Acquisition of a superantigen pathogenicity island (SaPI) significantly increases virulence in Staphylococcus aureus. Horizontal transfer of SaPIs occurs at high frequency and depends upon a helper bacteriophage, either through direct infection or SOS-mediated induction of a lysogen. SaPIs hijack the packaging machinery of the helper phage, leading to the formation of SaPI-containing transducing particles that can introduce the pathogenicity island into neighboring SaPI-negative cells. All SaPIs contain a conserved core of genes, some of which are co-transcribed as an operon and encode functions involved in helper exploitation. The goal of this study was to more fully understand the intricate relationships between the SaPI elements and their helper bacteriophages, specifically any regulatory crosstalk that might occur between them. We demonstrated phage-host interactions in 80 and 80α, and SaPI1 and SaPIbov1-mediated crosstalk with helper phage 80α. The phage Sri protein was shown to be a bi-functional protein that both derepresses SaPI1 and interferes with host chromosome replication. Incoming SaPI1 experiments showed that SaPI1 modulates the levels of the N-terminal part of orf14 mRNA. Induction experiments using the 80α ΔrinA phage as a genetic tool, reveal several new phage genes that SaPI1 targets for expression modulation. Finally, a novel SaPI1 interference mechanism was identified. In an 80α ΔrinA mutant, which cannot activate its late operon, SaPI1 can directly turn on expression of the packaging and structural genes in a noncanonical manner, initiating from the 2nd gene in the operon, the large terminase subunit.

La mammite bovine est une inflammation de la glande mammaire causée par un traumatisme ou par une infection. Au Canada, l’agent pathogène le plus souvent retrouvé dans les cas de mammites bovines est Staphylococcus aureus (S. aureus). Cet agent pathogène peut causer des mammites cliniques avec des symptômes graves et apparents, mais il peut aussi provoquer des mammites sous-cliniques ne présentant aucun symptôme. Cette infection engendre des pertes économiques importantes dans le secteur de la production laitière, principalement par le retrait du lait lors d’infection clinique et lorsqu’un antibiotique est utilisé. Il est ainsi important d’apporter de nouveaux outils aux producteurs laitiers pour contrer la mammite bovine. Prédire les symptômes associés à la mammite bovine causée par S. aureus pourrait améliorer la gestion des mammites en identifiant rapidement les souches problématiques. Pour ce faire, une caractérisation génotypique et phénotypique de 587 souches de S. aureus isolées dans l’ensemble du Canada a été effectuée dans le but de prédire l’apparition de symptômes cliniques et la durée de l’infection. Les séries d’isolats provenant du même quartier suivi sur une longue période de temps avec la manifestation d’au moins une mammite clinique ont été comparées aux isolats de séries sans aucune mammite clinique. Cette comparaison a été effectuée dans le but de différencier les deux types d’infection selon les caractéristiques de la souche de S. aureus impliqués. Les facteurs comparés sont la présence de gènes de virulence (seg, lukM et tst), la formation de biofilm in vitro et l’origine clonale des souches de S. aureus selon le typage spa. Des différences significatives ont été trouvées dans la distribution des types spa (p=0,018) et la présence du gène lukM (p=0,009) entre les souches causant des mammites cliniques ou sous-cliniques. Une régression logistique, qui prend en compte tous les facteurs de la comparaison, a été effectuée. Cela a permis de faire l’importante observation qu’à chaque log additionnel de biofilm, les chances d’avoir des signes cliniques augmentent de 97% (p=0,05). Pour déterminer quels facteurs influençaient la durée de l’infection, chaque facteur a été utilisé individuellement dans des tests d’analyse de survie. Aucun facteur n’est ressorti significatif dans ces tests lorsque pris individuellement, mais lorsque tous les facteurs sont pris en compte dans une régression logistique, quelques différences peuvent être observées. Nous avons déterminé que les types spa t529 et t359 ont respectivement 3,5 à 4,5 fois plus de chance de se faire éliminer en comparaison au type spa t13401 (p = 0,047 et 0,019). De nombreux facteurs ont été corrélés avec les types spa, tels que la possession de seg par t529 et la formation de biofilm variable selon le type spa. Ces résultats tendent à montrer qu’un outil de prédiction pourrait être développé avec une caractérisation plus poussée des types spa. L’outil de prédiction pourra alors permettre une meilleure gestion des cas de mammite bovine causés par S. aureus.

Staphylococcus aureus ist einer der häufigsten Erreger von nosokomialen Infektionen. Diese grampositiven Bakterien verursachen neben harmlosen oberflächlichen Hautinfektionen auch lebensbedrohliche Systeminfektionen. Ein großes Problem in der Therapie von S. aureus-Infektionen stellen die zunehmenden Multiresistenzen dar. Die Entwicklung neuer Antibiotika wird zukünftig wahrscheinlich nicht ausreichen, da immer wieder neue Resistenzen der Bakterien zu erwarten sind. Es besteht daher dringender Bedarf an der Entwicklung alternativer Therapieformen im Kampf gegen multiresistente Problemkeime wie S. aureus. Eine Möglichkeit besteht in der Immuntherapie, zum Beispiel durch Gewinnung von monoklonalen Antikörpern gegen geeignete Targetstrukturen von S. aureus. Ziel dieser Arbeit war es, zunächst zwei Proteine IsaA und IsaB herzustellen, um diese Proteine für Immunisierungsstudien zu nutzen. Zunächst wurde das gereinigte IsaA-Protein verwendet, um ein Kaninchen zu immunisieren. Mit den daraus gewonnenen Antikörpern wurden dann erste Tierversuche begonnen, um die Bedingungen für den therapeutischen Einatz von gegen IsaA-gerichteten Antikörpern zu ermitteln und die Wirksamkeit einer Antikörper-Behandlung zu evaluieren. Für die Herstellung der gewünschten Proteine wurden die Gensequenzen zunächst aus verschiedenen S. aureus-Stämmen mittels PCR amplifiziert und in den kommerziellen Expressionsvektor pQE30 kloniert. Die amplifizierte Gensequenz stammt aus den klinischen Stämmen 418 (IsaA) bzw. 134 (IsaB). Nach der Klonierung wurden geeignete Expressions- und Reinigungsstrategien entwickelt. Dabei wurden folgende Bedingungen als optimal für Wachstum und Überexpression herausgearbeitet: IsaA: Induktion der Überexpression mit 100 µM IPTG, 3 h Wachstum bei 37°C. IsaB: Induktion der Überexpression mit 100 µM IPTG, 4 h Wachstum bei 37°C. Es stellte sich auch heraus, dass IsaA zunächst in nur unzureichender Quantität vorhanden bzw. exprimiert worden war. Die Vermutung, dass IsaA überwiegend im Pellet in sogenannten Einschlusskörpern (inclusion bodies) eingeschlossen war, erklärte dieses Phänomen. Das Protein konnte erfolgreich aus dem Pellet isoliert werden. Die Produktion und Aufreinigung beider Proteine IsaA und IsaB unter optimierten Bedingungen ergab, dass beide Proteine nun in ausreichender Menge und Konzentration für die folgende Immunisierung und die weiteren Arbeiten vorlagen. Aus Kaninchen, die mit IsaA immunisiert wurden, konnten polyklonale Antikörper gewonnen werden, die die Grundlage für einen ersten Tierversuch mit 24 Ratten bildeten. Hierbei zeigte sich, dass die Tiere, die mit 1.000.000.000 Bakterien infiziert worden waren deutlich stärkere Infektionszeichen aufwiesen als diejenigen, die mit 100.000.000 Bakterien infiziert worden waren. Weiterhin wurde deutlich, dass die Tiere, die Serum (mit Antikörper gegen IsaA) erhalten hatten, gegenüber den Vergleichstieren mit Placebo einen deutlichen Vorteil hinsichtlich Infektionszeichen und Immunantwort hatten. Somit belegen die tierexperimentiellen Ergebnisse in dieser Arbeit erstmalig den therapeutischen Nutzen von Antikörpern gegen IsaA. IsaA ist demnach ein geeignetes Target für eine Immuntherapie gegen S. aureus. / Staphylococcus aureus is one of the most common pathogens of nosocomial infections. These grampositive bacteria not only cause harmless superficial skin infections but also life threatening systemic infections. A huge problem in therapy of S. aureus infections is the increasing rate of multiresistance. The development of new antibiotics will probably not be sufficient in the future because new resistance in bacteria is to expect. Therefore there is urgent need for alternative therapies fighting multiresistant bacteria such as S. aureus. One approach is immunotherapy, e.g. by production of monoclonal antibodies against adequate targets of S. aureus. The purpose of this paper was to produce two proteins, IsaA and IsaB, to use these for immunisation studies. First purified IsaA was used to immunise a rabbit. The extracted antibodies were used for early animal experiments to evaluate conditions for the therapeutic use and efficiency of antibodies against IsaA. For production of the wanted proteins gene sequences from various S. aureus strains were amplified by PCR and cloned into pQE30, a commercial expression vector. The amplified gene sequences come from strain 418 (IsaA) and strain 134 (IsaB). After cloning appropriate conditions for expression and purifiing were elaborated: IsaA: induction of overexpression with 100 µM IPTG, 3 h growth at 37°C. IsaB: induction of overexpression with 100 µM IPTG, 4 h growth at 37°C. First IsaA emerged to be present respectively expressed of low quantity only. The presumption that IsaA was predominantly enclosed in so called inclusion bodies explained this phenomenon. The protein could successfully isolated from the pellet. Production and purification of both proteins IsaA and IsaB under optimised conditions led to sufficient quantitiy and concentration for the immunisation following and further research. From a rabbit, immunised with IsaA, polyclonal antibodies were obtained and provided a basis for the first animal experiment with 24 rats. It showed that animals infected with 1.000.000.000 bacteria had considerably more signs of infection than those infected with 100.000.000 bacteria. It could also be shown that animals treated with serum (with antibodies against IsaA) had clear advantage regarding signs of infections and immune response compared to those animals treated with placebo. These results of the animal experiment document the therapeutic benefit of antibodies against IsaA for the first time. Therefore IsaA is an adequate target for immunotherapy against S. aureus.

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 colonização nasal por Staphylococcus aureus e a infecção pelo HIV representam problemas de saúde pública de preocupação mundial. O objetivo geral foi identificar os fatores de risco para a colonização nasal por Staphylococcus aureus em pessoas vivendo com HIV/aids. Para tanto, foi realizado um estudo tipo caso-controle, com pessoas vivendo com HIV/aids internadas nas unidades especializadas na assistência às doenças infecciosas de um hospital de ensino no interior paulista. A coleta de dados ocorreu de janeiro/2013 a fevereiro/2015, por meio de entrevista individual contemplando dados sociodemográficos e clínicos, além da coleta da secreção nasal com auxílio do swab em meio Stuart, ambos nas primeiras 24 horas de internação. As amostras foram encaminhadas e processadas pelo Laboratório de Microbiologia da própria instituição. Os critérios de inclusão foram: ter idade acima de 18 anos, ser soropositivo ao HIV, estar internado. Nas análises estatísticas foram realizados os testes qui-quadrado de Pearson, Exato de Fisher, t-Student, Wilcoxon e Regressão Logística Univariada e Multivariada, por meio do software SAS®. Os dados estão apresentados em tabelas e figuras. O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto (No CAAE 38990114.5.0000.5393) e pela instituição co-participante (No CAAE 38990114.5.3001.5440). Participaram do estudo 240 pessoas vivendo com HIV/aids, sendo 120 Casos e 120 Controles, houve predominância do sexo masculino em 65,0% dos Casos e 55,0% dos Controles, 35,8% dos Casos estavam na faixa etária de 30 a 39 anos e 45,8% dos Controles tinham idade de 40 a 49 anos, a etnia predominante foi a branca para Casos e Controles, 74,2% e 64,2%, respectivamente. Os grupos foram homogêneos entre si em relação ao sexo, etnia e escolaridade. A média do tempo de diagnóstico foi de 9 anos para Casos e 8,8 anos para Controles. O modelo final de regressão logística evidenciou como fatores de risco associados à colonização nasal por Staphylococcus aureus em pessoas vivendo com HIV/aids, ser da etnia branca, p=0,05 (OR:1,85; IC95% 1,00 - 3,57); ter carga viral >40 cópias/mL, p= 0,03 (OR: 2,90; IC95% 1,15 - 7,30); estar com contagem de LT-CD4+ <200 células/mm3 p=0,001 (OR: 2,71; IC95% 1,53 - 4,81); e apresentar doença oportunista p=0,014 (OR: 2,09; IC95% 1,20 - 3,67). Além disso, foi evidenciado como fator de proteção para a colonização nasal pelo Staphylococcus aureus em pessoas vivendo com HIV/aids o uso de antirretroviral p=0,008 (OR: 0,45; IC95% 0,25 - 0,81). Concluímos que a colonização nasal por Staphylococcus aureus nas pessoas vivendo com HIV/aids foi associada aos fatores: etnia, carga viral, contagem de LT-CD4+ , infecção oportunista e uso de antirretroviral / Staphylococcus aureus nasal colonization and HIV infection represent public health problems of global concern. The overall objective was to identify the risk factors for nasal colonization by Staphylococcus aureus in people living with HIV / AIDS. Therefore, a case-control study was conducted, with people living with HIV / AIDS hospitalized at the units specialized in infectious disease care at a teaching hospital in the interior of São Paulo. Data were collected from January / 2013 to February / 2015 by means of an individual interview, including sociodemographic and clinical data, as well as the collection of nasal secretions with the aid of swab in Stuart\'s medium, both during the first 24 hours of hospitalization. The samples were sent and processed by the Laboratory of Microbiology of the institution itself. The inclusion criteria were: to be over 18 years of age, to be known as infected HIV, to be hospitalized. Statistical analyzes were performed using the Pearson chi-square test, Fisher\'s exact test, Student t-test, Wilcoxon test, and Univariate and Multivariate logistic regression using the SAS® software. The data are presented in tables and figures. The present study was approved by the Research Ethics Committee of the Ribeirão Preto College of Nursing (CAAE 38990114.5.0000.5393) and by the co- participating institution (CAAE 38990114.5.3001.5440). A total of 240 people living with HIV / AIDS participated in the study, of which 120 were Cases and 120 Controls; 65.0% of Cases and 55.0% of Controls were male: 35.8% of Cases were in the age group of 30 at 39 years and 45.8% of the Controls were aged from 40 to 49 years, the predominant ethnicity was white for Cases and Controls, 74.2% and 64.2%, respectively. The groups were homogeneous among themselves in relation to gender, ethnicity and schooling. The mean time of diagnosis was 9 years for Cases and 8.8 years for Controls. The final logistic regression model showed that the risk factors associated with Staphylococcus aureus nasal colonization in people living with HIV / AIDS were white, p = 0.05 (OR: 1.85, 95% CI: 1.00 - 3.57); having viral load> 40 copies / mL, p = 0.03 (OR: 2.90; IC95% 1.15 - 7.30); being with LT-CD4+ <200 cells / mm3 p = 0.001 (OR: 2.71; IC95% 1.53 - 4.81); and present opportunistic disease p = 0.014 (OR: 2,09; IC95% 1,20 - 3,67). In addition, it was also obtained by the final regression final model that the use of antiretroviral therapy is a protection factor of p = 0.008 (OR: 0.45; 95% CI 0.25 - 0.81) for nasal colonization by Staphylococcus aureus. We conclude that nasal colonization by Staphylococcus aureus in people living with HIV/AIDS was associated with factors: ethnicity, viral load, LT-CD4+ count, opportunistic infection, and antiretroviral use

Thesis advisor: Jianmin Gao / With the rapid evolution of antibiotic resistance, the need for more effective antibiotics is imminent. Bacterial membranes are an appealing target due to their accessibility and relatively conserved structures. Membrane targeting antibiotics, especially cationic antimicrobial peptides (CAMPs) such as host defense peptides, have been increasingly explored as novel antibiotics and tunable innate antimicrobials. The latter could be achieved by treatment with an antibiotic adjuvant: a compound that would increase the potency of host CAMPs without killing the bacteria on its own. Boosting the host’s own immune system with an adjuvant is beneficial over using antibiotics and would theoretically avoid triggering bacterial resistance. One mechanism of bacterial resistance is increasing the cationic charge of the membrane. As CAMPs are electrostatically attracted to anionic bacterial membranes, making the membrane more cationic decreases that attraction, rendering CAMPs less effective. To target this resistance mechanism chemically, two antibiotic adjuvant strategies were explored as co-treatments with various CAMPs: membrane targeting peptides used to bind and block surface amines, and small molecules used to either acetylate surface amines or convert a cationic membrane phospholipid to an anionic phospholipid. Co-treatment of the Staphylococcus aureus (S. aureus) membrane targeting peptide KAM-CT and various CAMPs increased S. aureus susceptibility to those CAMPs. Bacterial surface acetylation using sulfo-NHS-acetate followed by CAMP treatment caused up to 10 times increased CAMP potency. Hydrazine and hydroxylamine were shown to cleave the lysine moiety from the lysyl-phosphatidylglycerol (Lys-PG) phospholipid to generate phosphatidylglycerol (PG) in liposome models. S. aureus was treated with a hydroxylamine-CAMP conjugate, but it showed decreased antibiotic activity compared to the CAMP alone. To better understand what was happening in the bacteria, a novel Lys-PG quantification protocol was created by fluorophore labeling Lys-PG and quantifying the labeled Lys-PG via normal phase high-performance liquid chromatography (NP-HPLC). Cyclic peptides, such as KAM-CT, represent complex yet synthetically attainable moieties that could be used as novel antibiotics adjuvants. Expanding the repertoire of reversible covalent chemistries, especially those applied to peptide cyclization, is desirable due to the high potency and selectivity of such interactions. Herein, we also describe a novel reversible covalent chemistry between 2-formylphenylboronic acid (FPBA) and 2,3-diaminopropionic acid (Dap): the imidazolidino boronate (IzB) conjugate. It was found to be potent (Kd = 100 μM) and quickly reversible (t1 = ~6 sec) under physiological conditions. IzB formation was successfully employed as a peptide cyclization strategy as there was little interference from biologically relevant small molecules, except cysteine. Cysteine interference was utilized to create “smart” peptides that can linearize upon increasing cysteine concentrations via thiazolidino boronate (TzB) formation with the FPBA moiety in the peptide. Such “smart” peptides could be used as pH-responsive peptides or cysteine sensors able to report on the cysteine concentration in complex media. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Na última década, Staphlylococcus aureus resistentes à meticilina não multidroga resistente (NM-MRSA) tem sido descrito como um importante agente de infecção de corrente sanguínea em nosso serviço. Este estudo de coorte prospectivo, realizado entre fevereiro de 2009 e janeiro de 2010 na unidade neonatal, avaliou 403 recém-nascidos (RN), suas 382 mães e 148 profissionais da área da saúde (PS). Duzentos e dezessete NB (54%), 187 mães (48%) e 87 PS (59%) foram colonizados por S. aureus (SA). A colonização por S. aureus resistente à meticilina (MRSA) foi maior entre RN (15%) do que entre mães (4.7%) e PS (3.4%). Embora a transmissão da mãe para seu RN tenha ocorrido, na maior parte dos casos, a mãe não foi a responsável pela colonização do RN. Houve dois padrões predominantes de polimorfismo do DNA por eletroforese em campo pulsado (PFGE) entre os RN, e algumas mães e PS foram colonizados por eles. Fatores estatisticamente associados com colonização por MRSA foram baixo nível de escolaridade materna (fator de risco - OR: 2.99; 95%CI: 1.10-8.07) e rinossinusite materna (fator protetor - OR: 0.33; 95%CI: 0.12-0.88). Entre os Rn que permaneceram hospitalizados mais do que 72 horas, o aleitamento materno foi protetor (OR: 0.22; 95%CI: 0.05-0.98). Todos os isolados foram NM-MRSA, portavam poucos fatores de virulência e Staphylococcal Cassete Chromossome mec (SCCmec) tipos IVa e IVd predominaram. Embora não tenham ocorrido casos de infecção, a transmissão nosocomial de MRSA claramente ocorreu na unidade neonatal e aponta para a necessidade de implementação de práticas de controle de infecção, como higienização das mãos para prevenção de infecção cruzada. Outras práticas de promoção à saúde, básicas, mas abrangentes, podem ser fundamentais, como educação e aleitamento materno / In the last decade non-multiresistant methicillin-resistant S. aureus (NM-MRSA) has been described as an important agent in bloodstream infections in our hospital. This prospective cohort study, conducted from February 2009 through January 2010 in the neonatal unit, evaluated 403 newborns (NB), their 382 mothers and 148 health care workers (HCW). 217 NB (54%), 187 mothers (48%) and 87 HCW (59%) were colonized by S. aureus (SA). Methicillin-resistant S. aureus (MRSA) colonization was greater among NB (15%) than mothers (4.7%) and HCW (3.4%). Although mother-to-NB transmission occurred, in most cases mothers were not responsible for NB colonization. There were two predominant PGFE patterns among the NB and some mothers and HCW became colonized by them. Factors significantly associated with MRSA carriage by NB were lower level of maternal schooling (risk factor: OR: 2.99; 95%CI: 1.10-8.07) and maternal rhinosinusitis (protective factor: OR: 0.33; 95%CI: 0.12-0.88). Among NB who remained hospitalized for more than 72 hours, breast feeding was protective (OR: 0.22; 95%CI: 0.05-0.98). All the isolates were NM-MRSA, carried few virulence factors and Staphylococcal Cassete Chromossome mec (SCCmec) types IVa and type IVd predominated. Although there were no cases of infection, nosocomial transmission of MRSA clearly occurred in the neonatal unit and this highlights the need for infection control practices such as hand hygiene to prevent cross-dissemination. Other healthcare practices, which are very basic but also ample in scope, may play a role, such as general education of women and breast feeding

Orientador: Antonio Nader Filho / Banca: Luiz Augusto do Amaral / Banca: Elisabeth Loshchagin Pizzolitto / Resumo: Estudou-se 94 estirpes de Staphylococcus aureus obtidas do leite de vacas com mastite subclínica em duas propriedades rurais no estado de São Paulo. Essas estirpes foram caracterizadas fenotipicamente quanto a produção de biofilmes pelos testes do agar vermelho congo e pelo teste de aderência em microplacas e também foram genotipicamente identificadas pela presença dos genes icaA e icaD responsáveis pela produção do polissacarídeo de adesão intercelular. Além disso, todas as estirpes foram também submetidas ao teste de sensibilidade aos antimicrobianos. Os resultados obtidos revelaram que 85% das estirpes produziram biofilmes "in vitro" para o teste do agar vermelho congo. No teste de aderência em placas foi verificado que 98,9% das estirpes produziram biofilme, devido a forte aderência nas placas de polietileno. A presença dos genes icaA e icaD foi encontrada em 95,7% das estirpes de S. aureus. No antibiograma foi observado que sete estirpes foram resistentes aos seguintes antimicrobianos: cloranfenicol, clindamicina, eritromicina, gentamicina, oxacilina e penicilinaG, sendo que as maiores resistências ocorreram frente a gentamicina (2,2%) e a penicilina G (6,6%). Na avaliação dos testes fenotípicos com os genotípicos o teste de aderência em microplacas foi o mais sensível (100%), sendo indicado para identificar estirpes produtoras de biofilmes. Todos os testes, com exceção do antibiograma, foram estatisticamente significativos (p<0,05). / Abstract: 94 Staphylococcus aureus strains obtained from milk samples of cows suffering from subclinical mastitis in dairy herd, in two properties, in the state of São Paulo were evaluated. These strains were characterized by the in vitro slime production on the Congo red agar, biofilm formation and by the presence of icaA and icaD genes which are responsible for the intercellular adhesion. All strains were too subjected to in vitro susceptibility to 12 different antibiotics. The results revealed that 85% of isolates tested produced slime on the Congo red agar and 98,9% of the isolates produced biofilm in vitro by the adherence in sterile 96-well "U" bottom polystyrene tissue culture plates. 95,7% of isolates possessed the icaA and icaD genes. The results of in vitro antibiotic susceptibility assay to 12 different antibiotics revealed that seven isolates were resistance to follow antibiotics: clorphenicol, clindamicin, erythromycin, gentamicin, oxacillin and penicillin, the higher resistance occurred to penicillin (6,6%) and gentamicin (2,2%). In the study of phenotypic tests compared with genotypic test, the 96-well polystyrene tissue culture plates assay was the most sensitivity (100%) and is recommended with genotypic test for the investigation biofilm formation in S. aureus. All the tests, exception of antibiotic susceptibility assay were statistically significant (p<005). / Mestre

Community-associated methicillin-resistant Staphylococcus aureus has been an increasing public health problem since its emergence in the 1990s and incarcerated populations are at disproportionately high-risk for colonization and infection. However, few studies have investigated why levels of S. aureus remain endemic in correctional settings in the absence of an outbreak. The overall objective of this dissertation was to evaluate S. aureus transmission in two maximum-security prisons using a molecular epidemiologic approach and data collected on over 2,700 inmates from 2009 – 2013. The objective of this dissertation was met using three aims. First, a systematic literature review was conducted to identify studies that used social network analysis (SNA) to evaluate infectious disease transmission via non-sexual/non-injection drug use contact pathways to detect influences of social networks on disease risk. Results of the review demonstrated that SNA approaches in infectious disease epidemiology are flexible and can be used to enhance traditional contact investigations, reveal granular patterns of transmission, evaluate influences of high-risk behaviors and activities, and identify both protective and causal effects resulting from context-specific social interactions. Second, changes in the distribution and diversity of S. aureus isolates with increasing length of incarceration were assessed. The results revealed some evidence for S. aureus transmission based on greater representation of certain strains; however, the genetic diversity of S. aureus was high regardless of length of time served. Third, the influence of social interactions among prison inmates on S. aureus colonization status was examined using SNA. The results showed that S. aureus colonized inmates were more likely to spend time in social groups and that the mechanisms of transmission differed for men and women. For women, the association was driven by being centrally located in the social network and for men it was driven by higher proportions of colonized inmates in close proximity. Overall, the results of this dissertation support the hypothesis that S. aureus is transmitted within prisons as a result of direct skin-to-skin contact and/or exposure to contaminated environmental surfaces. However, the results also demonstrate that, in the absence of an outbreak, S. aureus transmission within prisons is low indicating that endemic levels of S. aureus are primarily maintained by the constant introduction of clones into prisons from jails and the community.

No description available.