, there were similarities with the Brazilian program RM Network and even greater sensitivity of P. aeruginosa to cefepime and carbapenems. Two-hundred-two sequenced microorganisms were identified by BLASTn at GenBank and RDP banks. They provided identification, respectively: 88,1% and 88,6% in species; 7,9% and 6,4% on genus; 3,5% and 4% in family; and 0,5% and 1% in class. There was agreement among banks in 84,7% to species; 93,7% in genus and 99% in family. The microorganisms most frequently were P. aeruginosa (22,8%), A. baumannii (18,8%) and Klebsiella pneumoniae (11,9%). Among the rarest microorganisms are Brevibacillus agri, Acinetobacter radioresistens, Corynebacterium amycolatum, Corynebacterium striatum, Pseudomonas cedrina subsp. fulgida, Pseudomonas putida and Staphylococcus sciuri. In some cases there was a high similarity between different species not allowing assignment of identification. The differentiation was accomplished through the creation of consensus sequences with reference sequences for CLUSTALW alignment for each microorganism that has similarity. Among 10 cases identified only as Klebsiella, 7 were K. pneumoniae, 2 K. granulomatis and 1 without definition. Eleven Enterobacteriaceae were defined as: 3 Escherichia coli, 2 Enterobacter cancerogenus, 2 Enterobacter sp., 2 group E. coli / Shigella and 2 without definition in genus. The 2 sequences with the same score to Serratia marcescens, Pseudomonas fluorescens strains were identified as S. marcescens. Identification by sequencing is superior to phenotypic tests, especially for atypical biochemist profile, slow growth, rare species and microorganisms of difficult cultivation. Despite difficulties for service deployment and the need for caution in attributing identification, sequencing remains as a promising technique for use in routine laboratories, guiding proper medical team for a better management of the patient. / As infecções hospitalares são as mais frequentes complicações ocorridas em pacientes hospitalizados principalmente em hospitais de trauma, onde a gravidade da lesão é fator preditivo para o surgimento de infecções. O presente estudo avaliou o perfil das infecções na Unidade de Emergência do Agreste (UEA) através de dados da CCIH e análise de amostras clínicas. O perfil de resistência aos antimicrobianos também foi determinado. Os isolados foram submetidos ao sequenciamento do gene rRNA 16S e análise no bancos GenBank e RDP para identificação. Dos 271 pacientes analisados, 154 (57%) exibiram positividade em cultura (CP). Acidentes automobilísticos foram os principais motivos de internação (47,2%). A mediana de internação foi de 26 dias, sendo 10 dias a mais em pacientes CP. O uso de sonda vesical foi de 15±13 dias, sendo 9,5 dias a mais em pacientes CP, em média. Das 252 CP, em 43 foram isolados mais de um microrganismo, totalizando 269 bactérias e 26 leveduras. Pseudomonas aeruginosa (16,9%) e Acinetobacter baumannii (12,9%) foram os mais frequentes. Estavam em uso empírico de antimicrobianos, 76,6% dos pacientes, sendo 58,1% por cefalosporinas. A vancomicina (100%) e o cloranfenicol (82,4%) foram os antimicrobianos de melhor atividade frente aos Gram positivos, e os carbapenêmicos (90,1%), entre Gram negativos. Os resultados da UEA, comparados aos programas de vigilância internacionais, exibiram maior resistência. No entanto, houve semelhanças com o programa brasileiro Rede RM, e ainda maior sensibilidade de P.aeruginosa frente aos carbapenêmicos e cefepime. Dos 202 microrganismos sequenciados, a atribuição de identificação por BLASTn nos bancos genéticos GenBank e RDP forneceram identificação, respectivamente de 88,1% e 88,6% em espécie; 7,9% e 6,4% em gênero; 3,5% e 4% em família e 0,5% e 1% em classe. Houve concordância entre os bancos de 84,7% em espécie; 93,7% em gênero e 99% em família. Os microrganismos mais frequentes foram P. aeruginosa (22,8%), A. baumannii (18,8%) e Klebsiella pneumoniae (11,9%). Entre os mais raros estão Brevibacillus agri, Acinetobacter radioresistens, Corynebacterium amycolatum e Corynebacterium striatum, Pseudomonas cedrina subsp. fulgida, Pseudomonas putida e Staphylococcus sciuri. Em alguns casos, houve elevada similaridade entre diferentes espécies, não permitindo a atribuição de identificação. A diferenciação foi realizada através da criação de sequências consenso de referência por alinhamento no CLUSTALW para cada microrganismo que apresentou similaridade. Dos 10 casos determinados somente como Klebsiella, 7 eram K. pneumoniae, 2 K. granulomatis e 1 sem definição. Das 11 Enterobacteriaceae, 3 foram Escherichia coli, 2 Enterobacter cancerogenus, 2 Enterobacter sp., 2 E. coli / Shigella e 2 sem definição. As 2 sequências com mesma pontuação para Serratia marcescens e Pseudomonas fluorescens foram identificadas como S. marcescens. A identificação por sequenciamento é superior aos testes fenotípicos, sobretudo para isolados com perfil bioquímico atípico, crescimento lento, espécies raras e microrganismos de difícil cultivo. Apesar das dificuldades para implantação do serviço e a necessidade de cautela na atribuição de identificação, o sequenciamento permanece como uma técnica promissora para a utilização em laboratórios de rotina, orientando adequadamente a equipe médica para um melhor manejo do paciente.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-29Bitstream added on 2014-06-13T19:14:41Z : No. of bitstreams: 1 silva_lf_me_sjrp.pdf: 986291 bytes, checksum: e879f8c79aac66ac197646cb214878eb (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No Brasil, o queijo Mussarela elaborado com leite de búfala, tem uma boa aceitação pelos consumidores e mercado em expansão. Entretanto, poucas são as pesquisas em âmbito nacional sobre a microbiota e influência das bactérias ácido-láticas utilizadas na produção, sobre a qualidade tecnológica deste queijo. O objetivo deste trabalho foi compor um banco de culturas representativo da microbiota isolada de queijo Mussarela fabricado com leite de búfala e efetuar a caracterização das bactérias ácido-láticas (BAL). Foram realizadas três coletas em dois laticínios (Laticínios A e B), em diferentes etapas do processo de fabricação, assim como no produto acabado (queijo Mussarela e soro de conservação) recém processado e com 14 e 28 dias de estocagem. Foi feita a contagem de colônias viáveis, isolamento dos mesófilos e termófilos, caracterização morfológica por coloração de Gram e teste de catalase. Foram obtidos 313 isolados que apresentaram características de BAL. As culturas isoladas das amostras do queijo do Laticínio B foram identificadas pela técnica de RAPD e sequenciamento do gene 16S rRNA e caracterizadas quanto à atividade acidificante, capacidade de utilizarem o citrato, atividade proteolítica e capacidade de produzirem compostos voláteis precursores de aromas. Para os dois laticínios, a população de microorganismos termófilos prevaleceu sobre os mesofilos. Os isolados foram identificados como Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus e Leuconostoc mesenteroides. A velocidade máxima de acidificação para os isolados variou de 0,0005 e 0,0305 unidades de pH por minuto após 20 min e 18 h 50 min do início do processo de fermentação, respectivamente para termófilos e mesófilos. O tempo... / In Brazil, the Mozzarella cheese prepared with buffalo milk has a good acceptance and market expansion. However, there are few national studies about microflora and influence of lactic acid bacteria, used in production, on the technological quality of this cheese. The aim this study was to compose a representative bank of the microbial cultures isolated from Mozzarella cheese produced with buffalo milk and to characterize the lactic acid bacteria (LAB). Three collections were performed in two dairy (Dairy A and B) at different stages of the manufacturing process as well as the finished product (Mozzarella cheese and whey conservation) newly processed and with 14 and 28 days of storage. It was followed a count of viable colony, isolation of mesophiles and thermophiles, morphological characterization by Gram staining and catalase test. It was obtained 313 isolates that exhibited characteristics of LAB. The cultures isolated of the cheese samples of the cheese from the Dairy B were identified by RAPD and 16S rRNA gene sequencing and characterized by acidifying activity, ability to utilize citrate, proteolytic activity and ability to produce volatile compounds that are flavor precursors. At two dairies, the population of thermophilic microorganisms was higher than mesophylic. The isolates were identified as Lactobacillus fermentum, Lactobacillus casei, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus and Leuconostoc mesenteroides. The top speed of acidification for the isolates ranged from 0.0005 and 0.0305 pH units per minute after 20 minutes and 18:50 of the beginning of the fermentation process, respectively for thermophiles and mesophiles. The time required to reach the pH 5.0 ranged from 4h50min to 60h the beginning of... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T18:31:32Z : No. of bitstreams: 1 furlan_b_me_rcla.pdf: 3556255 bytes, checksum: b8dadd1a50eb8caa4d6eb51e31dc8f1e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O gênero Burkholderia compreende um grupo de bactérias muito diversificado, ocupando vários nichos ecológicos e possui espécies que causam doenças, mas outras espécies apresentam habilidades importantes para os ramos da agricultura, biotecnologia e do ambiente, como a biodegradação. Essas bactérias são morfologicamente semelhantes e o gênero está dividido em dezessete genomovars, onde espécies semelhantes geneticamente são agrupadas, formando o Complexo Burkholderia cepacia (CBC). Por meio de estudos moleculares do gene 16S rRNA e do gene recA foi possível fazer a classificação adequada em espécies dos 450 isolados obtidos da Terra Preta Antropogênica (TPA) e adjacentes, em quatro sítios (TPA Caldeirão Cultivado; TPA Caldeirão Capoeira; TPA Hatahara e TPA Mina-I), foram obtidos 177 isolados do gênero Burkholderia. Desses isolados, pela análise do gene 16S rRNA foi possível classificar 157 isolados ao nível de espécie e pela análise do gene recA somente 105 isolados foram classificados neste mesmo nível. Esses isolados foram utilizados no teste de biodegradação dos hidrocarbonetos. Os hidrocarbonetos poliaromáticos (HPAs) são compostos orgânicos resultantes da combustão incompleta da matéria orgânica e dos derivados de petróleo, são pouco solúveis em água e muito tóxicos para as células, por isso, não são metabolizados por muitos micro-organismos e acabam contaminando e inviabilizando os ambientes. Nos testes de biodegradação os substratos utilizados foram o naftaleno, o fenantreno e o diesel, juntamente com o indicador redox 2,6- diclorofenol-indofenol (DCPIP), que sofre descoloração (de azul para incolor) quando as células utilizam os substratos como fonte de carbono para o crescimento celular, gerando elétrons que vão reduzir o indicador. Pela análise do teste, 19 isolados degradaram o naftaleno, 16 degradaram... / The genus Burkholderia comprises a very diverse group of bacteria occupying various ecological niches and has species that cause disease, but other species have important skills to the branches of agriculture, biotechnology and the environment, as biodegradation. These bacteria are morphologically similar genus and is divided into seventeen genomovars, where genetically similar species are grouped together, forming the Burkholderia cepacia complex (BCC). Through molecular studies of 16S rRNA and recA gene was possible to make the appropriate classification of species in 450 isolates of Terra Preta Antropogênica (TPA) and adjacent at four sites (TPA Caldeirão Cultivado; TPA Caldeirão Capoeira; TPA Hatahara e TPA Mina-I) were obtained 177 isolates of the genus Burkholderia. These isolates by 16S rRNA gene analysis was possible to classify 157 isolates to species level and the recA gene analysis only 105 isolates were classified in the same level. These isolates were used to test the biodegradation of hydrocarbons. Polyaromatic hydrocarbons (PAHs) are organic compounds resulting from incomplete combustion of organic matter and petroleum derivatives, are not very soluble in water and very toxic to cells, therefore, are not metabolized by many microorganisms to contaminate and render environments. In biodegradation tests the substrates used were naphthalene, phenanthrene and the diesel, along with the redox indicator 2,6- dichlorophenol-indophenol (DCPIP), who suffers discoloration (blue to colorless) when cells use the substrates as a source of carbon for cell growth, generating electrons that will reduce the indicator. By analysis of the test, 19 isolates degraded naphthalene, phenanthrene degraded the 16 and 126 degraded diesel, generating a total of 132 isolates of the genus Burkholderia that have the ability to degrade at least... (Complete abstract click eletronic access below)

Made available in DSpace on 2015-04-01T14:16:02Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1597259 bytes, checksum: eaba4f2d4108bede1b543c3806717952 (MD5) Previous issue date: 2013-05-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bacteria play a fundamental role in the health of corals. The interest in the study of microorganisms associated with corals has increased since the confirmation that they can be pathogenic or mutualistic. In the coastal reefs of the State of Paraiba the cases of the pigmentation changes of scleractinian Siderastrea stellata, that probably occurs in the process of coral bleaching, are observed. The aim of this work was to analyze the density and diversity of culturable bacteria associated with healthy and with pattern pigmentation altered (pink) colonies of coral S. stellata of coral reefs of Cabo Branco, João Pessoa - PB, as well as the physico-chemical and microbiological parameters of seawater in the study area over one year. Among the environmental variables (temperature, salinity, pH, dissolved oxygen, turbidity) of the reefs and beach water of Cabo Branco, only turbidity showed higher differences among the sites studied. The thermotolerant fecal coliforms, enterococci and Escherichia coli of seawater at the study sites were within the limits recommended for saline water class I (CONAMA 274/00). In general, the values of density of total heterotrophic bacteria and Vibrio spp. were significantly higher in the seawater during the months of December, January and February. According to the results of the partial sequencing of the 16S rRNA gene was found that bacteria isolated from healthy and pink S. stellata belonged to the classes Alpha-Proteobacteria and Gamma-Proteobacteria, and the variety of genera of bacteria were very different among the isolates from the two colonies. The high percentage of Vibrio spp., bacteria that are usually related to the diseases of corals, were observed among the isolates from pink colony. / Bactérias desempenham um papel fundamental na saúde dos corais. Devido à confirmação de que elas podem ser patogênicas ou mutualistas, aumentou o interesse no estudo de microrganismos associados aos corais. Nos recifes costeiros do Estado da Paraíba observam-se casos de alteração de pigmentação no escleractíneo Siderastrea stellata, que provavelmente ocorre no processo de branqueamento de corais. Neste trabalho objetivou-se analisar a quantidade e diversidade de bactérias cultiváveis associadas ao coral S. stellata sadio e com coloração alterada (roxo) dos recifes de corais de Cabo Branco, João Pessoa PB, bem como os parâmetros físico-químicos e microbiológicos de água do mar da área estudada durante um ano. Entre as variáveis ambientais (temperatura, salinidade, pH, oxigênio dissolvido, turbidez) da água dos recifes e da praia de Cabo Branco apenas a turbidez apresentou maiores diferenças entre os locais estudados. Na base das análises de coliformes termotolerantes, Escherichia coli e enterococos foi constatado que a água dos locais analisados se enquadra dentro dos parâmetros para águas salinas de classe I (CONAMA 274/00). Em geral, os valores da densidade de bactérias totais e Vibrio spp. foram significativamente maiores em água do mar nos meses de dezembro, janeiro e fevereiro. Na base de dados de sequenciamento parcial do gene RNAr 16S foi constatado que as bactérias isoladas de S. stellata sadia e roxa pertenceram ás classes de Alfa-proteobactéria e Gama-proteobactéria, sendo que a variedade dos gêneros de bactérias foi bastante distinta entre os isolados das duas colônias. Os isolados da colônia roxa apresentaram um alto percentual de Vibrio spp., que são bactérias geralmente relacionadas com as doenças de corais.