A pesquisa de Staphylococcus spp. coagulase negativa (CNS) em alimentos não é prevista na legislação, entretanto, estas bactérias têm emergido como patógenos oportunistas e sua capacidade enterotoxigênica já foi documentada. O queijo destaca-se entre os principais derivados lácteos associados a intoxicações alimentares e a presença de cepas enterotoxigênicas do gênero Staphylococcus neste alimento representa um risco ao consumidor. O queijo colonial, tradicionalmente consumido pelos gaúchos, não possui regulamento técnico específico e poucos são os estudos relacionados ao risco do consumo desse alimento, bem como, à identificação de micro-organismos presentes nessa matriz. Sendo assim, os objetivos do presente estudo foram: (i) identificar as espécies de Staphylococcus spp. coagulase negativa, presentes em queijo colonial inspecionado; (ii) pesquisar a presença de genes codificadores de enterotoxinas clássicas (SE), bem como de resistência à penicilina e à meticilina nas cepas isoladas desta matriz. Para tanto, no período de novembro de 2014 a maio de 2015, foram analisadas 205 amostras de queijos coloniais inspecionados, sendo 121 adquiridas em Feiras Modelo e 84 em bancas do Mercado Público de Porto Alegre, compreendendo 17 marcas distintas. O isolamento inicial de Staphylococcus spp. foi realizado de acordo com o protocolo ISO 6888-1:1999, adicionado da triagem fenotípica para CNS. A identificação genotípica dos isolados foi realizada pela amplificação da região V1-V2 do gene 16S rRNA, seguida de sequenciamento e comparação das sequências obtidas no GenBank. A pesquisa de genes de enterotoxinas clássicas foi realizada por amplificação dos genes sea, seb, sec, sed e see. A determinação de resistência à penicilina foi avaliada a partir da amplificação do gene blaZ. Para resistência à meticilina, foi realizado teste de triagem frente à cefoxitina, e confirmação pela pesquisa do gene mecA. O armazenamento sob refrigeração foi observado em quase 90% das amostras coletadas. Entre as 179 colônias retiradas do ágar Baird-Parker, 59 apresentaram-se fenotipicamente compatíveis com CNS e foram identificadas genotipicamente. Treze espécies foram identificadas, sendo Macrococcus caseolyticus (40%) a mais frequente. Das 35 cepas confirmadas como CNS, S. equorum e S. vitulinus foram as espécies predominantes seguidas de S. hyicus, S. saprophyticus e S. epidermidis. O gene blaZ foi detectado em cinco cepas de CNS e em uma cepa de M. caseolyticus, sendo relativamente mais frequente em S. hyicus e S. epidermidis. O gene mecA não foi detectado. Oito cepas de CNS amplificaram algum gene para SE, sendo seb o mais frequente, seguido por sed, sea e see. O gene para enterotoxina C não foi detectado. Onze cepas apresentaram pelo menos um dos genes investigados, das quais, seis cepas apresentaram genes para SE e blaZ, concomitantemente. Os perfis seb/blaZ (n=4) e blaZ (n=3) foram os mais frequentes. Foi possível confirmar a diversidade de Staphylococcus spp. coagulase negativa em queijos coloniais inspecionados, além da baixa frequência de cepas carreadoras de genes para enterotoxinas clássicas e resistência à penicilina e à meticilina. / The investigation of coagulase-negative Staphylococcus spp. (CNS) is not included in food monitoring; although these bacteria have emerged as significant opportunistic pathogens and their toxigenic capacity has been documented. Among the dairy products, cheese features as one of the most involved in food poisoning outbreaks. The presence of enterotoxigenic strains of Staphylococcus in this food therefore represents a hazard for the consumers. Colonial cheese, which is a traditionally consumed cheese type in Rio Grande do Sul, does not have a specific technical regulation, and there are few studies targeting the risk for consumers, or aiming to identify its typical microbiota. Thus, the objectives of this study were: (i) to identify coagulase-negative Staphylococcus spp. species in inspected colonial cheese (ii) to investigate the presence of genes encoding classical enterotoxins (SE), and resistance to penicillin and methicillin in strains obtained from this food. For this purpose, from November 2014 to May 2015, 205 cheese samples were analyzed, 121 of which were acquired in street fairs and 84 in Central Market. The samples belonged to 17 different brands. The isolation of Staphylococcus spp. was performed according to the ISO 6888-1: 1999 protocol, followed by the phenotypic screening of CNS. The genotype identification of the isolates was performed by amplification of the V1-V2 region of the 16S rRNA gene, followed by sequencing and the sequences comparison with the GenBank database. Classical enterotoxin genes were investigated by amplification of sea, seb, sec, sed and see genes. The determination of penicillin resistance was evaluated by the amplification of blaZ gene. For methicillin resistance, a screening test with cefoxitin was conducted followed by confirmation through the mecA amplification. The majority (89,7%) of the collected samples were stored under refrigeration. Among the 179 atypical colonies obtained from Baird-Parker agar, 59 were phenotypically compatible with CNS and were further subjected to genotyping. Thirteen bacterial species were identified, being Macrococcus caseolyticus the most frequent (40%). Thirty-five strains were confirmed as CNS, being S. equorum and S. vitulinus the most prevalent followed by S. hyicus, S. saprophyticus and S. epidermidis. The gene blaZ was detected in five strains of CNS and in one strain of M. caseolyticus, being relatively more frequent in S. hyicus and S. warneri. The mecA gene was not detected. Eight CNS strains amplified SE gene: SEB was the most frequent, followed by SED, SEA and SEE. There was no enterotoxin C gene detected. Eleven strains carried at least one of the genes investigated; six strains presented genes for SE and blaZ, concomitantly. The profiles SEB/blaZ (n = 4) and blaZ (n = 3) were the most frequent. The diversity of CNS in inspected colonial cheeses was confirmed. In addition, the low frequency of strains carrying genes for enterotoxins and resistance to penicillin and methicillin was observed.

A pesquisa de Staphylococcus spp. coagulase negativa (CNS) em alimentos não é prevista na legislação, entretanto, estas bactérias têm emergido como patógenos oportunistas e sua capacidade enterotoxigênica já foi documentada. O queijo destaca-se entre os principais derivados lácteos associados a intoxicações alimentares e a presença de cepas enterotoxigênicas do gênero Staphylococcus neste alimento representa um risco ao consumidor. O queijo colonial, tradicionalmente consumido pelos gaúchos, não possui regulamento técnico específico e poucos são os estudos relacionados ao risco do consumo desse alimento, bem como, à identificação de micro-organismos presentes nessa matriz. Sendo assim, os objetivos do presente estudo foram: (i) identificar as espécies de Staphylococcus spp. coagulase negativa, presentes em queijo colonial inspecionado; (ii) pesquisar a presença de genes codificadores de enterotoxinas clássicas (SE), bem como de resistência à penicilina e à meticilina nas cepas isoladas desta matriz. Para tanto, no período de novembro de 2014 a maio de 2015, foram analisadas 205 amostras de queijos coloniais inspecionados, sendo 121 adquiridas em Feiras Modelo e 84 em bancas do Mercado Público de Porto Alegre, compreendendo 17 marcas distintas. O isolamento inicial de Staphylococcus spp. foi realizado de acordo com o protocolo ISO 6888-1:1999, adicionado da triagem fenotípica para CNS. A identificação genotípica dos isolados foi realizada pela amplificação da região V1-V2 do gene 16S rRNA, seguida de sequenciamento e comparação das sequências obtidas no GenBank. A pesquisa de genes de enterotoxinas clássicas foi realizada por amplificação dos genes sea, seb, sec, sed e see. A determinação de resistência à penicilina foi avaliada a partir da amplificação do gene blaZ. Para resistência à meticilina, foi realizado teste de triagem frente à cefoxitina, e confirmação pela pesquisa do gene mecA. O armazenamento sob refrigeração foi observado em quase 90% das amostras coletadas. Entre as 179 colônias retiradas do ágar Baird-Parker, 59 apresentaram-se fenotipicamente compatíveis com CNS e foram identificadas genotipicamente. Treze espécies foram identificadas, sendo Macrococcus caseolyticus (40%) a mais frequente. Das 35 cepas confirmadas como CNS, S. equorum e S. vitulinus foram as espécies predominantes seguidas de S. hyicus, S. saprophyticus e S. epidermidis. O gene blaZ foi detectado em cinco cepas de CNS e em uma cepa de M. caseolyticus, sendo relativamente mais frequente em S. hyicus e S. epidermidis. O gene mecA não foi detectado. Oito cepas de CNS amplificaram algum gene para SE, sendo seb o mais frequente, seguido por sed, sea e see. O gene para enterotoxina C não foi detectado. Onze cepas apresentaram pelo menos um dos genes investigados, das quais, seis cepas apresentaram genes para SE e blaZ, concomitantemente. Os perfis seb/blaZ (n=4) e blaZ (n=3) foram os mais frequentes. Foi possível confirmar a diversidade de Staphylococcus spp. coagulase negativa em queijos coloniais inspecionados, além da baixa frequência de cepas carreadoras de genes para enterotoxinas clássicas e resistência à penicilina e à meticilina. / The investigation of coagulase-negative Staphylococcus spp. (CNS) is not included in food monitoring; although these bacteria have emerged as significant opportunistic pathogens and their toxigenic capacity has been documented. Among the dairy products, cheese features as one of the most involved in food poisoning outbreaks. The presence of enterotoxigenic strains of Staphylococcus in this food therefore represents a hazard for the consumers. Colonial cheese, which is a traditionally consumed cheese type in Rio Grande do Sul, does not have a specific technical regulation, and there are few studies targeting the risk for consumers, or aiming to identify its typical microbiota. Thus, the objectives of this study were: (i) to identify coagulase-negative Staphylococcus spp. species in inspected colonial cheese (ii) to investigate the presence of genes encoding classical enterotoxins (SE), and resistance to penicillin and methicillin in strains obtained from this food. For this purpose, from November 2014 to May 2015, 205 cheese samples were analyzed, 121 of which were acquired in street fairs and 84 in Central Market. The samples belonged to 17 different brands. The isolation of Staphylococcus spp. was performed according to the ISO 6888-1: 1999 protocol, followed by the phenotypic screening of CNS. The genotype identification of the isolates was performed by amplification of the V1-V2 region of the 16S rRNA gene, followed by sequencing and the sequences comparison with the GenBank database. Classical enterotoxin genes were investigated by amplification of sea, seb, sec, sed and see genes. The determination of penicillin resistance was evaluated by the amplification of blaZ gene. For methicillin resistance, a screening test with cefoxitin was conducted followed by confirmation through the mecA amplification. The majority (89,7%) of the collected samples were stored under refrigeration. Among the 179 atypical colonies obtained from Baird-Parker agar, 59 were phenotypically compatible with CNS and were further subjected to genotyping. Thirteen bacterial species were identified, being Macrococcus caseolyticus the most frequent (40%). Thirty-five strains were confirmed as CNS, being S. equorum and S. vitulinus the most prevalent followed by S. hyicus, S. saprophyticus and S. epidermidis. The gene blaZ was detected in five strains of CNS and in one strain of M. caseolyticus, being relatively more frequent in S. hyicus and S. warneri. The mecA gene was not detected. Eight CNS strains amplified SE gene: SEB was the most frequent, followed by SED, SEA and SEE. There was no enterotoxin C gene detected. Eleven strains carried at least one of the genes investigated; six strains presented genes for SE and blaZ, concomitantly. The profiles SEB/blaZ (n = 4) and blaZ (n = 3) were the most frequent. The diversity of CNS in inspected colonial cheeses was confirmed. In addition, the low frequency of strains carrying genes for enterotoxins and resistance to penicillin and methicillin was observed.

A pesquisa de Staphylococcus spp. coagulase negativa (CNS) em alimentos não é prevista na legislação, entretanto, estas bactérias têm emergido como patógenos oportunistas e sua capacidade enterotoxigênica já foi documentada. O queijo destaca-se entre os principais derivados lácteos associados a intoxicações alimentares e a presença de cepas enterotoxigênicas do gênero Staphylococcus neste alimento representa um risco ao consumidor. O queijo colonial, tradicionalmente consumido pelos gaúchos, não possui regulamento técnico específico e poucos são os estudos relacionados ao risco do consumo desse alimento, bem como, à identificação de micro-organismos presentes nessa matriz. Sendo assim, os objetivos do presente estudo foram: (i) identificar as espécies de Staphylococcus spp. coagulase negativa, presentes em queijo colonial inspecionado; (ii) pesquisar a presença de genes codificadores de enterotoxinas clássicas (SE), bem como de resistência à penicilina e à meticilina nas cepas isoladas desta matriz. Para tanto, no período de novembro de 2014 a maio de 2015, foram analisadas 205 amostras de queijos coloniais inspecionados, sendo 121 adquiridas em Feiras Modelo e 84 em bancas do Mercado Público de Porto Alegre, compreendendo 17 marcas distintas. O isolamento inicial de Staphylococcus spp. foi realizado de acordo com o protocolo ISO 6888-1:1999, adicionado da triagem fenotípica para CNS. A identificação genotípica dos isolados foi realizada pela amplificação da região V1-V2 do gene 16S rRNA, seguida de sequenciamento e comparação das sequências obtidas no GenBank. A pesquisa de genes de enterotoxinas clássicas foi realizada por amplificação dos genes sea, seb, sec, sed e see. A determinação de resistência à penicilina foi avaliada a partir da amplificação do gene blaZ. Para resistência à meticilina, foi realizado teste de triagem frente à cefoxitina, e confirmação pela pesquisa do gene mecA. O armazenamento sob refrigeração foi observado em quase 90% das amostras coletadas. Entre as 179 colônias retiradas do ágar Baird-Parker, 59 apresentaram-se fenotipicamente compatíveis com CNS e foram identificadas genotipicamente. Treze espécies foram identificadas, sendo Macrococcus caseolyticus (40%) a mais frequente. Das 35 cepas confirmadas como CNS, S. equorum e S. vitulinus foram as espécies predominantes seguidas de S. hyicus, S. saprophyticus e S. epidermidis. O gene blaZ foi detectado em cinco cepas de CNS e em uma cepa de M. caseolyticus, sendo relativamente mais frequente em S. hyicus e S. epidermidis. O gene mecA não foi detectado. Oito cepas de CNS amplificaram algum gene para SE, sendo seb o mais frequente, seguido por sed, sea e see. O gene para enterotoxina C não foi detectado. Onze cepas apresentaram pelo menos um dos genes investigados, das quais, seis cepas apresentaram genes para SE e blaZ, concomitantemente. Os perfis seb/blaZ (n=4) e blaZ (n=3) foram os mais frequentes. Foi possível confirmar a diversidade de Staphylococcus spp. coagulase negativa em queijos coloniais inspecionados, além da baixa frequência de cepas carreadoras de genes para enterotoxinas clássicas e resistência à penicilina e à meticilina. / The investigation of coagulase-negative Staphylococcus spp. (CNS) is not included in food monitoring; although these bacteria have emerged as significant opportunistic pathogens and their toxigenic capacity has been documented. Among the dairy products, cheese features as one of the most involved in food poisoning outbreaks. The presence of enterotoxigenic strains of Staphylococcus in this food therefore represents a hazard for the consumers. Colonial cheese, which is a traditionally consumed cheese type in Rio Grande do Sul, does not have a specific technical regulation, and there are few studies targeting the risk for consumers, or aiming to identify its typical microbiota. Thus, the objectives of this study were: (i) to identify coagulase-negative Staphylococcus spp. species in inspected colonial cheese (ii) to investigate the presence of genes encoding classical enterotoxins (SE), and resistance to penicillin and methicillin in strains obtained from this food. For this purpose, from November 2014 to May 2015, 205 cheese samples were analyzed, 121 of which were acquired in street fairs and 84 in Central Market. The samples belonged to 17 different brands. The isolation of Staphylococcus spp. was performed according to the ISO 6888-1: 1999 protocol, followed by the phenotypic screening of CNS. The genotype identification of the isolates was performed by amplification of the V1-V2 region of the 16S rRNA gene, followed by sequencing and the sequences comparison with the GenBank database. Classical enterotoxin genes were investigated by amplification of sea, seb, sec, sed and see genes. The determination of penicillin resistance was evaluated by the amplification of blaZ gene. For methicillin resistance, a screening test with cefoxitin was conducted followed by confirmation through the mecA amplification. The majority (89,7%) of the collected samples were stored under refrigeration. Among the 179 atypical colonies obtained from Baird-Parker agar, 59 were phenotypically compatible with CNS and were further subjected to genotyping. Thirteen bacterial species were identified, being Macrococcus caseolyticus the most frequent (40%). Thirty-five strains were confirmed as CNS, being S. equorum and S. vitulinus the most prevalent followed by S. hyicus, S. saprophyticus and S. epidermidis. The gene blaZ was detected in five strains of CNS and in one strain of M. caseolyticus, being relatively more frequent in S. hyicus and S. warneri. The mecA gene was not detected. Eight CNS strains amplified SE gene: SEB was the most frequent, followed by SED, SEA and SEE. There was no enterotoxin C gene detected. Eleven strains carried at least one of the genes investigated; six strains presented genes for SE and blaZ, concomitantly. The profiles SEB/blaZ (n = 4) and blaZ (n = 3) were the most frequent. The diversity of CNS in inspected colonial cheeses was confirmed. In addition, the low frequency of strains carrying genes for enterotoxins and resistance to penicillin and methicillin was observed.

La thèse est divisée en trois chapitres indépendants. Chimie du β-lactames : Synthèse d'α-éthylène et d'α-alkylidène-β-lactames en utilisant la réaction de Kinugasa. Chimie de l'acylsilane : application d'une réaction aldolique intramoléculaire asymétrique sur un dérivé d'acylsilane nouvellement synthétisé. - Chimie médicinale: synthèse de nouvelles molécules à but anticancéreux.Dans le premier chapitre la réaction de Kinugasa a été appliquée pour la première fois à des alcynes vrais, portant en position propargylique un groupe partant permettait d'accéder directement et en une étape aux méthylène- et alkylidene β-lactames recherchés. Dans le second chapitre, la synthèse de molécules originales possédant à la fois une fonction acylsilane et un aldéhyde en position éloignée, et l'aldolisation intramoléculaire asymétrique a été explorée. Dans le dernier chapitre, notre objectif était de restaurer les propriétés apoptotiques au sein des cellules cancéreuses afin d'obtenir de nouveaux composés à activité antitumorale. A partir de données obtenues par modélisation moléculaire, nous avons fait le design de plusieurs séries d'analogues d'un inhibiteur connu(MIM-1) de la protéine anti-apoptotique Mcl-1. Huit composés ont été synthétisés et testés pour trois types de cellules cancéreuses (sein, ovaire et le mélanome). / The thesis is divided into three chapters:- β-lactams chemistry: synthesis of α-methylene and α-alkylidene-β-lactams using the Kinugasa reaction.- Acylsilane chemistry : applying asymmetric intramolecular aldol reaction on a newly synthesized acylsilane derivatives. - Medicinal chemistry: synthesis of new molecules with anticancer aimes. In the first chapter, Kinugasa reaction was applied for the first time with an alkyne bearing a nucleofuge in propargylic position that allowed us to discover a way of synthesis of exoalkylidene β-lactams. In the second chapter, a new acylislane derivatives bearing an aldehyde functional group in a remote position of the molecule were prepared, and asymmetric intramolecular aldolization reaction was performed. In the last chapter, our goal was to reinduce the pro-apoptotic properties in cancer cells in order to obtain new antitumor compounds. Starting from data obtained through molecular modeling studies, we designed and prepared several series of analogs for a known inhibitor (MIM-1) of the anti-apoptotic protein Mcl-1. Eight compounds have been synthetized and screened towards three types of cancer cells (breast, ovarian and melanoma).

L’augmentation et la dissémination de la résistance aux β-lactamines chez les bacilles à Gram négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par ces bactéries multi-résistantes (BMR) ont conduit à une augmentation de la mortalité, de la morbidité et du coût de traitement. L’utilisation abusive et non contrôlée de ces antibiotiques a grandement contribué à la large diffusion de cette résistance. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et de surveiller la diffusion et la dissémination des BMR. Contrairement à de nombreuses régions dans le monde, il existe peu d’informations concernant la caractérisation moléculaire des gènes de résistance aux β-lactamines des bacilles à Gram négatif isolés en Tunisie et surtout en Libye. C’est dans cette optique que ce projet de Thèse de Doctorat s’articule avec comme objectifs: (i) mettre en évidence la prévalence des bacilles à Gram négatifs multi-résistants isolés aux niveaux des hôpitaux tunisiens et libyens (ii) identifier le support génétique de la résistance aux β-lactamines de ces souches cliniques (iii) étudier la diversité clonale des souches multi-résistantes par typage moléculaire. / The increase and spread of β-lactam resistance in gram negative bacteria especially Enterobacteriaceae, Pseudomonas and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led to an increase in mortality, morbidity and cost of treatment. The frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of these MDR. Unlike many parts of the world, there is little information concerning the molecular characterization of the β-lactam resistance genes of Gram-negative bacilli isolated in Tunisia and especially in Libya. Therefore, it is in this context that the project of this thesis was conducted with essential objectives: (i) highlight the prevalence of multi-resistant Gram negative bacilli isolated in Tunisian and Libyan hospitals (ii) identify the genetic support of resistance to β-lactams of these clinical strains (iii) study the clonal diversity of the multi-resistant strains by molecular typing (iii) study the molecular epidemiology of these BMRs in these countries in order to control the decision-making process of the treatment and the rapid identification of epidemics by implementing appropriate control measures for the spread of infections and especially developing new tools and software for the diagnosis and monitoring of potential MDR bacteria in Mediterranean countries.