Staphylococcus aureus (S. aureus) est responsable de divers types d’infections chez l’homme et les animaux. Dans l’industrie laitière, ce pathogène est régulièrement mis en cause dans les infections intra-mammaires sous-cliniques qui persistent chez la vache et qui est à l’origine d’importantes pertes économiques. Le caractère chronique de cette infection lié aux échecs de l’antibiothérapie, sa difficile détection et l’inexistence de vaccin efficace ont suscité des recherches plus approfondies sur les S. aureus à l’origine de la mammite sous-clinique qui persiste afin de les diagnostiquer et de mieux les contrôler. Dans mon étude, j’ai caractérisé des marqueurs génétiques et phénotypiques de S. aureus persistant pour les utiliser comme un outil de diagnostique. La découverte de caractéristiques inhérentes aux souches causant des mammites bovines persistantes permettrait l'identification rapide de souches potentiellement problématiques en début d'infection et la prise de décisions justes prenant en compte la gestion thérapeutique et la gestion des troupeaux. La caractérisation des souches persistantes a commencé par la détection du profil des superantigènes qui sont des immuno-modulateurs de l’hôte. Ce profile nous a permis d’observer l’absence de l’îlot génomique νSaβ-1 chez les souches sous-cliniques. Cependant, la présence de seg augmente la probabilité de causer une infection non-persistante. L’étude du phénotype a permis d’observer une corrélation entre la production de biofilm et le niveau d’activation du système agr chez les souches persistantes et non persistantes. Nous avons observé une surexpression du biofilm suivi d’une sous-expression d’hld chez les souches persistantes et le phénomène inverse chez les souches non persistantes. Le typage par le gène spa a révélé que deux lignées bactériennes (t267 et t529) sont responsables de la majorité des cas de mammites sous-cliniques persistantes et cliniques au Canada.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-10-03T16:25:38Z No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) / Made available in DSpace on 2017-10-03T16:25:38Z (GMT). No. of bitstreams: 1 2016 - Viviane Figueira Marques.pdf: 1883187 bytes, checksum: 9996d5ea41ecd4f93424c808c4d82ba6 (MD5) Previous issue date: 2016-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Staphylococcus spp. plays an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant specie due to the production of virulence factors such as ?slime?, which is required for biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolates from bovine mastitis, to detect and quantify the expression of genes involved in its production and regulation, as well as to detect the phenogenotypic resistance to beta-lactam in order to evaluate the possible relation between biofilm production and antimicrobial resistance. The isolates were characterized by MALDI-TOF and phenogenotypic identification assays. Also they were submitted to the phenotypic tests to evaluate biofilm production and the susceptibility to beta-lactams. Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration in the Biofilm (MICB) were determined to three isolates presenting distinct biofilm production. Futherly, a PCR for the detection of ?slime? production genes (icaA and icaD), Bap protein (bap), beta-lactamase (blaZ) and protein altered penicillin-binding (mecA). Also, the Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Scanning Electron Microscopy (SEM) was performed to determine the most suitable time interval for biofilm analysis. Real-time PCR (qPCR) was performed at the chosen times to quantify the expression of icaA, icaD and hld genes in the three studied isolates. All 20 isolates were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II presented a prevalence of 70%. The SEM showed gradual changes in bacterial arrangement during the biofilm formation along the phases of the growth curve. The peak was reached at the stationary phase. Transcriptional analysis revealed increased expression of ica genes at 8 h of growth and of hld at 24 h. However, for the N-365 strain the ica expression was of low yield. For this study, the penicillin resistance was related to the production of beta-lactamase otherwise the high MBC detected for cefoxitin may be associated to biofilm protection, evidentiated by the fact that the isolates have MICB values higher than MICs tested for planktonic cells / Staphylococcus spp. tem papel importante na etiologia da mastite bovina. Staphylococcus aureus ? considerada a esp?cie mais relevante devido ? produ??o de fatores de virul?ncia, tais como ?slime?, o que favorece a forma??o do biofilme. O presente trabalho teve por objetivo detectar a express?o fenot?pica da forma??o de biofilme em 20 isolados de S. aureus oriundos de mastite bovina, detectar e quantificar a express?o dos genes envolvidos na sua produ??o e regula??o, al?m de detectar a resist?ncia fenogenot?pica aos beta-lact?micos para avalia??o da poss?vel rela??o da produ??o de biofilme com a resist?ncia antimicrobiana. Os isolados foram caracterizados atrav?s de testes fenogenot?picos e MALDI-TOF, submetidos ?s provas fenot?picas de detec??o da forma??o de biofilme e avalia??o da suscetibilidade aos beta-lact?micos. A Concentra??o Inibit?ria M?nima (CIM), Concentra??o Bactericida M?nima (CBM) e a Concentra??o Inibit?ria M?nima no Biofilme (CIMB) foram determinadas para tr?s isolados selecionados com base na varia??o da intensidade da produ??o de biofilme. Posteriormente, todos os isolados foram submetidos ? t?cnica de PCR para detec??o dos genes de produ??o de ?slime? (icaA e icaD), prote?na Bap (bap), beta-lactamase (blaZ) e prote?na ligante de penicilina alterada (mecA). Al?m de detec??o do sistema regulador Agr (agr RNAIII) e da tipifica??o do sistema Agr (agr I, agr II, agr III e agr IV). Foi realizada Microscopia Eletr?nica de Varredura (MEV) para determinar o intervalo de tempo mais adequado para a an?lise do biofilme. A PCR em tempo real (qPCR) foi realizada nos tempos selecionados para quantificar a express?o dos genes icaA, icaD e hld em tr?s isolados com produ??o variada de biofilme. Todos os isolados foram produtores de biofilme e a maioria apresentou os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr tipo II mostrou preval?ncia de 70%. A MEV mostrou mudan?as graduais no arranjo bacteriano durante a forma??o de biofilme ao longo das fases da curva de crescimento que atingiu seu pico de forma??o na fase estacion?ria. A an?lise transcricional evidenciou maior express?o dos genes ica no tempo de 8 h de crescimento e hld em 24 h. Contudo, a cepa N-365 mostrou baixa produ??o dos genes ica. Para este estudo, a resist?ncia ? penicilina foi relacionada com a produ??o de beta-lactamase, enquanto a elevada CBM detectada para cefoxitina pode estar associada ? prote??o que o biofilme oferece, epis?dio evidenciado pelo fato dos isolados apresentarem valores de CIMB superiores aos CIMs testados para as c?lulas planct?nicas.

S. aureus est un pathogène majeur de l’Homme capable de produire une grande variété de facteurs de virulence tels que les phénol-solubles modulines alpha (PSM) et l’hémolysine delta (Hld). La transmission de S. aureus est essentiellement manu-portée mais les éléments favorisant sa dissémination dans la population restent inconnus. Les mastocytes étant connus pour libérer des médiateurs pruritogènes, nous avons suspecté leur implication dans la physiopathologie et la transmission des infections cutanées staphylococciques. Sur une lignée de mastocytes humains, l’Hld et les PSM1, montrés pour être produits in vivo, déclenchaient la libération de tels médiateurs. Chez S. aureus, la production des toxines est sous la dépendance du système de régulation globale Agr. Les souches de S. aureus appartenant au type Agr1, produisant significativement plus d’Hld et de PSM que les autres souches, ont été les plus fréquemment retrouvées au cours de l’année 2017 dans les infections cutanées staphylococciques. Ceci corrobore l’hypothèse selon laquelle une souche de S. aureus produisant des toxines capables d’interagir avec les mastocytes et induisant un prurit, diffuse plus facilement dans la population. Nous avons ensuite étudié la modulation de l’expression des PSM et d’Hld par des concentrations sub-inhibitrices d’antibiotiques. L’oxacilline induisait une inhibition de l’expression des PSM et d’Hld alors que la clindamycine entraînait plus fréquemment une induction de leur expression. Ces observations nous ont interrogé sur l’utilisation de la clindamycine considérée habituellement comme anti-toxinique et sur l’effet bénéfique ou délétère de l’effet inhibiteur de l’oxacilline / S. aureus is a major human pathogen able to produce several virulence factors such as phenol-solublemodulins alpha (PSMalpha) and delta hemolysin (Hld). S. aureus is essentially spread through hand butthe elements promoting its spreading stay unsolved. Mast cells release several soluble mediatorstriggering itching behavior. We suspect the mast cell involvement in spreading of S. aureus strains andin physiopathology of staphylococcal skin infections. Upon human mast cell line, we showed thatPSMalpha1 and Hld induced the release of mediators triggering itching behavior. Moreover, these toxinswere produced in vivo during staphylococcal skin infections. Expression of staphylococcal virulencefactors is regulated by global regulatory system Agr. Interestingly, we observed that S. aureus strainsbelonging in Agr1 produced higher quantity of PSMalpha and Hld than those belonging to Agr2 and Agr3,and were more frequently responsible to skin infections during the last year. This observation supportsour hypothesis whereby a strain producing toxins able to trigger mast cell mediator inducingscratching behavior, spreads electively in the community. Thereafter, we studied modulation of PSMalphaand Hld expression by sub-inhibitory concentration of antibiotics. We reported that oxacillin inducedan inhibitory effect on PSMalpha and Hld expression, while clindamycin resulted in more frequently aninducer effect. These results are discordant with these observed with Panton-Valentine leucocidin andalpha hemolysin and interrogate on clindamycin use for its anti-toxin activity and on benefic ordeleterious effect of oxacillin inhibitory effect

Approved for public release, distribution is unlimited / The need for a well thought out, planned, and rehearsed command and control organization to conduct special operations in the US Northern Command Area of Responsibility is vital to success in defending the Homeland. Currently, USNORTHCOM does not have an apportioned or assigned command and control structure for the conduct of special operations. This thesis analyzes three courses of action to fulfill this requirement: use the current USNORTHCOM battle staff command structure including the integration of the Standing Joint Force Headquarters-North; rely on the newly formed US Special Operations Command's Joint Task Force Structures; and establish a Theater Special Operations Command North assigned to USNORTHCOM. Through the conduct of analysis and research this thesis recommends that The Joint Staff direct the reorganization required to establish a Theater Special Operations Command North to exercise command and control of special operations forces conducting operations in the USNORTHCOM AOR. / Lieutenant Colonel, United States Army