Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus. / Master of Science

The antimicrobial effects of silver nanoparticles were studied. Silver nanoparticles were synthesized through wet chemistry method, and were dispersed in aqueous suspension. With nanoscale silica particles served as heterogeneous nucleation sites, silver nanoparticles were formed anchoring on the silica surface. Suspensions were found to be stable at high silver concentrations as well as over a broad pH range. By varying the processing conditions, diameter of the silver nanoparticles could be controlled between ~2 nm to ~25 nm. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to reveal the formation and the corresponding morphology of the silver-silica coupling nanoparticles. Ultra-violet visible (UV-vis) scanning spectrophotometer was used to detecting the distinct absorption spectrum of silver nanoparticles. The antimicrobial activities of these silver-silica coupling nanoparticles were investigated. E. coli and S aureus were used as representatives of Gram-negative and Gram-positive bacteria, respectively. Bacteriological tests showed either bacterial growth inhibition or cell death occurred, corresponding to different concentrations of silver nanoparticles and the type of bacteria that were testing on. Fluorescent microscopic images were also provided to confirm the bacterial viability after several hours' treatment with silver nanoparticles. / Master of Science

Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses. Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization. Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes. Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. / Ph. D. / Dairy cows’ health and productivity is negatively impacted by mastitis, infection starting at the mucosal surfaces of the udder. <i>Staphylococcus aureus</i> is a bacterium that can cause mastitis and there is no efficacious vaccine available. I explored the use of weakened <i>S. aureus</i> as a source of vaccine components and the α-toxins role in stimulating the immune cells like dendritic cells (DC) and lymphocytes. <i>S. aureus</i> was weakened using gamma irradiation to conserve the structural components of the bacterium and render it unable to secrete α-toxin. The DC were collected from dairy cows and stimulated with irradiated <i>S. aureus</i> and live <i>S. aureus</i> before lymphocytes were added to the cultures. The DC signaling, lymphocytes’ pro-inflammatory interferon gamma and mucosal immunity related interleukin responses were measured from RNA production. Memory cell formation and production of interferon gamma were measured from whole cells. The role of α-toxin in lymphocyte stimulation was further studied using a strain of bacterium that does not produce the toxin. Irradiated <i>S. aureus</i> induced low production of inflammatory interferon gamma compared to the live <i>S. aureus</i>. The α-toxin played no role in this, even if other components produced under the same regulatory element likely did, as shown by reduced interferon production in response to bacteria without the regulatory element. Irradiation of the bacterium did not reduce mucosal immunity related cytokine production or formation of memory cells. The irradiated <i>S. aureus</i> is a source for vaccine components that stimulate immune cells like DC and immunity to <i>S. aureus</i> on mucosal surfaces of the udder.

Staphylococcus aureus is a Gram-positive bacterium and part of the human bacterial microflora but it also is an opportunistic pathogen and a notorious cause of hospital – acquired and epidemic infections. S. aureus shows a remarkable adaptation to a range of niches within its human host.Previously viewed as an exclusively extracellular pathogen, S. aureus has been demonstrated to replicate inside virtually all cell types after cell invasion. S. aureus thereby either multiplies within phagosomal compartments in professional phagocytes or escapes into the cytosol prior to replication in non-professional phagocytic cells such as epithelial cells. Besides α-type phenol soluble modulins(PSMα, an important role in phagosomal escape was attributed to the non-ribosomal peptide synthase (NRPS) AusAB. AusAB incorporates the aromatic amino acids (AAAs) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAAs) valine and leucine into three cyclic dipeptides collectively called aureusimines: Phevalin, Tyrvalin and Leuvalin. The role of AusAB in S. aureus infection is not entirely understood. BCAAs are essential amino acids for S. aureus and serve as protein building blocks,precursors for the biosynthesis of branched-chain fatty acids, as well as regulatory molecules for the transcriptional regulator CodY. Severe BCAA depletion triggers stringent response. It is therefore counterintuitive that AusAB would incorporate valine into aureusimines thereby depleting BCAAs in host niches where available nutrients are scarce. The present study therefore analysed the role of the AusAB NRPS and its BCAA-derived monoketopiperazine products, in the interaction of S. aureus with various host niches and with other bacterial species. By using genetic tools and metabolomic approaches, it could be established that the AusAB NRPS preferentially incorporates only the exogenous aromatic amino acids phenylalanine and tyrosine, while the source of valine can be either endogenous or exogenous. By using promoter reporter assays, an effect of the global SaeR regulator on ausAB expression was observed. Additionally, a possible role of the NRPS in modulatory metabolic processes of overflow metabolism emerged. Finally, while the role of AusAB in infection is still unclear, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. By employing bioluminescence and biofilm assays in both Gram-negative (Vibrio harveyi and Pseudomonas aeruginosa) and Gram-positive (CoNS Staphylococcus spp.) model species, a potential role as a Quorum Quenching molecule arose for Phevalin, but not Tyrvalin. While studying a potential connection of AusAB and stringent response, an unexpected role for the BrnQ1 BCAA transporter was revealed. BrnQ1 is known as the major BCAA import protein in S. aureus. The bacteria therefore rely on its activity in environments with low concentrations of available peptides or free BCAAs. The present study shows that BrnQ1 is necessary for efficient phagosomal escape of S. aureus in epithelial cells and that BrnQ1-mediated BCAA uptake is crucial for intracellular bacterial replication in epithelial cells, macrophages and whole human blood. Moreover, BrnQ1 loss of function allows bacteria to survive indefinitely inside macrophages without causing phenotypic changes in colony morphology, size and pigmentation as well as haemolysis after recovery. A dual RNA-sequencing approach further showed that, while no major host transcriptomic rearrangements occurred in human macrophages infected with brnQ1 mutants compared to wild-type S. aureus, intracellular brnQ1 mutants do not respond to local host iron depletion and thus fail to upregulate iron uptake systems. In summary, the present work elaborates on multiple functions of BCAA-based small molecules as well as BCAA-transporters in the S. aureus host and bacterial interactions. This work provides a consolidation of the role of the AusAB NRPS in S. aureus lung infection, gives an insight into the unique mode of action of the NRPS and attributes a novel function for the NRPS-product Phevalin in bacterial communication providing, to my knowledge, the first example of a natural monoketopiperazine involved in bacterial communication. Further, experiments using ex vivo human lung tissue suggest, for the first time, that AusAB NRPS might be involved in the staphylococcal virulence against human lung tissue. Additionally, this work describes a potential mechanism of S. aureus long-term survival in macrophages involving a BCAA-iron metabolism axis. / Staphylococcus aureus ist ein Gram-positives Bakterium und Teil der menschlichen bakteriellen Mikroflora, aber auch ein opportunistisches Pathogen und eine allgemein bekannte Ursache für Krankenhaus- und epidemische Infektionen. S. aureus zeigt eine bemerkenswerte Anpassung an eine Reihe von Nischen innerhalb seines menschlichen Wirtes. Früher wurde der Erreger ausschließlich als extrazelluläres Pathogen angesehen. Doch es hat sich gezeigt, dass S. aureus nach einer Zellinvasion in praktisch allen Zelltypen replizieren kann. Dabei vermehrt sich S. aureus in professionellen Phagozyten innerhalb phagosomaler Kompartimente, in nicht-professionellen phagozytischen Zellen wie Epithelzellen entweicht das Bakterium vor der Replikation in das Zytosol. Neben α-Typ Phenollöslichen Modulinen (PSMα) wurde der nicht-ribosomalen Peptidsynthase (NRPS) AusAB eine wichtige Rolle beim phagosomalen Ausbruch zugeschrieben. AusAB baut die aromatischen Aminosäuren (AAAs) Phenylalanin oder Tyrosin, sowie die verzweigtkettigen Aminosäuren (BCAAs) Valin und Leucin zu den drei zyklische Dipeptiden Phevalin, Tyrvalin und Leuvalin zusammen, die unter dem Namen Aureusimine zusammengefasst werden. Die Rolle von AusAB bei der S. aureus-Infektion ist nicht vollständig geklärt. BCAAs sind essentielle Aminosäuren für S. aureus und dienen als Proteinbausteine, Vorstufen für die Biosynthese von verzweigtkettigen Fettsäuren sowie als regulatorische Moleküle für den Transkriptionsregulator CodY. Eine starke BCAA-Depletion löst eine stringente Antwort aus. Es ist daher kontraintuitiv, dass AusAB Valin in Aureusimine einbaut und damit BCAAs in Wirtsnischen, in denen die verfügbaren Nährstoffe knapp sind, dezimiert. In der vorliegenden Studie wurde daher die Rolle der AusAB NRPS und seiner BCAA-abgeleiteten Monoketopiperazin-Produkte bei der Interaktion von S. aureus mit verschiedenen Wirtsnischen und mit anderen Bakterienarten analysiert. Durch den Einsatz von genetischen Werkzeugen und metabolomischen Ansätzen konnte festgestellt werden, dass die AusAB NRPS bevorzugt nur die exogenen aromatischen Aminosäuren Phenylalanin und Tyrosin einbaut, während die Quelle für Valin entweder endogen oder exogen sein kann. Mit Hilfe von Promotor-Reporter-Assays wurde ein Effekt des globalen Regulators SaeR auf die ausAB-Expression beobachtet. Zusätzlich zeigte sich eine mögliche Rolle des NRPS bei modulatorischen Stoffwechselvorgängen des Überlaufmetabolismus. Schließlich, während die Rolle von AusAB bei der Infektion noch unklar ist, deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das NRPS von AusAB an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Durch den Einsatz von Biolumineszenz- und Biofilm-Experimenten bei repräsentativen Gramnegativen (Vibrio harveyi und Pseudomonas aeruginosa) als auch Gram-positiven (CoNS Staphylococcus spp.) Spezies ergab sich für Phevalin, nicht aber für Tyrvalin, eine mögliche Rolle als Quorum Quenching-Molekül. Bei Untersuchung einer möglichen Verbindung von AusAB und der stringenten Antwort wurde eine unerwartete Rolle für den BrnQ1 BCAA-Transporter entdeckt. BrnQ1 ist als das wichtigste BCAAImportprotein in S. aureus bekannt. Die Bakterien sind daher auf seine Aktivität in Umgebungen mit niedrigen Konzentrationen an verfügbaren Peptiden oder freien BCAAs angewiesen. Die vorliegende Studie zeigt, dass BrnQ1 für einen effizienten phagosomalen Ausbruch von S. aureus in Epithelzellen notwendig ist und dass die BrnQ1-vermittelte BCAA-Aufnahme für die intrazelluläre bakterielle Replikation in Epithelzellen, Makrophagen und menschlichem Vollblut entscheidend ist. Darüber hinaus ermöglicht der Funktionsverlust von BrnQ1 den Bakterien ein unbegrenztes Überleben im Inneren von Makrophagen ohne phänotypische Veränderungen in Koloniemorphologie, -größe und -pigmentierung sowie in Hämolyse nach der Rückgewinnung der Bakterien. Ein dualer RNASequenzierungsansatz zeigte darüber hinaus, dass in humanen Makrophagen, die mit brnQ1- Mutanten infiziert waren, im Vergleich zu Wildtyp S. aureus keine größeren Transkriptomänderungen im Wirt auftraten. Jedoch reagierten intrazelluläre brnQ1-Mutanten nicht auf eine lokale Eisenverarmung des Wirts und regulieren somit keine Eisenaufnahmesysteme hoch. Zusammenfassend zeigt die vorliegende Arbeit die vielfältigen Funktionen von BCAA-basierten kleinen Molekülen sowie von BCAA-Transportern in der Interaktion zwischen dem S. aureus-Wirt und -Bakterium auf. Diese Arbeit liefert eine Konsolidierung der Rolle des AusAB NRPS in der S. aureus Lungeninfektion, gibt einen Einblick in die einzigartige Wirkweise des NRPS und schreibt dem NRPSProdukt Phevalin eine neuartige Funktion in der bakteriellen Kommunikation zu, die meines Wissens das erste Beispiel eines an der bakteriellen Kommunikation beteiligten natürlichen Monoketopiperazins darstellt. Darüber hinaus deuten Experimente mit ex vivo menschlichem Lungengewebe zum ersten Mal darauf hin, dass das AusAB NRPS an der Virulenz von Staphylokokken gegenüber menschlichem Lungengewebe beteiligt sein könnte. Zusätzlich beschreibt diese Arbeit einen möglichen Mechanismus für das Langzeitüberleben von S. aureus in Makrophagen, der eine BCAA-Eisenmetabolismus-Achse beinhaltet.

Staphylococcus aureus et ses entérotoxines constituent un risque pour l’industrie alimentaire ainsi que pour les consommateurs. Environ la moitié des souches de S. aureus peuvent libérer des toxines menant à des symptômes de nausées, de diarrhées et de vomissements chez la personne ayant ingéré un produit contaminé. Une des solutions envisagées pour éliminer S. aureus et éviter la production d’entérotoxines, est l’utilisation d’un cocktail de phages. Au cours de ce projet de doctorat, trois objectifs ont été développés afin de poursuivre une stratégie de sélection d’un cocktail de phages anti-S. aureus. Tout d’abord, deux phages isolés du milieu laitier, adaptés à celui-ci et respectant des critères de sélection, ont été caractérisés. Ensuite, afin d’éviter un transfert de facteurs de virulence, des myophages anti-S. aureus ont été produits sur une souche de Staphylococcus xylosus, une espèce non-pathogène utilisée en transformation alimentaire et ont été caractérisés afin de confirmer leur identité lorsqu’amplifiés sur les deux espèces. D’un autre côté, l’efficacité contre une panoplie de souches de S. aureus isolées de sources distinctes, l’absence de gènes de virulence dans les génomes phagiques et la résistance à différentes conditions environnementales ont permis de sélectionner des phages différents. Ceux-ci ont fait l’objet de deux cocktails de phages efficaces menant à une réduction significative de la concentration de S. aureus dans des fromages de type Cheddar produits en laboratoire. De plus, ces phages n’ont pas déclenché une surproduction d’entérotoxines staphylococciques C, confirmant la sécurité de leur utilisation dans le milieu laitier. Enfin, la conservation des phages sous forme encapsulée et congelée dans des microbilles de gel d’alginate/calcium semble une approche à approfondir. Les phages staphylococciques virulents analysés dans cette thèse constituent d’excellents agents de biocontrôle étant non nocifs et infectant spécifiquement une espèce bactérienne donnée. Ayant confirmé l’efficacité de deux cocktails de phages, l’utilisation du produit phagique permettra de diminuer le risque d’apparition de souches bactériennes résistantes aux phages. De plus, entreprendre une mise à jour constante du cocktail phagique permettra de réduire la contamination causée par S. aureus, préservant ainsi l’innocuité et la qualité des aliments. / Staphylococcus aureus and its enterotoxins pose a risk to the food industry and for consumers. Approximately half of the S. aureus strains can release toxins leading to symptoms of nausea, diarrhea and vomiting to the person who ingests a contaminated product. One emerging solution to eliminating and preventing S. aureus enterotoxin production is the use of a phage cocktail. Through this doctoral project, three objectives were developed to pursue a strategy for selecting an anti-S. aureus phage cocktail based on well-defined criteria. First, a methodology was developed leading to the isolation and characterization of two phages from raw milk. Then, in order to avoid a transfer of virulence factors, anti-staphylococcal myophages were produced on a strain of Staphylococcus xylosus, a non-pathogenic species used in food processing. Their genomic identity when propagated separately on the two species was confirmed. Moreover, the host range of these phages was tested against a panel of S. aureus strains isolated from different sources. Their genome was analyzed to confirm the lack of virulence genes. In addition, their resistance to different environmental conditions was used to select different phages for application purposes. These data helped design two efficient phage cocktails leading to a significant drop of S. aureus concentration in small-scale laboratory-based Cheddar cheeses. In addition, they did not trigger the overproduction of staphylococcal enterotoxin C confirming the safety of their use in the dairy environment. Finally, in the aim of commercializing the product, conservation methods were investigated and the encapsulation and freeze of phages in micro-beads consisting of alginate/calcium gel particles appear promising. Staphylococcal virulent phages seem to be excellent biocontrol agents, being harmless and infecting specifically a given bacterial species. Having confirmed the efficacy of two phage cocktails, the use of the phage product could help decreasing the risk of emergence of phage resistant bacteria. Furthermore, undertaking a constant update of the phage cocktail could also help reducing contamination caused by S. aureus and thereby preserving safety and food quality.

Multiresistenta bakterier så som meticillinresistenta Staphylococcus aureus (MRSA), bakterier som producerar extended-spectrum beta-lactamase (ESBL) och vancomycinresistenta enterokocker (VRE) är ett problem sedan årtionden tillbaka och som ökar för varje år. Idag på mikrobiologen, Unilabs Skövde, isoleras bakteriestammar från selektiva medier för just MRSA, ESBL och VRE på blodagar innan resistensbestämningarna utförs. Syftet med studien var därför att undersöka möjligheten att göra diskdiffusionstest direkt från de selektiva medierna och således kunna svara ut resultaten tidigare. Utvärdering av detta gjordes genom att undersöka om storleken på antibiotikazonerna för sammanlagt 64 isolat påverkades av att bakterierna som användes vuxit på ett selektivt agarmedium i förhållande till om de vuxit på blodagar. Resultatet visade vad som ansågs vara en normal variation på maximalt ±2 mm för alla parvisa zoner utom en på 3 mm. Av alla zoner som undersöktes för MRSA, ESBL och VRE var majoriteten identiska i antal millimeter, 62 %, 89 % och 98 % respektive. Baserat på det goda resultatet ansågs materialet vara tillräckligt stort för att göra bedömningen att metoden är utförbar. Med tanke på de positiva effekterna av att göra resistensbestämningar direkt från de selektiva agarmedierna görs rekommendationen till mikrobiologen, Unilabs Skövde, att övergå till denna metod. / Multiresistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL) producing bacteria and vancomycin-resistant enterococci (VRE) have been a problem for decades with an increasing rate. Today, at mikrobiologen, Unilabs Skövde, bacterial strains are isolated from selective media for MRSA, ESBL and VRE onto blood agar before the susceptibility testing. The aim of the study was to examine the possibility of disk diffusion susceptibility testing directly from the selective media and thus be able to reply the findings earlier. The zones of inhibition were examined for a total of 64 isolates after disk diffusion testing from both the selective and blood agar plates in order to evaluate if the zone sizes were affected. The results showed what was considered a normal variation of ±2 mm for all pairwise zones except for a difference in 3 mm. The majority of all zones tested for MRSA, ESBL and VRE had equally large zones, 62%, 89% and 98% respectively. Based on the good results, the material was considered enough to make the conclusion that the method is feasible. Considering the positive effects of making susceptibility testing directly from selective agar, a change to this method is recommended to mikrobiologen, Unilabs Skövde.

Historiskt sett har personer med smittsamma infektioner uteslutits från samhället. Personerna har setts med avsky och rädsla från omgivningen med risk för att överföra smittan. Multiresistenta bakterier (MRB) är ett ökande problem världen över och orsakar stort lidande för patienter. Syftet med litteraturstudien var att undersöka patienters upplevelse av att vara smittbärare av meticillinresistenta staphylococcus aureus (MRSA). Litteraturstudiens resultat baseras på nio vetenskapliga artiklar, där resultatet utföll i två kategorier. I kategorin känslan av att vara smittsam framkom underkategorierna att vara smutsig, skuld och skam samt rädsla och oro. I kategorin känslan av att vara annorlunda framkom underkategorierna känna sig kränkt, ilska och frustration samt känna sig stigmatiserad. För att patienter med MRSA-smitta ska få en god vård krävs det att vårdpersonalen har evidensbaserad kunskap. Därför skulle det vara av stort intresse att forskning i framtiden fokuserar på patienters upplevelse av att vara smittbärare. Ytterligare forskning behövs inom området på grund av ett ökat globalt problem med MRB. / Historically, people with contagious infections has been excluded from society. The characters have been seen with disgust and fear from the environment with the risk of transmitting the infection. Multi-drug resistant bacteria (MRB) is a growing problem worldwide and causes great suffering for patients. The purpose of this study was to investigate patients' experience of being carriers of methicillin-resistant staphylococcus aureus (MRSA). Literature study results are based on nine scientific articles, which precipitated the result of two categories. In the category of feeling of being contagious emerged subcategories to be dirty, guilt and shame and fear and anxiety. In the category of the feeling of being different subcategories emerged feel hurt, anger and frustration, and feel stigmatized. For patients with MRSA infection should get good care requires that health professionals have evidence-based knowledge. Therefore, it would be of great interest to future research focusing on patient experience of being contaminated. Further research is needed in this area because of the increasing global problem of MRB.

O estudo objetivou identificar a colonização nasal por Staphylococcus aureus em PVHA hospitalizadas e relacionar com adesão a TARV. Trata-se de um estudo transversal, realizado em duas unidades de cuidados especializados às doenças infecciosas de um hospital universitário, do interior do Estado de São Paulo. A coleta de dados ocorreu no período entre 01 de agosto de 2011 e 31 de janeiro de 2015, procedida do levantamento dos dados sociodemográficos, clínicos e imunológicos; os quais foram obtidos através do prontuário e entrevista individual; da coleta de duas amostras de swab nasal; e da identificação da retirada dos medicamentos antirretrovirais, através do SICLOM. Todos os aspectos éticos foram contemplados. Os swabs foram coletados, encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia onde foram utilizados cartões AST-P585 para avaliar a sensibilidade dos Staphylococcus aureus aos antibióticos. A organização dos dados foi feita no Microsoft® Office Excel® 2010 for Windows 8 e transportada para o software IBM® SPSS®, versão 23.0 for Windows para a análise descritiva e analítica. Utilizouse o procedimento PROC LOGISTIC do software SAS 9.4®, para a análise exploratória pela variável resposta. Adotou-se o nível de significância de todos os testes de 5%. Dos 236 participantes elegíveis, a maioria era do sexo masculino (59,3%), na faixa etária entre 40 e 49 anos (48,3%), de etnia branca (66,1%), procedente de Ribeirão Preto-SP (59,7%), com ensino primário incompleto (40,2%), com ocupação profissional (52,5%), heterossexual (81,8%) e não teve parceria sexual nos últimos 6 meses (57,6%). Houve a predominância do tempo de diagnóstico pelo HIV em > 5 e <= 10 anos (44,4%), a contaminação se deu pela via sexual (66,1%), a admissão foi via o ambulatório da intituição (45,5%), a carga viral foi detectável (56,4%), a contagem de linfócitos T+ CD4 foi menor que 350 células/mm3 (61,8%), a antibioticoterapia estava prescrita (65,3%), assim como a terapia antirretroviral (51,7%) e haviam recebido procedimentos invasivos (67,4%). Quanto a colonização nasal por Staphylococcus aureus, 36,0% foram identificados como colonizados, no primeiro dia de internação hospitalar. Dos 137 participantes que permaneceram internados no sétimo dia, em 37 (27,0%) houve a identificação da colonização nasal por Staphyloccocus aureus. Quanto a classificação da adesão aos medicamentos antirretrovirais, houve o predomínio dos participantes na categoria de adesão indesejável (67,8%). Concluiu-se que a prevalência da colonização nasal por Staphylococcus aureus foi de 38,13%. A classificação quanto a categoria da adesão aos antirretrovirais interfere na presença/ausência da colonização nasal por Staphylococcus aureus, assim as PVHA hospitalizadas e categorizadas em adesão indesejável possuem 2,597 vezes o risco de obter presença de colonização nasal por Staphylococcus aureus, em relação àquelas classificadas na categoria de adesão desejável / The research aimed to identify nasal colonization by Staphylococcus aureus in hospitalized PLWHA and to correlate with ART adherence. It was a cross-sectional study carried out in two specialized care units for infectious diseases at a university hospital in the interior of the State of São Paulo. The data collection took place between August 1, 2011 and January 31, 2015, proceeding from the survey of sociodemographic, clinical and immunological data, which were obtained through the medical record and individual interview; the collection of two samples of nasal swab and the identification of the withdrawal of antiretroviral drugs through SICLOM. All ethical aspects have been contemplated. The swabs were collected, sent and processed by the Laboratory of Microbiology and Serology, AST-P585 cards were used to evaluate the sensitivity of Staphylococcus aureus to antibiotics. Data organization was done in Microsoft® Office Excel® 2010 for Windows 8 and shipped to IBM® SPSS® software, version 23.0 for Windows for descriptive and analytical analysis. The PROC LOGISTIC procedure of the SAS 9.4® software was used for the exploratory analysis by the response variable. The level of significance was 5%. Results: From the 236 eligible participants, the majority were male (59.3%), aged 4049 (48.3%), white (66.1%), from Ribeirão Preto-SP (59.7%), with incomplete primary education (40.2%), with occupational (52.5%), heterosexual (81.8%) and had no sexual partner in the last 6 months (57.6% ). There was a predominance of HIV diagnosis time in > 5 and <= 10 years (44.4%), contamination occurred through the sexual route (66.1%), admission was from the outpatient clinic (45.5%), the viral load was detectable (56.4%), the T + CD4 count was lower than 350 cells / mL (61.8%), antibiotic therapy was prescribed (65.3%), and antiretroviral therapy (51.7%) and had received invasive procedures (67.4%). Regarding nasal colonization by Staphylococcus aureus, 36.0% were identified as colonized, on the first day of hospital admission. From the 137 participants who remained hospitalized on the seventh day, 37 (27.0%) were identified the nasal colonization by Staphylococcus aureus. Regarding the classification of adherence to antiretroviral drugs, the participants were predominant in the category of undesirable adherence (67.8%). Concludes that the prevalence of nasal colonization by Staphylococcus aureus was 38.13%. The classification as to the category of antiretroviral adherence interferes with the presence/absence of nasal colonization by Staphylococcus aureus, so the hospitalized PLWHA and categorized as undesirable adherence have 2,597 times the risk of nasal colonization by Staphylococcus aureus in relation to those classified in desirable membership category

A formação de biofilme representa preocupação à indústria de alimentos pois é uma fonte crônica de contaminação. Encontrar estratégias eficientes para controlar o crescimento de microrganismos continua a ser um importante desafio. Uma delas é o uso dos ramnolipídeos (RLs), um biossurfatante produzido tipicamente por P. aeruginosa que apresenta potencial como agente antimicrobiano, anti-adesivo e dispersivo. Sua baixa toxicidade, biodegradabilidade, eficiência e especificidade em comparação aos surfatantes sintéticos podem torná-los promissores agentes de biocontrole. O presente estudo teve como objetivo estudar o potencial de uso de ramnolipídeos, em diferentes condições de concentração e temperatura, no controle e remoção de biofilmes de patógenos alimentares formados em meio de cultura e leite. Foram utilizadas Escherichia coli ATCC 43895, Listeria monocytogenes ATCC 19112, Staphylococcus aureus ATCC 8095, reconhecidos patógenos alimentares. Os biofilmes foram formados em placas de microtitulação de poliestireno nos meios de cultivo: caldo nutriente (CN), extrato de levedura com triptona de soja (TSYE) e matriz alimentar (leite) à 37 &deg;C, por 24 h (E. coli) e 48 h (S. aureus e L. monocytogenes). Os biofilmes foram avaliados pela quantificação da biomassa, viabilidade celular, hidrofobicidade de superfície e análises qualitativa (microscopia eletrônica de varredura e de fluorescência) e quantitativa (caracterização da matriz polimérica). O ramnolipídeo foi submetido à análise físico-química de espalhamento dinâmico de luz (DLS), espalhamento de raios-X a baixo ângulo (SAXS). Os resultados obtidos para E. coli mostraram que a concentração de RL que mais removeu o biofilme foi 2 &permil;, porém em temperaturas diferentes, para o CN à 25 &deg;C e para o leite à 37 &deg;C, com 33 &permil; e 80 &permil; de remoção, respectivamente. Para o biofilme de S. aureus em caldo nutriente os resultados mais eficientes foram à 25 &deg;C, na concentração de 0,1 &permil; de RL e em leite 4 &deg;C, na concentração de 0,05 &permil; de RL, com remoção de 35 &permil; e 89 &permil;, respectivamente. O biofilme de L. monocytogenes em TSYE mostrou-se mais sensível à 37 &deg;C, na concentração 0,5 &permil; de RL, o qual foi possível remover 35,3 &permil; da biomassa. Enquanto que em leite a 4 °C e 0,5 &permil; de RL, com remoção de 63,6 &permil; .Quanto à redução das células viáveis foi observado que para as bactérias Gram-positivas o tratamento mais efetivo foi à 4 &deg;C com 0,05 &permil; de RL, nos meios CN e TSYEe 1 &permil; em leite. Para os biofilmes de E. coli a maior redução da viabilidade ocorreu em leite, após tratamento com RL 0,05 &permil; à 37 &deg;C. As imagens de microscopia mostraram uma morfologia heterogênea na presença dos diferentes meios de cultivos, com destaque para os biofilmes de S. aureus (leite) e L. monocytogenes (TSYE), nos quais houve grande produção de matriz polimérica extracelular (MPE), e também apresentaram as maiores quantidades de carboidratos e proteínas. O tratamento com o ramnolipídeo reduziu a hidrofobicidade dos biofilmes. As análises de DLS e SAXS mostraram uma predominância em número de micelas com diâmetro entre 1-10 nm, independente das concentrações e temperaturas analisadas. De modo geral, a aplicação de ramnolipídeo promoveu remoção da biomassa celular como também redução de células viáveis presentes no biofilme. As evidências obtidas aqui, podem ser importantes subsídios para futuras investigações sobre as interações físico-químicas entre ramnolipídeos e a camada de biofilme visando aplicação como agentes sanitizantes em indústria de alimentos. / Biofilm formation is a concern to the food industry because it is a chronic source of contamination. Finding effective strategies to control the growth of microorganisms remains a major challenge. One strategy is the use of rhamnolipids (RLs), a biosurfactant typically produced by P. aeruginosa that has potential as antimicrobial, anti-adhesive and biofilm disrupting agent. RLs low toxicity, biodegradability, efficiency and specificity comparatively to synthetic surfactants, makes them promising biocontrol agents. This work aimed to study the potential use of rhamnolipid at different conditions of concentration and temperature, to control and removal of biofilms of food pathogens established in culture medium and milk. The bacterial strain utilized Escherichia coli ATCC 43895, Listeria monocytogenes ATCC 19112, Staphylococcus aureus ATCC 8095, are well-recognized food pathogens. The biofilms were formed in polystyrene microtiter plates in culture media: nutrient broth (NB), yeast extract and tryptone soya (TSYE) and in food matrix (milk) at 37 &deg;C for 24 h (E. coli) and 48 h (S. aureus and L. monocytogenes). Biofilms were assessed by biomass quantification, cell viability, surface hydrophobicity, qualitative (scanning electron microscopy and fluorescence) and quantitative (characterization of polymer matrix) analysis. The rhamnolipid was subjected to physical and chemical analysis of dynamic light scattering (DLS) and X-ray small angle scattering (SAXS). E. coli biofilms were removed more efficiently using 2 &permil; RL, but at different temperatures for NB (25 °C) and milk (37 &deg;C) showing 33 &permil; and 80 &permil; respectively. For the biofilm of S. aureus in NB the best results was obtained at 25 &deg;C and 0.1 &permil; RL and in milk medium at 4 &deg;C with 0.05 &permil; RL showing 35 &permil; and 89 &permil; of biofilm disruption, respectively. The biofilm of L. monocytogenes in TSYE was more sensitive to the treatment at 37 &deg;C with 0.5 &permil; RL, removing 35.3 &permil; of the biofilm; while in milk at 4 °C and 0.5 &permil; RL, biofilm removal reached 63.6 &permil;. Reduction on cell viability was more effective for Gram-positive bacteria at 4 &deg;C with 0.05 &permil; RL, for NB and TSYE and at 1 &permil; in milk. For E. coli biofilms the largest reduction of viability occurred in milk after treatment with 0.05 &permil; RL at 37 &deg;C. The microscopy images showed a heterogeneous morphology in the presence of different media, especially biofilms of S. aureus (milk) and L. monocytogenes (TSYE), in which there was a great production of extracellular polymeric matrix (EPM), and also the highest amounts of carbohydrates and protein. The treatment with RL reduced the hydrophobicity of biofilms. The DLS and SAXS analysis of RL showed a predominance of micelles with diameters between 1-10 nm, independent of the concentrations and temperatures utilized. In general, the application of rhamnolipid promoted a reduction in biofilm mass as well in cell viability. The evidences obtained can provide a basis for future research on the physical and chemical interactions between rhamnolipid and biofilm layer aiming their application as sanitizers in food industry.

Biofilmes de estafilococos têm se tornado uma das grandes preocupações da indústria de lácteos, principalmente em decorrência do ritmo de produção intenso, automação das plantas de processamento e exigência cada vez maior quanto à qualidade microbiológica de leite e derivados. O presente estudo teve por objetivo identificar cepas de S. aureus potencialmente produtoras de biofilmes isoladas de 3 laticínios produtores de queijo Minas frescal, avaliar a influência da temperatura e da superfície de contato (aço inoxidável e polipropileno) no processo de adesão bacteriana, bem como a eficácia de um protocolo simulado de limpeza e sanificação na remoção das células aderidas. Para as análises genotípicas pesquisaram-se os genes icaA e icaD nos isolados, relacionados à produção de polissacarídeos de adesão celular e exopolissacarídeos da matriz de biofilmes. Os ensaios de biofilmes foram realizados em cupons incubados em um reator de biofilmes, comparando-se a adesão frente a duas temperaturas (5°C e 35°C), duas superfícies (aço inoxidável e polipropileno) e quatro tempos de contato (3, 6, 12 horas e após processo de limpeza e sanificação). Para avaliação da eficácia do processo na remoção das células aderidas, foram utilizados detergente neutro (3,5% v/v) e sanificante à base de hipoclorito de sódio (1000 mg.L-1), de modo a simular a situação observada em um dos laticínios estudados. Foi detectada a presença dos genes icaA e icaD em 74% e 77% dos isolados, respectivamente; 70% dos isolados apresentaram ambos os genes, enquanto 19% não apresentaram nenhum. O número de células aderidas em ambas as superfícies foi em torno de 3 e 6 log10 UFC.cm-2 nas temperaturas de 5°C e 35°C, respectivamente, para a maioria das situações avaliadas, com aumento significativo no decorrer dos períodos avaliados. De maneira geral, a temperatura de 35°C favoreceu uma maior adesão de S. aureus. A 5°C, houve considerável número de células aderidas, mas em populações significativamente inferiores às observadas a 35°C. O protocolo de limpeza e sanificação mostrou-se ineficaz na remoção das células aderidas; uma melhor atuação do hipoclorito de sódio foi observada a 5°C, o que deve estar relacionado à menor adesão observada nessa temperatura. De qualquer forma, o processo não foi capaz de reduzir a níveis seguros a quantidade de S. aureus aderida a ambas as superfícies, nas condições avaliadas. O estudo demonstrou a capacidade de adesão de S. aureus isolados de laticínios em superfícies comumente encontradas na produção de queijo Minas frescal, situação que pode favorecer o desenvolvimento de biofilmes em equipamentos e utensílios, conferindo risco à saúde dos consumidores. / Staphylococci biofilms have become a major concern for the dairy industry, mainly due to the intensive production flow, automation of processing plants and increased demand on the microbiological quality of dairy products. This study aims to identify isolated S. aureus strains potentially producers of biofilms from 3 dairies of Minas fresh cheese, evaluate the influence of temperature and the contact surface (stainless steel and polypropylene) in the bacterial adhesion process, as well as the efficacy of an hygiene and sanitation simulated protocol in removing the adhered cells. For genotypic analyzes, the presence of icaA and icaD in strains were sought related to polysaccharide production in cell adhesion and exopolysaccharide of biofilm matrix. Biofilms trials were performed in biofilm coupons incubated in biofilm reactor, comparing the adhesion with two temperatures (5°C and 35°C), two surfaces (stainless steel and polypropylene) and four contact times (3, 6, 12 hours and after cleaning and sanitizing process). To evaluate the process effectiveness in removing the adhered cells, neutral detergent (3.5% v/v) and sanitizing based on sodium hypochlorite (1000 mg.L-1) were used in order to simulate the observed situation in one of the dairy products studied. The presence of icaA and icaD genes was detected in 74% and 77% of strains, respectively; 70% of the isolates showed both genes, whereas 19% of it showed no genes. The number of cells adhered on both surfaces was about 3 and 6 log10 FCU.cm-2 in temperatures of 5 °C and 35 °C, respectively, for most situations evaluated, with a significant increase over the evaluation period. In general, the temperature of 35°C favored a greater adherence of S. aureus. At 5°C, there was a considerable number of adhered cells, but in populations significantly lower than those observed at 35°C. The cleaning and sanitizing protocol was ineffective in removing adhered cells; a better performance of sodium hypochlorite was observed at 5°C, which should be related to lower adherence observed at this temperature. At all, the process was not able to reduce the amount of S. aureus adhered on both surfaces to safe levels under the conditions evaluated. The study demonstrated the ability of adhesion of isolated S. aureus from dairy on surfaces commonly found in Minas fresh cheese production, a situation that may favor the development of biofilms on equipment and utensils, indicating health risk to the consumers.