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Staphylococcus aureus est une bactérie pathogène des animaux et de l'homme causant plusieurs symptômes chez ceux-ci. De plus, elle est responsable d'intoxications alimentaires liées à l'ingestion de nourritures contaminées par des entérotoxines staphylococciques. Avec l'émergence de souches staphylococciques résistantes aux .antibiotiques, l'utilisation de phages virulents est maintenant envisagée comme alternative pour lutter contre les souches pathogènes de S. aureus. Au cours de ce projet, un nouveau phage virulent (LH1) a été isolé à partir d'échantillons de lait cru. L'utilisation de ce dernier et du phage K dans du lait UHT a montré qu'ils sont capables d'éliminer les souches de S. aureus mais que leur activité lytique est inhibée dans le lait cru par un composé encore inconnu. La présence de deux gènes codant pour la toxine leucocidine de Panton-Valentin dans le génome du phage LH1 compromet son utilisation comme agent de biocontrôle dans les applications alimentaires et médicales. Finalement, un arbre phylogénétique a été créé en comparant et regroupant 54 génomes de phages différents de S. aureus disponibles dans GenBank en plus de deux nouveaux phages isolés incluant LH1. L'avancement des connaissances sur des phages de S. aureus pourrait favoriser leur utilisation dans la prévention ou le contrôle des intoxications alimentaires.

published_or_final_version / Physiology / Master / Master of Philosophy

Staphylococcus aureus und Staphylococcus epidermidis gehören zu den häufigsten Erregern nosokomialer Infektionen bei immunsupprimierten Patienten. Gleichzeitig bilden diese Bakterien einen wesentlichen Teil der gesunden Hautflora des Menschen. Bisher ist wenig darüber bekannt, ob es Unterschiede in der genetischen Ausstattung zwischen klinischen und kommensalen Isolaten gibt und welche Faktoren zur Etablierung von Staphylokokken im Hospitalmilieu beitragen. Die Ergebnisse der vorliegenden Arbeit zeigen, daß die Fähigkeit zur Biofilmbildung offensichtlich ein wesentliches Merkmal pathogener Staphylokokken ist. Die Expression dieses Virulenzfaktors ist dabei hochvariabel und hängt von der genetischen Ausstattung der Stämme mit dem für die Biofilmbildung verantwortlichen ica-Operon, bestimmten Umweltfaktoren und dem Einfluß von Insertionssequenzen ab. In einer epidemiologische Untersuchung wurde gezeigt, daß in S. epidermidis das ica-Operon häufiger in klinischen als in kommensalen Stämmen vorkommt. Der überwiegende Teil dieser ica-positiven Stämme bildete phänotypisch einen Biofilm aus. Im Unterschied dazu enthielten alle untersuchten S. aureus-Stämme, unabhängig von ihrer Herkunft, das vollständige ica-Gencluster, wobei jedoch keiner dieser Stämme unter Laborbedingungen einen Biofilm bildete. Durch subinhibitorischen Konzentrationen bestimmter Antibiotika bzw. durch Osmostress ließ sich die Biofilmbildung in 30 Prozent der S. aureus-Stämme induzieren. Ebenso konnte in ica-positiven S. epidermidis-Stämmen die Biofilmbildung dirch diese Umweltfaktoren stimuliert werden. Die Studie ergab auch, daß es einen Zusammenhang zwischen der Biofilmbildung, der Antibiotikaresistenz und dem Vorkommen der Insertionssequenz IS256 gibt. So war IS256 signifikant häufig in klinischen S. epidermidis und S. aureus-Stämmen nachweisbar, während es keinen Unterschied im Auftreten von IS257 zwischen klinischen und saprophytären Isolaten gab. Die IS256-positiven S. epidermidis-Stämme wiesen überdurchschnittlich oft das ica-Operon auf und waren gegen mindestens zwei Antibiotika gleichzeitig resistent. Weiterhin konnte gezeigt werden, daß IS256 an der Phasenvariation der Biofilmbildung in vivo beteiligt ist. Bei einem klinischen S. epidermidis-Stamm, der von einem Patienten mit einer Katheter-assoziierten Harnwegsinfektion isoliert wurde, wurde die Insertion des Elementes im icaC-Gen nachgewiesen, was in einem Biofilm-negativen Phänotyp resultierte. Subkultivierung der Insertionsmutante führte nach wenigen Passagen zur Ausbildung eines Biofilms. Die Nukleotidsequenzierung ergab die vollständige Exzision von IS256 aus dem icaC-Gen einschließlich der duplizierten Zielsequenz von sieben Basenpaaren. Diese Daten stimmen vollständig mit den zuvor in einer in-vitro-Studie erhaltenenen Ergebnissen überein und sie zeigen, daß IS256 die Expression des ica-Operons offensichtlich auch in vivo während einer Infektion beeinflußt. Bei S. aureus konnte in dieser Arbeit ebenfalls eine Phasenvariation der Biofilmexpression nachgewiesen werden. Durch Mehrfachpassagen wurden aus ehemals Biofilm-negativen Einzelkolonien mehrere Biofilmproduzenten gewonnen, die auch wieder zum Biofilm-negativen Phänotyp revertieren konnten. Die DNA-Analyse mittels Pulsfeldgelelektrophorese zeigte, daß es in den varianten Stämmen zu größeren DNA-Rearrangements gekommen war, die neben der variablen Biofilmbildung auch mit Unterschieden in der Expression des alternativen Transkriptionsfaktors SigmaB einhergingen. Die Nukleotidsequenzierung des sigB-Systems ergab in den Varianten mehrere Punktmutationen in den SigB-Regulatorgenen rsbU und rsbW. Dies legt nahe, daß der SigB-Genlokus einer starken genetischen Variabilität unterliegt, die wiederum pleiotrope Effekte auf die Genexpression in S. aureus ausübt. Durch Northern-Blot-Analysen konnte allerdings gezeigt werden, daß die Biofilmbildung in den S. aureus-Varianten nicht mit der veränderten SigB-Expression in Zusammenhang steht. / Staphylococcus aureus and Staphylococcus epidermidis belong to the most frequent causes of nosocomial infections in immunocompromised patients. These bacteria form an essential part of the healthy skin flora of human beings. Little is known, whether there are differences in the genetic equipment between clinical and commensal isolates and which factors contribute to the setup of staphylococci in the hospital environment. The results of the presented work show that the ability to form biofilms is an essential feature of pathogenic staphylococci. The expression of this virulence factor is highly variable and depends on the presence of the ica operon which is responsible for biofilm formation, specific environmental factors and the influence of insertion sequences. In an epidemiological investigation, it was shown that the ica operon in S. epidermidis is more often present in clinical strains than in commensal ones. The predominant part of these ica-positive strains formed phenotypically a biofilm. In contrast, all examined S. aureus contained, independent of their origin, the complete ica gene clusters, while, however, none of these strains formed a biofilm under laboratory conditions. Biofilm formation could be induced by subinhibitory concentrations of specific antibiotics or osmotic stress in 30 percent of the S. aureus strains. Also, biofilm formation could be stimulated in ica-positive S. epidermidis strains through these environmental factors. The study also revealed that there is an association between biofilm formation, antibiotic resistance and the occurrence of the insertion sequence IS256. Thus, IS256 was significantly more often detected in clinical S. epidermidis and S. aureus strains, while there was no difference in the occurrence of IS257 between clinical and saprophytic isolates. Most of the IS256-positive S. epidermidis strains carried the ica operon and were simultaneously resistant against at least two antibiotics. Furthermore, it was shown that IS256 is involved in phase variation of biofilm formation in vivo. In case of a clinical S. epidermidis strain that was isolated from a patient with a catheter-associated urinary tract infection, the insertion of the element in the icaC gene was detected resulting in a biofilm-negative phenotype. Subcultivation of the insertion mutant resulted in biofilm-forming variants after a few passages. Nucleotide sequencing indicated the complete excision of IS256 from the icaC gene including the duplicated target site sequence of seven base pairs. These data are in agreement with the results received in a recent in vitro study and show that IS256 has an influence on the ica-expression during an infection. In this study, phase variation of biofilm formation was also shown in S. aureus. After serial passages, several biofilm producers were derived from formerly biofilm-negative single colonies which could also revert to the biofilm-negative phenotype again. DNA analysis by pulsed-field gel electrophoresis showed that in the variants large DNA-rearrangements took place. In addition to the variable biofilm production, differences in the expression of the alternative transcription factor SigmaB were observed in the variants. Nucleotide sequencing of the sigB system indicated several point mutations in the SigB regulatory genes rsbU and rsbW of the variants. This implies that the SigB gene locus is subject to a strong genetic variability that results, in turn, in pleiotropic effects on gene expression in S. aureus. However, Northern blot analysis revealed that the biofilm formation in the S. aureus variants are not associated with the varying SigB expression.

A piodermite bacteriana afeta cães de todas as idades, podendo ser recorrente por toda a vida. O principal agente etiológico é o Staphylococcus pseudintermedius, que é um micro-organismo saprófita da pele, agindo como oportunista frente a alguma fragilidade na barreira imunológica. A imunização dos cães através de uma vacina eficaz tem sido um desafio, pois a vacina comercial atualmente utilizada em clínicas veterinárias é de uso terapêutico e não tem caráter preventivo. Um dos problemas da vacina de uso terapêutico para piodermite é a dependência de um bom diagnóstico diferencial em relação a outras doenças de pele ou sistêmicas que tem como sequelas a erupção cutânea. Os animais que recebem a vacina terapêutica e não a preventiva são os que já apresentam problemas de pele reincidentes e são tratados concomitantemente com antibióticos. O objetivo do presente trabalho é desenvolver um antígeno vacinal com possibilidade de aplicação por via intradérmica de modo a tratar preventivamente a piodermite canina causada por Staphylococcus spp:. A via de administração intradérmica tem como finalidade estimular uma resposta imune sistêmica, principalmente por ativação das células dendríticas da derme, de modo a estimular estas células apresentadoras de antígeno no local onde ocorrem as infecções piodérmicas. Os resultados apresentados neste trabalho demonstraram que a inoculação intradérmica foram estatisticamente igual, quando comparada com a inoculação intraperitoneal, no que se refere à inibição por anticorpos: da atividade hemolítica, da atividade da catalase, da atividade da coagulase e da atividade citotóxica, bem como na reação antígeno-anticorpo determinado por ensaio imunoenzimático. Este trabalho também demonstra que a melhor formulação do antígeno estafilocóccico foi preparado a partir de sobrenadante de cultura diafiltrado por filtração tangencial com \"cut off\" de 5.000 Da. Outras preparações utilizando somente células bacterianas ou associação de sobrenadante com células bacterianas, apesar de produzirem antigenicidade, apresentaram títulos inferiores do que quando empregado somente sobrenadante de cultura diafiltrado. / The bacterial pyoderma affects dogs of all ages and can be recurrent for a lifetime. The main etiologic agent is Staphylococcus pseudintermedius, which is a saprophytic organism of the skin, acting as an opportunistic opposite to some weakness in the immune barrier. The immunization of dogs through an effective vaccine has been a challenge, since currently it has been used commercially in veterinary clinics is therapeutic and has a preventive character. One of the problems of the therapeutic use of the vaccine for pyoderma is dependent on a good differential diagnosis in relation to other skin diseases or systemic consequences which is the rash, so treatment often does not reach its goal. Animals receiving this vaccine are those who already have skin problems again and are receiving concomitant antibiotics. The objective of this study is to develop a vaccine antigen with the possibility of implementing a preventive treatment for canine pyoderma caused by Staphylococcus spp: through pre-clinical studies, for use in young and adults, preferably before they develop any type of skin disorder primary or secondary. The administration route is intradermal, stimulating an immune response primarily by dendritic cells to become a barrier to microorganisms, as well as systemic, by the production of antibodies, to last for at least one year until needed in the body booster vaccination. The results presented here demonstrated that the intradermal inoculation was statistically equivalent when compared to intraperitoneal inoculation with regard to inhibition by antibody: of the hemolytic activity, catalase activity, the coagulase activity and cytotoxic activity, as well as antigen-antibody reaction determined by enzyme immunoassay. This work also demonstrates that the best estafilococcico antigen formulation was prepared from the culture supernatant diafiltered by tangential filtration with \"cut off\" of 5,000 Da. Other preparations using only the bacterial cells or the association of supernatant with bacterial cells, although producing antigenicity, showed lower titles than when used dialyzed culture supernatant alone.

A intoxicação alimentar causada por toxinas de Staphylococcus aureus é uma das principais causas de Doenças Transmitidas por Alimentos, principalmente em locais onde se preparam grandes quantidades de refeições, sendo o homem considerado a principal fonte de contaminação dos alimentos por este patógeno, pois de 30 a 50 % das pessoas sadias são portadoras desse microrganismo. O presente trabalho teve como objetivo comparar geneticamente cepas de S. aureus isoladas das mãos de manipuladores de alimentos, com cepas da mesma bactéria, recuperadas de alimentos e veiculadas por formigas. As cepas recuperadas dos manipuladores foram obtidas a partir das mãos destes utilizando-se a técnica de suabe e as cepas recuperadas de alimentos e veiculadas por formigas pertenciam à coleção de culturas do Laboratório de Microbiologia e Biologia Molecular do Departamento de Prática de Saúde Publica da Faculdade de Saúde Publica da USP. Todas as amostras foram submetidas às técnicas de PCR para verificação da presença de plasmídios e do gene sea. As análises microbiológicas revelaram que 18,4% dos manipuladores albergavam S. aureus em suas mãos. A confirmação da espécie aureus por PCR revelou somente 15,4% das cepas recuperadas dos manipuladores e 62,5% e 100% das recuperadas de alimentos e veiculadas por formigas, respectivamente. O perfil plasmidial revelou a existência de 2 grupos de cepas com perfis semelhantes e a PCR para o gene sea, que codifica a produção da enterotoxina SEA, demonstrou sua presença em 78,6% das cepas. A caracterização das cepas por ERIC-PCR evidenciou que todas as cepas apresentaram uma porcentagem de semelhança maior que 84% e que, as cepas de maior similaridade, apresentaram cerca de 90% de semelhança. Conclui-se que é de extrema importância a confirmação da espécie por método molecular, uma vez que este apresenta maior especificidade e sensibilidade do que os métodos microbiológicos; cepas isoladas de manipuladores, alimentos e veiculadas por formigas apresentavam certas semelhanças em relação ao perfil plasmidial, entretanto, a presença de plasmídio não esteve ligada necessariamente à presença do gene sea, normalmente relacionados; a técnica de ERIC-PCR constitui ferramenta importante para caracterização genética de cepas e elucidação de surtos de DTAs. / The food poisoning caused by toxins of Staphylococcus aureus is one of the main causes of illnesses transmitted by foods, mainly in places where there is prepare of great amounts of meals, since human being is considered the main source of contamination of foods for this microrganism, therefore of 30% to 50 % of the healthy people are S. aureus carriers. The present work had as objective to compare genetically strains of S. aureus isolated from hands of food handlers, with strains of the same bacteria, recovered from foods and carried by ants. Strains recovered from food handlers were obtained by technique of swab and strains recovered from foods and carried by ants belong to collection of cultures of the Laboratory of Microbiology and Molecular Biology of Department of Practical of Public Health of College of Public Health - USP. All strains were submitted to PCR for verification of the presence of plasmids and sea gene. The microbiological analyses had observed that 18,4% of the food handlers were S. aureus carriers. The confirmation of aureus species by PCR had shown only 15.4% of strais recovered from food handlers and 62.5% and 100% recovered from foods and propagated for ants, respectively. The plasmidial profile revealed presence of 2 groups of strains with similar profiles and PCR for verification of sea gene, that it codifies the production of SEA enterotoxin, demonstrated that 78,6% of strains were positive. The caracterization of strains by ERIC-PCR revealed that all of them had similarity above 84% and, strains that were very similar, presented 90% of similarity. It is concluded that it´s extremely important confirmation of species by molecular methods, since performed higher especificity and sensitivity than microbiological methods; strains isolated from food handlers, foods and carried by ants presented relative similarities in relation to the plasmidial profile, however, plasmid presence was not linked, necessarily, to presence of sea gene, normally related; ERIC-PCR is important tool to genetic diferentiating strains and to solve outbreaks.