Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-07-29T13:36:35Z No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) / Made available in DSpace on 2016-07-29T13:36:35Z (GMT). No. of bitstreams: 1 2011 - Bruno Rocha Pribul.pdf: 1463809 bytes, checksum: ba7b3b0f00c0b4469b45fe02ec9f8a7f (MD5) Previous issue date: 2011-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / PRIBUL, Bruno Rocha. Evaluation of Vancomycin Resistance and Susceptibility Profile Bacteriocins of staphylococci isolated from bovine mastitis in Southern State of Rio de Janeiro. 41p Thesis (Master of Veterinary Science). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Bacteria of the genus Staphylococcus are among the most implicated in the etiology of mastitis. An additional difficulty in controlling bacterial infections by this genus is represented by its frequent resistance to antibiotics. The glycopeptides (eg vancomycin) appear as an alternative therapy because of the increasing strains of staphylococci resistant to beta-lactams, which are the drugs of choice in these infections. Thus, glycopeptide resistance represents a threat to the future of antimicrobial therapy in humans and animals. This study aimed to evaluate the profile of vancomycin resistance pheno-genotypically in Staphylococcus spp. milk samples from cows with mastitis. All 150 isolates (50 isolates of Staphylococcus aureus, 50 isolates of Staphylococcus intermedius and 50 isolates of Coagulase-negative Staphylococcus spp. (CNS) were evaluated using the tests of disk diffusion, agar microdilution and detection of vanA e vanB genes. The antimicrobial susceptibility test showed high resistance to beta-lactam antibiotics for all groups. In the disk diffusion test, 24% of coagulase-positive isolates showed resistance to teicoplanin (24/100) and 23% to vancomycin (23/100). In the agar microdilution test for detection of CIM, 16% of the isolates were resistant to vancomycin (16/100). Among the resistant isolates in the agar microdilution testing, 56.25% of the isolates (9 / 16) had MIC values ranging from 4-8?g/ml. Staphylococcus coagulase-negative showed no resistance to the evaluated glycopeptides. Genes for resistance to vancomycin were detected in 37.5% of vancomycin-resistant isolates (6 / 16). To seek a correlation between the presence of glycopeptide resistance and the ability of producing biofilm, tests were performed to characterize growth in Congo red agar and also detection of the biofilm adherence test in microplates. It were detected 66.66% and 90, 00%, respectively, of isolates producing biofilm. The production of "slime" presented no statistical correlation with resistance to glycopeptides. The sensitivity profile of Staphylococcus spp. was assessed against the bacteriocins produced by Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus fermentum. Lactobacillus paracasei bacteriocins presented the highest inhibition capacity. / PRIBUL, Bruno Rocha. Avalia??o da Resist?ncia a Vancomicina e do Perfil de Suscetibilidade a Bacteriocinas de Staphylococcus spp Isolados de Mastite Bovina da Regi?o Sul do Estado do Rio de Janeiro. 41p Disserta??o (Mestrado em Ci?ncias Veterin?rias). Instituto de Veterin?ria, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. As bact?rias do g?nero Staphylococcus est?o entre as mais implicadas na etiologia das mastites. Uma dificuldade adicional no controle das infec??es bacterianas por este g?nero ? representada por sua freq?ente resist?ncia aos antimicrobianos. Os glicopept?deos, como a vancomicina, aparecem como uma alternativa terap?utica devido ao crescente aumento de cepas estafiloc?cicas resistentes aos betalact?micos, que s?o os f?rmacos de elei??o nestas infec??es. Desse modo, a resist?ncia aos glicopept?deos representa uma amea?a ao futuro da terapia antimicrobiana em humanos e animais. O presente trabalho buscou avaliar o perfil de resist?ncia ? vancomicina em isolados de Staphylococcus spp. oriundos de amostras de leite de vacas com mastite. Todos os 150 isolados estudados (50 Staphylococcus aureus, 50 Staphylococcus intermedius e 50 Staphylococcus spp. coagulase negativos), foram avaliados atrav?s dos testes de difus?o em disco simples, microdilui??o em ?gar e detec??o dos genes vanA e B. O teste de suscetibilidade antimicrobiana revelou elevada resist?ncia aos beta-lact?micos para todos os grupos avaliados. No ensaio de difus?o em disco, os isolados coagulasepositivos apresentaram um perfil de resist?ncia de 24% a teicoplanina (24/100) e 23% a vancomicina (23/100). No teste de microdilui??o em ?gar para detec??o da CIM, 16% dos isolados apresentaram resist?ncia ? vancomicina (16/100). Entre os isolados resistentes no teste de microdilui??o em ?gar, 56,25% dos isolados (9/16) apresentaram valores de CIM que variaram entre 4-8?g/ml. Os Staphylococcus coagulase-negativos n?o apresentaram resist?ncia aos glicopeptideos. Os genes de resist?ncia ? vancomicina foram detectados em 37,5% dos isolados vancomicina-resistentes (6/16). Para se buscar uma correla??o entre a presen?a de resist?ncia aos glicopeptideos e a capacidade de forma??o de biofilme foram realizados os testes de caracteriza??o de crescimento em ?gar vermelho congo e detec??o de biofilme pelo teste de ader?ncia em microplacas, onde foram detectados 66,66% e 90,00% respectivamente, de isolados produtores de biofilme. A produ??o de ?slime? n?o apresentou correla??o estat?stica com a resist?ncia aos glicopept?deos. O perfil de sensibilidade dos isolados de Staphylococcus spp. foi avaliado frente as bacteriocinas produzidas pelos Lactobacillus paracasei, Lactobacillus acidophillus e Lactobacillus fermentum. As bacteriocinas que apresentaram maior capacidade de inibi??o frente aos Saphylococcus spp. testados foram as produzidas pelos Lactobacillus paracasei

Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Bioanalytical laboratory diagnostics in health care Candidate: Bc. Martina Malíková Thesis supervisor: PharmDr. Ondřej Janďourek, Ph.D. Consultant: RNDr. Irena Hanovcová, CSc. Title of diploma thesis: Effectiveness of disinfectants for hygienic hand disinfection against clinically important strains of enterococci Alcohol-based hand disinfectants for hygienic hand disinfection are a key tool for the control of nosocomial infections worldwide. The aim of this study was to verify the effectiveness of these products, which are used in healthcare facilities, against vancomycin-sensitive and vancomycin-resistant enterococci isolated from clinical materials. Testing of the efficacy of disinfectants was performed according to the Czech technical standard ČSN EN 1040 - Chemical disinfectants and antiseptics - Quantitative test using suspension to determine the basic bactericidal effect of chemical disinfectants and antiseptics - Test method and requirements (phase 1). We used the dilution method with a neutraliser for our testing. There were used a total of 35 strains of enterococci to determine efficacy, 12 of which were vancomycin sensitive (four E. faecium strains and eight E. faecalis...

Empiric antimicrobial therapy often consists of the combination of Gram-positive coverage with vancomycin (VAN) and Gram-negative coverage, specifically an anti-pseudomonal beta-lactam, such as piperacillin-tazobactam (PTZ). Nephrotoxicity is commonly associated with VAN therapy; however, recent reports demonstrate increasing nephrotoxicity rates among patients treated with the combination of VAN and PTZ. This study evaluated the effect of the VAN/PTZ combination on acute kidney injury (AKI), as defined by the RIFLE criteria, compared to VAN and PTZ monotherapies. Overall, 11,650 patients were analyzed, with 1,647 (14.1%) AKI cases occurring. AKI was significantly more frequent in the VAN/PTZ group (21%) compared to either monotherapy group (VAN 8.3%, PTZ 7.8%, p<0.001 for both). Combination therapy was independently associated with higher AKI odds compared to monotherapy with either agent (aOR=2.03; 95% CI 1.74-2.39; aOR=2.31; 95% CI 1.97-2.71, for VAN and PTZ, respectively). Receipt of concomitant nephrotoxic drugs were independently associated with increased AKI rates, as were increased duration of therapy, length of hospital stay, increasing severity of illness, and increasing baseline renal function. VAN combined with PTZ was associated with twice the odds of AKI development compared to either agent as monotherapy. This demonstrates the need for judicious use of combination empiric therapy.

Les nouveau-nés et jeunes enfants forment une population spécifique pour laquelle les études cliniques sont rares et difficiles à mettre en œuvre. La modélisation pharmacocinétique permet de réaliser des études non invasives et est donc particulièrement bien adaptée à cette population. Tout comme pour l'expérimentation scientifique qui nécessite plusieurs études pour conduire à un consensus, plusieurs bases de données pharmacocinétiques sont nécessaires afin d'arriver à un modèle optimal généralisable. Néanmoins, de nombreux modèles pharmacocinétiques sont publiés pour une même molécule, indépendamment les uns des autres, sans que l'on sache clairement lequel est le plus adapté. Inversement de nombreuses molécules ne sont pas ou peu étudiées et ne permettent pas de conduire à des recommandations fiables.L'objectif de ce travail de thèse était de rechercher une stratégie pour privilégier un modèle par rapport à un autre et ainsi, conduire à un modèle optimal permettant d'émettre des recommandations. Nous avons choisi de centrer notre intérêt sur les études pharmacocinétiques de population en néonatalogie et plus précisément sur deux molécules: la vancomycine et le phénobarbital. / Neonates and young infants are a specific population in which clinical studies are rare and difficult. Pharmacokinetic modeling allows to realize non invasive studies and is therefore particularly well suited for this population.As well as for scientific experimentation which requires several studies to lead to a consensus, several pharmacokinetic databases are needed to achieve a generalizable optimal model.Nevertheless, many pharmacokinetic models are published for the same molecule, independently of each other, without knowing clearly which is the most suitable. Conversely many molecules are not or little studied and can not lead to reliable recommendations.The aim of this thesis was to find a strategy to prefer a model over another, and thus lead to an optimal model to make recommendations. We chose to focus our interest on population pharmacokinetic studies in neonatology and more specifically on two molecules: vancomycin and phenobarbital.

O ensaio turbidimétrico tem como princípio a redução da densidade óptica em suspensões microbianas, decorrente o incremento na concentração do antibiótico. No ensaio convencional trabalha-se com tubos de ensaio que demandam longo tempo para visualização da resposta. Assim, o método empregando microplacas com leitura cinética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizado. O objetivo deste trabalho é desenvolver o método de doseamento microbiológico de vancomicina empregando microplacas e modo de leitura cinético, assim como validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, exatidão e precisão. Adicionalmente foi contemplado desenvolvimento paralelo entre o método convencional e o cinético, de forma a permitir respaldo positivo quanto à validação. Sendo assim, através do estudo dos parâmetros de validação do método cinético, trabalhou-se em condições estabelecidas abrangendo curva-padrão de vancomicina com concentrações entre 0,6 e 1,0 &#181;g/mL, e emprego de meio de cultura caldo de caseína-soja inoculado com Bacillus subtilis (ATCC 6633) na proporção de 5%. Foram obtidos resultados satisfatórios em 5 horas de incubação. Conclui-se que o ensaio em microplacas permite avaliar, com vantagens, a atividade do antibiótico frente ao microrganismo-teste, permitindo facilidade operacional e maior rapidez na obtenção dos resultados. / The principle of the turbidimetric assay is to reduce the optical density of microbial suspensions, due to the increase in the concentration of antibiotic. In the conventional test works with tubes that require long time to display the answer. Thus, the method using microplates with kinetic reading for the dosage of antibiotics is of considerable interest, since it allows possible to reduce the amount of material and analysis time required and allows for testing of large numbers of samples simultaneously, with automated reading and calculating. The aim of this work is to develop the method of microbiological assay of vancomycin using microplates and kinetic reading mode, and to validate the method developed by evaluating the parameters of specificity and selectivity, linearity, linear range, accuracy and precision. Also included was the parallel development between the conventional method and the kinetic, to enable positive support for the validation. Thus, by studying the validation parameters of the kinetic method, worked under conditions laid down covering vancomycin standard curve with concentrations between 0.6 and 1.0 mg / mL, and soybean-casein broth inoculated with 5% of Bacillus subtilis (ATCC 6633). Satisfactory results were obtained with 5 hours incubation. In conclusion, the microplate assay allows to evaluate, with advantages, the activity of the antibiotic against the microorganism, allowing greater operational ease and rapidity of obtaining results.

Enterococcus são cocos gram-positivos que ocorrem isolados, aos pares (diplococos) ou em cadeias e pertencem à microbiota intestinal de uma grande variedade de hospedeiros, de mamíferos a insetos. Enterococcus foram originalmente considerados organismos de pouca importância clínica, mas têm se revelado importantes patógenos nosocomiais. O fato dos Enterococcus possuírem formas de resistência intrínseca e adquirida a vários antibióticos dificulta o tratamento de infecções causadas por eles. Enterococcus faecalis é geralmente a espécie predominante entre os enterococos isolados, sendo de 80% a 90% das amostras clínicas. O surgimento de Enterococcus resistentes à vancomicina (VRE- do inglês vancomycin resistant enterococci) reduziu significativamente as opções de tratamento. O objetivo deste estudo foi identificar e comparar amostras de E. faecalis resistentes à vancomicina (VREfs) isolados em pacientes nos anos de 2009 e 2011, durante um programa de vigilância no Hospital Risoleta Tolentino Neves, em Belo Horizonte, MG. A identificação das espécies foi feita por multiplex-PCR com primers espécie-específicos e os E. faecalis foram selecionados para estudo. Foram realizadas a pesquisa da presença dos genes elrA, cylLL, esp e gelE, a determinação do genótipo responsável pela resistência e a caracterização do transposon que contém o gene de resistência, todos por meio de PCR. Foi também realizada a determinação da concentração inibitória mínima (CIM) de acordo com o CLSI (2013) para vancomicina, linezolida, tigeciclina e daptomicina e testes para resistência a altas concentrações de aminoglicosídeos. Por fim, a tipagem das amostras foi feita através de eletroforese em campo pulsado (PFGE- do inglês Pulsed Field Gel Eletroforesis) e MLST (Multilocus sequence typing). Foi encontrado que 22,2% dos VRE isolados em 2009 e 61,7% dos isolados em 2011 pertencem à espécie E. faecalis e estes foram utilizados no estudo. Houve surgimento de resistência à tigeciclina nos isolados de 2011, sendo 10 isolados resistentes, logo após o início do uso deste antimicrobiano no hospital. Foi encontrado um isolado de 2011 com resistência intermediária à linezolida. Todos os isolados foram sensíveis à daptomicina e altamente resistentes à vancomicina, com CIM maior que 256 &mu;g/mL. Essa alta resistência à vancomicina é condizente com o genótipo Vana, encontrado em todas as amostras. O perfil de virulência prevalente nos VREfs em 2009 era elrA+gelE+, sendo dos pulsotipos A1 e A4 e ST103. Apenas um isolado de 2009, com pulsotipo A1 apresentou o perfil de virulência cyl+elrA+gelE+. Em 2011, o perfil de virulência prevalente foi cyl+esp+elrA+gelE+, sendo que os 12 isolados com esse perfil pertenciam aos pulsotipos B e C e ST6, que ocorreram somente em isolados de 2011. Onze amostras de 2011 apresentaram o perfil elrA+gelE+ e foram classificadas no pulsotipo A e seis amostras tem perfil cyl+elrA+gelE+ e são dos pulsotipos B e D. Resultados de PFGE mostram a inserção no hospital da linhagem ST6, um clone multirresistente amplamente disseminado em hospitais da Itália, Portugal, Espanha e Estados Unidos. Concluiu-se que houve mudanças no perfil de E. faecalis resistentes à vancomicina no hospital, ao longo dos dois anos, com aumento de resistência e linhagens mais virulentas, sendo estes motivos de preocupação. / Enterococci are Gram-positive cocci occurring isolated, in pairs (diplococci) or chains and belong to the intestinal tract of a wide variety of hosts, from mammalian to insect. Enterococcus organisms were originally considered of little clinical importance, but have proved important nosocomial pathogens. The fact that Enterococcus possess intrinsic and acquired resistance to multiple antibiotics complicates the treatment of infections caused by them. Enterococcus faecalis is usually the predominant species among enterococci isolates, with 80% to 90% of the clinical samples. The emergence of vancomycin-resistant Enterococcus (VRE) significantly reduced the treatment options. The aim of this study was to identify and compare samples of vancomycin- resistant E. faecalis (VREfs) in patients in the years 2009 and 2011, during a surveillance program in Hospital Risoleta Tolentino Neves, Belo Horizonte, MG. Species identification was done by multiplex-PCR with primers species-specific and E. faecalis were selected for this study. Tests were conducted for the presence of genes elrA, cylLL, esp and gelE, the determination of the genotype responsible for the resistance and characterization of transposon containing the resistance gene, all by PCR. The minimum inhibitory concentration (MIC) was determined according to the CLSI (2013) to vancomycin, linezolid, daptomycin and tigecycline and tests for resistance to high concentrations of aminoglycosides. Finally, the samples typing was performed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). It was found that 22.2 % of VRE isolates in 2009 and 61.7 % of isolates in 2011 belong to the species E. faecalis and these were used in the study. There was emergence of tigecycline resistance in 2011, with 10 resistant isolates, after the introduction of this drug in the hospital. One isolate from 2011 showed intermediate resistance to linezolid. All isolates were sensitive to daptomycin and highly vancomycin resistant, with MICs greater than 256 mg / mL. This high resistance to vancomycin is consistent with the vanA genotype found in all samples. The virulence profile prevalent in VREfs in 2009 was elrA+gelE+, belonging to the pulsotypes A1 and A4 and ST103. Only one isolate from 2009, presented pulsotype A1 and the virulence profile cyl+elrA+gelE+. In 2011, the prevalent virulence profile was cyl+esp+elrA+gelE+, and the 12 isolates with this profile belonged to pulsotypes B and C and ST6, which occurred only in 2011 isolates. Eleven isolates from 2011 have the profile elrA+gelE+ and were classified as pulsotype A, six isolates have profile cyl+elrA+gelE+ and are of pulsotypes B and D. PFGE results show the insertion of ST6 lineage in the hospital, a multiresistant clone widely disseminated in hospitals in Italy, Portugal, Spain and the United States. It was concluded that there were changes in the profile of E. faecalis resistant to vancomycin in the hospital over the two years, with increased resistance and more virulent strains, which are of concern.

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 &micro;g/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores. / Demands for sanitary conditions in animal farming have been increasing progressively given the need to combine productivity and high quality products to support increasingly demanding consumer markets. In this context, antimicrobial drugs used in prevention as well as in therapy remain as the control strategy for several pathogenic microorganisms, not important only in animal production but also in human health, although restrictions for the indiscriminate use of these drugs have been intensified. However, the excessive use of these products has been associated to the selection of resistant microorganisms in production areas. On the other hand, investigation on strains of public health importance circulating in animal herds is still limited in Brazil. Enterococcus genus bacteria, usually present in animal and human gastrointestinal microbiota, are environmental indicators of fecal contamination and have become a concerning subject in public and veterinary health given the occurrence of strains resistant to vancomycin (VRE). The present study aimed to isolate, quantify and characterize VRE present in stool samples of sheep and goats from several farms in the center-east and northeast regions of São Paulo State. Swabs collected one hundred and thirty-two stool samples either directly from the animal\'s rectum or from the ground. Samples were plated onto m-Enterococcus agar plates and subcultivated in Bile esculin agar with 6 &micro;g/mL of vancomycin (BEAV) to confirm Enterococcus spp and detect resistant samples. Colonies were identified by colonial morphology, Gram\'s staining, biochemical, and molecular profile. The highest colony count was equal to 2.6 × 105 and 1.7 × 105 CFU/g of feces from environmental and animal samples, respectively. Regarding biochemical characterization, Enterococcus faecalis e Vagococcus fluvialis were the most prevalent species. VRE was detected on BEAV in 33 out of 84 sheep-goat samples and in 21 out of 48 ambient samples, indicating a positivity rate of 46.7% and 29.3% respectively in the investigated samples. Analysis by multiplex PCR of the obtained 54 VRE strains indicated that 23 (43%), 22 (41%) 2 (3.5%) and 2 (3.5%) were positive, respectively, for the vanC2/C3, vanC1, vanA and vanB genes, and no product was amplified for 5.3% of the isolates, suggesting the possible occurrence of other known van gene groups among the isolates. The results obtained in this study indicate, for the first time in the studied areas, the circulation of VRE in sheep and goat farms, with no occurrence of apparent clinical signs in the animals, but with possible health risks to the farmers and workers involved, as well as potential consumers.

Orientador: Daniela Ponce / Resumo: A vancomicina é um antiobiótico chave no tratamento de infecções graves por germes gram positivos multirresistentes e deve ser administrada com segurança, buscando minimizar o risco de nefrotoxicidade e garantir a sua eficácia. Entretanto, são escassos os estudos que avaliaram a monitorização de seu nível plasmático e sua associação com desfechos clínicos, como desenvolvimento de lesão renal aguda (LRA) e óbito. OBJETIVO: Ao avaliar pacientes com sepse em uso de vancomicina admitidos em unidade de terapia intensiva (UTI), os objetivos deste estudo foram avaliar a prevalência de vancocinemia em níveis adequados, subterapêuticos e tóxicos e o impacto da adequação dos níveis séricos de vancomicina sobre a LRA e mortalidade precoce. METODOLOGIA: Estudo prospectivo observacional do tipo coorte de pacientes em uso de vancomicina admitidos em duas UTIs do Hospital das Clínicas da Faculdade de Medicina de Botucatu de 01 de agosto de 2016 a 31 de julho de 2017 de modo diário e ininterrupto. RESULTADOS: Foram avaliados 189 pacientes, dos quais foram incluídos 63. Os principais motivos de admissão na UTI foram afecções pulmonares (52,4%) e pós-operatório (15,9%). LRA foi diagnosticada em 44,4% dos pacientes, sendo que a maioria foi classificada como LRA KDIGO III (46,4%). Os pacientes que desenvolveram LRA apresentaram, comparativamente àqueles sem LRA, maiores valores de creatinina basal, maior uso de diurético e de droga vasoativa e maior porcentagem de nível sérico tóxico. A regressã... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Vancomycin is a key antiobiotic in the treatment of severe infections caused by multiresistant gram-positive germs and should be administered safely in order to minimize the risk for nephrotoxicity and ensure its efficacy. However, there are few studies evaluating the monitoring of its plasma level and its association with clinical outcomes, such as the development of acute kidney injury (AKI) and death. OBJECTIVE: The aim of this study was to evaluate the prevalence of vancocinemia at appropriate levels, subtherapeutic and toxic levels, and the impact of the adequacy of vancomycin levels on the AKI and early mortality. METHODS: Prospective observational cohort study of vancomycin patients admitted to two intensive care units of the Clinical Hospital of Botucatu Medical School from August 1, 2016 to July 31, 2017 in a daily and uninterrupted manner. RESULTS: A total of 189 patients were evaluated and 63 were included. The main reasons for admission to the ICU were pulmonary (52.4%) and postoperative (15.9%) conditions. AKI was diagnosed in 44.4% of the patients, most of them were classified as AKI KDIGO III (46.4%). Patients who developed AKI showed higher values of baseline creatinine, greater use of diuretics and vasoactive drugs, and a higher percentage of toxic serum levels compared to those without AKI. Logistic regression showed that the highest level of vancocinemia between the 2nd and 4th days was predictor of AKI, preceding its diagnosis in at least 48 hours (OR= 1,1... (Complete abstract click electronic access below) / Mestre

Orientador: Daniela Ponce / Resumo: Introdução: O controle da infecção no ambiente de terapia intensiva por patógenos hospitalares, frequentemente, inclui a utilização de vancomicina. Ressalta-se que profundas alterações ocorrem na farmacocinética dos antimicrobianos prescritos aos pacientes criticamente doentes e que os diferentes métodos dialíticos podem removê-los parcial ou totalmente. Objetivo: Avaliar a redução do antimicrobiano vancomicina por diferentes métodos de diálise em pacientes com lesão renal aguda (LRA) associada à sepse e identificar as variáveis associadas a concentrações terapêuticas. Metodologia: Estudo transversal que avaliou pacientes sépticos com LRA em hemodiálise convencional intermitente (HDI) ou hemodiálise prolongada (HDP) e em tratamento com vancomicina internados em Unidade de Terapia Intensivas (UTI) do Hospital das Clínicas da Faculdade de Medicina de Botucatu-UNESP. Foram colhidas amostras seriadas de sangue no início da terapia dialítica, após 2 e 4 horas do tratamento e ao final da terapia. A concentração sérica de vancomicina foi aferida por cromatografia líquida de alta eficiência (CLAE) ou por Imunoensaio enzimático homogêneo (Enzyme Multiplied Immunoassay Technique - EMIT). A partir desses dados foi realizado avaliação farmacocinética e Modelagem PK/PD. Resultados: De março de 2015 a agosto de 2017 foram incluídos 27 pacientes tratados por HDI, 17 por HDP 6h e 11 por HDP 10h. O volume de distribuição, assim como o tempo de meia vida e clearence dialítico da vancomicina fo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Infection control in the intensive care environment by hospital pathogens often includes the use of vancomycin. It is noteworthy that profound changes occur in the pharmacokinetics of antimicrobials prescribed to critical patients and that different dialytic methods may partially or totally remove them. Objective: To evaluate the reduction of antimicrobial vancomycin by different dialysis methods in patients with AKI associated with sepsis and to identify the variables associated with its therapeutic level. Methodology: A cross-sectional study evaluating septic patients with AKI on intermittent conventional hemodialysis (IHD) and prolonged hemodialysis (PHD) and vancomycin treatment in intensive care unit (ICU) of Clinics Hospital of the Faculty of Medicine of Botucatu - UNESP. Serial blood samples were collected at the start of dialysis therapy after 2 and 4 hours of treatment and at the end of therapy. The serum level of vancomycin was measured by high performance liquid chromatography (HPLC) or by Enzyme Multiplied Immunoassay Technique (EMIT). From these data, pharmacokinetic evaluation and PK / PD modeling were performed. Results: From March 2015 to August 2017, 27 patients treated for IHD, 17 for PHD 6h and 11 for PHD 10h were included. The volume of distribution was higher in the groups PHD 6h and PHD 10h (p <0.001), as well as half-life (p <0.001) and dialytic clearance of vancomycin (p <0.001). The reduction of vancomycin after 2 hours of therapy was 26... (Complete abstract click electronic access below) / Doutor

C. difficile est une bactérie anaérobie du tube digestif reconnue comme l’agent responsable de 25% des diarrhées nosocomiales post antibiotiques et de la plupart des colites pseudomembraneuses. Le métronidazole et la vancomycine sont les traitements de référence pour les infections liées à ce pathogène. A ce jour, aucune souche de C. difficile résistante à la vancomycine n’a été rapportée. Paradoxalement, 85 % des souches de cette espèce hébergent dans leur génome un groupe de gènes cryptiques vanGCd homologue à l’opéron de résistance à la vancomycine vanG de E. faecalis. Dans ce travail, nous avons étudié la capacité des gènes vanGCd à conférer la résistance à la vancomycine. L’étude transcriptionnelle a révélé que le groupe de gènes vanGCd est transcrit en deux opérons (i) l’un de régulation exprimé de façon constitutive et (ii) l’autre de résistance, inductible par la vancomycine. L’analyse enzymatique de VanGCd, VanXYCd et VanTCd in vitro a montré que ces protéines possèdent respectivement des activités ligase, D,D-peptidase et racémase nécessaires au pontage D-Ala-D-Ser et à l’hydrolyse des dipeptides naturels D-Ala-D-Ala. L’analyse des précurseurs du peptidoglycane par spectrométrie de masse en présence de vancomycine a permis l’identification de l’UDP-MurNAc-pentapeptide [Ser] témoin de l’activité in vivo de ces protéines. L’opéron de résistance vanGcd cloné chez E. coli NR698 sensible à la vancomycine exerce un très faible effet sur la CMI de cet antibiotique. En revanche, le clonage chez E. faecalis n’a pas été obtenu en raison de mutations spontanées. Pour ces raisons, nous avons introduit l’opéron vanC de E. gallinarum chez C. difficile et observé une faible augmentation de la CMI de la vancomycine. Ce résultat indique que l’expression de la résistance à cet antibiotique chez C. difficile implique des facteurs intrinsèques liés à la bactérie. Nous avons observé que la ligase MurF possède une meilleure affinité vis-à-vis du dipeptide D-Ala-D-Ala que pour le dipeptide D-Ala-D-Ser ce qui pourrait impacter la synthèse de l’UDP-MurNAc-pentapeptide[Ser]. Par ailleurs, nous avons observé qu’en présence de vancomycine, les précurseurs du peptidoglycane sont amidés. Cette modification affecte également le peptidoglycane mature. Toutefois, le rôle physiologique de l’amidation chez C. difficile reste à élucider. En conclusion, (i) l’absence d’expression de la résistance à la vancomycine de C. difficile est multifactorielle, (ii) le groupe de gènes vanGCd a peu de potentiel pour contribuer à l’émergence de la résistance à la vancomycine. / Clostridium difficile is a major enteric pathogen responsible for 25% of post antibiotics diarrhea and most cases of pseudomembranous colitis. Metronidazole and vancomycin are the standard treatments for infected patients. vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer vancomycine resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd, VanXYCd and VanTCd are functional, exhibiting D-Ala:D-Ser ligase, D,D-dipeptidase and D-Ser racemase activities, respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans-complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sub-lethal concentration of vancomycin led to production of UDP-MurNAc-pentapeptide [D-Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP-MurNAc-tetrapeptide, UDP-MurNAc-pentapeptide[D-Ala] and UDP-MurNAc-pentapeptide[D-Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra-Tetra is amidated on the meso-diaminopimelic acid. We also demostrated that the ligase MurF has a better affinity for the D-Ala-D-Ala dipeptide than for the D-Ala-D-Ser, which could impact on the synthesis of UDP-MurNAc-pentapeptide [Ser].In conclusion, the lack of vancomycin resistance expression may be due to several factors that, in combinination with a gene cluster conferring a low level vancomycin resistancemay prevent the emergence of vancomycin resistance based on D-Ala- D-Ser modification of peptidoglycane precursors in C. difficile.