O glioblastoma multiforme, tumor de alta malignidade é conhecido por ser um câncer de difícil cura devido sua resistência ao tratamento de quimioterapia. Neste contexto sabe-se a existência de um gene responsável pelo fenótipo de resistência (MDR) e produção de proteínas associadas à resistência a uma grande variedade de drogas (MRPs). As proteínas de resistência a múltiplas drogas funcionam como bombas de efluxo, capazes de retirar das células compostos citotóxicos, deixando o tratamento quimioterápico sem o efeito esperado. Atualmente a suposta relação entre as ciclooxigenases e as proteínas de resistência a múltiplas drogas tem sido estudada em alguns tipos de câncer e na maioria deles observa-se uma relação positiva no sentido da COX poder participar da super-expressão de proteínas de resistência, fazendo com que as células fiquem ainda mais resistentes ao tratamento. O objetivo do projeto é analisar o possível efeito das COX 1 e 2 na expressão e atividade de MDRs e MRPs. / Glioblastoma multiforme, a highly malignant tumor is difficult to cure because of its resistance to chemotherapy treatment. A well-established cause of multidrug resistance (MDR) involves the increased expression of members of the ATP binding cassette (ABC) transporter superfamily, many of which transport chemotherapeutic compounds from cells. Multidrug resistance proteins can act as efflux pumps allowing. The cells to remove cytotoxic compounds and often leaving the chemotherapy treatment without the expected effect. The possible relationship between cyclooxygenase and MDRPs has been studied in several cancers and in most there is a positive relationship. The role of cyclooxygenases (COX) has been extensively studied, especially COX-2, which is expressed in many human cancers. Recently studies have been performed to identify whether the presence of COXs has some involvement with the expression of MDRPs. A positive correlation between COXs and MDRPs has been identified in certain cancers. To analyze the possible relationship between COX 1 and 2 and the expression and activity of MDRs and MRPs in gliomas. The following family members of ABC transporters were studied: MDR1, MRP1, MRP2, MRP3, MRP4 e MRP5. Our analysis show e da constitutive expression of Cox-2 in U138MG and U251 cells, as well as the expression of all the MDRPs studied (MDR1 and MRPs1-6). However, in the U138MG cell line where COX-1 was not is expressed there was a large decrease in the expression of MDR1 in comparison with the COX 1 positive U251 cell line.

O aumento da resistência do parasita da malária a maioria da drogas antimaláricas disponíveis, tornandose necessário pesquisar novos compostos com potencial atividade antimalárica. O objetivo desta tese foi inicialmente caracterizar a atividade do risedronato contra as formas intraeritrocitárias do parasita in vivo, além de identificar seu possível mecanismo de ação. A IC50 do risedronato foi de 20 <font face=\"Symbol\">mM em culturas de Plasmodium falciparum. Risedronato reduziu a biossíntese de FOH e GGOH e a isoprenilação de proteínas, inibindo a transferência do grupo FPP para as proteínas farnesiladas, entretanto, a transferência do GGPP para as proteínas geranilgeraniladas não foi inibida, isto também ocorreu quando proteínas ras e rab foram analisadas, sugerindo que a droga está inibindo a enzima FPPS. A enzima FPPS de P. falciparum foi expressa e obtivemos uma proteína recombinante fusionada a GST (rPfFPPS). Os substratos IPP, DMAPP, GPP e FPP foram utilizados para determinação da atividade catalítica da enzima, demonstrando FPP e GGPP como principais produtos. Os valores de Km para os diferentes substratos foi determinado. Demonstramos também que rPfFPPS é inibida por risedronato, podendo ser explorado como potencial alvo antimalárico. / The increased resistance of the malaria parasite most of antimalarial drugs are available, making it necessary to search for new compounds with potential antimalarial activity. The aim of this thesis was initially characterize the activity of risedronate against intraerythrocytic forms of the parasite in vivo, and identify its possible mechanism of action. The IC50 of risedronate was 20 <font face=\"Symbol\">mM in cultures of Plasmodium falciparum. Risedronate reduced biosynthesis and FOH, GGOH and protein isoprenylation, inhibiting the transfer of FPP group for farnesylated proteins, however, the transfer of GGPP to geranygeranylated proteins was not inhibited, this also occurred when ras and rab proteins were analyzed, suggesting that the drug is inhibiting the enzyme FPPS. The FPPS enzyme from P. falciparum was expressed and obtained a recombinant protein fused to GST (rPfFPPS). The substrates IPP, DMAPP, GPP and FPP were used to determine the catalytic activity of the enzyme, demonstrating FPP and GGPP as main products. The Km values for the various substrates were determined. We also demonstrate that rPfFPPS is inhibited by risedronate, which can be exploited as potential antimalarial target.

O glioblastoma multiforme, tumor de alta malignidade é conhecido por ser um câncer de difícil cura devido sua resistência ao tratamento de quimioterapia. Neste contexto sabe-se a existência de um gene responsável pelo fenótipo de resistência (MDR) e produção de proteínas associadas à resistência a uma grande variedade de drogas (MRPs). As proteínas de resistência a múltiplas drogas funcionam como bombas de efluxo, capazes de retirar das células compostos citotóxicos, deixando o tratamento quimioterápico sem o efeito esperado. Atualmente a suposta relação entre as ciclooxigenases e as proteínas de resistência a múltiplas drogas tem sido estudada em alguns tipos de câncer e na maioria deles observa-se uma relação positiva no sentido da COX poder participar da super-expressão de proteínas de resistência, fazendo com que as células fiquem ainda mais resistentes ao tratamento. O objetivo do projeto é analisar o possível efeito das COX 1 e 2 na expressão e atividade de MDRs e MRPs. / Glioblastoma multiforme, a highly malignant tumor is difficult to cure because of its resistance to chemotherapy treatment. A well-established cause of multidrug resistance (MDR) involves the increased expression of members of the ATP binding cassette (ABC) transporter superfamily, many of which transport chemotherapeutic compounds from cells. Multidrug resistance proteins can act as efflux pumps allowing. The cells to remove cytotoxic compounds and often leaving the chemotherapy treatment without the expected effect. The possible relationship between cyclooxygenase and MDRPs has been studied in several cancers and in most there is a positive relationship. The role of cyclooxygenases (COX) has been extensively studied, especially COX-2, which is expressed in many human cancers. Recently studies have been performed to identify whether the presence of COXs has some involvement with the expression of MDRPs. A positive correlation between COXs and MDRPs has been identified in certain cancers. To analyze the possible relationship between COX 1 and 2 and the expression and activity of MDRs and MRPs in gliomas. The following family members of ABC transporters were studied: MDR1, MRP1, MRP2, MRP3, MRP4 e MRP5. Our analysis show e da constitutive expression of Cox-2 in U138MG and U251 cells, as well as the expression of all the MDRPs studied (MDR1 and MRPs1-6). However, in the U138MG cell line where COX-1 was not is expressed there was a large decrease in the expression of MDR1 in comparison with the COX 1 positive U251 cell line.

Orientador: Ana Julia Fernandes Cardoso Oliveira / Resumo: A ocupação desordenada de áreas costeiras e os efeitos das ações antropogênicas tem causado diferentes impactos aos ambientes costeiros, com o aumento da descarga de efluentes domésticos como resultado do aumento da população local e flutuante. Para o monitoramento da qualidade da água destes ambientes é utilizado o padrão microbiológico definido pelas resoluções Conama 274/2000 e 357/2005, com as densidades de Escherichia coli e Enterococcus spp. Os efeitos do uso de antibióticos e os mecanismos de resistência microbiana ao longo dos anos são preocupações de saúde pública quanto ao risco de disseminação desta resistência além dos hospitais, sendo reportada no ambiente. Este trabalho teve como objetivo avaliar a qualidade microbiológica da água e dos sedimentos da Bacia Hidrográfica do Rio Itanhaém, isolar bactérias do gênero Staphylococcus spp e avaliar o perfil de resistência aos antibióticos. Foram selecionados seis pontos de coleta de amostras e identificados três espécies sendo S. aureus, S. saprophyticus e S. schleiferi, evidenciando-se a resistência a diferentes antibióticos, dentre os quais a vancomicina, utilizada em ambiente hospitalar. Esta resistência foi observada nos isolados da água, no ponto de coleta onde ocorre o lançamento do efluente da ETE, e no sedimento na área utilizada por banhistas. / Abstract: The disordered occupation of coastal areas and the effects of anthropogenic actions have caused different impacts to coastal environments with the increase of the discharge of domestic effluents as a result of the increase of the local population transitory. For the monitoring of the water quality of these environments the microbiological standard defined by the resolutions Conama 274/2000 and 357/2005, with the densities of Escherichia coli and Enterococcus spp. The effects of antibiotic use and the mechanisms of microbial resistance over the years are public health worry about the risk of dissemination of resistance beyond hospitals, being reported in the environment. The objective of this work was to evaluate the microbiological quality of water and sediments of the Itanhaém river hydrographic basin, isolate bacteria of the genus Staphylococcus spp and evaluate the antibiotic resistance profile. Six sample collection points were selected and identified three species being S. aureus, S. saprophyticus e S.schleiferi, evidencing the resistance to different antibiotics, in between the vancomycin, used in hospital. This resistance was observed in water isolates, at the collection point where the posting takes place of the ETE effluent, and sediment in the area used by bathers. / Mestre

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Surface water used for irrigation is often highly contaminated on a microbial level. Using contaminated surface water for the irrigation of fresh produce can lead to foodborne disease outbreaks and Escherichia coli has been a major cause of foodborne outbreaks associated with fresh produce over the past few years. There are many possible on-farm treatment options available to decrease the high microbial loads present in surface water, one of these is H2O2 and various factors can influence its use. The aim of this study was to determine the efficacy of H2O2 on different E. coli strains. Water from the Plankenburg River was sampled and treated with (250, 300 and 350 mg.L-1) H2O2 and the impact at 0, 30, 60, 90 and 120 min was then evaluated. It was found that the log reductions differed between samples. Log reductions ranged between 1.60 – 2.63 for Aerobic colony counts (ACC), total coliforms and Escherichia coli. The water was not considered safe for irrigation use although it had been treated with H2O2. Reference (ATCC) and environmental E. coli strains were individually treated with H2O2 (250, 300 and 350 mg.L-1) at 0, 30, 60, 90 and 120 min. Log reductions for the ATCC strains ranged between 2.13 – 5.48. This indicated a variation in H2O2 resistance between the different reference strains tested. Log reductions for the environmental E. coli strains ranged between 2.17 – 3.93. Escherichia coli M53 and MJ56 were the most resistant and most sensitive environmental strains to the H2O2 treatment, respectively. Once again it was observed that variations existed between the log reductions achieved for different strains. Overall, it was observed that the ATCC E. coli strains were more sensitive to the H2O2 treatments when compared the environmental strains. This indicates that ATCC strains should not be used for H2O2 treatment optimisation. Certain factors can influence the efficacy of H2O2 such as concentration and organic matter (chemical oxygen demand) present in the water. Different H2O2 concentrations were evaluated (50, 350, 700 and 1 000 mg.L-1) on two E. coli strains (M53 and W1371). Results indicated that 50 mg.L-1 was not effective as less than 1 log reduction was achieved after 120 min. When 350 and 700 mg.L-1 were used similar log reductions were achieved (1.78 – 2.27), which was not expected. Using 1 000 mg.L-1 was considered an effective concentration that resulted in no growth present after 120 min. Escherichia coli strain W1371 carried EPEC virulence factors (potential pathogen). This was included in the study in order to determine how a strain carrying virulence factors would react to H2O2. Escherichia coli W1371 was considered resistant to the H2O2 treatment and log reductions were similar to that achieved for M53. The catalase activity of the E. coli strains was studied to determine if a link existed between catalase activity and H2O2 resistance. Although a trend was observed between heat-stable catalase activity and H2O2 resistance, there were exceptions. It was concluded that high catalase activity does not always coincide with H2O2 resistance and that other mechanisms might also contribute to E. coli survival. Overall, it was observed that there are certain factors that influence the efficacy of H2O2 as a treatment option. It can be concluded that environmental E. coli strains are generally more resistant to the H2O2 treatment compared to ATCC E. coli strains, this needs to be considered when using H2O2 or other chemical disinfectants as a treatment option. / AFRIKAANSE OPSOMMING: Oppervlakwater wat gebruik word vir besproeiing is dikwels op ‘n mikrobiese vlak hoogs gekontamineer. Die gebruik van oppervlakwater vir die besproeiing van vars produkte kan tot die uitbraak van voedselgedraagde siektes lei. Escherichia coli was een van die hoofoorsake van voedselgedraagde uitbrake geassosieerd met vars produkte gedurende die laaste paar jaar. Daar is verskeie moontlike behandelingsmetodes op plaasvlak beskikbaar om die hoë mikrobiese las in oppervlakwater te verlaag. Een hiervan is waterstofperoksied (H2O2) en verskeie faktore kan die gebruik hiervan beïnvloed. Die doel van hierdie studie was om die doeltreffendheid van H2O2 op verskillende E. coli isolate te bepaal. Watermonsters uit die Plankenburg Rivier is behandel met drie konsentrasies H2O2 (250, 300 en 350 mg.L-1) en die impak is na 0, 30, 60, 90 en 120 minute geëvalueer. Daar is gevind dat die log reduksies tussen monsters verskil het. Log reduksies het gewissel tussen 1.60 en 2.63 vir aerobiese kolonietellings (AKT), totale kolivorme en E. coli. Selfs na H2O2 behandeling, is die water nie as veilig vir besproeiing beskou nie. Verwysingsisolate (ATCC) en omgewingsisolate van E. coli is afsonderlik met H2O2 behandel (250, 300 en 350 mg.L-1) vir 0, 30, 60, 90 en 120 minute. Log reduksies vir die ATCC isolate het gewissel tussen 2.13 en 5.48. Hierdie verskille dui op die variasies wat tussen die getoetste verwysingsisolate voorkom. Log reduksies vir die omgewingsisolate het gewissel tussen 2.17 en 3.93. Escherichia coli M53 en MJ56 was onderskeidelik die mees weerstandbiedende en mees sensitiewe verwysingsisolate wat getoets is. Verskille in log reduksies het daarop gedui dat isolaat variasies voorkom. In geheel is dit gevind dat die ATCC E. coli isolate meer sensitief was vir die H2O2 behandelings vergeleke met die omgewingsisolate. Dit toon dat die ATCC isolate nie gebruik moet word vir H2O2 behandeling optimering nie. Sekere faktore, soos die konsentrasie en organiese materiaal (chemiese suurstof vereiste) in die water, kan die doeltreffendheid van H2O2 behandeling beïnvloed. Verskillende H2O2 konsentrasies is geëvalueer (50, 350, 700 en 1000 mg.L-1) op twee E. coli isolate (M53 en W1371). Resultate dui daarop dat 50 mg.L-1 nie effektief was nie omdat minder as 1 log reduksie behaal is na 120 minute. Toe 350 en 700 mg.L-1 gebruik is, is soortgelyke log reduksies (1.78 – 2.27) teen verwagting in behaal. Die gebruik van 1000 mg.L-1 is as ‘n effektiewe behandeling beskou aangesien daar geen groei na 120 minute teenwoordig was nie. Escherichia coli isolaat W1371 besit EPEC virulensie faktore (potensiële patogeen). Dit is in die studie ingesluit ten einde te bepaal hoe ‘n isolaat met virulensie faktore sou reageer op H2O2. Escherichia coli W1371 is as weerstandbiedend teen die H2O2 behandeling beskou en log reduksies was soortgelyk aan die van M53 . Die katalase aktiwiteit van die E. coli isolate is bestudeer om te bepaal of ʼn skakel bestaan tussen katalase aktiwiteit en H2O2 weerstandbiedendheid. Alhoewel ‘n tendens waargeneem is tussen hitte-stabiele katalase aktiwiteit en H2O2 weerstandbiedendheid, was daar uitsonderings. Die gevolgtrekking was dat hoë katalase aktiwiteit nie altyd saamval met H2O2 weerstandbiedendheid nie en dat ander meganismes ook mag bydra tot E. coli oorlewing. In geheel is dit waargeneem dat daar sekere faktore is wat die doeltreffendheid van H2O2 as ‘n behandelingsmetode beïnvloed. Daar is gevind dat omgewingsisolate van E. coli in die algemeen meer weerstandbiedend is teenoor H2O2 behandeling in vergelyking met ATCC E. coli isolate. Dit moet in ag geneem word wanneer H2O2 of ander chemiese ontsmettingsmiddels oorweeg word as ʼn behandelingsopsie.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Biofilms are biological communities with a high degree of organization in which microorganisms are adhered to a surface. Microorganisms, when in biofilm, become a target of concern in the clinical field due to the low response to antimicrobial treatments and ease of colonization of surfaces such as implants, catheters and surgical instruments. Several studies have shown that antimicrobial and biocides have their effectiveness decreased against biofilms. Chlorhexidine is a powerful antiseptic widely used in hospitals especially applied in hand antisepsis, disinfection of environments, and sterilization of surgical instruments used in invasive procedures. Thus, the present study aimed to determine whether bacterial and fungal biofilms are able to resist the antimicrobial action of chlorhexidine. Disk diffusion and susceptibility tests were performed according to Clinical Laboratory Standards Institute (CLSI), 2013. To determine the Biofilm Inhibition Concentration (BIC), chlorhexidine was tested at concentrations of Minimum Inhibitory Concentration (MIC) and at higher concentrations when necessary. The plates were developed with a solution of 0.1% crystal violet and the optical density (OD) was obtained at 570 nm. Results showed that chlorhexidine has excellent antimicrobial activity against most organisms tested in its free form; however, it was less effective against biofilms of Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus resistant to Methicillin (MRSA) and Pseudomonas aeruginosa, developed in isolation for each species. Thus, chlorhexidine is likely to have its antimicrobial activity decreased when exposed to microorganisms in biofilms. This probably occurs due to resistance mechanisms attributed to the biofilm structure exopolysaccharide matrix, quorum sensing (QS), genetic diversity and to the inappropriate use of this biocide. / Biofilmes são comunidades biológicas com elevado grau de organização, onde os microrganismos se encontram aderidos a uma superfície. Os microrganismos, quando em biofilme, tornam-se alvo de preocupação na área clínica devido à baixa resposta aos tratamentos antimicrobianos e à facilidade de colonização de superfícies como próteses, cateteres e instrumentos cirúrgicos. Vários estudos demonstram que os antimicrobianos e biocidas têm sua eficácia diminuída frente aos biofilmes. A clorexidina é um poderoso antisséptico largamente empregado no ambiente hospitalar, aplicado especialmente na antissepsia de mãos, desinfecção de ambientes cirúrgicos e esterilização de instrumentos utilizados em procedimentos invasivos. Deste modo, o presente trabalho teve como objetivo verificar se biofilmes bacterianos e fúngico são capazes de resistir à atuação antimicrobiana da clorexidina. Testes de disco-difusão e de suscetibilidade foram conduzidos de acordo com CLSI (Clinical Laboratory Standards Institute), 2013. Para a determinação da CIB (Concentração de Inibição de Biofilme), a clorexidina foi testada nas concentrações da CIM (Concentração Inibitória Mínima) e em concentrações maiores, quando necessário. As placas foram reveladas com solução de 0.1% de Cristal Violeta e a densidade óptica (D.O.) obtida em 570nm. Os resultados demostraram que a clorexidina possui uma excelente atividade antimicrobiana para a maioria dos microrganismos testados em suas formas livres, porém, mostrou-se menos eficaz contra os biofilmes de Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus resistentes a Meticilina (MRSA) e Pseudomonas aeruginosa, desenvolvidos de forma isolada para cada espécie. Assim, sugere-se que a clorexidina apresenta sua atividade antimicrobiana diminuída quando exposta a microrganismos em biofilme. Provavelmente isso ocorra devido aos mecanismos de resistência atribuídos à estrutura do biofilme - matriz exopolissacarídica, quorum sensing (QS), diversidade genética - e também ao uso inadequado deste biocida.

O aumento da resistência do parasita da malária a maioria da drogas antimaláricas disponíveis, tornandose necessário pesquisar novos compostos com potencial atividade antimalárica. O objetivo desta tese foi inicialmente caracterizar a atividade do risedronato contra as formas intraeritrocitárias do parasita in vivo, além de identificar seu possível mecanismo de ação. A IC50 do risedronato foi de 20 <font face=\"Symbol\">mM em culturas de Plasmodium falciparum. Risedronato reduziu a biossíntese de FOH e GGOH e a isoprenilação de proteínas, inibindo a transferência do grupo FPP para as proteínas farnesiladas, entretanto, a transferência do GGPP para as proteínas geranilgeraniladas não foi inibida, isto também ocorreu quando proteínas ras e rab foram analisadas, sugerindo que a droga está inibindo a enzima FPPS. A enzima FPPS de P. falciparum foi expressa e obtivemos uma proteína recombinante fusionada a GST (rPfFPPS). Os substratos IPP, DMAPP, GPP e FPP foram utilizados para determinação da atividade catalítica da enzima, demonstrando FPP e GGPP como principais produtos. Os valores de Km para os diferentes substratos foi determinado. Demonstramos também que rPfFPPS é inibida por risedronato, podendo ser explorado como potencial alvo antimalárico. / The increased resistance of the malaria parasite most of antimalarial drugs are available, making it necessary to search for new compounds with potential antimalarial activity. The aim of this thesis was initially characterize the activity of risedronate against intraerythrocytic forms of the parasite in vivo, and identify its possible mechanism of action. The IC50 of risedronate was 20 <font face=\"Symbol\">mM in cultures of Plasmodium falciparum. Risedronate reduced biosynthesis and FOH, GGOH and protein isoprenylation, inhibiting the transfer of FPP group for farnesylated proteins, however, the transfer of GGPP to geranygeranylated proteins was not inhibited, this also occurred when ras and rab proteins were analyzed, suggesting that the drug is inhibiting the enzyme FPPS. The FPPS enzyme from P. falciparum was expressed and obtained a recombinant protein fused to GST (rPfFPPS). The substrates IPP, DMAPP, GPP and FPP were used to determine the catalytic activity of the enzyme, demonstrating FPP and GGPP as main products. The Km values for the various substrates were determined. We also demonstrate that rPfFPPS is inhibited by risedronate, which can be exploited as potential antimalarial target.

Rhipicephalus (Boophilus) microplus é o parasito de maior importância econômica para a produção bovina. Há suspeita de resistência disseminada a ivermectina (IVM), droga amplamente utilizada para seu controle e seu diagnóstico preciso se faz necessário. Neste trabalho foram padronizados testes diagnósticos in vitro que, ao serem aplicados a campo no Brasil, diagnosticaram a resistência em 18 de 30 populações testadas. A resistência in vitro foi confirmada por teste in vivo. Testes com sinergistas sobre cepa resistente isolada indicaram que a destoxificação enzimática tem papel secundário na resistência. Não foram encontradas mutações associadas à resistência no trecho analisado do gene GluCl. Informações obtidas sobre evolução da resistência a campo e em laboratório poderão ser úteis para o uso de IVM no controle de R. (B.) microplus. Os estudos conduzidos sobre mecanismos de resistência podem servir para o desenvolvimento de marcadores moleculares diagnósticos de resistência a IVM. / Rhipicephalus (Boophilus) microplus is the most economically important parasite for cattle production. There is suspicion of widespread resistance to ivermectin (IVM), a drug widely used for their control and being necessary its accurate diagnosis. In this study were standardized in vitro diagnostic tests that, when applied to the field in Brazil, diagnosed resistance in 18 of 30 populations tested. The in vitro resistance was confirmed by a field trial. Tests with synergists on an isolated resistant strain indicated that enzyme detoxification has a secondary role in resistance. There were no mutations associated with resistance in the analyzed fragment of the gene GluCl. Information obtained about the evolution of resistance in field and laboratory may be useful for the use of IVM in the control of R. (B.) microplus. The conducted studies on resistance mechanisms may serve for the development of diagnostic molecular markers of resistance to IVM.

Pseudomonas aeruginosa é um dos principais agentes de infecção hospitalar que tem adquirido um caráter endêmico decorrente a sua resistência intrínseca e/ou adquirida aos antibacterianos comercialmente disponíveis. O objetivo do estudo foi avaliar, in vitro, opções terapêuticas baseadas na atividade sinérgica. Dez cepas de P. aeruginosa clonalmente não relacionadas, previamente caracterizadas como produtoras de metalo-beta-lactamase (M<font face=\"Symbol\">bL) do tipo SPM-1, VIM-1 e PA GIM-1, foram avaliadas. O efeito sinérgico foi investigado por Checkerboard e Time-Kill. As combinações [Piperacilina/Tazobactam x Aztreonam] e [Tigeciclina x nanofragmentos de bicamada de brometo de dioctadecildimetilamônio (DDA)] mostraram atividade sinérgica para 90 e 100% das cepas, respectivamente. Os resultados respaldam o uso terapêutico combinado de [Piperacilina/Tazobactam x Aztreonam], contra infecções produzidas por cepas de P. aeruginosa multirresistentes produtoras de M<font face=\"Symbol\">bLs, além disso, [DDA/Tigeciclina] pode constituir a base para consolidar uma nova forma farmacêutica de uso clínico. / Pseudomonas aeruginosa is a leading cause of nosocomial infections, which it has acquired an endemic status due to their intrinsic or acquired resistance to antibacterial agents commercially available. The aim of the study was to evaluate in vitro therapeutic options based on the synergistic activity. Ten clonally unrelated strains of P. aeruginosa, previously characterized as metallo-beta-lactamase (M<font face=\"Symbol\">bL) producers (i.e., SPM-1, VIM-1 and PA GIM-1) were evaluated. The synergistic effect was investigated by checkerboard and Time-Kill assays. The combinations [Piperacillin/Tazobactam x Aztreonam] and [Tigecycline x bilayer fragments of dioctadecyldimethylammonium bromide (DDA)] showed synergistic activity for 90 and 100% of the strains, respectively. The results support the use of combined therapy by using [Piperacillin/Tazobactam x Aztreonam] against infections produced by strains of P. aeruginosa producing M<font face=\"Symbol\">bLs, moreover, [DDA / Tigecycline] may be the basis for build a new pharmaceutical form for clinical use.

As populações de aves antárticas têm sido estudadas e consideradas indicadoras da qualidade do ecossistema marinho, especialmente dos oceanos do sul, ao longo dos últimos 50 anos. Existem cerca de 40 espécies de aves marinhas que se reproduzem em áreas descobertas de gelo. Dentre as aves marinhas antárticas, os pinguins são os que têm a maior representatividade ecológica e são considerados espécies sentinelas para o estudo das mudanças ambientais nesse continente. Esses animais representam 90% da biomassa de aves nos oceanos do sul, e suas colônias estão distribuídas nas ilhas antárticas e subantárticas bem como sobre o Continente Antártico. Há três espécies de pigoscelídeos mais representativos desta biomassa, são eles: Pygoscelis papua (Pinguim gentoo), Pygoscelis adeliae (Pinguim-de-adélia) e Pygoscelis antarcticus (Pinguim-de-barbicha). Os pinguins antárticos estão entre as aves de menor contato com humanos, o que os torna possíveis indicadores da presença natural de genes de resistência a antimicrobianos na microbiota intestinal de aves e no ambiente. O objetivo desta dissertação foi avaliar a presença de resistência a antimicrobianos na microbiota intestinal de P. antarcticus e de P. papua, e compará-las à microbiota de Spheniscus magellanicus (pinguins-de-magalhães). Os S. magellanicus habitam a Argentina, Chile e Ilhas Malvinas, locais em que há variadas atividades humanas. Foram coletadas amostras de fezes aparentemente frescas de 46 pinguins P.antarcticus e de 12 pinguins P. papua, nas suas respectivas colônias na Ilha Elefante, em dezembro de 2014. De S. magellanicus foram coletadas, com auxílio de suabes cloacais, amostras de 19 indivíduos, encontrados na costa norte do Rio Grande do Sul, de Quintão a Torres, durante os meses de inverno de 2015 e de 2016. As amostras de microbiota dos pinguins foram cultivadas em ágar LB e os isolados bacterianos foram triados para os seguintes antimicrobianos: eritromicina (≥ 8μg/mL), estreptomicina (≥ 2.000 μg/mL), tetraciclina (≥ 16 μg/mL) e vancomicina (≥ 32 μg/mL). Em 10 amostras de P. antarcticus e em 15 amostras de S. magellanicus foram identificadas bactérias resistentes a pelo menos um dos antimicrobianos testados. Todas as amostras de P. papua foram sensíveis a esses antimicrobianos. As espécies dos isolados resistentes foram identificadas pelo sequenciamento do rRNA 16S, que revelou sete gêneros, sendo os mais recorrentes Enterococcus sp. e Staphylococcus sp. Esses isolados resistentes também foram triados para a presença de genes de resistência aos antimicrobianos. O tet(M) foi mais abundante em S. magellanicus (5) do que em P. antarcticus (3), ao passo que o int e van(B) foram identificados somente em P. antarcticus (três e um, respectivamente). O gene erm(B) não foi encontrado em nenhum dos isolados. Uma vez que a fração não cultivável das fezes também pode apresentar genes de resistência, foi realizada a extração do DNA das fezes de pinguins antárticos para obtermos DNA de todos os micro-organismos presentes. Os genes mais recorrentes nas amostras de DNA total das fezes de P. antarcticus e P. papua foram, respectivamente, int (5 e 7), seguido de tet(M) (1 e 5). O van(B) foi encontrado em amostras das duas espécies de pinguins, enquanto que o erm(B) foi encontrado somente nas amostras de P. papua. De acordo com esses resultados, houve mais resistência a antimicrobianos na fração cultivável da microbiota de pinguins-de-magalhães do que em pinguins antárticos. Na fração não-cultivável, foram encontrados mais genes de resistência nas amostras de P. papua do que de P. antarcticus. / Antarctic seabird populations have been studied as bioindicators of the nature variability in the Southern Ocean marine ecosystems over the last 50 years. Among the Antarctic seabirds, the most representative species are penguins; they represent 90% of total biomass of birds in the Southern Ocean, and are considered sentinels for environmental changes in the Antarctic region. Pygoscelis antarcticus and P. papua are the most prevalent species in Antarctida. Because they remain among the wild birds with least contact with humans, their microbiota may serve as indicators of antimicrobial resistance in the environment. The aim of this work was to evaluate the antimicrobial resistance present in the microbiota of P. antarcticus and P. papua, and compare it with the microbiota of Spheniscus magellanicus (Magellanicus penguins). Magellanicus penguins inhabit Argentina, Chile and Falkland Islands, and therefore have more contact with humans. We have collected samples of apparently fresh feces from P. antarcticus (n = 46) and from P. papua (n = 12) in their respective colonies located in the Elephant Island in December 2014. From S. magellanicus, we have collected cloacal swabs (n = 19) from specimens found in the northern coast of Rio Grande do Sul, from Quintão to Torres, during the winter months of 2015 and 2016. All samples were evaluated for the presence of resistant bacteria to the following antimicrobials: erythromycin (≥ 8μg/mL), streptomycin (≥ 2.000 μg/mL), tetracycline ( ≥ 16 μg/mL) and vancomycin (32 μg/mL). We have isolated resistant bacteria from 10 samples of P. antarcticus and from 15 samples of S. magellanicus; there was no bacterial growth in the presence of any of these antimicrobials from samples of P. papua feces. The species of resistant bacteria were identified by 16S rRNA sequencing: among the 7 genera identified, the most frequent were Enterococcus sp. and Staphylococcus sp. Resistant bacteria were screened for the resistance genes ermB, tet(M), int and van(B). tet(M) was more frequent in S. magellanicus (5) than in P. antarcticus (3), while int and van(B) were identified only in P. antarcticus (3 and 1, respectively). The erm(B) gene was not identified in any isolate. Considering that the non-cultivable fraction from feces can also harbor resistance genes, we extracted DNA from feces of the Antarctic penguins in a attempt to obtain DNA from all micro-organisms of their microbiota. The most abundant genes present in the microbiota of P. antarcticus and P. papua were, respectively: int (5 and 7) and tet (M) (1 and 5). The van(B) gene was found in one sample of each species, while erm(B) was found in only one sample of P. papua. According to our results, antimicrobial resistance is more frequent in the cultivable microbiota of S. magellanicus than of P. antarcticus. In the non-cultivable fraction, resistance genes were more frequent in samples from P. papua than from P. antarcticus.