Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection. / Master of Science

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

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.

Bacteria adapt to stress conditions by altering their physiology, behavior, and gene expression in re- sponse to external challenges. They use evolved mechanisms to thrive in environments with various stress factors including temperature changes, nutrient shortages, and toxins. Specific stress-response pathways are activated upon encountering stress, regulating genes to cope and maintain cellular func- tions. This thesis focuses on a bioinformatical analysis of two bacterial pathogens: a) Staphylococcus aureus and b) Chlamydia trachomatis during stress. To understand the role of S. aureus proteins during infection, gene expression data from two strains (NewHG and NCTC 8325) were analyzed, and flux changes were examined during the middle and late exponential growth phases. The aim was to identify metabolic variations between the wild-type (WT) and various knockout mutants, including the Ser/Thr Kinase PknB, its phosphatase Stp, and the double knockout PknB/Stp. Both S. aureus strains were cultured in nutrient-poor medium to simulate infection conditions similar to those in an abscess of infected individuals. Subsequently, a comprehensive meta- bolic model was constructed using time-resolved transcriptome data, which was validated by qRT-PCR. This study highlights the critical role of PknB-mediated phosphorylation on Ser/Thr residues in regulat- ing amino acid catabolism and promoting gluconeogenesis, ensuring the cell's supply of essential com- ponents. Both PknB and Stp play crucial roles in multiple cellular processes, such as peptidoglycan, nucleotide, and aromatic amino acid synthesis, as well as aspartate transaminase catabolism. Deletion of stp signif- icantly impaired pyrimidine synthesis, while functional loss of PknB had a minor impact. In double knock-out experiments, genes responsible for synthesizing peptidoglycan, purines, and aromatic amino acids from glucose showed increased activity, while pyrimidine synthesis from glucose was less active compared to the WT. Furthermore, this thesis extensively explores the regulatory modules associated with the glmR/yvcK regulon and the cdaA-cdaR-glmM-glmS module. The study emphasizes the conservation of the glmR/yvcK regulon and its core genes, namely yvcJ, glmR/yvcK, and whiA, across various bacteria and cellular processes. Notably, the presence of structural and phosphorylation site similarities in glmR sequences among different bacteria suggests a complex interactome involving PknB/Stp and these regulatory modules, potentially leading to broader implica- tions for bacterial adaptation and virulence. The aim of the second part of this thesis was to investigate how Chlamydia trachomatis adapts to stress induced by the host and evades immune defenses by manipulating human cells. The study introduces an innovative in silico model that captures the dynamic regulatory processes of C. trachomatis and its in- teractions with host signaling proteins during infection. The network model gives particular emphasis to chlamydial chaperones like ClpB, ClpC, and Dnak, along with related proteins such as UhpC, PyrG, inclusion membrane proteins, and the host proteins associated with various signaling pathways. The analysis of multiple time points and subsequent statistical data analysis pinpointed significantly up- and down-regulated genes and proteins, which were used as the basis for dynamic modeling and interac- tome analysis. Network models of predicted and validated protein interactions were used to study the time course of pathogen-host interactions, particularly focusing on chlamydial membrane proteins and chaperones. Through transcriptomic data (GSE104166, GSE147538, and GSE165628) analysis and pathway enrichment, the study identifies key host proteins, including PI3K, MAP kinases, MDM2, c- Myc, and hexokinases, that play significant roles in C. trachomatis pathogenesis. The infection substan- tially modulates multiple signaling pathways, including Interferon-α and -γ response, TNF-α signaling via NFκB, and inflammatory responses. Gene set enrichment analysis provides valuable insights into the host's response to C. trachomatis in- fection, showing distinct gene enrichment patterns at different time points. The integration of pathways from multiple transcriptome datasets enhances the specificity of the network model. Additionally, the research underscores the importance of metabolic reprogramming, particularly involving glutamine uti- lization, for chlamydial survival and the transition between the two different morphological forms. Developed bioinformatic pipeline and systems biology model used in this study offer valuable tools for identification of essential genes/proteins for bacterial survival in the stress environment, virulence profiling, and understanding host-pathogen interactions, with potential applications in various research areas beyond the scope of this study. / Bakterien passen sich an Stressbedingungen an, indem sie ihre Physiologie, ihr Verhalten und ihre Genexpression als Reaktion auf externe Herausforderungen verändern. Sie nutzen weiterentwickelte Mechanismen, um in Umgebungen mit verschiedenen Stressfaktoren wie Temperaturschwankungen, Nährstoffmangel und Toxinen zu gedeihen. Beim Auftreten von Stress werden spezifische Stressreaktionswege aktiviert, die Gene regulieren, um mit dem Stress fertig zu werden und die zellulären Funktionen aufrechtzuerhalten. Diese Arbeit konzentriert sich auf eine bioinformatische Analyse zweier bakterieller Krankheitserreger: a) Staphylococcus aureus und b) Chlamydia trachomatis. Um die Rolle von S. aureus-Proteinen während der Infektion zu verstehen, wurden Genexpressionsdaten von zwei Stämmen (NewHG und NCTC 8325) analysiert und Flussänderungen während der mittleren und späten exponentiellen Wachstumsphase untersucht. Ziel war es, metabolische Variationen zwischen dem Wildtyp und verschiedenen Knockout-Mutanten zu identifizieren, nämlich Ser/Thr-Kinase PknB, ihre Phosphatase Stp und der Doppel-Knockout PknB/Stp. Beide S. aureus-Stämme wurden in nährstoffarmem Medium kultiviert, um Infektionsbedingungen zu simulieren, die denen in einem Abszess infizierter Personen ähneln. Anschließend wurde anhand von zeitaufgelösten Transkriptom-Daten ein umfassendes Stoffwechselmodell erstellt, das durch qRT-PCR Messungen validiert wurde. Diese Studie unterstreicht die kritische Rolle der PknB-vermittelten Phosphorylierung an Ser/Thr-Resten bei der Regulierung des Aminosäurekatabolismus und der Förderung der Gluconeogenese, wodurch die Versorgung der Zelle mit essentiellen Komponenten gewährleistet wird. Sowohl PknB als auch Stp spielen eine entscheidende Rolle bei zahlreichen zellulären Prozessen wie der Peptidoglykan-, Nukleotid- und aromatischen Aminosäuresynthese sowie dem Aspartat-Transaminase-Katabolismus. Die Deletion von stp beeinträchtigte die Pyrimidin-Synthese erheblich, während der Funktionsverlust von PknB nur geringe Auswirkungen hatte. In doppelten Knock-out-Experimenten zeigten die Gene, die für die Synthese von Peptidoglykan, Purinen und aromatischen Aminosäuren aus Glukose verantwortlich sind, eine erhöhte Aktivität, während die Pyrimidinsynthese aus Glukose im Vergleich zum Wildtyp weniger aktiv war. Darüber hinaus werden in dieser Arbeit die regulatorischen Module im Zusammenhang mit dem glmR/yvcK-Regulon und dem cdaA-cdaR-glmM-glmS-Modul eingehend untersucht. Die Studie unterstreicht die Konservierung des glmR/yvcK-Regulons und seiner Kerngene, nämlich yvcJ, glmR/yvcK und whiA, über verschiedene Bakterien und zelluläre Prozesse hinweg. Insbesondere das Vorhandensein von Struktur- und Phosphorylierungsstellen in glmR-Sequenzen zwischen verschiedenen Bakterien deutet auf ein komplexes Interaktionssystem hin, an dem PknB/Stp und diese regulatorischen Module beteiligt sind, was möglicherweise zu umfassenderen Auswirkungen auf die bakterielle Anpassung und Virulenz führt. Ziel des zweiten Teils dieser Arbeit war es, zu untersuchen, wie sich C. trachomatis an den vom Wirt ausgelösten Stress anpasst und der Immunabwehr entgeht, indem es menschliche Zellen manipuliert. In der Studie wird ein innovatives in silico Modell vorgestellt, das die dynamischen Regulationsprozesse von C. trachomatis und seine Interaktionen mit Wirtssignalproteinen während der Infektion abbildet. Das Netzwerkmodell legt besonderes Augenmerk auf Chlamydien-Chaperone wie ClpB, ClpC und Dnak sowie auf verwandte Proteine wie UhpC, PyrG, Einschlussmembranproteine und Wirtsproteine, die mit die mit verschiedenen Signalwegen assoziiert sind. Mehrere Zeitpunkte und statistische Datenanalysen ermittelten signifikant hoch- und herunterregulierte Gene und Proteine, die als Grundlage für die dynamische Modellierung und Interaktomanalyse verwendet wurden. Mit Hilfe von Netzwerkmodellen für vorhergesagte und validierte Proteininteraktionen wurde der zeitliche Verlauf von Pathogen-Wirt-Interaktionen untersucht, wobei der Schwerpunkt auf chlamydialen Membranproteinen und Chaperonen lag. Die Analyse von Transkriptom-Daten (GSE104166, GSE147538 und GSE165628) und die Anreicherung von Signalwegen identifizierte wichtige Wirtsproteine, darunter PI3K, MAP-Kinasen, MDM2, c-Myc und Hexokinasen, die eine wichtige Rolle in der Pathogenese von C. trachomatis spielen. Die Infektion moduliert im Wesentlichen mehrere Signalwege, darunter die Interferon-α- und -γ-Antwort, die TNF-α -Signalisierung über NFκB und Entzündungsreaktionen. Die Analyse der Anreicherung von Gensätzen liefert wertvolle Einblicke in die Reaktion des Wirts auf die Infektion mit C. trachomatis und zeigt unterschiedliche Muster der Genanreicherung zu verschiedenen Zeitpunkten. Die Integration von Signalwegen aus mehreren Transkriptom-Datensätzen erhöht die Spezifität des Netzwerkmodells. Darüber hinaus unterstreicht die Forschung die Bedeutung der metabolischen Reprogrammierung, insbesondere der Glutaminverwertung, für das Überleben von Chlamydien und den Übergang zwischen den beiden morphologisch unterschiedlichen Lebensformen. Sowohl die entwickelten bioinformatischen Pipelines als auch die systembiologischen Modelle bieten wertvolle Werkzeuge zur Identifizierung wesentlicher Gene/Proteine für das Überleben von Bakterien in der Stressumgebung, zur Erstellung von Virulenzprofilen und das Verständnis von Wirt-Pathogen-Interaktionen, mit potenziellen Anwendungen in verschiedenen Forschungsbereichen, die über den Rahmen dieser Studie hinausgehen.

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.

En milieu hospitalier, des mesures de prévention et contrôle des infections sont mises en place. Dans le cas du SARM, l'isolement sur une unité dédiée où les patients ne sont pas confinés à leur chambre semble bénéfique. Une étude qualitative d'inspiration naturaliste a été réalisée afin de décrire l'effet de l'acquisition de la bactérie SARM et de l'isolement sur une unité dédiée. Les résultats ont mis en évidence que l'unité dédiée, par son mode de fonctionnement, n'entraine pas les effets négatifs attendus et décrits dans la littérature. Ces conclusions guident le choix d'interventions cliniques et structurelles permettant de limiter les effets de l'isolement relié à la colonisation par une bactérie dont l'acquisition est avant tout nosocomiale. Ainsi, les milieux de soins pourront, en contrepartie, offrir un milieu de soins moins contraignant à cette clientèle.

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.