Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-19T12:28:29Z No. of bitstreams: 1 claudiaoliveirafontes.pdf: 4732336 bytes, checksum: b5385191fb0ea13de3e2e756b49d0981 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-06-29T12:22:06Z (GMT) No. of bitstreams: 1 claudiaoliveirafontes.pdf: 4732336 bytes, checksum: b5385191fb0ea13de3e2e756b49d0981 (MD5) / Made available in DSpace on 2017-06-29T12:22:06Z (GMT). No. of bitstreams: 1 claudiaoliveirafontes.pdf: 4732336 bytes, checksum: b5385191fb0ea13de3e2e756b49d0981 (MD5) Previous issue date: 2013-04-12 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste estudo, os padrões de susceptibilidade a antimicrobianos, ocorrência de marcadores moleculares de resistência aos antimicrobianos e biocidas, bem como as características de virulência associadas aos Staphylococcus spp., foram avaliados tanto em SCN quanto em S. aureus isolados de queijo Minas Frescal no Brasil. Um total de 246 isolados bacterianos foram recuperados de 35 amostras de queijo, as quais foram obtidas em cinco lotes de cada uma das sete marcas comerciais diferentes avaliadas neste estudo. As contagens bacterianas variaram de 103 a 107 UFC/g de queijo. Os percentuais elevados de resistência a antimicrobianos foram observados nos dois grupos bacterianos com relação à oxacilina, penicilina e eritromicina. Um percentual baixo de resistência aos antimicrobianos foi encontrado com relação ao cloranfenicol, e todas as bactérias avaliadas se mostraram susceptíveis à vancomicina e linezolida. No total, o índice de resistência múltipla aos antimicrobianos (MAR) > 0,2 foi observado em 80,6 % e 84,2 % dos isolados de SCN e S. aureus, respectivamente. Entretanto, o índice MAR variou de 50 % a 100 % considerando apenas os isolados bacterianos estudados por marca comercial de queijo. Com relação à prevalência de SCN e S. aureus que carregam o gene mecA, respectivamente, 81,5 % e 47,4 % das linhagens isoladas foram consideradas mecA+, e 76,2 % e 42,1 % destas se mostraram fenotipicamente resistentes à oxacilina. Três isolados bacterianos carregavam o gene para produção de enterotoxina A (sea), 30,5 % produziram biofilme em um teste de laboratório, e a α ou ß-hemólise foi observada em 2,8 % e 7,2 %, respectivamente, em todos os Staphylococcus spp. avaliados. Os marcadores moleculares de resistência aos antimicrobianos e biocidas mais prevalentes encontrados nas linhagens de SCN e S. aureus foram blaZ, mecA, msrA, msrB, linA, aacA-aphD, e smr. Este estudo destaca a importância do fenômeno da resistência aos antimicrobianos com relação aos microrganismos negligenciados que são veiculados por alimentos, e os prováveis riscos para a saúde pública relacionados ao consumo de queijo contendo bactérias do gênero Staphylococcus. / In this study, antimicrobial susceptibility patterns, molecular markers of antimicrobial and biocide-resistance occurrence and virulence-associated characteristics were evaluated in CoNS and S. aureus isolated from soft cheese in Brazil. A total of 246 bacterial isolates were recovered from 35 cheese samples belonging to five batches with seven different trademarks. The bacterial counts ranged from 103 to 107 CFU g1. High antimicrobial resistance percentages were observed for oxacillin, penicillin and erythromycin for both S. aureus and SCN. A low antimicrobial resistance percentage was observed for chloramphenicol, and all of the tested bacteria were susceptible to vancomycin and linezolid. In total, a multiple antibiotic resistance (MAR) index of > 0.2 was observed for 80.6 % and 84.2 % of the isolated CoNS and S. aureus, respectively. However, the MAR index ranged from 50 % to 100 % when only bacterial cheese isolates belonging to the same trademark were considered. Regarding to the prevalence of SCN and S. aureus carrying mecA gene, respectively, 81.5 % e 47,4 % of the isolated strains were mecA+, and 76.2 % and 42.1 % of these were phenotypically resistant to oxacillin. Three isolates carried the enterotoxin A gene (sea), 30.5 % produced biofilm in a laboratory test, and α- or ßhemolysis was observed for 2,8 % and 7.2 %, respectively, for all the Staphylococcus spp.. blaZ, mecA, msrA, msrB, linA, aacA-aphD, and smr were most prevalent molecular markers found both in SCN and S. aureus strains. This study highlights the extent of the antimicrobial resistance phenomenon in neglected foodborne microorganisms and the potential public health risks that are related to the consumption of Staphylococci-contaminated soft cheese.

Charles University Faculty of Pharmacy in Hradec Králové Study program: Healthcare Bioanalytics Author: Bc. Adéla Diepoltová Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Optimization of the method for sensitivity evaluation of biofilm- forming staphylococci against candidate antimicrobial compounds Background: The aim of this thesis was to optimize approach for in vitro formation of staphylococcal biofilms on the pegs and on the wells of the 96-well panel as an analogous approach to commercially available Calgary Biofilm Device system. The aim of the Experiment 1 was to evaluate incubation conditions (such as impact of a growth medium, incubation mode, optical density of the starting bacterial inoculum and type of surface) leading to maximal biofilm formation of two biofilm producer strains with unknown biofilm phenotype and one staphylococcal strain known as strong biofilm producer. The most advisable conditions were used in incubation process of Experiment 2. This work should propose the approach leading to in vitro formation of the most voluminous staphylococcal biofilms exploitable for candidate drug antimicrobial activity testing. Methods: Spectrophotometric measurement of the crystal violet colour extracted from wells with fixed and stained Staphylococci to evaluate the ability to...

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.