The study felld into three sections. The first section that a liquid chromatography method with fluorescence detection was developed for simultaneous determination of 11 quinolones (QNs; marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in chicken, pork, fish and shrimp. The analytes were extracted with 0.3% metaphosphoric acid: acetonitrile (1:1, v/v), followed by a HLB cartridge clean-up procedure. The HPLC separation was carried out on a symmetry column C18 (250 mm x 4.5 mm i.d., 5 £gm) with linear gradient elution of 0.1% formic acid: acetonitrile as mobile phase and programmable fluorescence detection. The method was validated by spiking blank animals tissues at three different levels (25, 50 and 250 ng/g; except 6.25, 12.5 and 62.5 ng/g for DAN) and linearity, detection limit, quantification limit, precision and accuracy were checked. Mean recoveries of 11 QNs from edible animal tissues were 71.7-105.3%. The limits of quantification in different muscle tissues ranged from 5.0 to 28.0 ng/g. The results showed it was simple, rapid, sensitive and suitable for routine test. The second section that a LC-ESI-MS/MS method was developed for determining 18 (fluoro)quinolone (QNs) residues in milk, chicken, pork, fish and shrimp. This method is capable of screening and confirming the presence of 12 amphoteric QNs (marbofloxacin, norfloxacin, enrofloxacin, ciprofloxacin, desethylene ciprofloxacin, lomefloxacin, danofloxacin, sarfloxacin, difloxacin, ofloxacin, orbifloxacin and enoxacin) and 6 acidic QNs (oxolinic acid, nalidixic acid, flumequine, cinoxacin, piromidic acid and pipemidic acid). The drugs were extracted from matrix using acetonitrile with 1% formic acid, diluted in 10% acetonitrile and defatted by extraction with hexane. The LC separation was conducted on a XDB C8 (150 x 4.6 mm, 5£gm) column with gradient elution of 20 mM ammonium formate with 0.1% formic acid¡Vacetonitrile as the mobile phase. Mass spectral acquisition was completed in the positive ion mode by applying multiple reaction mode (MRM). The decision limit (CC£\) and detection capability (CC£]) stated in the Decision No. 2002/657/EC and the ISO standard No.11843, has been calculated in the case of the nonauthorized substance. The values of CC£\ ranged from 0.18 to 0.68 ng/g and CC£] ranged from 0.24 to 0.96 ng/g under specified conditions. The third section that the pharmacokinetics of ENR and its active metabolite (CIP and des-CIP) were estimated in tilapia after intravenous (i.v.) and oral (p.o.) administration of a single dose of 2.5 and 10 mg/kg body weigh, respectively. At prefixed time points, from 0.25 h to 7 days after administration, whole blood and main tissue (liver, kidney, bile and muscle) from 4 individuals in each were collected. The concentration of ENR and its active metabolites in the main tissue were simultaneously detected by LC/MS/MS method. Limited of quantitation (LOQ) of this method were 0.01£gg/g. Pharmacokinetic parameters from both routes were described to have a two- compartment open model with first-order elimination. After i.v. administration, the area under the drug concentration-time (AUC), elimination half-life (t1/2£]), maximum plasma concentration (Cmax ), total body clearance (Cltot) and apparent volume of distribution at steady-state (Vss) of ENR were 109.6 ¡Ó 31.33 £gg.h/mL, 55.17 ¡Ó 22.84 h, 4.70 ¡Ó 0.36 £gg/mL, 14.82 ¡Ó 4.24 L/h/kg, 1105 ¡Ó 223.40 L/kg ,respectively. After oral administration, the AUC , t1/2£], Tmax , Cmax of ENR were 599.42 ¡Ó 76.19£gg.h/mL , 75.95 ¡Ó 12.94 h, 0.601¡Ó0.06h, 9.75 ¡Ó 0.46£gg/mL, respectively. After p.o. administration, CIP could be detected in liver, kidney and bile. Regarding des-CIP, the main active metabolite of CIP, could be detected in 120¡ã168 h bile among tissue. ENR and CIP had significance enterohepatic cycle in Tilapia and easily accumulated in bile. It seems reasonable to explain the phenomenon of ENR and CIP maintenance of high concentration in blood and muscle during the test time.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-06-22T13:49:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE DOUTORADO MARIA CECILIA SANTOS C MELO.pdf: 1417870 bytes, checksum: b618daedc0a0d5458d1a1e355c421b86 (MD5) / Made available in DSpace on 2016-06-22T13:49:01Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE DOUTORADO MARIA CECILIA SANTOS C MELO.pdf: 1417870 bytes, checksum: b618daedc0a0d5458d1a1e355c421b86 (MD5) Previous issue date: 2015-12-23 / CAPEs / Objetivo: Investigar as alterações dos encéfalos e olhos de conceptos recém-nascidos de ratas que se submeteram a peritonite fecal autógena e avaliar as respostas com a intervenção de dois esquemas terapêuticos com moxifloxacino-dexametasona e meropenem realizados durante a prenhez. Métodos: Foram incluídas, de forma aleatória, 30 ratas Wistar para acasalamento. Dessas, 15 ratas tiveram esfregaço vaginal positivo para prenhez. O experimento foi realizado em duas fases: 1. Desenvolvimento do modelo experimental: As ratas foram distribuídas em : AGrupo estudo com cinco ratas após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de três ml/Kg no nono dia de prenhez, B- Grupo estudo com cinco ratas após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez e C- grupo de cinco ratas prenhes sem peritonite fecal autógena (controle). Após o parto, foi realizada a eutanásia e o inventário das cavidades abdominal e torácica das ratas dos grupos estudo A e B e os dados de seus conceptos. 2.Intervenção com esquemas terapêuticos: Grupo1: Duas ratas prenhes após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez que receberam moxifloxacino - dexametasona intraperitoneal em 48 e 72 horas e Grupo 2: Duas ratas prenhes após peritonite fecal autógena com suspensão de fezes a 10 %, na dose de quatro ml/Kg no nono dia de prenhez que receberam meropenem intravenoso em 48 e 72 horas. Após o parto, foi realizada a eutanásia e o inventário das cavidades abdominal e torácica destas e a decapitação com inspeção do crânio, da consistência dos cérebros e os olhos de todos os conceptos. O projeto foi aprovado pelo Comitê de Ética da Faculdade de Ciências Médicas de Campina Grande- Paraíba. P ≤ 0.05 foi usado para rejeição da hipótese de nulidade. Resultados: O modelo de peritonite fecal autógena selecionado foi o que utilizou a dose de quatro ml/Kg da suspensão de fezes a 10%. Das 15 ratas com esfregaço vaginal positivo dez estavam prenhas, sendo três no grupo A, quatro no grupo B e três no grupo C. Os cérebros dos conceptos das ratas que receberam quatro ml/kg da suspensão de fezes a 10% se mostraram, significantemente, menores e com consistência menos firme que aqueles do grupo controle, assim como, comparados com os das intervenções terapêuticas. Cataratas congênitas foram observadas em nove de 26 (34,6%) conceptos das ratas que receberam quatro ml/Kg da suspensão de fezes a 10% e não receberam intervenção terapêutica, sendo sete bilateral e dois unilateral. Nenhuma catarata congênita foi observada nos 20 recém-nascidos das ratas que receberam a combinação de moxifloxacino - dexametasona intraperitoneal. Catarata congênita foi observada em três (13.6%) dos 22 recém-nascidos das ratas que receberam meropenem intravenoso. Conclusões: Septicemia gestacional em ratas pode produzir alteração cerebral e catarata congênita nos conceptos. Com a utilização dos esquemas terapêuicos, nas ratas prenhas submetidas à peritonite fecal autógena com a dose de quatro ml/Kg da suspensão de fezes a 10%, houve menor número de casos de conceptos com alterações encefálicas e oculares. / Purpose: To investigate the abnormalities of brains and eyes of newborn fetuses of rats that underwent autologous fecal peritonitis and evaluate the responses with the intervention of two treatment regimens with moxifloxacin and meropenem - dexamethasone performed during pregnancy. Methods: Randomly, 30 Wistar rats for mating. Of these, 15 rats had a positive vaginal smear for pregnancy. The experiment was conducted in two phases: 1. Development the experimental model: The rats were divided into: Group A- study with five rats after fecal peritonitis autogenous with faeces of a 10% suspension at a dose of three ml/kg on the ninth day of pregnancy, group B study with five rats after fecal peritonitis autogenous with faeces of a 10% suspension at a dose four ml/kg on the ninth day of pregnancy and C-group of five pregnant rats without autologous fecal peritonitis (control). After delivery, euthanasia and inventory of abdominal and thoracic cavities of study groups A and B and the data of their fetuses. 2.Intervention with therapeutic regimens : Group 1 : Two pregnant rats after fecal peritonitis autogenous suspension of faeces to 10 % at a dose four ml / kg on the ninth day of pregnancy who received moxifloxacin - intraperitoneal dexamethasone at 48 and 72 hours and Group 2 : Two pregnant rats, autogenous after peritonitis with stool suspension at 10 % in doses four ml / kg on the ninth day of pregnancy who received intravenous meropenem in 48 and 72 hours. After delivery, euthanasia and inventory of abdominal and chest cavities of these and the beheading with skull inspection was carried, the consistency of the brain and the eyes of all fetuses . The project was approved by the Ethics Committee of the Faculty of Medical Sciences of Campina Grande - Paraíba. P ≤ 0.05 was used to reject the null hypothesis. Results: The fecal peritonitis autogenous model selected was that using 10% faeces suspension at a dose four ml/kg. Of the 15 rats with positive vaginal smear ten were pregnant, three in group A, four in group B and three in group C. The brains of fetuse of rats given four ml/kg of 10% faeces suspension is shown, significantly, smaller and less firm consistency than those of the control group, as compared with the therapeutic interventions. Congenital cataract was observed in nine of 26 (34.6%) fetuses of rats given four ml/kg of 10% faeces suspension received no therapeutic intervention, seven bilateral and two unilateral. No congenital cataract was observed in 20 newborns of rats given the combination of intraperitoneal moxifloxacin - dexamethasone. Congenital cataract was observed in three (13.6%) of 22 infants of rats given intravenous meropenem. Conclusions: Gestational septicemia in rats can produce brain abnormalities and congenital cataracts in fetuses. With the use of treatment regimens, in pregnant rats submitted autologous fecal peritonitis with a dose of four ml/kg of 10% faeces suspension, there were few cases of fetuses with brain and eye abnormalities.

学位プログラム名: 京都大学大学院思修館 / 京都大学 / 0048 / 新制・課程博士 / 博士(総合学術) / 甲第21231号 / 総総博第3号 / 新制||総総||1(附属図書館) / 京都大学大学院総合生存学館総合生存学専攻 / (主査)教授 竹本 佳司, 特定教授 大嶌 幸一郎, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Philosophy / Kyoto University / DFAM

El objetivo principal del estudio fue establecer el nivel de resistencia a antimicrobianos en un total de 222 cepas comensales de E. coli de origen fecal, en Perú. Las frecuencias de resistencia encontrados, frente los antimicrobianos evaluados, fueron: ampicilina (62,6%), cotrimoxazol (48,6%), tetraciclina (43,0%) y cloranfenicol (15,8%). Destacan los elevados niveles de resistencia a quinolonas: 32% al ácido nalidíxico (NAL) y 12% a ciprofloxacino (CIP). Estos elevados niveles hacia las quinolonas en cepas comensales aisladas en niños de esta franja de edad, realzan el uso extendido y el impacto de consumo de este tipo de antimicrobianos en la comunidad, mostrando el riesgo potencial de su pérdida de utilidad en el área. / The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. Coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area.

Enterobactérias produtoras de AmpC cromossômica, especialmente Citrobacter, Serratia, Providencia, Proteus e Morganella, entre outros, são patógenos oportunistas e estão implicados em infecção relacionada a assistência à saúde. Uma vez que mecanismos de resistência adquiridos a antibióticos são cada vez mais frequentemente encontrados nesses micro-organismos, o gerenciamento das infecções causadas por eles tem sido desafio para a escolha da antibioticoterapia, pois existem poucos dados fenotípicos e moleculares sobre essas espécies. O objetivo deste trabalho foi a investigação de genes de resistência adquiridos (mediados por plasmídeos) aos antibióticos beta-lactâmicos de amplo espectro e quinolonas, bem como a determinação da epidemiologia molecular das enterobactérias produtoras de AmpC cromossômica, isoladas de pacientes ambulatoriais e internados em hospital universitário. Foram estudadas e comparadas bactérias isoladas em 2007 e 2016 durante o período de cinco meses em cada ano. Foi investigada fenotipicamente a produção de ESBL e AmpC associadas à resistência aos beta-lactâmicos de amplo espectro. Adicionalmente, também foram pesquisados genes de resistência adquiridos aos beta-lactâmicos de amplo espectro e quinolonas tão bem como plasmídeos carreando tais genes. Foram encontrados dois genes blaSHV-5 e um blaCTX-M-2, além de, um gene qnrS2, dois qnrB6, dezenove genes qnrD1 e vinte e um aac(6\')-Ib, sendo que desses oito apresentaram a variante aac(6\')-Ib-cr. O gene qnrD1 já estava presente no hospital estudado antes do primeiro relato do gene no Brasil. Sequenciamento de plasmídeos carreando gene qnrD1 mostra que pelo menos dois plasmídeos distintos estão envolvidos em sua disseminação. Por PFGE foi possível observar que não houve disseminação clonal dos isolados bacterianos no hospital nos períodos estudados. Foi determinada a epidemiologia molecular comparativa das bactérias do estudo. Este conhecimento torna-se fundamental para que, haja informações consistentes sobre as bactérias do estudo, fornecendo subsídio para o tratamento dos pacientes e contribuindo para o melhor prognóstico e gerenciamento das infecções bacterianas. / Enterobacteria producing chromosomal AmpC, especially Citrobacter, Serratia, Providencia, Proteus and Morganella, among others, are opportunistic pathogens implicated in nosocomial infections. Since antibiotic resistance mechanisms are increasingly found in these microorganisms, the management of infections caused by them has been challenging on choosing the antibiotic therapy, as there are few phenotypic and molecular data on these species. The aim of this study was the investigation of acquired (plasmid-mediated) resistance genes to broad-spectrum beta-lactam antibiotics and quinolones, as well as the determination of the molecular epidemiology of chromosomal AmpC-producing enterobacteria isolated from outpatients and inpatients in a university education hospital. Bacteria isolated in 2007 and 2016 during the five-month period each year were studied and compared. The production of ESBL and AmpC associated with resistance to broad-spectrum beta-lactams was phenotypically investigated. In addition, acquired resistance genes from broad-spectrum beta-lactams and quinolones were also screened as well as plasmids carrying such genes. Two blaSHV-5 genes and one blaCTX-M-2 were found, in addition to one qnrS2, two qnrB6, nineteen genes qnrD1 and twenty-one aac(6\')-Ib, of which eight presented the aac(6\')-Ib-cr variant. qnrD1 gene was already present in the hospital studied before the first report of such gene in Brazil. Sequencing of plasmids carrying qnrD1 gene shows that at least two distinct plasmids are involved in its dissemination. Through PFGE it was possible to observe that there was no clonal dissemination of the bacterial isolates in the hospital during the periods studied. Comparative molecular epidemiology of the bacteria in the study was determined. This knowledge becomes critical for consistent information about the bacteria in the study, providing subsidy for the treatment of patients and contributing to the better prognosis and management of bacterial infections.

As grandes quantidades de antimicrobianos utilizados na produção de aves e suínos é um problema de saúde pública em todo mundo. O surgimento e disseminação de novos mecanismos de resistência a antibióticos de importância clínica em medicina humana e veterinária é fato que tem apontado a entrada de uma era \"pós-antibióticos\". Considerando a importância da avicultura e suinocultura na economia brasileira e os riscos para saúde humana e dos animais, o objetivo deste estudo foi determinar a ocorrência de genes emergentes de resistência aos antibióticos das classes dos β-lactâmicos, quinolonas, fosfomicina e colistina. Foram selecionados 1.152 isolados de Escherichia coli de origem clínica, fecal e de carne de suínos, frangos de corte, poedeiras e perus proveniente dos principais estados produtores e exportadores do Brasil (SP, RS, SC, PR, MG e GO). Através de métodos clássicos de biologia molecular e sequenciamento do genoma completo foi realizada a caracterização genotípica dos isolados, assim como a caracterização dos elementos genéticos móveis que carreavam esses genes. Dentre os isolados de aves (n=773), 103 (13,4%) foram produtores de ESBL: CTX-M-2 (n=61); CTX-M-55 (n=26); CTX-M-8 (n=12); CTX-M-2 + CTX-M-55 (n=3); CTX-M-2+CTX-M-8 (n=1); CTX-M-8+CTX-M-55 (n=1). 44 (5,7%) apresentaram CMY-2, presente em plasmídeos IncI1/ST12 e IncK. Em relação à resistência às quinolonas e colistina mediada por plasmídeos, os genes encontrados foram qnrA1 (1%), qnrB19 (6%) e qnrS1 (0,8%), além de 41 isolados positivos para mcr-1. A ocorrência de genes plasmidiais de resistência à fosfomicina ( fosA3) foi de 3,4%, que estiveram relacionados a plasmídeos epidêmicos IncN/F33:A-:B-. Dentre os isolados de E. coli patogênicas (ETEC, STEC), comensais e de carne de suínos ( n =378) foi encontrada uma alta prevalência de cepas positivas para mcr-1 (33,8%). Foram encontrados três isolados positivos para mcr-3 dentre os isolados de ETEC e comensais. O gene qnrS1 também teve alta prevalência (24,6%). Nos isolados de aves e suínos, o gene mcr-1 esteve presente em plasmídeos IncX4. Análises da sequência completa de DNA de vários plasmídeos mostrou relação com plasmídeos que circulam em criações animais na Ásia. Este estudo contribui para o estabelecimento de um cenário em relação à resistência antimicrobiana na produção animal no Brasil. Levando em conta que o Brasil está entre os maiores exportadores de carne de aves e suínos do mundo, é urgente a elaboração de estratégias para o controle da disseminação de bactérias resistentes a antibióticos clinicamente importantes. / The large amounts of antimicrobials used in the poultry and swine production is a public health concern worldwide. The emergence and spread of novel resistance mechanisms to important antibiotics in human and veterinary medicine is a fact that has pointed to entry into a \"post-antibiotic\" era. Considering the importance of poultry and pig farming in the Brazilian economy and the risks to human and animal health, the aim of this study was to determine the occurrence of emerging resistance genes to β-lactam, quinolones, fosfomycin and colistin antibiotics. A total of 1.152 of clinical, fecal and retail meat isolates of Escherichia coli from from swine, broilers, laying hens and turkeys from the main producing and exporting states of Brazil (SP, RS, SC, PR, MG and GO) were selected. Through classical molecular biology methods and whole genome sequencing, the characterization of the isolates was performed, as well as the characterization of the mobile genetic elements that carried these genes. Among avian isolates (n = 773), 103 (13.4%) were ESBL-producers: CTX-M-2 (n = 61); CTX-M-55 (n = 26); CTX-M-8 (n = 12); CTX-M-2 + CTX-M-55 (n = 3); CTX-M-2 + CTX-M-8 (n = 1); CTX-M-8 + CTX-M-55 (n = 1). 44 (5.7%) showed pAmpC CMY-2, present in IncI1/ST12 and IncK plasmids. An isolate belonging to ST117 harboring bla CMY2 and bla CTX-M-2 integrated into the chromosome. Regarding plasmid-mediated resistance to quinolones and colistin, the genes found were qnrA1 (1%), qnrB19 (6%) and qnrS1 (0.8%), in addition to 41 mcr-1 positive isolates. The occurrence of plasmid-mediated fosfomycin resistance ( fosA3) was 3.4%, which were related to the IncN/F33: A-: B- epidemic plasmids. A high prevalence of mcr-1 positive strains (33.8%) was found among pathogenic (ETEC, STEC) and commensal porcine E. coli isolates (n = 378). Three mcr-3 positive isolates were found among the ETEC and commensal isolates. The qnrS1 gene also had a high prevalence (24.6%). In avian and porcine isolates, the mcr-1 gene was present in IncX4 plasmids. Analyzes of the complete DNA sequence of various plasmids showed relation to plasmids circulating in animal production in Asia. This study contributes to the determination of a national scenario of antimicrobial resistance in animal production in Brazil. Taking into account that Brazil is among the largest exporters of poultry and pork in the world, it is urgent to devise strategies to control the spread of multidrug resistant bacteria to clinically important antibiotics.

DE LA TESIS:Desde la aparición de las quinolonas, las resistencias bacterianas a las mismas han evolucionado de forma paralela. Conforme han ido apareciendo nuevas moléculas con mayor capacidad bactericida y mejores parámetros farmacodinámicos y farmacocinéticos, también se han ido identificando nuevos y más sofisticados mecanismos de resistencia, que se han ido adaptando a las necesidades de cada momento. Es por ello que se hace necesaria una nueva metodología para el diseño y la síntesis de nuevos agentes antibacterianos con el objetivo de aumentar su potencia antibacteriana y, al mismo tiempo, disminuir la probabilidad de generar a corto plazo nuevos mecanismos de resistencia. Con este fin, es fundamental conocer todos los mecanismos de resistencia y cuál es, en detalle, su mecanismo de acción, para poder diseñar nuevas moléculas capaces de, manteniendo su capacidad bactericida, eludir dichos mecanismos.En esta tesis nos propusimos como objetivos fundamentales, en primer lugar, investigar las bases moleculares de los mecanismos de resistencia a quinolonas en bacterias Gram-negativas. En concreto, en Escherichia coli, Yersinia enterocolitica y Citrobacter freundii. Por otro lado, se llevaron a cabo cálculos de acoplamiento o "docking" mediante la utilización de programas informáticos con el fin de incrementar nuestros conocimientos respecto al modelo de interacción entra ADN-ADN girasa-quinolona, para de esta manera mejorar nuestra comprensión de los mecanismos de resistencia a fluoroquinolonas asociados a las mutaciones más comúnmente encontradas en el gen gyrA. Finalmente, el último de los objetivos que nos propusimos fue el de diseñar, sintetizar y evaluar diferentes derivados de ciprofloxacino y norfloxacino con el fin de encontrar entre ellos alguno que tuviera la capacidad de interaccionar con la enzima ADN girasa, poseyendo ésta uno o dos cambios aminoacídicos en su subunidad A.

Las quinolonas son un grupo de antimicrobianos sintéticos, con un amplio espectro de acción y utilizados con gran éxito para el tratamiento de muy diversas patologías. Es también un grupo en plena fase de desarrollo, donde continuamente están apareciendo nuevas moléculas más activas que las preexistentes en muchos casos.Lamentablemente, la resistencia a quinolonas presenta entre la mayoría de sus moléculas lo que se conoce como resistencia cruzada, o susceptibilidad reducida. Por ello y para poder desarrollar nuevas moléculas dentro de esta familia es importante conocer cuales son los mecanismos de resistencia que presentan los microorganismos, en concreto las bacterias Gram-positivas, frente a estos compuestos. De este modo se podrían diseñar nuevas moléculas que no se vean afectadas por los mecanismos de resistencia ya conocidos. Otro de los parámetros importantes es conocer como las propias quinolonas seleccionan la aparición de mutantes resistentes.Por todos estos motivos, para el presente trabajo se plantearon los siguientes objetivos:1- Investigar las bases moleculares de los mecanismos de resistencia en bacterias Gram-positivas. En concreto, a Staphylococcus aureus un importante patógeno nosocomial, a Streptococcus pneumoniae un patógeno extrahospitalario y a Corynebacterium spp. un patógeno oportunista emergente, perteneciente a la flora habitual de la piel.2- Estudiar la selección in vitro de mutantes resistentes a quinolonas en aislamientos clínicos de S. aureus y S. pneumoniae.3- Determinar el grado de mutagenicidad de las diversas fluoroquinolonas y correlacionarla con la selección de mutantes resistentes. / The quinolones constitutes a group of synthetic antimicrobial agents, with broad spectrum, and have been used with great success for the treatment of a wide variety of pathologies. This group of antimicrobials still under development, and continuously, new molecules more active than the pre-existing ones in many cases are appearing. The resistance to quinolones is present between most of their molecules, this phenomenon is called crossed resistance, or reduced susceptibility. For that reason, to develop new molecules within this family, it is important to know the mechanisms of resistance that the microorganisms presented in front of these compounds, in particular in Gram-positive bacteria. In this way, new molecules could be designed to circumvent the mechanisms of resistance already known. Another important parameter is to know how the quinolones select for resistant mutants. By all these reasons, for the present work the following objectives were considered: 1 - To investigate the molecular bases of the mechanisms of resistance in Gram-positive bacteria. In particular, to Staphylococcus aureus an important nosocomial pathogen, Streptococcus pneumoniae a pathogen causing mainly community-acquired infections and to Corynebacterium spp. an emergent opportunistic pathogen, pertaining to the habitual skin flora. 2 - To study the "in vitro" selection of resistant mutants to quinolones in clinical isolates of S. aureus and S. pneumoniae. To determine the power of mutagenicity of different fluoroquinolones and to correlate it with the selection of resistant mutants.

Natural products have long been known for their broad range of useful therapeutic properties, and have been widely utilised in the field of medicine. This dissertation describes work towards the total synthesis of natural products possessing biological activity in two important areas. The first section concerns the total synthesis of six 4-quinolone natural products, four of which had never been synthesised before. These compounds were originally isolated from a soil bacterium of the genus Pseudonocardia, and bear intriguing structural resemblance to the Pseudomonas Quinolone Signal. This signalling molecule is vital to the quorum sensing activity of the human pathogen Pseudomonas aeruginosa, which is a phenomenon by which it regulates many of its virulence factors. These natural products possess the potential to disrupt this system, hence attenuating the pathogenicity of the bacteria. The routes that were developed are highly divergent, efficiently giving access to multiple natural products from mutual late stage intermediates. Key steps included regioselective epoxidation, palladium-catalysed heterocylisation and acid catalysed 1,3-transposition of an allylic alcohol. In the second section, attention turns towards the total synthesis of the complex sesquiterpene lactone 1β-Hydroxyalantolactone. The compound possesses five stereogenic centres, one of which is quaternary, alongside a challenging tricyclic core scaffold. Previous biological studies have revealed a range of intriguing properties, including anti-inflammatory and anti-tumour activity. The chosen route utilises as its key step the catalytic desymmetrisation of a diene which was itself accessed by Birch reduction chemistry. Whilst the synthesis is as yet incomplete, access was granted to a key intermediate encompassing around half of the stereocentres present in the natural product.

AIG, CFL, SM, JR participaron en la concepción y diseño del estudio; AIG, CFL, MM, TJO y JR en el aporte de pacientes o material de estudio; CFL, TJO y JR en la obtención del financiamiento; TJO, SM, MJP, JR, y LJdV participaron el análisis e interpretación de los datos. Todos los autores participaron de la recolección de resultados, revisión crítica del manuscrito, aprobación de su versión final. / The main aim of this study was to establish the resistance levels to antimicrobial agents, in 222 non-pathogenic E. Coli strains of fecal origin in Peru. The proportion of resistance found to the evaluated antimicrobials was ampicillin (62.6%), cotrimoxazole (48,6%), tetracycline (43,0%) and chloramphenicol (15,8%). We emphasize the high resistance levels found for quinolones: 32% for nalidixic acid (NAL) and 12% for ciprofloxacin (CIP). These high levels of quinoloneresistance in non-pathogenic strains isolated from children in this age group highlight the extensive use and the impact of the intake of this kind of antimicrobials in the community, showing the potential risk of the loss of their utility in the area. / Este trabajo fue parcialmente financiado por Agència Catalana de Cooperació al Desenvolupament proyecto U2006 (LJdV), Centre de Cooperació per al Desenvolupament - Universitat Politècnica de Catalunya (LJdV), Agencia Española de Cooperación Internacional al Desarrollo proyectos numero A/4892/06 (LJdV), D/019499/08 y D/024648/09 (JR), Fogarty International Center, National Institute of Health, USA, proyecto 1K01TW007405 (TJO) Sanofi Pasteur y fondos de investigación del Dr. Lanata, Instituto de Investigación Nutricional, Lima, Perú. La investigación de JR es financiado por el proyecto CP05/0130 del FIS (Fondo de Investigaciones Sanitarias, España). / Revisión por pares.