No description available.

Staphylococcus aureus is a notorious human pathogen associated with severe nosocomial and community-acquired infections. In addition, S. aureus is a major cause of animal diseases including skeletal infections of poultry and bovine and ovine mastitis, which are a large economic burden on the broiler chicken and dairy farming industries. The population structure of S. aureus associated with humans has been well studied. However, despite the prevalence of S. aureus infections in broiler flocks, our understanding of the diversity of poultry S. aureus is very limited. In this study, multilocus sequence typing was performed on 48 strains of S. aureus isolated from broiler chickens on farms in 6 countries on 4 different continents, in addition to 9 isolates from different species of reared game and wild birds in Scotland. This was followed by fine scale population genetic analysis of a subset of strains by single nucleotide polymorphism discovery. These studies reveal that the majority of S. aureus isolates from broiler chickens are the descendants of a single human-to-poultry host jump by a subtype of the worldwide human clonal complex 5 (CC5) clonal lineage unique to Poland. In contrast to human subtypes of the CC5 radiation, which demonstrate strong geographic clustering, the poultry CC5 clade was distributed in different continents, consistent with wide dissemination via the global poultry industry distribution network. In order to establish the molecular basis for avian specificity in the CC5 poultry clade, whole genome sequences were determined for a sequence type 5 (ST5) poultry isolate from Ireland and a basal human associated ST5 MRSA strain from Poland. Sequence analysis revealed that the poultry CC5 clade has undergone genetic diversification from its human progenitor strain by acquisition of novel mobile genetic elements from an avian-specific accessory gene pool, and by the inactivation of several proteins important for human disease pathogenesis. In order to examine the importance of positive selection in the adaptation of S. aureus to poultry and for S. aureus evolution, in general, genome-wide analysis of the ratio of synonymous to non-synonymous substitutions was performed on 30 strains from 3 humans and other animals, from diverse lineages. Positive selection has affected proteins from the majority of functional categories, resulting in diversification of the proteome, metabolome and replication capacity, which may be associated with adaptation of S. aureus to diverse environments. For several proteins, an elevated rate of non-synonymous substitutions unique to animal-associated lineages is consistent with a role for these proteins in host adaptation. Taken together, the results of this study have determined the evolutionary history of a major new animal pathogen that has undergone rapid avian host adaptation and intercontinental dissemination. The data highlight the importance of gene acquisition and loss and positive selection in the adaptive evolution of S. aureus.

Orientador: Ana Claúdia Pavarina / Banca: Eunice Teresinha Giampaolo / Banca: Ana Paula Dias Ribeiro / Resumo: A necessidade de superar o desafio criado pelos biofilmes resistentes aos tratamentos antimicrobianos convencionais tem levado à busca por tratamentos alternativos, como terapia fotodinâmica antimicrobiana (aPDT). Este estudo avaliou in vitro a eficácia da aPDT na inativação de biofilmes de Staphylococcus aureus suscetíveis e resistentes à meticilina (MRSA e MSSA), mediado pelos fotossensibilizadores (PSs) Curcumina (Cur) e Photodithazine® (PDZ). Biofilmes foram formados e tratados com diferentes concentrações de Cur (0, 20, 40 e 80 μM) e PDZ (0, 50 e 75 mg/L), e iluminados ou não por fonte de luz LED (Cur 455 ± 3 nm/ 5,28 J/cm2; PDZ 660 ± 3 nm/ 5,28 J/cm2 ou 50 J/cm²). Os grupos Controle Positivo (CP) não receberam nenhum PS e também não foram iluminados. A viabilidade dos micro-organismos após a aPDT foi avaliado pelo número de colônias viáveis, pelo ensaio de XTT e pela utilização do kit LIVE/DEAD® na Microscopia Confocal de Varredura à Laser (MCVL). Os resultados foram avaliados por análises de variância de dois fatores de efeitos fixos (ANOVA) e complementados por comparações múltiplas de médias pelo teste de Tukey. Para ambas as cepas, todas as concentrações de Cur e PDZ testadas reduziram significativamente a atividade metabólica e o UFC/mL para ambos micro-organismos quando comparado com os grupos CN (p0,05). Os resultados foram otimizados para a Cur quando utilizou-se a maior concentração (80 μM), para a PDZ, a maior redução nos micro-organismos foi observada quando associou-se a maior concentração de PDZ (75 mg/L) com a maior dose de luz (50 J/cm²). Os biofilmes submetidos a aPDT demostraram pela MCVL um maior número de células coradas em vermelho, indicando que a aPDT foi eficaz para promover danos ou morte às células bacterianas. Assim, a aPDT pode ser considerada promissora para atuar de forma sinérgica no tratamento de infecções bacterianas / Abstract: The need to overcome the challenge created by biofilms regarding conventional antimicrobial approaches has lead to search of alternative treatments such as Antimicrobial Photodynamic Therapy (aPDT). This in vitro study evaluated the efficacy of aPDT using the photosensitizer (PS) Curcumin (Cur) and Photodithazine® (PDZ) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA). Biofilms were treated with different Cur (0, 20, 40 or 80 μM of Cur) and PDZ concentrations (0, 50 or 75 mg/L) and illuminated or not with LED source (Cur 455 ± 3 nm/ 5.28 J/cm2; PDZ 660 ± 3 nm/ 5.28 J/cm2 or 50 J/cm²). Positive control samples were not exposed to PS or light. The microorganisms viability after aPDT were evaluated by counting the number of colonies, the XTT assay and LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). The results were evaluated by analysis of variance, two-factor fixed effects (ANOVA) and complemented by multiple comparisons by Tukey test. For both strains, all the tested Cur and PDZ concentrations reduced significantly both biofilm metabolic activity and CFU/mL compared to the negative control (p0.05). Moreover, the results were optimized for Cur when the higher concentration was used (80 μM); For PDZ, the best results were obtained when it was associated a higher concentration of PDZ (75 mg/L) with the higher dose of light (50 J/cm²). Biofilms submitted to aPDT showed a large number of red-stained colonies, indicating that this therapy was efficient in disrupting the bacterial membrane. It can be concluded that PS was efficient in reducing viable colonies of both S. aureus strains by damaging cell membrane and causing cell death. Thus, the aPDT is can be considered promising to act synergistically in the treatment of bacterial infections / Mestre

Hintergrund: Zunehmend wird der Eigenschaft von Staphylococcus aureus als fakultativ intrazellulärem Erreger Bedeutung zugemessen. Ein direkter Nachweis der in vivo Relevanz von fakultativ intrazellulärem S. aureus bleibt allerdings bisher aus. Der Mechanismus zellulärer Invasivität ist bekannt und korreliert mit verschiedenen molekularen Markern (spa-Typ, SCCmec-Typ und pls/Pls). In dieser Studie wurde die Zuverlässigkeit und Ausweitbarkeit dieser Marker getestet. Des Weiteren wurde überprüft, ob sich die zelluläre Invasivität von kolonisierenden und Infektions-assoziierten MRSA-Isolaten unterscheidet und, ob die alleinige Bestimmung molekularer Marker in vitro die Virulenz eines Isolats in vivo abzuschätzen vermag. Methoden:Insgesamt wurden 109 MRSA-Isolate gesammelt, molekular charakterisiert (spa-Typ, BURP-Analyse, SCCmec-Typ, pls, agr-Typ, Hämolyseverhalten) und das Potential zellulärer Invasivität in vitro ermittelt. Die Assoziation eines Isolates mit einer Infektion in vivo wurde nachverfolgt (93 Kolonisierer versus 16 Infektions-assoziierte-Isolate). Zusätzlich wurde eine Referenzgruppe aus 13 S. aureus-Isolaten etabliert, die klinisch mit vergleichsweise invasiven Infektionen assoziiert waren (12 Osteomyelitis-Isolate und 1 Endokarditis-Isolat). Ergebnisse: Die bekannten molekularen Marker zellulärer Invasivität korrelieren zuverlässig in einer Population klinischer MRSA-Isolate und lassen sich auch auf bisher nicht bekannte (spa- und SCCmec-) Typen ausweiten. Das Hämolyseverhalten korrelierte nicht mit der zellulären Invasivität. Der agr-Typ wurde als weiterer molekularer Marker identifiziert. Die zelluläre Invasivität war unabhängig von der Etablierung einer Infektion in vivo (mediane Invasivität der Kolonisierer 100% versus 108% der Infektions-assoziierten Studienisolate und 110% der externen Referenzisolate). Des Weiteren waren die molekularen Marker spa- und agr-Typ nicht in der Lage, die Virulenz eines MRSA-Isolats in vivo abzuschätzen. Diskussion: Die zelluläre Invasivität klinischer MRSA-Isolate korreliert zuverlässig mit molekularen Markern. Allerdings vermögen weder die zelluläre Invasivität, noch mit ihr assoziierte molekulare Marker die Etablierung einer Infektion in vivo vorherzusagen. Beide scheinen also als Surrogat-Parameter zur Abschätzung der klinischen Virulenz eines Isolats ungeeignet. Zur Klärung der Frage, ob molekulare Marker zellulärer Invasivität in anderen Abschnitten der Pathogenese von S. aureus- Infektionen eine Rolle spielen, bedarf es weiterer Studien. / Background: Fibronectin-binding of S. aureus is reported to correlate with the propensity to cause invasive infections. Cellular invasion of S. aureus is Fn dependent and clusters with molecular markers (spa type, SCCmec, Pls). Here, we tested the hypothesis that cellular invasiveness and corresponding molecular markers predict the propensity to cause infections in a prospective cohort. Methods: 109 clinical MRSA isolates were characterized (agr, spa, BURP, SCCmec, pls) and cellular invasiveness was determined. Association with clinical infection was assessed (93 colonizing vs. 16 infecting isolates). Further 13 S. aureus isolates from patients with severe S. aureus infections served as an external comparison. Results: The agr type was identified as a new marker for invasiveness and known molecular markers were corroborated. Establishment of infection was independent from cellular invasiveness of MRSA (mean colonizing vs. infecting isolates 100% and 89%, respectively; external infecting isolates: 109%). Furthermore agr and spa types were unable to predict clinical behavior. Conclusion: Cellular invasiveness of MRSA isolates clusters reproducibly with molecular markers. However, cellular invasiveness and molecular markers cannot predict establishment of infection. This suggests that both criteria may not be used as surrogate parameters for the virulence potential of human MRSA isolates.

Mastitis is a syndrome manifested by mammary gland inflammation which is thought to cause between $300 and $400 million in annual losses to the Canadian Dairy Industry. Studies have indicated that <i>S. aureus</i> may cause the production of anti-inflammatory cytokines which may enhance its survival within the bovine mammary gland. However, other studies have reported differing results following S. aureus intramammary infection (IMI). This thesis tested the hypothesis that S. aureus generated anti-inflammatory cytokine responses at the site of infection. In the first objective, different S. aureus isolates were screened for their effects on cytokine production (IFN-γ, TNF-α, IL-4 and IL-10) by bovine peripheral blood mononuclear cells (PBMCs) in vitro. Nine S. aureus isolates were co-cultured with PBMCs from lactating dairy cattle. Cattle used in the study had recall immune responses to <i>S. aureus</i>. The majority (6/9) of S. aureus isolates had minor effectors on cytokine production. The three remaining isolates generated large cytokine responses with both pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-4 and IL-10) characteristics. Two of these three isolates were tested in vivo by experimentally infecting lactating ewes. Cytokine production was characterized in the teat end, the mammary parenchyma and the supramammary lymph nodes (SMLNs). One isolate generated anti-inflammatory responses <i>in vivo</i> (IL-4 and IL-10) whilst the other generated both pro-inflammatory (IFN-γ) and anti-inflammatory (IL-10) responses in vivo. Given that some studies have suggested a role of staphylococcal enterotoxin C (sec) in the generation of anti-inflammatory responses, the role of sec was also investigated using bovine PBMCs. When purified SEC protein was co-cultured with PBMCs from beef steers, anti-inflammatory cytokines were produced. However, a <i>S. aureus</i> strain which was transformed for the sec gene did not affect cytokine production when co-cultured with PBMCs from lactating dairy cattle. The results of this thesis suggest that <i>S. aureus</i> infection can cause anti-inflammatory cytokine production but the response depends on the isolate causing the infection. Furthermore, the role of sec appears to be minimal.

Mastitis is a syndrome manifested by mammary gland inflammation which is thought to cause between $300 and $400 million in annual losses to the Canadian Dairy Industry. Studies have indicated that <i>S. aureus</i> may cause the production of anti-inflammatory cytokines which may enhance its survival within the bovine mammary gland. However, other studies have reported differing results following S. aureus intramammary infection (IMI). This thesis tested the hypothesis that S. aureus generated anti-inflammatory cytokine responses at the site of infection. In the first objective, different S. aureus isolates were screened for their effects on cytokine production (IFN-γ, TNF-α, IL-4 and IL-10) by bovine peripheral blood mononuclear cells (PBMCs) in vitro. Nine S. aureus isolates were co-cultured with PBMCs from lactating dairy cattle. Cattle used in the study had recall immune responses to <i>S. aureus</i>. The majority (6/9) of S. aureus isolates had minor effectors on cytokine production. The three remaining isolates generated large cytokine responses with both pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-4 and IL-10) characteristics. Two of these three isolates were tested in vivo by experimentally infecting lactating ewes. Cytokine production was characterized in the teat end, the mammary parenchyma and the supramammary lymph nodes (SMLNs). One isolate generated anti-inflammatory responses <i>in vivo</i> (IL-4 and IL-10) whilst the other generated both pro-inflammatory (IFN-γ) and anti-inflammatory (IL-10) responses in vivo. Given that some studies have suggested a role of staphylococcal enterotoxin C (sec) in the generation of anti-inflammatory responses, the role of sec was also investigated using bovine PBMCs. When purified SEC protein was co-cultured with PBMCs from beef steers, anti-inflammatory cytokines were produced. However, a <i>S. aureus</i> strain which was transformed for the sec gene did not affect cytokine production when co-cultured with PBMCs from lactating dairy cattle. The results of this thesis suggest that <i>S. aureus</i> infection can cause anti-inflammatory cytokine production but the response depends on the isolate causing the infection. Furthermore, the role of sec appears to be minimal.

published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / Public Health / Master / Master of Public Health

No description available.

Staphylococcus aureus (S. aureus) is a medically important bacterial pathogen associated with many diseases and infections of the respiratory system, wound sites, surgical incisions, and other portals of entry and exit. S. aureus is able to invade cells via mechanisms that have yet to be fully characterized. Vimentin, a protein filament of the animal cell cytoskeleton, and phosphoinositide 3-kinase (PI3K), a family of kinases responsible for initiating several cell signaling events, were found to be associated with S. aureus invasion. Confocal microscopy revealed that the vimentin network in human umbilical vein endothelial cells (HUVECs) undergoes dynamic rearrangement in steady state under control conditions. However, cells infected with S. aureus demonstrated peri-nuclear collapse of the vimentin network. Pre-treatment with LY294002, a drug that inhibits PI3K activity, decreased invasion of S. aureus and paralyzed the vimentin network. These data suggest that PI3K mediates S. aureus infection and vimentin rearrangement. / Department of Biology