pC221 belongs to a family of staphylococcal plasmids, including pT181, pS194, pC223, pUB112 and pCW7 . All possess open reading frames with 70-80% homology to the pC221 rapD gene. REP D has sequence specific topoisomerase activity at the pC221 origin (or iD) which is thought to be involved in replication initiation. DNase I footprinting has been carried out, showing that REP D binds to a region of oriD downstream of the nick site. The pattern of DNase I cleavage suggests that REP D contacts one face of the DNA helix, which may be bent around the protein. Extracts of S. aureus support incorporation of radioactive dNTPs into pC221 in the presence of REP D. Labelling with a[32P] dATP shows that replication initiates within the region containing oriD and proceeds in the direction expected for elongation of a 3' OH generated by nicking at oriD. With supercoiled DNA, REP D initiates replication of other members of this plasmid family in Vitro. However, with relaxed DNA, REP D is specific for oriD, suggesting that a change in the DNA, stabilised by supercoiling of the DNA or by binding of REP D, may be required for nicking. Of three inverted repeat sequences (ICRI, II & III) at the origin, ICRII has the greatest predicted hairpin stability and is almost totally conserved. Nicking takes place within the loop of this proposed hairpin. Disruption of base pairing within this hairpin has been investigated by mutagenesis of cloned oriD and using oligonucleotides based on the ICRII sequence. These experiments show that the 3' side of ICRII is more important for nicking than the 5' side. This is in agreement with footprinting data which shows that REP D binds the 3' side of ICRII, along with the whole of ICRIII. However, there is no evidence for hairpin formation at ICRII being required for nicking.

O objetivo do presente estudo foi avaliar a ocorrência de cepas de Staphylococcus aureus no ambiente de ordenha, analisar seu perfil genético e a produção de biofilme, provenientes de 10 propriedades localizadas nas regiões de Franca e Ribeirão Preto, estado de São Paulo, Brasil. Foram analisadas 220 amostras de leite individual de vacas positivas no teste CMT (California Mastitis Test), 120 amostras de leite de tanque de expansão, 389 swabs de utensílios e equipamento relacionados com ordenha e 120 swabs de mãos de manipuladores. As coletas de amostras foram realizadas mensalmente durante o período de agosto/2010 a janeiro/2011. Das 849 amostras analisadas, 56 cepas de S. aureus (6,6%) foram isoladas, sendo 12 (5,4%) de leite individual de vacas, 26 (21,6%) de leite de tanque de expansão, 14 (3,6%) de utensílios e equipamentos e 4 (6,9%) de mãos de manipuladores. Os resultados indicam uma baixa prevalência de S. aureus nas propriedades analisadas, não havendo diferença significativa entre as frequências encontradas nas duas regiões analisadas. A técnica de PFGE (pulsed-field gel electrophoresis) permitiu identificar 31 perfis genéticos (pulsotipos), utilizando-se a enzima de restrição SmaI. Nos ensaios de produção de biofilmes em microplaca, 19 (63,3%) de 30 pulsotipos avaliados foram considerados produtores de biofilme. Nos ensaios conduzidos em aço inox, 13 (43,3%, N=30) foram positivos e, para o silicone, não houve produção de biofilme. O elevado percentual de isolados no leite de tanque de expansão evidencia um problema de saúde pública, considerando que no Brasil muitas vezes o leite é consumido sem pasteurização. A ocorrência de pulsitipos de S. aureus formadores de biofilmes indica a persistência do patógeno em diversos pontos no ambiente de ordenha, bem como provável envolvimento dos mesmos com casos de mastite e sua eliminação no leite, ressaltando a necessidade de práticas higiênicas para prevenir a formação de biofilmes nas propriedades estudadas. / The objective of the present study was to evaluate the occurrence of biofilm-producing strains of Staphylococcus aureus in the milking environment from 10 farms located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Twohundred twenty samples of milk from individual cows previously tested for CMT (California Mastitis Test), 120 samples of bulk tank milk, 389 swabs of equipments and utensils related to milking and 120 swabs of milk\'s handlers hands were analyzed. A total of 56 (6.6%) S. aureus strains were isolated out of 849 samples analyzed, being 12 (5.4%) from milk samples from individual cows, 26 (21.6%) from bulk tank milk, 14 (3.6%) from swabs of equipments and utensils, and 4 (3.3%) from swabs hands of milk\'s handlers. Results indicated a low prevalence of S. aureus in the dairy farms analyzed, and there was no significant difference between the percentages found in the two regions evaluated. On the basis of PFGE results (using SmaI enzyme), 31 profiles (pulsotypes) were found. In the biofilm-production assay using microplates, 19 (63.3%) of 30 pulsotypes tested were considered positive (biofilm producers). For assays conducted in stainless steel, 13 (43.3%) of pulsotypes were biofilm producers, although no pulsotype was able to produce biofilms on the surface of silicon. Results of this trial showed a high percentage of bulk tank milk samples contaminated with S. aureus, hence indicating a public health problem especially in Brazil were milk is often consumed without pasteurization. The occurrence of S. aureus pulsotypes which were also biofilm-producers suggests the persistence of the pathogen in several sites at the milking environment, as well the probable involvement of S. aureus biofilms with mastitis and its excretion in the milk. The need for adoption of hygienic practices to prevent the formation of biofilms of S. aureus in the dairy farms evaluated is stressed.

O objetivo do presente estudo foi avaliar a ocorrência de cepas de Staphylococcus aureus no ambiente de ordenha, analisar seu perfil genético e a produção de biofilme, provenientes de 10 propriedades localizadas nas regiões de Franca e Ribeirão Preto, estado de São Paulo, Brasil. Foram analisadas 220 amostras de leite individual de vacas positivas no teste CMT (California Mastitis Test), 120 amostras de leite de tanque de expansão, 389 swabs de utensílios e equipamento relacionados com ordenha e 120 swabs de mãos de manipuladores. As coletas de amostras foram realizadas mensalmente durante o período de agosto/2010 a janeiro/2011. Das 849 amostras analisadas, 56 cepas de S. aureus (6,6%) foram isoladas, sendo 12 (5,4%) de leite individual de vacas, 26 (21,6%) de leite de tanque de expansão, 14 (3,6%) de utensílios e equipamentos e 4 (6,9%) de mãos de manipuladores. Os resultados indicam uma baixa prevalência de S. aureus nas propriedades analisadas, não havendo diferença significativa entre as frequências encontradas nas duas regiões analisadas. A técnica de PFGE (pulsed-field gel electrophoresis) permitiu identificar 31 perfis genéticos (pulsotipos), utilizando-se a enzima de restrição SmaI. Nos ensaios de produção de biofilmes em microplaca, 19 (63,3%) de 30 pulsotipos avaliados foram considerados produtores de biofilme. Nos ensaios conduzidos em aço inox, 13 (43,3%, N=30) foram positivos e, para o silicone, não houve produção de biofilme. O elevado percentual de isolados no leite de tanque de expansão evidencia um problema de saúde pública, considerando que no Brasil muitas vezes o leite é consumido sem pasteurização. A ocorrência de pulsitipos de S. aureus formadores de biofilmes indica a persistência do patógeno em diversos pontos no ambiente de ordenha, bem como provável envolvimento dos mesmos com casos de mastite e sua eliminação no leite, ressaltando a necessidade de práticas higiênicas para prevenir a formação de biofilmes nas propriedades estudadas. / The objective of the present study was to evaluate the occurrence of biofilm-producing strains of Staphylococcus aureus in the milking environment from 10 farms located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Twohundred twenty samples of milk from individual cows previously tested for CMT (California Mastitis Test), 120 samples of bulk tank milk, 389 swabs of equipments and utensils related to milking and 120 swabs of milk\'s handlers hands were analyzed. A total of 56 (6.6%) S. aureus strains were isolated out of 849 samples analyzed, being 12 (5.4%) from milk samples from individual cows, 26 (21.6%) from bulk tank milk, 14 (3.6%) from swabs of equipments and utensils, and 4 (3.3%) from swabs hands of milk\'s handlers. Results indicated a low prevalence of S. aureus in the dairy farms analyzed, and there was no significant difference between the percentages found in the two regions evaluated. On the basis of PFGE results (using SmaI enzyme), 31 profiles (pulsotypes) were found. In the biofilm-production assay using microplates, 19 (63.3%) of 30 pulsotypes tested were considered positive (biofilm producers). For assays conducted in stainless steel, 13 (43.3%) of pulsotypes were biofilm producers, although no pulsotype was able to produce biofilms on the surface of silicon. Results of this trial showed a high percentage of bulk tank milk samples contaminated with S. aureus, hence indicating a public health problem especially in Brazil were milk is often consumed without pasteurization. The occurrence of S. aureus pulsotypes which were also biofilm-producers suggests the persistence of the pathogen in several sites at the milking environment, as well the probable involvement of S. aureus biofilms with mastitis and its excretion in the milk. The need for adoption of hygienic practices to prevent the formation of biofilms of S. aureus in the dairy farms evaluated is stressed.

Bien que l'initiation de la traduction soit un processus conservé entre les bactéries, nous avons montré que le mécanisme par lequel les ARNm structurés sont reconnus et adaptés sur le ribosome diffère chez Staphylococcus aureus, un micro-organisme avec un bas taux de G+C et chez Escherichia coli. Une particularité du ribosome de S. aureus est l'absence de la protéine ribosomale S1, qui non seulement est plus courte que celle de E. coli mais qui possède également une organisation distincte des domaines. Mes expériences suggèrent que la protéine S1 (SauS1) favorise spécifiquement l'initiation de la traduction de l'opéron α-psm 1-4 en liant son ARNm hautement structuré. En outre, il influence aussi l'expression et la production de facteurs de virulence comme les exotoxines (α-haemolysine, δ-hémolysine et γ- hémolysine) et les exoenzymes (protéases et lipases). En plus de son rôle dans la traduction, SauS1 pourrait être impliquée dans d'autres processus cellulaires tels que le métabolisme de l'ARN et la régulation par des ARN non-codants (ARNnc). Elle forme des complexes in vivo avec plusieurs ARNnc dont la stabilité serait affectée dans la souche délétée du gène rpsA codant S1. SauS1 a donc une activité chaperonne favorisant la cinétique d’appariement entre deux molécules d'ARN et au moins dans un cas, elle stimule la reconnaissance entre un ARNnc et son ARN cible. Ainsi, SauS1 appartient à une nouvelle classe de chaperons d'ARN qui jouent un rôle clé dans la régulation du virulon de S. aureus. / Even if translation initiation is a conserved process among bacteria, we have recently shown that low G+C content Gram-positive, such as Staphylococcus aureus, differ from E. coli on the mechanism by which structured mRNAs are recognized and adapted on the ribosome. One peculiarity of the S. aureus ribosome is the absence of ribosomal protein S1, which is shorter than E. coli S1 and has different domains organization. My work could demonstrate that S. aureus S1 (SauS1) specifically promotes translation initiation of the α-psm 1-4 operon by binding its highly structured mRNA. Moreover, it influences the expression and production of other exotoxins (α-haemolysin, δ-haemolysin and γ-haemolysins) and exoenzymes (proteases and lipases). Besides its role in translation, SauS1 could be implicated in other cellular processes such as RNA maturation/degradation and sRNA-mediated regulation. It forms in vivo complexes with several sRNAs whose level is affected in a strain deleted of rpsA gene, coding for S1. Preliminary results show that SauS1 has a chaperone activity promoting the kinetic of annealing of two model RNA molecules and at least in one case, we could demonstrate that it stimulates the recognition between a sRNA and its target RNA. Taken together, SauS1 belongs to a new class of RNA chaperones that play key roles in the regulation of S.aureus virulon.

No description available.

Orientador: Vera Lúcia Mores Rall / Resumo: Staphylococcus aureus é um agente comum de mastite bovina, responsável por grandes perdas econômicas na pecuária mundial. Esse micro-organismo possui fatores de virulência bastante conhecidos como a produção de hemolisinas, leucotoxinas e superantígenos como a toxina do síndrome do choque tóxico e enterotoxinas. O objetivo do estudo foi caracterizar molecularmente os isolados de S. aureus provenientes de leite de vacas com mastite subclínica, de várias regiões do estado de São Paulo, através de Multilocus Sequence Typing (MLST), spa typing, Pulse Field Gel Electrophoresis (PFGE) e agr. Também fizeram parte dos objetivos, testes fenótipicos e genotípicos de resistência a antimicrobianos, além da pesquisa de genes de alguns fatores de virulência como o pvl (toxina de Panton Valentine), tst (toxina da Síndrome do Choque Tóxico, , sea-see, seg, seh e sei, a fim de verificar quais os fatores de virulência mais envolvidos nesse quadro. Todos os isolados de S. aureus foram sensíveis a meticilina (MSSA), pois os genes mecA e mecC não foram encontrados. Entre os 12 antibióticos testados, observou-se resistência intermediaria à eritromicina e nenhuma das cepas foram resistentes à oxacilina, vancomicina e gentamicina. Os isolados resistentes à tetraciclina apresentaram o gene tetK . Na caracterização molecular, observou-se 23 spa types diferentes com prevalência dos tipos t605 e t127. A maioria das cepas (48,1%) eram pertencentes ao grupo agr II, seguido de 20,1% do grupo III e 8,1%... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Staphylococcus aureus is a common agent of bovine mastitis, causing economic losses in Brazilian livestock. This microorganism has well known virulence factors such as the production of hemolysins, leukotoxins and superantigens such as toxic shock syndrome toxin and enterotoxins. The objective of the study was to characterize molecularly isolates of S. aureus from milk of cows with subclinical mastitis, of the several regions of São Paulo State, through Multilocus sequence typing (MLST), spatyping, Pulse Field Electrophoresis Gel (PFGE) and agr. Phenotypic and genotypic tests of antimicrobial resistance, as well as the search for genes of some virulence factors such as pvl (Panton Valentine toxin), tst (Toxic Shock Syndrome toxin). In the present study, all isolates of S. aureus were sensitive to methicillin (MSSA), as the mecA and mecC genes were not found. About 12 antibiotics tested, intermediate resistance to erythromicyn was observed and none resistance to oxacillin, vancomycin and gentamicin. The tetracycline resistant isolates showed the tetK gene. Molecular characterization showed 23 different spa types with prevalence of t605 and t127. The most of the strains (48.1%) presented as belonging to the agr II group, followed by 20.1% of the agr III group and 8.1% of the agrI group, and the agr IV group was not found. About the virulence factors studied, the pvl gene was not observed. In relation to super antigens, the tst gene was observed in 105 of the 285 strains (37.1%)... (Complete abstract click electronic access below) / Doutor

Bacterial superantigens (SAgs) are virulence factors that induce nonspecific T-cell proliferation contributing to host immune avoidance, and occasionally severe life-threatening toxinoses such as toxic shock syndrome. In the current study, the multiple functions of 3 superantigens named staphylococcal enterotoxin-like toxins X, Y and Z are investigated. SElX and SElZ were non-emetic in a musk shrew model of emesis. SElX is structurally and phylogenetically related to staphylococcal superantigen-like proteins (SSls) which are non-mitogenic but exhibit a variety of immune modulatory properties. We carried out protein and gene expression analysis of mutants of different S. aureus gene regulators and demonstrated that selx expression is controlled by saeRS, a two-component regulator linked to the bacterial response to phagocytic signals. Considering the co-regulation of SElX with known mediators of innate immune evasion we investigated a potential role for SElX in both humoral and cellular innate immune modulation and discovered that SElX strongly binds to human, bovine, murine, and laprine neutrophils and interferes with IgG-mediated phagocytosis, independently of Fcγ receptor signalling. Bacterial survival assays with neutrophils demonstrated that the deletion of selx significantly reduced the ability of S. aureus to resist neutrophil killing. Site-directed mutagenesis in the conserved sialic acid-binding motif of SElX abolished its neutrophil binding capacity, which is consistent with a critical role for glycosylated receptors in this interaction. Importantly, the sialic-acid binding mutants of SElX retained the ability to induce T-cell proliferation demonstrating that the distinct functions of SElX are mechanistically independent. Affinity precipitation experiments identified potential glycoprotein receptors for SElX and the interaction with protein ICAM-3, an important ligand for MAC-1 integrins, was validated suggesting SElX may interfere with cell signalling. Taken together, we present the first example of a bi-functional SAg that can manipulate two distinct arms of the human immune system and contribute to S. aureus survival during infection.

Made available in DSpace on 2014-06-12T22:58:20Z (GMT). No. of bitstreams: 2 arquivo2804_1.pdf: 2519864 bytes, checksum: a581d50e83db0c9412ea646764813b08 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O reino vegetal é uma das principais fontes de substâncias orgânicas de interesse terapêutico. A espécie Hymenaea stigonocarpa Mart. ex Hayne, família Leguminosae- Caesalpinioideae conhecida popularmente como Jatobá-do-cerrado é uma ―madeira de lei‖ utilizada na medicina popular como antibiótico e antiinflamatório. Os resultados promissores a cerca da fotoquímica e farmacologia de espécies do gênero Hymenaea inspiraram o presente estudo, que teve por objetivo avaliar a atividade antimicrobiana do extrato de Hymenaea stigonocarpa Mart. ex Hayne frente a 28 cepas de S. aureus com fenótipo de resistência a vários antimicrobianos. Os resultados in vitro, concentração inibitória mínima das 28 cepas, revelaram que tanto o extrato acetato de etila quanto o hidroalcoólico apresentaram atividade antimicrobiana, sendo o último o mais eficaz, e escolhido para a avaliação da atividade antimicrobiana in vivo. Para isso, foi empregado o modelo de ferida aberta infectada utilizando grupos de ratos diabéticos e não-diabéticos. As feridas foram infectadas com uma cepa de S. aureus resistente a meticilina e tratadas diariamente com um gel hidrofílico a 2% do extrato hidroalcoólico por 14 dias. Todas as feridas dos animais tratados com o gel de H. stigonocarpa, sendo eles diabéticos ou não, cicatrizaram ao fim do experimento. Os animais não-diabéticos tratados com o gel de H. stigonocarpa apresentaram um percentual de cicatrização maior que os controles e esta diferença foi estatisticamente significativa. Embora as feridas dos animais diabéticos tratados com o gel não tenham apresentado uma diferença estatisticamente significativa percentual da área de cicatrização em relação aos controles, os resultados histológicos das áreas das feridas revelaram não apenas reepitelização, como também formação de tecido em fase de maturação mais avançada que os controles diabéticos. A avaliação da toxicidade dérmica aguda do gel do extrato de H. stigonocarpa não revelou ação tóxica ou irritante. Nosso estudo aponta a eficácia microbiológica dos extratos de H. stigonocarpa e desperta para a necessidade de estudos adicionais que contribuam para o desenvolvimento de um novo medicamento

Die Innenausbauflächen von Krankentransport- bzw. Rettungswagen unterliegen dem Risiko einer Verunreinigung durch Methicillin-resistenten S. aureus (MRSA). In der vorliegenden Arbeit wurden Krankentransport- bzw. Rettungswagen unmittelbar nach dem Transport von MRSA-kolonisierten oder -infizierten Patienten auf MRSA untersucht. Zu diesem Zweck wurden an 2 Stellen der Trage und an 3 Stellen der Innenausbauflächen Proben entnommen. 89 von 100 untersuchten Transporten, welche das Einschlusskriterium einer Transportzeit von weniger als 20 Minuten erfüllten, wurden weitergehend analysiert. 8 der untersuchten Kranken- bzw. Rettungswagen (7,1%) wiesen eine Kontamination auf (90% Konfidenzintervall: 4-14 %), wobei die Transportzeit keinen Einfluss auf die Kontamination hatte. MRSA wurde nur an der Trage nachgewiesen und zwar ausschließlich am Kopfteil der Trage und an den Tragegriffen. Die beprobten Stellen der Innenausbauflächen waren nicht kontaminiert. Bei Kurzzeittransporten von MRSA-positiven Patienten sollte daher der Fokus der Desinfektion auf die Oberflächen in unmittelbarer Patientennähe gelegt werden. Eine weitere Untersuchung von 60 Transporten MRSA-negativer Patienten blieb ohne MRSA-Befund. In 12 dieser Krankentransportwagen wurde jedoch Methicillin-sensibler S. aureus nachgewiesen, der sich ebenfalls vorwiegend an Kopfteil und Handgriffen der Trage fand. Auch dieses Ergebnis unterstreicht die Bedeutung der Desinfektion patientennaher Flächen unabhängig vom MRSA-Status der transportierten Patienten. / Patients carrying MRSA provide a risk of surface contamination with MRSA in ambulance cars. This study analysed whether surfaces of ambulance cars, in which MRSA-colonised or -infected patients were transported, were contaminated with MRSA and where the contamination occurred. 89 transportation events lasting less than 20 minutes were included in the analysis. 8 ambulance cars (7,1%) were found to be contaminated with MRSA following transport (90% confidence interval: 4-14%). The transport time was not relevant for the contamination rate. MRSA was exclusively found on the stretcher, i.e. the headrest and the handles. This finding sug¬gests that disinfection measures after transport should be focused on the patients' contact surfaces. Consecutive investigation included 60 transport events in the absence of MRSA-notification and demonstrated that MSSA, but not MRSA, could be detected in 12 cars, again mostly at handles and headrests. This study shows the importance of disinfection of surfaces in the vicinity of patients transported in an ambulance irrespective of the patients' MRSA-status

No description available.