Orientador: Carlos Magno Castelo Branco Fortaleza / Coorientador: Lenice do Rosário de Souza / Banca: Anna Sara S. Levin / Banca: Elisei Alves Waldman / Banca: Maria de Lourdes Ribeiro de Souza da Cunha / Banca: Adriano Dias / Resumo: Estudos recentes identificaram comportamento sazonal de alguns microrganismos implicados na etiologia das Infecções Hospitalares ou Relacionadas à Assistência em Saúde (IH/IRAS). A maior parte desses estudos foi conduzida em países de clima temperado. Para identificar a sazonalidade de patógenos hospitalares em uma região tropical, nós conduzimos um estudo ecológico e um caso-controle, envolvendo hemoculturas positivas de presumível origem hospitalar (colhidas após o terceiro dia de internação) do Hospital das Clínicas da Faculdade de Medicina de Botucatu no período de 2005 a 2010. O estudo foi realizado em duas fases. No estudo ecológico, foram realizadas: (a) comparações de incidência de bacteremias por microrganimos ou grupos específicos em meses "quentes/úmidos" (outubro-março) e "frios/secos" (abril a setembro); (b) análise de regressão para identificar relação entre temperatura e umidade médias e incidência mensal de bacteremias; (c) análise de séries temporais para identificação de sazonalidade.No "casocontrole", foram abordados fatores climáticos (temperatura e umidade médias dos sete dias que precederam a coleta de cada hemocultura) para identificar preditores do isolamento de microrganismos e grupos específicos, em modelo de regressão logística. Os resultados demonstraram consistência entre as diversas abordagens, identificando para três grupos (Gram-negativos como um todo, Acinetobacter baumannii e Enterobacter spp) um aumento de incidência nos meses "quentes/úmidos", relação significante entre temperatura média mensal e incidência e sazonalidade em modelos estocásticos. Neste último teste, também se detectou sazonalidade para estafilococos coagulase-negativa, aparentemente não relacionada a fatores climáticos. No estudo de base individual ("caso-controle") identificamos correlação entre temperaturas elevadas na semana anterior ... / Abstract: Recent studies identified seasonal behavior of some microorganisms implicated in the etiology of Healthcare-associated Infections (HAIs). Most of these studies were conducted in developed countries with temperate climate. In order to identify the seasonality of nosocomial pathogens in a tropical region, we conducted an ecological study involving positive blood cultures of suspected nosocomial origin (collected after the third day of hospitalization) in the Hospital das Clinicas from Faculdade de Medicina de Botucatu in the period 2005-2010. The study was conducted in two phases. In the first ("ecologic study") we performed: (a) comparisons of the incidence of bacteremia caused by specific organisms or groups during "warm" (October to March) and "cold" (April to September) months; (b) regression analysis, aimed at identifying the association between temperature and humidity and the average monthly incidence of bacteremia, (c) time series analysis. In the second phase, ("casecontrol"), weather factors (temperature and humidity averages of the seven days preceding the collection of each blood cultures) were addressed in order to identify predictors of isolation of specific microorganisms and groups in the logistic regression model. The results showed consistency between the various approaches. Three groups (Gram-negative as a whole, Acinetobacter baumannii and Enterobacter spp) presented increased incidence in "warm" months, significant relation between mean temperature and monthly incidence and seasonality in stochastic models. Those models also detected seasonality for coagulase-negative staphylococci, apparently not related to climatic factors. In individualbased analysis ("case-control") we identified correlation between high temperatures in the past week and recovery of Gram-negatives in general, and among these, A. baumannii. The findings are consistent with the literature, showing ... / Mestre

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-20T17:09:26Z No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-20T17:39:39Z (GMT) No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-20T17:39:39Z (GMT). No. of bitstreams: 2 Dissertação - Cássia Silva de Miranda Godoy - 2012.pdf: 2252636 bytes, checksum: cdb569738a5d271a5bc6ac26e7d4dc4e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-03-23 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Acinetobacter baumannii (Ab), has an important role in healthcare-associated infections, present a rapid global and emerging multidrug-resistant (MDR) strains, affecting many countries. In Brazil, Ab is responsible for outbreaks infections in intensive care units (ICUs) since 1996, with high rates of antimicrobial resistance. This was a descriptive cohort study of adult infected with Ab during the period of June to December of 2010, that evaluated the clinical and epidemiological profile of infections caused by Ab and analyzed genetically, by pulsed field gel electrophoresis (PFGE), clinical isolates from patients admitted in five ICUs of the Municipality of Goiania. We identified 64 cases of patient infected or colonized with Ab during the study period, 84 samples culture positive for Ab with a global infection rate of 4.8%. Infection incidence at each hospital was as follows: 10 % in ICU-1, 4.3% in ICU-2, 7.8% in ICU-3, 1.3% in ICU-4, and 7.5 % in ICU-5. The mean age of patients was 53.2 years (sd=19) and 59.4% (38) were male. Symptomatic infections occurred in 90.6% of the cases. The most frequent site of infection was pulmonary (53.1%), followed by surgical site (10.9%), and urinary tract (7.8%). The most common underlying diseases were neoplasia (34.4%) and AIDS (17.2%). Most of the patients infected with Ab (98.4%) had received antimicrobial therapy previously. The most frequently used drugs were cephalosporins (71.4%), carbapenems (50.8%), glycopeptides (46.0%), and fluoroquinolones (33.3%). Among the invasive procedures realized prior to Ab infection, intravascular catheters and vesical catheters where the most frequent (93.7%). The culture results from the isolate of each patient revealed that carbapenems resistance was 73.4%, ampicillin-sulbactam 60.9%, amikacin 20.3%, polymyxins 6.2%, tigecycline 3.1%. The overall mortality rate was 79.7% and related mortality rate to Ab infection was 67.2%. PFGE analysis of the isolates from 56 patients demonstrated the dissemination of genetically related clones within the ICU and between the different ICUs, and the identification of a major outbreak of MDR Ab in four out of the five analyzed ICUs involving 12 patients. In conclusion a high rate of antimicrobial resistance was detected as well as a high mortality rate among ICU patients infected with Ab, requiring stronger and more efficient measures to control this agent, as well as urgent measures are needed for the rational utilization of antibiotics in ICUs. / O Acinetobacter baumannii (Ab) tem importante papel nas infecções relacionadas à assistência à saúde e apresenta emergência rápida e global de cepas multidrogarresistentes (MDR), atingindo vários países. No Brasil, o Ab é responsável por surtos de infecções em unidades de terapia intensiva (UTIs) desde 1996, com elevadas taxas de resistência aos antimicrobianos. Esse estudo foi delineado como uma coorte descritiva de pacientes adultos infectados por Ab no período de junho a dezembro de 2010. Avaliou o perfil clínico e epidemiológico das infecções causadas por Ab e analisou geneticamente, por eletroforese em gel de campo pulsado (PFGE), os isolados clínicos destes pacientes em cinco UTIs de Goiânia e Aparecida de Goiânia. Foram identificados 64 casos de pacientes infectados ou colonizados por Ab no período do estudo, 84 amostras de cultura positivas para Ab e taxa global de infecção de 4,8 %. A frequência de infecções por UTIs foi de: 10% na UTI-1; 4,3% na UTI-2; 7,8% na UTI-3 e 1,3% na UTI-4 e 7,5% na UTI-5. A média de idade dos pacientes foi de 53,2 anos (dp=19) sendo 59,4% (38) do sexo masculino. Infecção sintomática ocorreu em 90,6% dos casos. O sítio de infecção mais frequente foi o pulmonar com 53,1%, seguido de infecção do sítio cirúrgico 10,9% e trato urinário 7,8%. As doenças de base mais comuns foram neoplasia (34,4%) e AIDS (17,2%). Dos pacientes com infecção pelo Ab, 98,4% receberam antibacteriano prévio. Os antibacterianos mais usados foram: cefalosporinas 71,4% (45); carbapenêmicos 50,8% (32), glicopeptídeos 46,0% (29) e fluorquinolonas 33,3% (21). Dos procedimentos invasivos realizados previamente à infecção pelo Ab, cateter vascular central e sondagem vesical de demora (93,7%) foram os mais comuns. A letalidade global foi de 79,7% (51/64), e a relacionada às infecções pelo Ab foi de 67,2% (39/58). Avaliando os resultados de cultura positiva, a resistência aos carbapenêmicos foi de 73,4%, à ampicilina/sulbactam de 60,9% (39/64), amicacina de 20,3% (13/64), polimixinas de 6,2% (4/64) e tigeciclina de 3,1% (2/64). A análise por PFGE das isolados bacterianos dos pacientes (56), demonstrou a disseminação clonal de Ab dentro das UTIs, e entre as diferentes UTIs, com identificação de um surto por Ab MDR entre quatro das cinco UTIs investigadas no período do estudo, envolvendo 12 pacientes. Constatamos alto índice de Ab MDR e alta letalidade nas UTIs avaliadas, com necessidade de intensificar o controle deste agente, além de racionalização do uso de antimicrobianos nas UTIs.

Acinetobacter baumannii é um patógeno oportunista, geralmente resistente a muitos antimicrobianos, que causa surtos de infecções hospitalares. Assim, a sua habilidade de formar biofilme pode explicar a capacidade de sobreviver no ambiente hospitalar e em utensílios médicos. O objetivo desse estudo foi avaliar a capacidade de duas cepas clínicas de A. baumannii obtido de hospitais de Porto Alegre, Brasil (abC e abH) e uma cepa controle ATCC 19606 (abA) formar biofilme e realizar a análise transcricional dos genes possivelmente envolvidos na produção e manutenção do biofilme: bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA. A capacidade de formação de biofilme foi avaliada pelo método cristal violeta em superfície plástica a 25°C e 37°C nos meios LB, LB + 1% glicose, urina pura e LB + 10% sangue de carneiro. A análise transcricional foi feita por real time PCR. A cepas AbH, AbC e AbA foram fortes formadoras de biofileme em LB e LB+glicose à 25 e 37°C. Em LB+sangue, as cepas AbH e AbA foram fortes formadoras e AbC foi fraca formadora de biofilme. Em urina, a cepa AbH foi moderadamente formadora, AbC e AbA foram fracas formadoras de biofilme. Os genes wspR, pgaA, ompA, bap, abaI de AbA, csdA de AbH e o operon IscRSU foram superexpressos em biofilme; os genes eagg de AbH, pilZ e csuAB foram inibidos e os genes bfmRS, eal, eagg de AbA, csdA de AbA e abaI de AbH não possuíram uma variação significativa na expressão durante o biofilme. Portantanto, a regulação positiva dos genes wspR, pgaA, ompA, bap, csdA e do operon IscRSU é um indício de sua importância na formação e manutenção do biofilme por esse patógeno. / Acinetobacter baumannii is an opportunistic pathogen which causes a wide range of nosocomial infections, being usually multirresistent to drugs. Their ability to form biofilm can explain the feature of surviving inside hospital environment and on medical devices. The aim of the present study was to evaluate the ability of two clinically isolated MDR A. baumannii strain, obtained from a general hospital of Porto Alegre, RS, Brazil (AbH and AbC), and the reference ATCC 19606 strain (AbA), to form biofilm and to analyze the relative expression of genes putatively involved in biofilm formation: bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA. The biofilm formation capability was evaluated by the crystal-violet staining method on plastic surfaces at 25°C and 37°C using the broth LB, LB + 1% glucose, pure urine and LB + 10% sheep blood. The trancritional profiling of the genes was analyzed through real time PCR methodologies. The strains AbH, AbC e AbA were strong biofilm producers in LB e LB+glucose at 25 and 37°C. In the broth LB+blood, the strains AbH and AbA were strong biofilm producers and AbC was week biofilm producer. In urine, the strain AbH was moderated producer, AbC and AbA were week biofilm producers. The genes wspR, pgaA, ompA, bap, abaI from AbA, csdA from AbH and the operon IscRSU were overexpressed in biofilm; the genes eagg de AbH, pilZ e csuAB were suppressed and the genes bfmRS, eal, eagg from AbA, csdA from AbA e abaI from AbH did not vary their expression significantly during the biofilm condition. In conclusion, the factors wspR, pgaA, ompA, bap, csdA and of the operon IscRSU, that were positively regulated, appear to have an important role during the process of biofilm formation by A. baumannii.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

Um estudo de coorte prospectivo foi realizado com o objetivo de comparar a eficácia da polimixina B à de outros antibióticos. Foram estudados pacientes com pneumonia ou traqueobronquite associadas à ventilação mecânica causadas por Pseudomonas aeruginosa ou Acinetobacter baumannii. Critérios de inclusão para este estudo foram idade igual ou superior a dezoito anos e uso de terapia antimicrobiana apropriada por período igual ou superior a 48 horas. O desfecho primário avaliado foi mortalidade em 30 dias. Variáveis clínicas foram comparadas entre os pacientes que utilizaram polimixina B e os que utilizaram outras drogas. O modelo de regressão de Cox foi realizado. Um total de 67 episódios ocorridos em 63 pacientes foi analisado: 45(67,2%) foram tratados com polimixina B e 22 (32,8%) com comparadores. A maior parte dos comparadores (72,7%) era de beta-lactâmicos. A maioria dos episódios foi de pneumonia associada à ventilação mecânica (PAV). As infecções foram causadas por P. aeruginosa em 28 casos (41,8%), por A. baumannii em 35 casos (52,2%) e por ambos em 4 casos (6%). A mortalidade geral em 30 dias foi de 44,8% (30 de 67): 53,3% (24 de 45) no grupo da polimixina B e 27,3% (6 de 22) no grupo dos comparadores (p=0,08). A taxa de mortalidade no grupo da polimixina e comparadores foi de 65,6 e 12,0 por 1000 pacientes-dia, respectivamente (p=0,02). A análise multivariada mostrou que o uso de polimixina B foi fator independente associado à mortalidade em 30 dias (Hazard Ratio ajustada, 4,3; Intervalo de Confiança de 95%, 1,39-13,03), após ajuste para tempo de internação hospitalar antes da infecção e aumento > 100% da creatinina em relação ao valor basal durante o tratamento. O escore APACHE II no inicio da infecção foi mantido no modelo final, embora não tenha atingido significância estatística, para ajuste de possível fator confundidor residual. Não houve diferenças significativas nos desfechos secundários, incluindo tempo de ventilação mecânica após terapia adequada, superinfecção e erradicação bacteriana nas secreções respiratórias. A terapia com polimixina B foi inferior comparada a outras drogas na pneumonia e traqueobronquite associadas à ventilação mecânica causadas por P. aeruginosa e A. baumannii. / To compare the efficacy of polymyxin B with other antimicrobials in the treatment of ventilator-associated pneumonia (VAP) and tracheobronchitis (VAT) by Pseudomonas aeruginosa or Acinetobacter baumannii, a prospective cohort study was performed. Patients who received appropriate therapy for ≥48h were analyzed. The primary outcome was 30-day mortality. A total of 67 episodes were analyzed: 45 (67.2%) treated with polymyxin B and 22 (32.8%) with comparators. Thirty-day mortality was 44.8%: 53.3% (24 of 45) in the polymyxin B group and 27.3% (6 of 22) in the comparator group, P=0.08. The mortality rates in the polymyxin B and comparator group were 65.6 and 12.0 per 1000-patients-day, respectively (P=0.02) Treatment with polymyxin B was independently associated with 30-day mortality in multivariate model, with similar results in the subgroup of patients with VAP, suggesting that this antibiotic may be inferior to other drugs in the treatment of VAP and VAT by these organisms.

Thirty-three 1,4-dihydropyridine diastereomeric pairs were synthesized and the structure-activity relationship studied in a Plasmodium falciparum in vitro model. Twenty-nine of these derivatives contained a 6-position oxygen, with 2.31, 2.32, 2.52 and 2.53 having single and double digit nanomolar activities. This SAR study revealed some insightful information about the 1,4-dihydropyridine substitution pattern. Substitution at the 7-position other than 3,4-dimethoxy severely reduced the activity. 4-phenyl substitution with 2- or 4- halo or methyl formed active compounds while substitution at the 3-position or with methoxy or conjugated aryl systems resulted in inactive compounds. The 2-position was found to majorly affect the activity, with groups larger than methyl being the most active. The other four derivatives contained a 6-position methylene, with 2.1, 2.59 and 2.60 having single nanomolar activities. Lastly, stereochemistry was revealed to play an important role in the activity of 2.1. One stereoisomer, (+)-trans-2.1, had subnanomolar activity in two assays. Another stereoisomer, (4S,7S)-2.1, had nanomolar activity. The other two stereoisomers were inactive. A series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and Leishmania amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50s in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 3.23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg/kg/day for five consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N2,N4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents. A similar series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against methicilin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar or high nanomolar range were identified via SAR. In a murine model of MRSA infection, 1x the MIC for quinazoline 4.47 allowed for the survival of all tested mice at the end of a one week study. An in vivo model of A. baumannii was also undertaken using a Galleria mellonella model of infection. Quinazolines 4.74-4.76 afforded an increased protection of 87.5% when compared to the control experiments, with 70% of the wax worms surviving until day three. The observed potencies of frontrunner compounds in in vivo assays and their ease of synthesis make N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-bacterial agents.

The development of a scum in a commercial sports drink is of concern because the product would be of poor quality, which may result in financial losses due to consumer rejection of the product and hence a decrease in the firm’s market share. The scum could be harmful to health and as such the firm could be litigated. Several factors, including microbial proliferation, may be the cause of the development of a scum in sports drink, but the actual cause seems not to have been established. The aim of this study was to characterise the scum in sports drink and determine the effects of preservation factors (pasteurisation, chemical preservatives and refrigeration) on its development. Samples of the sports drink were taken at different stages of processing to determine the effect of preservatives, pasteurisation and storage temperature on scum development. Some samples were kept at room temperature (approx. 25°C) and others were kept in the refrigerator (approx. 4ºC) during the study. A total of 150 samples were analysed over a period of four months. The structural characteristics of the scum that developed in the sports drink were determined by scanning electron microscopy (SEM) and elemental analysis. The sports drink samples were analysed for their microbial load and microbial types. Consumer acceptability of pasteurised and non-pasteurised drink was compared by conducting sensory evaluation using a consumer panel of 60 panellists. Customer complaints recorded by the sports drink manufacture that were due to scum development in the drink were also reviewed to establish the impact of scum development on consumer acceptability of the drink. The results of the study indicated that scum development was due to microbial contamination of the drink. The causative organism of the scum was identified as Acinetobacter baumanii. Acinetobacter baumanii is a gram negative non-spore forming coccobacilli and does not ferment sucrose. Acinetobacter baumanii forms the scum in sports drink as a means of protection from environmental stresses. The scum was found to be a compound of C, Si and O. The non-pasteurised samples were slightly more acceptable to consumers compared to the pasteurised samples. The consumer acceptability of pasteurised drink samples was negatively affected by the loss of aroma and flavour during pasteurisation. The preservation factors (chemical preservatives, pasteurisation and refrigeration) had no effect on scum development. To prevent post pasteurisation contamination, it is recommended that the pasteurisation process be done at the filling stage instead of at the holding stage. The frequency of changing rubbers and gaskets on the filling line should be at least every two months. The drink is pasteurised at 90ºC for 20 seconds, this needs to be reduced to a level where it will not have an influence on the loss of taste and aroma of the pasteurised drink, but without reducing the effectiveness of pasteurisation. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Normally present in water, soil and waste water, Acinetobacter baumannii has become an important nosocomial pathogen, as causal agent of pneumonias, septicemias and urinary tract infections, among other complications in compromised patients from hospital’s intensive care units. One of its last acquired abilities is the resistance to colistin (polymixin E), the last therapeutic option for its infections. In this thesis, descriptive and quantitative differential expression proteomics is used in the study of acquired colistin resistance. As result of this research, 1,097 proteins belonging to the Acinetobacter genus have been identified by combined application of bidimensional gel electrophoresis (2DE), differential gel electrophoresis (DIGE), and peptide labeling with stable isobaric isotopes tags (iTRAQ). Analyses have been performed on the global expressed proteome of a reference, colistin-sensible strain (A. baumannii ATCC 19606) and, for comparative purposes, on a derived strain on which colistin resistance has been induced in vitro. The resistant phenotype shows reduced fitness, with significant differences in expression found in outer membrane proteins, membrane active transporters, diverse metabolic enzymes (fatty acids, citrate, phenylacetate, piruvate and nitrogen), proteins involved in stress response and biofilm formation, as well as in protein synthesis and folding pathways. The work has allowed to assess the strengths and weaknesses of the different techniques currently used in this type of proteomic analysis. / Acinetobacter baumannii, normalmente aislado en suelos y aguas (corrientes o residuales), se ha convertido en importante patógeno nosocomial, siendo agente causal de, entre otras complicaciones, neumonías, septicemias e infecciones del tracto urinario de pacientes comprometidos en unidades hospitalarias de cuidados intensivos. La más reciente de sus capacidades adquiridas es la resistencia a colistina (polimixina E), antibiótico peptídico considerado la última opción terapéutica en contextos clínicos. Esta tesis doctoral emplea la proteómica descriptiva y de expresión diferencial cuantitativa para investigar la resistencia adquirida por A. baumannii a dicho antibiótico. Los resultados han supuesto la identificación de 1.097 proteínas de Acinetobacter mediante el empleo combinado de electroforesis bidimensional convencional (2DE), 2DE diferencial (DIGE) y marcaje peptídico mediante isótopos isobáricos estables (iTRAQ). Los análisis se han realizado en el proteoma expresado por una cepa de referencia sensible a colistina (A. baumannii ATCC 19606), así como en una cepa derivada de ésta en la que se ha inducido, a efectos comparativos, resistencia a colistina in vitro. El fenotipo resistente manifestó reducida adaptabilidad biológica, encontrándose las principales diferencias en la estructura de la membrana externa, en la expresión de transportadores activos de membrana, en diversos enzimas metabólicos (ácidos grasos, citrato, fenilacetato, piruvato, nitrógeno) y de respuesta a condiciones de estrés, así como en la expresión de proteínas participantes en la formación de biopelículas y en el proceso de síntesis y plegamiento de proteínas. Además, el trabajo ha permitido evaluar los puntos fuertes y débiles de las técnicas empleadas actualmente en este tipo de análisis proteómicos.

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Obrigada Luciana Pizzani on 2018-04-03T18:47:46Z (GMT) / Submitted by MÔNICA DA SILVEIRA null (monysilveira@hotmail.com) on 2018-04-03T19:30:34Z No. of bitstreams: 1 TESE MONICA Envio corrigida final word modificada ok.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-04-03T19:40:57Z (GMT) No. of bitstreams: 1 silveira_m_dr_bot.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) / Made available in DSpace on 2018-04-03T19:40:57Z (GMT). No. of bitstreams: 1 silveira_m_dr_bot.pdf: 3526672 bytes, checksum: aa12e249981a8458333be778d0062faf (MD5) Previous issue date: 2018-03-02 / Estima-se que no Brasil ocorram centenas de milhares de Infecções Relacionadas à Assistência (IRAS) à Saúde todos os anos, e uma proporção significativa desses quadros é causada por Acinetobacter baumannii. Essa bactéria, hiperendêmica em hospitais brasileiros, é geralmente resistente a múltiplas drogas, restando um escasso arsenal terapêutico. Para além dos fatores de risco tradicionais (comorbidades, exposição ao ambiente hospitalar, procedimentos invasivos, uso de antimicrobianos), estudos recentes demonstraram sazonalidade e associação de sua incidência com altas temperaturas ambientais. No entanto, as razões para esses achados permanecem obscuras. Nós conduzimos estudos em três hospitais públicos conveniados do município de Bauru-SP, com o objetivo de colaborar para a compreensão da epidemiologia do A. baumannii, com ênfase nos isolados resistentes a carbapenêmicos (Carbapenem-resistant A. baumannii, CRAB). O primeiro deles, com delineamento ecológico, analisou o comportamento de taxas mensais de incidência de A. baumannii como um todo e em subgrupos (enfermarias versus Unidades de Terapia Intensiva [UTI], amostras clínicas totais versus hemoculturas) no período de 2006 a 2017. Estudamos o impacto de temperatura, umidade e pluviosidade sobre essa incidência. Como resultados, identificamos que temperaturas médias mensais acima do percentil 75 (24,7°C) estavam associadas a maior incidência de A. baumannii como um todo e de CRAB. Curiosamente, esses achados foram mais consistentes nas UTI e para isolados de hemoculturas. No segundo estudo, tipo “caso-controle” analisamos a clonalidade e fatores de risco para infecções da corrente sanguínea causadas por CRAB (CRABBloodstream infections, CRAB-BSI). Foram estudados 38 pacientes com CRABBSI e 76 controles. Fatores de risco significantes (p<0,05) em análise multivariada para aquisição de CRAB-BSI foram a inserção de cateter venoso central e o uso de cefepima ou meropenem. Na genotipagem por Pulsed-Field Gel Electrophoresis (PFGE), identificamos 12 perfis, sendo que 7 deles agrupavam mais de um isolado. Além disso, identificamos a presença de genes codificadores das enzimas OXA-23 e OXA-51, associadas à resistência aos carbapenêmicos. O terceiro estudo comparou os mesmos 38 casos de CRAB e 76 controles, em delineamento tipo “coorte pareada”, para o desfecho “óbito durante a internação”. Como esperado, os casos foram significantemente associados a maior risco de óbito em modelos de análise de sobrevida. Além disso, quando estudados somente os casos, o uso de Aminoglicosídeos e Polimixina após o diagnóstico se associou a melhor prognóstico. Concluímos que o A. baumannii é um agente potencialmente letal, que se transmite no interior de hospitais e entre diferentes serviços, com incidência aumentada em períodos quentes. / It is estimated that in Brazil hundreds of thousands of Healthcare Associated Infections (HCAI) occur every year, and a significant proportion of these are caused by Acinetobacter baumannii. This bacterium, hyperendemic in Brazilian hospitals, is generally resistant to multiple drugs, leaving behind a scarce therapeutic arsenal. In addition to the traditional risk factors (comorbidities, exposure to the hospital environment, invasive procedures, antimicrobial use), recent studies have demonstrated seasonality and association of A. baumannii incidence with high environmental temperatures. However, the reasons underlying these findings remain obscure. We conducted studies in three public hospitals in the city of Bauru-SP, Brazil, in order to contribute to understanding the epidemiology of A. baumannii, with emphasis on carbapenem resistant isolates (CRAB). The first one, with ecological design, analyzed the behavior of monthly rates of incidence of overall A. baumannii and several subgroups (non-critical wards vs. Intensive Care Units (ICU), overall clinical samples vs. blood cultures) from 2006 to 2017. We studied the impact of temperature, humidity and rainfall on this incidence. As a result, we identified that average monthly temperatures above the 75th percentile (24.7°C) were associated with a higher incidence of overall A. baumannii and CRAB. Interestingly, these findings were more consistent in ICUs and for isolates of blood cultures. In the second study (casecontrol), we analyzed the clonality and risk factors for CRAB bloodstream infections (CRAB-BSI). We studied 38 patients with CRAB-BSI and 76 controls. Significant risk factors (p <0.05) in multivariable analysis for CRAB-BSI acquisition were central venous catheter insertion and use of cefepime or meropenem. In the Pulsed-Field Gel Electrophoresis (PFGE) genotyping, we identified 12 patterns, 7 of which grouped more than one isolate. In addition, we identified the presence of genes encoding the enzymes OXA-23 and OXA-51, associated with resistance to carbapenems. The third study compared the same 38 cases of CRAB and 76 controls, in a "matched cohort" design, for the outcome "in-hospital death”. As expected, the cases were significantly associated with a higher risk of death in survival analysis models. In addition, when only cases were studied, the use of Aminoglycosides and Polimyxyns after diagnosis was associated with a better prognosis. We conclude that A. baumannii is a potentially lethal agent, which is transmitted within hospitals and between different services, with an increased incidence during warm periods.

Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: Estima-se que no Brasil ocorram centenas de milhares de Infecções Relacionadas à Assistência (IRAS) à Saúde todos os anos, e uma proporção significativa desses quadros é causada por Acinetobacter baumannii. Essa bactéria, hiperendêmica em hospitais brasileiros, é geralmente resistente a múltiplas drogas, restando um escasso arsenal terapêutico. Para além dos fatores de risco tradicionais (comorbidades, exposição ao ambiente hospitalar, procedimentos invasivos, uso de antimicrobianos), estudos recentes demonstraram sazonalidade e associação de sua incidência com altas temperaturas ambientais. No entanto, as razões para esses achados permanecem obscuras. Nós conduzimos estudos em três hospitais públicos conveniados do município de Bauru-SP, com o objetivo de colaborar para a compreensão da epidemiologia do A. baumannii, com ênfase nos isolados resistentes a carbapenêmicos (Carbapenem-resistant A. baumannii, CRAB). O primeiro deles, com delineamento ecológico, analisou o comportamento de taxas mensais de incidência de A. baumannii como um todo e em subgrupos (enfermarias versus Unidades de Terapia Intensiva [UTI], amostras clínicas totais versus hemoculturas) no período de 2006 a 2017. Estudamos o impacto de temperatura, umidade e pluviosidade sobre essa incidência. Como resultados, identificamos que temperaturas médias mensais acima do percentil 75 (24,7°C) estavam associadas a maior incidência de A. baumannii como um todo e de CRAB. Curiosamente, esses achados foram mais consi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is estimated that in Brazil hundreds of thousands of Healthcare Associated Infections (HCAI) occur every year, and a significant proportion of these are caused by Acinetobacter baumannii. This bacterium, hyperendemic in Brazilian hospitals, is generally resistant to multiple drugs, leaving behind a scarce therapeutic arsenal. In addition to the traditional risk factors (comorbidities, exposure to the hospital environment, invasive procedures, antimicrobial use), recent studies have demonstrated seasonality and association of A. baumannii incidence with high environmental temperatures. However, the reasons underlying these findings remain obscure. We conducted studies in three public hospitals in the city of Bauru-SP, Brazil, in order to contribute to understanding the epidemiology of A. baumannii, with emphasis on carbapenem resistant isolates (CRAB). The first one, with ecological design, analyzed the behavior of monthly rates of incidence of overall A. baumannii and several subgroups (non-critical wards vs. Intensive Care Units (ICU), overall clinical samples vs. blood cultures) from 2006 to 2017. We studied the impact of temperature, humidity and rainfall on this incidence. As a result, we identified that average monthly temperatures above the 75th percentile (24.7°C) were associated with a higher incidence of overall A. baumannii and CRAB. Interestingly, these findings were more consistent in ICUs and for isolates of blood cultures. In the second study (casecontrol), w... (Complete abstract click electronic access below) / Doutor