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.

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Staphylococcus aureus yra vienas iš dažniausių įvairių infekcijų sukėlėjų. Labai svarbu žinoti regioninius antimikrobinio jautrumo duomenis, kad būtų galima skirti veiklius vaistus prieš Staphylococcus aureus, dar nenustačius konkrečios kultūros jautrumo antibiotikams. Darbo tikslas. Staphylococcus aureus jautrumo antibiotikams analizė Klaipėdos vaikų ligoninėje 2008-2009 m. Tikslui įgyvendinti iškelti tokie uždaviniai: 1) išanalizuoti 2008-2009 m. Klaipėdos vaikų ligoninėje iš pasėlių išskirtas Staphylococcus aureus kultūras; 2) ištirti S. aureus kultūrų jautrumą antibiotikams ir atlikti jautrumo antibiotikams 2008-2009 metais analizę. Metodai. S. aureus kultūros išskyrimui ir identifikavimui naudoti metodai - auginimas specifinėse terpėse, dažymas gramo būdu, katalazės testas, plazmos koaguliazės testas. Jautrumas antibiotikams nustatytas diskų difuzijos metodu, β-laktamazės aptikimas – nitrocefino testu, MRSA nustatymas - peniciliną surišančio baltymo (PBP2‘) latekso agliutinacijos testu. Statistinė analizė buvo atlikta naudojant programą SPSS Statistics 17.0. Rezultatai. 2008-2009 m. Klaipėdos vaikų ligoninėje iš pasėlių išskirta 1691 S. aureus kultūrų. Statistiškai reikšmingai daugiau S. aureus kultūrų išskirta jaunesniems nei 1 metų amžiaus vaikams. Daugiausia kultūrų buvo išauginta iš išmatų (52.4%), pūlių (29.3%) ir tepinėlių iš gerklės (14.0%). Nustatyta, kad penicilinui atsparios 89.5% kultūrų; eritromicinui atsparios 20.5%, mažai jautrios – 0.7% kultūrų... [toliau žr. visą tekstą] / Staphylococcus aureus is one of the most common cause of infections. It’s relevant to know regional data of antimicrobial susceptibility to be able to prescribe effective drugs against S. aureus before knowing the suscpetibility of a specific culture. Objective. The analysis of Staphylococcus aureus’ susceptibility to antibiotics in Klaipėda’s children hospital in 2008-2009. In order to implement it, these tasks were established: 1) to analyse Staphylococcus aureus’ cultures isolated from clinical material in Klaipėda’s children hospital in 2008-2009; 2) to determine the antibiotic susceptibility of S. aureus’ cultures and perform the analysis of antibiotic susceptibility in 2008-2009. Methods. S. aureus was isolated by cultivating it on specific agars, catalase test, Gram staining; plasma coagulase test. Antibiotic susceptibility was determined by disk diffussion method; β-lactamase detection – by nitrocefin test; MRSA detection – by penicilin-binding protein (PBP’) late agglutination test. Statistical analysis was performed with program SPSS Statistics 17.0.' Results. In Klaipėda’s children hospital 1691 cultures of S. aureus were isolated in 2008-2009. Statistically relevant higher number of S. aureus’ cultures was isolated form children aged less than one year old. Most cultures were isolated from faeces (52.4%), pus (29.3%) and throat swabs (14.0%). 89.5% resistance to penicillin was determined. 20.5% and 0.7% of cultures were resistant and intermediate to... [to full text]

Staphylococcus aureus produces a family of at least 21 distinct superantigens (SAgs) which include staphylococcal enterotoxins (SEs), staphylococcal enterotoxin-like toxins (SEls), and toxic shock syndrome toxin-1 (TSST-1), and contribute to disease pathogenesis via modulation of the host immune response. Specific SAgs have been shown to cause toxinoses such as staphylococcal food poisoning and toxic shock syndrome, and have been implicated in immunological disorders such as rheumatoid arthritis, psoriasis, and Kawasaki syndrome. However the role of SAgs in disease pathogenesis, in general, is poorly understood. S. aureus is a common cause of bovine mastitis. Analysis of the genome sequence of the bovine strain RF122 revealed genes encoding bovine variants of characterized SAgs, TSST-1, SElL, SEC, SEG, SEI, SElU, SElO, SElN and a truncated form of SElM. In addition we identified 3 genes with sequence homology to characterized SAgs, which are predicted to encode novel SAgs, SElX, SElY and SElZ. Expression of all 11 predicted SAg genes was detected in vitro, including several with growth phase-dependent expression. Characterization of a novel SAg, SElX which is encoded in the core genome of 94% of phylogenetically diverse S. aureus strains from human and animal infections was carried out. In addition to its superantigenic properties, SElX has a unique predicted structure characterized by a truncated SAg B domain. At least 14 different alleles of the selx gene were identified among the common human and animal pathogenic clones, and evidence for assortive recombination of selx alleles between distinct clonal lineages was discovered. SElX was expressed by representative human, bovine and ovine strains in vitro, in a growth phase dependent manner, and during human, bovine and ovine infections, consistent with a broad role in the pathogenesis of different S. aureus diseases in multiple hosts. SElX produced by bovine- and ovine-specialized S. aureus strains had 10-fold greater mitogenic activity and a distinct V activation profile for bovine lymphocytes compared to SElX made by the human strain USA300 indicating functional diversification of selx alleles from different hosts. This is the first description of a core-genome encoded SAg of S. aureus. The discovery that the great majority of S. aureus clinical isolates have superantigenic capacity has important implications for our understanding of staphylococcal disease pathogenesis. To investigate the role of SAgs in disease pathogenesis, a SAg-deficient strain of S. aureus, RF122-8 was constructed by sequential allele replacement. RF122-encoded SAg genes were cloned into the pALC2073 plasmid, which has an inducible promoter allowing controlled expression in the SAg-deficient strain RF122-8. These constructs allowed us to determine that TSST-1bov, SElLbov, SECbov, SElNbov, SEIbov and novel SAgs, SElXbov and SElYbov were mitogenic for bovine Tcells, and stimulated T-cell receptor variable (TRBV) sub-family-specific activation. Preliminary experimental intra-mammary infections of dairy cows revealed that clinical symptoms were similar during infection with wild type RF122 and SAg-deficient strains, including high somatic cell counts (6 LogSCC), and elevated body temperature (106 °F). However a higher infectious dose was required to establish infection with the SAg-deficient strain RF122-8 in comparison to the wild type, RF122 indicating an attenuation of virulence. Overall, these data provide broad new insights into the importance of SAgs in staphylococcal disease pathogenesis.

El Staphylococcus aureus resistente a meticilina (SARM), es una bacteria grampositiva multirresistente, considerada un patógeno crítico en medicina humana, y es una de las causas que lideran las infecciones asociadas a hospitales (SARM-AH). Sin embargo en los últimos años se están incrementando los casos de infecciones asociadas a la comunidad (SARM-AC), y SARM asociado al ganado (SARM-AL).La crianza porcina se identificó como uno de los factores de riesgo emergentes en el incremento de portadores de S. aureus a nivel nasal en las infecciones por SARM-AL, probablemente favorecido por el extenso uso de antibióticos.El objetivo de este estudio fue determinar la presencia de SARM en crianza porcina de traspatio en el departamento de Tumbes. Para la ejecución del estudio se tomaron 325 muestras de hisopados nasales de cerdos de crianza de traspatio, previos a su sacrificio. Los hisopados de ambas fosas nasales se inocularon en agar manitol sal cefoxitina 4 µg/ml. Las colonias morfológicamente compatibles con SARM fueron inoculadas en agar sangre al 5% y fueron evaluadas con pruebas bioquímicas para realizar la identificación fenotípica de la bacteria.La reacción en cadena Polimerasa permitió la detección del gen mecA en 15 muestras, sin embargo ninguna de ellas fue positiva al gen nuc, gen propio de Staphylococcus aureus. Los resultados hallados, dieron como negativa la presencia de SARM. Sin embargo podemos estimar la presencia de resistencia a meticilina en 15/79 (coagulasa positiva) de muestras compatibles con Staphylococcus.La probabilidad de encontrar un cerdo con SARM se encontraba en el intervalo de confianza del 95% desde 0.00009 a 0.01251. Consecuentemente, se encontraba muy por debajo de la prevalencia límite. Palabras claves: Staphylococcus aureus, SARM, cerdos, meticilina, Tumbes / --- Methicillin-resistant Staphylococcus aureus (MRSA) is a gram positive bacteria multidrug considered a critical pathogen in human medicine, and is one of the leading causes associated to hospitals infections (HA-MRSA). However in recent years there are increasing cases of community-associated infections (CA-MRSA), and livestock associated MRSA (LA-MRSA). Swine production was identified as one of the emerging risk factors in the increase in nasal carriers of S. aureus in LA-MRSA infections, probably aided by the widespread use of antibiotics. The aim of this study was to determine the presence of MRSA in backyard pigs rearing in the department of Tumbes. For the execution of the study 325 samples of nasal swabs from backyard pigs before slaughter were taken. The swabs from both nostrils were inoculated mannitol salt agar-cefoxitin 4 µg / ml. The colonies that were morphologically compatible with MRSA were inoculated on blood agar 5% and were evaluated by biochemical tests for phenotypic identification of the bacteria. Polymerase chain reaction allowed the detection of mecA gene in 15 samples; however none of them were positive to the characteristic nuc gene of Staphylococcus. These results found, showed lack of evidence for MRSA. However, we can estimate the presence of methicillin resistance in 15/79 (coagulase positive) samples compatible with Staphylococcus. The probability of finding a MRSA positive pig was within the confidence interval of 95% from 0.00009 to 0.01251. Consequently, it was well below the threshold prevalence. Keywords: Staphylococcus aureus, MRSA, pigs, methicillin, Tumbes.

Introdução: a colonização de indivíduos com HIV/aids por microrganismos patogênicos tem sido associada a maior risco de morbidade e mortalidade, principalmente quando esse microrganismo é o Staphylococcus aureus. Identificar precocemente esta condição permite implementar medidas preventivas do adoecimento a ele relacionado, em nível individual e coletivo. Objetivo: avaliar a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids internados em um hospital escola. Metodologia: estudo de corte transversal, tendo como sujeito pessoas vivendo com HIV/aids, internadas em duas unidades especializadas em HIV/aids de um Hospital Escola do município de Ribeirão Preto- SP. Todos os preceitos éticos foram criteriosamente respeitados. No período de Agosto/2011 a Julho/2012, todos os indivíduos internados foram abordados e para aqueles que aceitaram participar, procedeu-se a coleta de amostra de saliva e secreção nasal, além da coleta de dados sociodemográficos, clínicos e imunológicos, obtidos por meio do prontuário e entrevista individual. As amostras foram encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia da instituição em estudo. Foram semeadas em meios de cultura ágar sangue e manitol, e após, transferidas para o sistema automatizado Vitek® 2 (BioMérieux(TM)), por meio dos cartões GP Test Kit Vitek® 2, para bactérias gram-positivas. Foram empregados cartões AST-P585 para avaliar a sensibilidade dos Staphylococcus aureus meticilina resistente (MRSA) aos antibióticos. Os dados foram armazenados em planilhas do Microsoft Office Excel 2011 for Mac e organizados por meio do software Statistical Package for the Social Sciences (SPSS), versão 17.0 for Windows. Resultados: De 229 indivíduos com HIV/aids internados nas unidades, 169 constituíram os sujeitos desta pesquisa, dos quais 57,4% eram do sexo masculino, 39,6% apresentaram idade de 40 a 49 anos e 45% tinham o primeiro grau completo. Foram obtidas 338 amostras (169 de secreção nasal e 169 de saliva). A prevalência de colonização por Staphylococcus aureus foi identificada em 20,4% das amostras, com 21,7% de resistência à oxacilina, sendo em secreção nasal 66,7% e em saliva 33,3%. Apresentaram contagem de linfócitos T CD4 abaixo de 200 células/mm3 60,0% dos indivíduos com MRSA nasal e 80,0% estavam em uso de antimicrobianos. Em 40,0% dos indivíduos com MRSA na saliva carga viral foi igual ou superior a 500.001 cópias/mL, e 80,0% destes também usavam antimicrobianos, MRSA nasal e saliva foi identificado em 60,0% dos indivíduos que não estavam em uso de antirretroviral. Conclusão: a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids foi predominante em secreção nasal, com baixa contagem de linfócitos T CD4, com história de internação prévia, uso de antimicrobiano e ausência do uso de antirretroviral, podendo representar importante fonte de infecção. / Introduction: colonization by pathogenic microorganisms in individuals with HIV/AIDS has been associated with increased risk of morbidity and mortality, especially when that organism is Staphylococcus aureus. Early identification of this condition allows implementing preventive measures of illness related to it, both individually and collectively. Objective: to evaluate the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS in a teaching hospital. Method: cross-sectional study; the subjects were people living with HIV/AIDS and hospitalized in two specialized HIV/AIDS care units of a Teaching Hospital in the city of Ribeirão Preto. All ethical principles were carefully observed. In the period from August 2011 to July 2012, all subjects hospitalized were approached and, for those who agreed to participate, the collection of saliva and nasal discharge sample was performed, in addition to collecting sociodemographic, clinical and immunological data, obtained through medical record and individual interviews. The samples were forwarded and processed by the Laboratory of Microbiology and Sorology of the institution. They were seeded in blood agar and mannitol-salt-agar culture medium, and thereafter, transferred to the automated system Vitek® 2 (BioMérieux(TM)) through Vitek® 2 Test Cards for Gram-positive bacteria. AST-P585 cards were used to assess the sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to the antibiotic. Data were stored in spreadsheets of Microsoft Office Excel 2011 for Mac and organized by the Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows. Results: of the 229 individuals with HIV/AIDS hospitalized in the units, 169 were the subjects in this study, of whom 57.4% were male, 39.6% were aged from 40 to 49 years, and 45% had completed elementary school. 338 samples were collected (169 of nasal discharge and 169 of saliva). The prevalence of Staphylococcus aureus colonization was identified in 20.4% of samples, with 21.7% of oxacillin resistance, being 66.7% in nasal discharge and 33.3% in saliva. 60.0% of individuals with MRSA in nasal had lymphocytes T CD4 count below 200 cells/mm3 , and 80.0% were taking antimicrobials. In 40.0% of the individuals with MRSA in saliva, the viral load was equal or higher than 500.001 copies/mL, and 80.0% of these also used antimicrobials; MRSA in nasal and in saliva were detected in 60.0% of individuals who were not taking antiretroviral. Conclusion: the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS was prevalent in nasal discharge, had lymphocytes T CD4 low count, with a history of previous hospitalization, antimicrobial use and the absence of antiretroviral use, and it may represent an important source of infection.

Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell. This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology. Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host. Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis. / Staphylococcus aureus ist ein fakultativ pathogener Kommensale des Menschen und besiedelt bei etwa einem Drittel der Bevölkerung überwiegend den Nasen-Rachenraum sowie die Haut ohne klinische Symptome auszulösen. Darüber hinaus zählen diese Bakterien zu den wichtigsten Vertretern der Kranken- hauskeime, die schwerwiegende Infektionen besonders im Bereich der Intensivstationen in Kranken- häusern hervorrufen können. Methicillin-resistente Staphylococcus aureus (MRSA) sind dabei resistent gegen übliche Antibiotika und daher schlecht therapierbar. Neuere Forschungsarbeiten zeigten, dass S. aureus von Zellen des Wirts aufgenommen wird und diese von innen heraus abzutöten vermag. Über die zugrunde liegenden molekularen Mechanismen dieser Zelltoxizität ist jedoch nicht viel bekannt. In der vorliegenden Arbeit sollten daher Faktoren von S. aureus identifiziert und charakterisiert wer- den, die die intrazelluläre Virulenz des Bakteriums und das darauf folgende Absterben der Wirtszelle beeinflussen. Dafür wurden mittels Transposon-Insertionsmutagenese S. aureus Mutanten-Bibliotheken erstellt, welche für genomweite Infektionsscreens in vitro und in vivo genutzt wurden. Die Auswertung dieser Analysen erfolgte dabei durch Hochdurchsatz-Sequenzierung der Transposon-Insertionsstellen (Tn-seq). In diesen Studien wurde neben zahlreichen bakteriellen Faktoren ein neuartiges Virulenzreg- ulator - System identifiziert. Dieses System besteht aus dem Transkriptionsregulator der AraC-Familie Repressor of surface proteins (Rsp) und einer nicht-kodierenden RNA, SSR42. rsp-Mutanten zeigten eine erhöhte intrazelluläre Überlebensrate in menschlichen Epithelzellen sowie eine verzögerte Cytotoxizität im Wirt. Durch RNA-Sequenzierung (RNA-seq) wurde der Einfluss von Rsp auf die globale Genexpres- sion ermittelt. Dabei zeigte sich, dass Rsp die Expression von SSR42, sowie Cytotoxinen und anderen immunmodulatorischen Faktoren von S. aureus kontrolliert. Wasserstoffperoxid, ein Molekül, welches durch Neutrophile zur Bekämpfung von Pathogenen gebildet wird, führt dabei Rsp-abhängig zu einer Erhöhung der bakteriellen Toxinproduktion. Die Abwesenheit von Rsp in bakteriellen Mutanten res- ultiert in einer Reduktion S. aureus-induzierter Zerstörung von Neutrophilen sowie zum Überleben von Versuchstieren im Lungeninfektionsmodell. Eine systemische Infektion ist dabei jedoch weiterhin mög- lich. Interessanterweise führt ein Fehlen des rsp zu einer erhöhten Überlebensrate von Makrophagen, welches auf eine Verbreitung der Bakterien im Organismus in diesem Zelltyp hindeuten könnte. Rsp ist demnach ein neuartiger globaler Regulator bakterieller Virulenz, der zwar die infektions- bedingte Letalität verzögert, jedoch damit eine Disseminierung der Infektion mit S. aureus begünstigt.