Das bei der Sanierung von großen segmentalen Knochendefekten bestehende Risiko einer fremdkörperassoziierten Infektion soll durch die Integration eines Wirkstofffreisetzungssystems in ein bestehendes textiles Knochenimplantat gemindert werden. Durch Immobilisierung des Wirkstoffs in eine degradierbare Polymermatrix wird eine zeitlich verzögerte Freisetzung bewirkt. Als Wirkstofffreisetzungssystem wird die Kombination von Polylactid (PLA) bzw. Poly(Lactid-co-Glycolid) (PLGA) als Matrixpolymer mit dem Antibiotikum Gentamicin als Wirkstoff untersucht, welches durch Beschichtung der textilen Scaffolds mittels Dip-Coating eingebracht werden soll. Es stehen die drei Beschichtungsmethoden „Suspension“, „Emulsion“ und „Schichtaufbau“ zur Auswahl, die jeweils über eigene Parameter zur Beeinflussung des Freisetzungsprofils verfügen. Die Methode „Suspension“ und die damit verbundenen Einflussfaktoren Korngröße, Korngrößenverteilung sowie Masseanteil des Antibiotikums und Schichtdicke der aufgetragenen Polymerschicht wurde als die günstigste herausgearbeitet. Im Teil II dieser Arbeit wird diese soweit optimiert, dass nahezu über den gesamten geforderten Zeitraum die festgelegte notwendige Dosierung aufrechterhalten werden kann. Erste in vitro Versuche weisen auf eine gute Zellverträglichkeit sowie eine ausreichende mikrobielle Wirksamkeit hin. / To reduce the risk of infection in the treatment of long bone defects, a novel embroidered bone implant is to be provided with an antibiotic drug delivery system. Prolonged and controlled drug release can be achieved by coating the thread material with antibiotics incorporated in a degradable polymer matrix. The chosen drug delivery system is composed of polylactide acid (PLA) or poly(lactide-co-glycolide) acid (PLGA) as matrix polymer and the antibiotic gentamicin. It is integrated into the textile structure by dip-coating providing the three different methods suspension, emulsion and layered. Each method bears its appropriate parameters to influence the releasing profile. The suspension-method and its parameters grain size and grain size distribution as well as mass fraction of the antibiotic and the coating thickness could be proved as the most feasible. In part II of this essay the chosen coating set-up gets optimized so that a drug release nearly along the whole required term can be achieved. Preliminary in vitro studies show a good cell tolerance besides a sufficient microbial efficacy.

Made available in DSpace on 2017-07-24T19:38:01Z (GMT). No. of bitstreams: 1 dissertacao - parte liberada.pdf: 1154549 bytes, checksum: d7a9809bce797a5c245ab7132bbf659b (MD5) Previous issue date: 2009-10-27 / Several studies have demonstrated that hydroxyapatite outstands amongst bioceramics, since it presents excellent biocompatibility with human bone mineral phase, stoichiometric formula (Ca10(PO4)6(OH)2 ), with Ca/P ratio equal 1,67. It also has the capability of bonding chemically with the tissue, which makes it a bioactive material. Despite the structure and properties of bone tissue representing one of the major fields of study, nowadays the interest is in the possibility of incorporating drugs to such biomaterials. In this work, HA and HA:Gel composites were prepared through the precipitation method, which consisted of the mixture of a Ca(OH)2 (2 mol.L-1 ) solution to a H3PO4 (1,2 mol.L-1) solution, in constant agitation, heating and pH control. The precipitate obtained were put to age for a day and later one part of them was dried in oven at 100ºC and another part lyophilized for 48 hours. Powders were then sieved, calcined and had gentamicin sulfate (C21H43N5O7.2,5H2SO4) added to them in 0,25, 0,5, 1,0 e 2,0% in mass concentrations. These powders were characterized through: infrared absorption spectroscopy, X-ray diffractometry, scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), granulometric analyses, specific superficial area by the BET method, differential thermal analysis (DTA) and thermogravimetry (TG). In all samples analyzed, the mineral part was characterized as carbonated hydroxyapatite. It was observed that the morphology and crystal size are influenced by the drying method and gelatin addition. The Ca/P ratio found was above the desired one for the stoichiometric hydroxyapatite. Through the SEM and TEM analysis, the presence of agglomerates, characteristic to apatites was noticed. The samples microbiological activity evaluation with saliva, with 0,5, 1,0 e 2,0% gentamicin sulfate in mass resulted in UFC equal zero, and samples with 0,25% of the same drug in mass presented sharp decrease in the colonies development, however, only the HA:Gel dried in oven, the UFC remained equal zero. Microbiological analysis results confirm the antibiotic inhibitor action when impregnating the hydroxyapatite. / Estudos demonstram que a hidroxiapatita, vem se destacando entre as biocerâmicas, pois apresenta excelente biocompatibilidade com a fase mineral do osso humano, de fórmula estequiométrica (Ca10(PO4)6(OH)2 ), com razão Ca/P igual a 1,67. Tem a capacidade de unir-se quimicamente com o tecido, o que a torna um material bioativo. Apesar da estrutura e propriedades do tecido ósseo representar um dos maiores campos de estudo, na atualidade o interesse está na possibilidade de incorporação de drogas a esses biomateriais. Neste trabalho foram preparados compósitos de HA e HA:Gel pelo método da precipitação, que consistiu na mistura de uma solução de Ca(OH)2 (2 mol.L-1) com uma solução de H3PO4 (1,2 mol.L-1), sob agitação constante,aquecimento e controle de pH. Os precipitados obtidos foram envelhecidos por um dia e posteriormente uma parte deles foi seco em estufa à 100ºC e a outra parte foi liofilizada por 48 horas. Os pós foram peneirados, calcinados e em seguida adicionados sulfato de gentamicina (C21H43N5O7.2,5H2SO4) em concentrações de 0,25, 0,5, 1,0 e 2,0% em massa. Os pós obtidos foram caracterizados por: espectroscopia de absorção na região do infravermelho; difratometria de Raios X; microscopia eletrônica de varredura (MEV-EDS), microscopia eletrônica de transmissão (MET), análises granulométricas, área superficial específica pelo método BET; análise térmica diferencial (DTA) e termogravimetria (TG). Em todas as amostras analisadas a parte mineral foi caracterizada como hidroxiapatita carbonatada. Foi verificado que a morfologia e o tamanho dos cristais são influenciados pelo tipo de secagem e pela adição de gelatina. A razão Ca/P encontrada foi superior a desejada para hidroxiapatita estequiométrica. Através das análises de MEV e MET observa-se a presença de aglomerados, característicos as apatitas. A avaliação da atividade microbiológica das amostras frente à saliva, com impregnação de 0,5, 1,0 e 2,0% em massa de sulfato de gentamicina obteve-se a contagem de Unidade Formadora de Colônia (UFC) igual a zero, sendo que nas amostras com 0,25% em massa do mesmo antibiótico houve uma grande diminuição no crescimento de colônias, porém apenas na amostra de HA:Gel seca em estufa, a contagem de UFC, permaneceu igual a zero. Os resultados das análises microbiológicas comprovam a ação inibidora do antibiótico quando impregnados a hidroxiapatita.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-16T17:27:57Z No. of bitstreams: 1 2012 - Adriana Cristina Pedroso Ferraz.pdf: 2396072 bytes, checksum: 4c8224201e7dfca3888239f1edd74e0f (MD5) / Made available in DSpace on 2017-05-16T17:27:57Z (GMT). No. of bitstreams: 1 2012 - Adriana Cristina Pedroso Ferraz.pdf: 2396072 bytes, checksum: 4c8224201e7dfca3888239f1edd74e0f (MD5) Previous issue date: 2012-05-02 / CNPq / The research was divided into chapters. The first assessed the post-embryonic development of Chrysomya albiceps (Widemann) on chicken gizzard (control: beef). There were four replications (100g each diet, 40 1st instar/2nd generation larvae) per treatment, each recipient was placed in a larger one containing sawdust and then sealed. The mature larvae were weighed and stored in test tubes. The variation among mature larva weight means and the duration of the larva, pupa and total stages were analyzed by the Student t test (?=5%), the viabilities by ANOVA and the sex ratio by the chi-square test. The following were recorded: mean temperature 25.6oC and 72.4% relative air humidity, larva-adult period mean duration of 8.1 days (meat) and 8.2 days (gizzard); 71% to 87% larva viability; 100% and 99% pupa viability 58% and 67% larva and adult viabilities, respectively. Chicken gizzard was shown to be satisfactory as diet for C. albiceps. The second chapter assessed the post embryonic development of Chysomya putoria (Widemann) in chicken gizzard and homogenized chicken gizzard in 65% agar (control: meat). Four replications (60 mL diet, 40 1st instar/5th generation larvae) were made per treatment. The homogenate was prepared in a mixer (gizzard, distilled water and agar). A mean temperature of 20.6 oC and 67.7% relative air humidity were recorded. The mean duration of the larva-adult period was 8.868 days (meat), 8.676 days (gizzard) and 9.067 days (homogenate); the larva viability was 98%; 92% and 73%; the pupa viability was 98%; 91% and 71%; the larva and adult viabilities were 93%; 83% and 64%, respectively. There were significant difference in the duration of the pupa period between meat and the homogenate. Both diets were shown to be satisfactory for C. putoria. The third chapter assessed different ciprofloxacin concentrations (3.33 ?g/mL; 6.66 ?g/mL and 13.33 ?g/mL in gizzard/65% agar homogenate) on C. putoria development (the control received distilled water). They were replicated four times (60 grams diet, 40 1st instar/3rd generation larvae) in an acclimatized chamber 30oC day/28 oC night, 70+10%relative air humidity and 14-hour photoperiod. There was no significant difference for: mean individual larva weight, mean duration of the larva inoculation until abandonment and the larva, pupa and total stages. Only treatment 2 differed significantly from the control in the larval and total viability. Ciprofloxacin seemed not to alter C. putoria development. The fourth chapter assessed different gentamicin concentrations (4.44?g/mL; 13.33?g/mL and 66.66?g/mL) on C. putoria. The materials and methods were similar to those of chapter III. There was no significant difference for: mean individual larva weight, mean duration of the larva inoculation until abandonment and the larva, pupa and total stages. Only treatment 2 differed significantly from the control for larva viability. Gentamicin seemed not to alter C. putoria development. The fifth chapter assessed different ampicillin concentrations (66?g/mL; 81.33?g/mL and 166.66?g/mL) on C. putoria. The materials and methods were similar to chapter III. There was no significant difference for: mean individual larva weight, mean duration of the larvae inoculation until abandonment and the larval, pupa and total stages. There was no significant difference for larva and total viability, but pupa viability in T1 differed significantly from the control and T2, and T3 differed from the control. Ampicillin seemed not to alter C. putoria development. / A pesquisa foi dividida em cap?tulos. O primeiro avaliou o desenvolvimento p?s-embrion?rio de Chrysomya albiceps (Widemann) em moela de frango (controle: carne bovina). Foram quatro repeti??es (100g de dieta cada, 40 larvas de 1? instar/2? gera??o) por tratamento, cada recipiente inserido em outro maior contendo serragem e vedado. As larvas maduras foram pesadas e armazenadas em tubos de ensaio. A varia??o entre m?dias de massa de larvas maduras e dura??es dos est?gios de larva, pupa e total foram analisadas por Teste t de Student (?=5%), as viabilidades por ANOVA, a raz?o sexual pelo qui-quadrado. Foram registradas temperatura m?dia 25,6?C e umidade relativa do ar m?dia 72,4%; dura??o m?dia do per?odo de larva a adulto 8,1 dias (carne) e 8,2 (moela); viabilidades de larva 71% e 87%; viabilidades de pupa 100% e 99%; viabilidades de larva a adulto 58% e 67%, respectivamente. Moela de frango se mostrou satisfat?ria como dieta para C. albiceps. O segundo cap?tulo avaliou desenvolvimento p?s-embrion?rio de Chysomya putoria (Widemann) em moela e homogenato de moela de frango em agar 65% (controle: carne). Foram quatro repeti??es (60 mL de dieta, 40 larvas de 1? instar/5?gera??o) por tratamento. O homogenato foi preparado em mixer (moela, ?gua destilada e agar). Foram registradas temperatura m?dia 20,6? C e umidade relativa do ar m?dia 67,7%. A dura??o m?dia do per?odo de larva a adulto foi 8,868 dias (carne), 8,676 (moela) e 9,067 (homogenato); as viabilidades larvais 98%; 92% e 73%; as viabilidades de pupa 98%; 91% e 71%; as viabilidades de larva a adulto 93%; 83% e 64%, respectivamente. Houve diferen?a significativa na dura??o do per?odo pupal entre carne e homogenato. Ambas dietas mostraram-se satisfat?rias para C. putoria. O terceiro cap?tulo avaliou diferentes concentra??es de ciprofloxacino (3,33 ?g/mL; 6,66 ?g/mL e 13,33 ?g/mL em homogenato de moela/agar 65%) sobre desenvolvimento de C. putoria (controle recebeu agua destilada). Foram replicados quatro vezes (60 gramas dieta, 40 larvas 1? ?nstar/3? gera??o) em c?mara climatizada 30?C dia/28?C noite, 70+10% U.R. e 14 horas fotoper?odo. N?o houve diferen?a significativa: massa individual m?dio das larvas, dura??o m?dia da inocula??o das larvas at? abandono e est?gios larval, pupal e total. Apenas tratamento 2 diferiu significativamente do controle nas viabilidades larval e total. Ciprofloxacino pareceu n?o alterar desenvolvimento de C. putoria. O quarto cap?tulo avaliou diferentes concentra??es de gentamicina (4,44?g/mL; 13,33?g/mL e 66,66?g/mL) sobre C. putoria. Os materiais e m?todos foram semelhantes ao do cap?tulo III. N?o houve diferen?a significativa: massa individual m?dia das larvas; dura??o m?dia da inocula??o das larvas at? abandono e dos est?gios larval, pupal e total. Apenas tratamento 2 diferiu significativamente do controle na viabilidade larval. Gentamicina pareceu n?o alterar o desenvolvimento de C. putoria. O quinto cap?tulo avaliou diferentes concentra??es de ampicilina (66?g/mL; 81,33?g/mL e 166,66?g/mL) sobre C. putoria. Os materiais e m?todos foram semelhantes ao cap?tulo III. N?o houve diferen?a significativa: massa individual m?dia das larvas, dura??o m?dia da inocula??o das larvas at? abandono e est?gios larval, pupal e total. N?o houve diferen?a significativa: viabilidades larval e total, por?m viabilidade pupal do T1 diferiu significativamente do controle e T2, e T3 diferiu do controle. Ampicilina pareceu n?o alterar desenvolvimento de C. putoria.

In cardiac surgery, sternal wound infection (SWI) continues to be one of the most serious postoperative complications. Coagulase-negative staphylococci (CoNS) have become the most common causative agents of SWI. Prophylaxis with intravenous beta-lactam antibiotics (cephalosporins or in Sweden most commonly isoxazolyl penicillins) is routinely practised. However, many CoNS species are resistant to beta-lactam antibiotics. Vancomycin is often the only effective antibiotic available for treatment of these infections, but its use for routine prophylaxis is strongly discouraged because of the risk of increasing the selection of resistant bacteria. The aim of this work was to develop and evaluate a new technique for antibiotic prophylaxis in cardiac surgery consisting of application of drug eluting collagen-gentamicin sponges in the sternal wound in addition to conventional intravenous antibiotics. The antibiotic concentrations in the wound and serum achieved by routine intravenous dicloxacillin prophylaxis and those after application of local collagen-gentamicin in the sternal wound were investigated. These studies showed dicloxacillin levels adequate for prevention of infections by methicillin-susceptible staphylococci, and extremely high gentamicin levels in the wound fluid, during the first 8-12 hours postoperatively with the local application. Two thousand cardiac surgery patients were then randomised to routine prophylaxis with intravenous isoxazolyl penicillin alone (control group) or to this prophylaxis combined with application of collagen-gentamicin (260 mg gentamicin) sponges within the sternotomy before wound closure. The primary end-point was any sternal wound infection within two months postoperatively. Evaluation was possible in 983 and 967 patients in the treatment and control groups, respectively. The incidence of any sternal wound infection was 4.3% in the treatment group and 9.0 % in the control group (relative risk = 0.47, (95% confidence interval 0.33 to 0.68); P<0.001). The most common microbiological agents were CoNS, followed by Staphylococcus aureus. Local gentamicin reduced the incidence of SWIs caused by all major, clinically important microbiological agents except Propionibacterium acnes. Assignment to the control group, high body mass index, diabetes mellitus, younger age, single or double internal mammary artery, left ventricular ejection fraction less than 35% and longer operation time were independent risk factors for SWI in a multivariable risk factor analysis. In patients with additional sternal fixation wires (> six wires) the collagen-gentamicin prophylaxis was associated with an approximately 70 % reduction in the incidence of SWI at all depths and the application of collagen sponges between sternal halves may require particular attention regarding the stability of fixation. A cost effectiveness analysis showed that the application of local collagen-gentamicin as prophylaxis was dominant, i.e. resulted in both lower costs and fewer wound infections. Routine use of the described prophylaxis in all adult cardiac surgery patients could be recommended.

Exposure to antibiotics is an important factor influencing the development of bacterial resistance.  In an era where very few new antibiotics are being developed, a strategy for the development of optimal dosing regimen and combination treatment that reduces the rate of resistance development and overcome existing resistance is of utmost importance. In addition, the optimal dosing in subpopulations is often not fully elucidated. The aim of this thesis was to develop pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PKPD) models that characterize the interaction of antibiotics with bacterial growth, killing and resistance over time, and can be applied to guide optimization of dosing regimens that enhance the efficacy of mono- and combination antibiotic therapy. A mechanism-based PKPD model that incorporates the growth, killing kinetics and adaptive resistance development in Escherichia coli against gentamicin was developed based on  in vitro time-kill curve data. After some adaptations, the model was successfully applied for similar data on colistin and meropenem alone, and in combination, on one wild type and one meropenem-resistant strain of Pseudomonas aeruginosa. The developed population PK model for colistin and its prodrug colistin methanesulfonate (CMS) in combination with the PKPD model showed the benefits for applying a loading dose for this drug. Simulations predicted the variability in bacteria kill to be larger between dosing occasions than between patients. A flat-fixed loading dose followed by an 8 or 12 hourly maintenance dose with infusion duration of up to 2 hours was shown to result in satisfactory bacterial kill under these conditions. Pharmacometric models that characterize the time-course of drug concentrations, bacterial growth, antibacterial killing and resistance development were successfully developed. Predictions illustrated how PKPD models based on in vitro data can be utilized to guide development of antibiotic dosing, with examples advocating regimens that (i) promote bacterial killing and reduce risk for toxicity in preterm and term newborn infants receiving gentamicin, (ii) achieve a fast initial bacterial killing and reduced resistance development of colistin in critically ill patients by application of a loading dose, and (iii) overcome existing meropenem resistance by combining colistin and meropenem

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina, azitromicina, claritromicina, eritromicina, levofloxacino, gentamicina, doxiciclina, cloranfenicol, clindamicina e vancomicina. Para Pseudomonas aeruginosa foram testados os mesmos, exceto rifampicina e vancomicina. A discrepância entre MIC e MBIC foi muito grande para vários fármacos, mostrando a necessidade de se avaliar estes parâmetros antes do início da farmacoterapia para uma escolha correta, especialmente em hospitais. Os fármacos que apresentaram melhores resultados foram a rifampicina e os macrolídeos, enquanto que os menos efetivos foram vancomicina e clindamicina. Isso foi atribuído ao perfil lipofílico, porém com alguma solubilidade em água das melhores moléculas. Em contra ponto, a elevada área polar, complexidade e massa molar foram características negativas para a penetração em biofilme, resultando numa ineficácia para essas moléculas. Além disso, também foi avaliado o tratamento de polímeros plásticos com EDTA, obtendo-se redução significativa da produção de biofilme nas placas tratadas com o agente químico. / The use of central venous catheters in medicine has brought benefits to the patients and represents a great advance in clinical practice, while on the other hand this device is related to an increase in the incidence of infections caused by multiresistant pathogens. Furthermore, frequently, the catheters get colonized by biofilm producing bacteria. These microorganisms adhere to the abiotic material of the catheters keeping themselves protected underneath the exopolysaccharide matrix of biofilm, this way the immune system and antimicrobials are incapable to fulfill their action and, many times, are unable to reach internal bacteria. This fact is explained by the fact that many of the biological and pharmacological agents have physical-chemical properties incompatible with the penetration into the matrix. Aiming to determine which antimicrobials are suitable for using when dealing with a biofilm producing microorganism and which physical-chemical properties are directly related to the agent penetration into the polysaccharide matrix, we used colorimetric method with crystal violet to obtain biofilm minimum inhibitory concentration (MBIC) and the already standardized method for minimum inhibitory concentration (MIC). To accomplish these 10 antimicrobials were tested in Staphylococcus spp.: rifampin, azithromycin, clarithromycin, erythromycin, levofloxacin, gentamicin, doxycycline, chloramphenicol, clindamycin and vancomycin. For Pseudomonas aeruginosa all antimicrobials except for rifampin and vancomycin were included. There was a great difference between MIC and MBIC for many drugs, showing the need to evaluate these parameters before beginning treatment. The drugs with better results were rifampin and macrolides, while the worse were vancomycin and clindamycin, which can be attributed to the lipophilic profile with some water solubility present in the molecules with better results. The characteristics associated with poor penetration into biofilm were high polar surface area, complexity e molecular weight. Furthermore, the previous treatment of the plastic polymers with EDTA was accessed resulting in statistically significant reduction of biofilm production.

Orientador: Humberto Santo Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:31:35Z (GMT). No. of bitstreams: 1 Perez_PaulaSpanopoulos_M.pdf: 4055081 bytes, checksum: 1297b13fc85bb6918c3785c93466dff5 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

There is a significant current need to elucidate the molecular mechanisms of the side-effects caused by widely-used pharmaceuticals. Examples include the acute nephrotoxicity and irreversible ototoxicity promoted by the cationic drugs gentamicin and cisplatin. Gentamicin is an aminoglycoside antibiotic used for the prevention and treatment of life-threatening gram-negative bacterial infections, such as tuberculosis and meningitis. Cisplatin is used to treat a broad spectrum of cancers including head and neck, ovarian, cervical, stomach, bladder, sarcoma, lymphoma, testicular cancer and others. The objective of this study is to design and synthesize rhodamine derivatives that can be used for the construction of geometrically well-defined cationic drug conjugates. The long-term goal is to use the conjugates as tools to aid in elucidating the properties and identities of ion channels involved in the uptake of cationic pharmaceuticals into kidney and cochlear hair cells. This will shed light on the origin and potential prevention of unwanted side effects such as nephrotoxicity and ototoxicity associated with specific cationic drugs. A series of extended rhodamine analogs with reactive groups for biomolecule conjugation has been synthesized. These fluorophores show similar spectral properties to their prototype, Texas Red succinimidyl ester (TR-SE). However, they contain rigid linkers between the fluorophore and amine-reactive moiety. The resultant gentamicin conjugates of these materials are rigidified enabling one to assess channel pore dimensions without the confounding issue of conjugate folding. Preliminary cell studies are promising, as one observes reduced gentamicin uptake in both kidney and sensory hair cell upon systematically increasing the dimension of the fluorophore. This work has enabled us to tentatively assign the maximum dilated MET channel pore size as between 1.44 nm to 1.56 nm. However, this preliminary finding, though encouraging, needs further validation via ongoing studies with larger diameter fluorophore conjugates, A cisplatin-Texas Red conjugate has also been synthesized to enable studies of cellular uptake mechanisms. This conjugate preserves not only the spectral properties of Texas Red after conjugation, but also the cytotoxicity of cisplatin. This has been validated in zebrafish. The series of rhodamine probes that have been conjugated to gentamicin should be similarly useful for cisplatin studies. These studies are planned. Additional future work includes the synthesis of semi-flexible (glycol) and flexible (alkyl) linkers to evaluate structure-activity relationships.