Antibiotic resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE), are an increasing burden on healthcare systems. Hospital acquired infections with these organisms leads to higher morbidity and mortality compared with the sensitive strains of the same species and both VRE and MRSA are on the rise worldwide including in Australian hospitals. Emerging community infectious diseases are also having an impact on hospitals. The Severe Acute Respiratory Syndrome virus (SARS Co-V) was noted for its propensity to spread throughout hospitals, and was contained largely through social distancing interventions including hospital isolation. A detailed understanding of the transmission of these and other emerging pathogens is crucial for their containment. The statistical inference and mathematical models used in this thesis aim to improve understanding of pathogen transmission by estimating the transmission rates of contagions and predicting the impact of interventions. Datasets used for these studies come from the Princess Alexandra Hospital in Brisbane, Australia and Shanxi province, mainland China. Epidemiological data on infection outbreaks are challenging to analyse due to the censored nature of infection transmission events. Most datasets record the time on symptom onset, but the transmission time is not observable. There are many ways of managing censored data, in this study we use Bayesian inference, with transmission times incorporated into the augmented dataset as latent variables. Hospital infection surveillance data is often much less detailed that data collected for epidemiological studies, often consisting of serial incidence or prevalence of patient colonisation with a resistant pathogen without individual patient event histories. Despite the lack of detailed data, transmission characteristics can be inferred from such a dataset using structured HiddenMarkovModels (HMMs). Each new transmission in an epidemic increases the infection pressure on those remaining susceptible, hence infection outbreak data are serially dependent. Statistical methods that assume independence of infection events are misleading and prone to over-estimating the impact of infection control interventions. Structured mathematical models that include transmission pressure are essential. Mathematical models can also give insights into the potential impact of interventions. The complex interaction of different infection control strategies, and their likely impact on transmission can be predicted using mathematical models. This dissertation uses modified or novel mathematical models that are specific to the pathogen and dataset being analysed. The first study estimates MRSA transmission in an Intensive Care Unit, using a structured four compartment model, Bayesian inference and a piecewise hazard methods. The model predicts the impact of interventions, such as changes to staff/patient ratios, ward size and decolonisation. A comparison of results of the stochastic and deterministic model is made and reason for differences given. The second study constructs a Hidden Markov Model to describe longitudinal data on weekly VRE prevalence. Transmission is assumed to be either from patient to patient cross-transmission or sporadic (independent of cross-transmission) and parameters for each mode of acquisition are estimated from the data. The third study develops a new model with a compartment representing an environmental reservoir. Parameters for the model are gathered from literature sources and the implications of the environmental reservoir are explored. The fourth study uses a modified Susceptible-Exposed-Infectious-Removed (SEIR) model to analyse data from a SARS outbreak in Shanxi province, China. Infectivity is determined before and after interventions as well as separately for hospitalised and community symptomatic SARS cases. Model diagnostics including sensitivity analysis, model comparison and bootstrapping are implemented.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

Inledning: Antibiotikaresistens är ett av de största hoten mot global folkhälsa. Meticillinresistent Staphylococcus aureus, tidigare främst associerat med sjukhusvård, överförs nu mellan individer i samhället (samhällsförvärvad meticillinresistent staphylococcus aureus). Att identifiera riskfaktorer är centralt för att kunna bedriva effektivt preventivt arbete mot smittöverföring. Syfte: Syftet med uppsatsen var att identifiera och beskriva riskfaktorer associerade med förekomst av samhällsförvärvad meticillinresistent staphylococcus aureus. Metod: Litteraturstudie baserad på 20 internationella artiklar. Huvudfynden i artiklarna kategoriserades i teman. Resultat: Riskfaktorer på samhälls-, hushålls- och individnivå kunde identifieras, bland annat rörande klimat, tidigare antibiotikaanvändning och samsjuklighet.  Diskussion: Ett fåtal enkelt påverkbara riskfaktorer kunde identifieras. Av de identifierade riskfaktorerna är troligen inte alla generaliserbara till en svensk kontext. I flera tidigare studier framhålls att samhällsförvärvad meticillinresistent staphylococcus aureus främst drabbar individer som sedan tidigare är friska, vilket fynden i föreliggande uppsats delvis motsäger då samsjuklighet i exempelvis HIV, diabetes och fetma identifierades som riskfaktorer associerade med samhällsförvärvad meticillinresistent staphylococcus aureus. / Introduction: Antibiotic resistance a threat to global public health. Methicillin resistant staphylococcus aureus, previously primarily associated with hospital care, is now being transmitted in the community (community acquired methicillin resistant staphylococcus aureus). Identifying risk factors is central to enable effective preventive efforts against transmission of community acquired methicillin resistant staphylococcus aureus.   Aim: The aim of this essay was to identify and describe risk factors associated with occurrence of community acquired methicillin resistant staphylococcus aureus.    Methods: Literature study based on 20 international studies. The main results from the articles were categorized into themes. Results: Risk factors at community-, household- and individual level could be identified, for instance concerning climate, previous antibiotic treatment and comorbidity. Discussion: A few easily affectable risk factors could be identified. Perhaps not all of the identified risk factors are generalizable to a Swedish context. Previous research demonstrates that community acquired methicillin resistant staphylococcus aureus usually affects healthy individuals, which the findings in this essay partly contradict: comorbidity, for instance with HIV, diabetes or obesity, is a risk factor associated with community acquired methicillin resistant staphylococcus aureus.

Microbial resistance to the current available antibiotics is considered a global health problem, especially for the Multi-Drug Resistant pathogens (MDR) including methicillin resistant Staphylococcus aureus. Recently nanoparticles (NPs) have been involved in variety of antimicrobial applications due to their unique properties of antibacterial effects. However, the molecular mechanisms behind their antibacterial activity are still not fully understood. In this study, we produced silver Ag NPs (average size 27 nm) and silver-Titanium Ag-TiO2 NPs (average size 47 nm) using picosecond laser ablation. Our results showed that both laser NPs had obvious size-dependent antibacterial activity. The laser Ag NPs with a size of 19 nm and Ag-TiO2 NPs with a size 20 nm presented the highest bactericidal effect. The laser generated Ag and Ag-TiO2 NPs with concentrations 20, 30, 40, and 50 Î1⁄4g/ml showed strong antibacterial effect against three bacterial strains: E. coli, P. aeruginosa, and S. aureus, and induced the generation of reactive oxygen species (ROS), lead to cell membrane interruption, lipid peroxidation, DNA damages, glutathione depletion and the eventual cell death. Both types of laser NPs at two concentrations (2.5 and 20 Î1⁄4g/ml) showed low cytotoxicity to the in vitro cultured five types of human cells originated from the lung (A549), kidney (HEK293), Liver (HepG2), skin (HDFc) and blood vessel cells (hCAECs). The antibacterial activity of the laser generated Ag and Ag-TiO2 NPs had lasted for over one year depending on the degree of air exposure and storage conditions. Frequent air exposure increased particle oxidation and reduced the antibacterial durability of the laser generated Ag NPs. The laser generated Ag NPs had lower antibacterial activity when stored in cold compared to that stored at room temperature. The antibacterial activity of laser generated Ag and Ag-TiO2 NPs were also compared with four types of commercial based-silver wound dressings (Acticoat TM, Aquacel® Ag, Contreet ®Foam, and Urgotul® SSD) against E. coli to inform future application in this area. In conclusion, laser generated Ag and Ag-TiO2 NPs have strong bactericidal effect and low toxicity to human cells which could be a type of promising antibacterial agents for future hygiene and medical applications.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-28T19:21:24Z No. of bitstreams: 1 rodrigo-muller.pdf: 1238864 bytes, checksum: 6c267fda3ccb992508b5424e77147783 (MD5) / Made available in DSpace on 2012-11-28T19:21:24Z (GMT). No. of bitstreams: 1 rodrigo-muller.pdf: 1238864 bytes, checksum: 6c267fda3ccb992508b5424e77147783 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / As infecções causadas por Staphylococcus aureusresistentes à meticilina (MRSA) são temidas em virtude da dificuldade de seu tratamentoe da elevada morbidade associada. A PBP2a (penicillin binding protein 2a), enzima responsável pela síntese da parede celular em MRSA, apresenta baixíssima afinidade porbeta-lactâmicos. Pelo fato de a PBP2a estar localizada na superfície externa, a mesma seria acessível ao sistema imune. Durante as infecções causadas por MRSA, pouco se sabe se o hospedeiro infectado ou colonizado desenvolve anticorpos anti-PBP2a e se osmesmos seriam protetores ou não. O presente projeto tem por finalidade avaliar a presença e níveis de anticorpos anti-PBP2a em um grupo de 56 pacientes colonizados por MRSA e investigar se estes anticorpos seriam protetores ou não. Os resultados gerados permitiram observar que 71% das amostras analisadas apresentaram anticorposanti-PBP2a pelo teste ELISA. Estas amostras foram submetidas à Western blot paraconfirmação, demonstrando que 46% das amostras possuíram anticorpos anti-PBP2a. Após estes resultados, as amostras positivas foram submetidas à purificação para avaliar a cinética de crescimento de MRSA. Foi observado que houve ligeira redução do crescimento bacteriano entre amostras de soro com anticorpos MRSA comparadas comas de soro com anticorpos anti-MSSA (PBP2a negativos). Por conseguinte avaliamos a curva de crescimento de MRSA em presença de imunoglobulinas purificadas de soro de pacientes colonizados por MRSA, (quantificação bacteriana). Observamos que houve uma redução drástica do crescimento bacteriano em presença destas imunoglobulinas. Os resultados obtidos indicaram que: (i) pacientes colonizados por MRSA podem produzir anticorpos anti-PBP2a e (ii) estes anticorpos podem conferir proteção contra MRSA. Estes dados parecem indicar que uma potencial vacina anti-PBP2apoderia ser efetiva para prevenção de infecções causadas por MRSA, como também para o emprego de imunoterapia passiva para o tratamento de pacientesinfectados por este patógeno. / Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are feared because of the difficulty of their treatment and the high associated morbidity. The PBP2a (penicillin binding protein 2a), the enzyme responsible for cell wall synthesis in MRSA, presents low affinity for beta-lactams. Because of the PBP2a is located on the external surface, it would be accessible to the immune system. During the MRSA infections, little is known whether the infected orcolonized host can produce antibodies anti-PBP2a and whether these antibodies would be protective or not. This project aims to assess the presence and levels of anti-PBP2a in a group of 56 patients colonized by MRSA and investigate whether these antibodies wouldbe protective or not. The results showed that 71% of the samples presented anti-PBP2aantibodies in ELISA. These samples which were subjected to Western blot for confirmation, it was demonstrated that 46% of the samples presented antibodies anti-PBP2a. After these results, the positive samples were subjected to purification to assess the MRSA growth kinetics. It was observed that there was a slight reduction in bacterial growth between serum samples with antibodies and MRSA compared to those of the serum with anti-MSSA (PBP2a negative). Therefore, the curve of growth ofMRSA was evaluated in the presence of purified immunoglobulins from the serumof patients colonized by MRSA (bacterial quantification). We observed that there was a drastic reduction in bacterial growth in the presence of immunoglobulins, thus demonstrating that these antibodies could have a protective action. These results indicated that: (i) MRSA colonized patients can produce antibodies against PBP2a and (ii) these antibodies can be protective against MRSA. These data suggest that a potential vaccine anti-PBP2a could be effective for prevention of infections caused byMRSA and for the use of passive immunotherapy in the treatment of patients infectedby this pathogen.

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais microrganismos envolvidos nas Infecções relacionadas à Assistência à Saúde (IrAS). Porém, um clone de MRSA, o CA-MRSA, emergiu na comunidade e atualmente vem sendo agente de IrAS. O objetivo desta dissertação é avaliar fenotípica e genotipicamente 111 amostras de Staphylococcus aureus resistentes à meticilina e sensíveis a antibióticos não ß-lactâmicos de pacientes atendidos em cinco hospitais no município do Rio de Janeiro. Utilizando os critérios padronizados pelo CLSI 2012, foram determinadas as susceptibilidades a 11 antimicrobianos pelo método de disco difusão em ágar e concentração inibitória mínima para vancomicina e oxacilina pelo método da microdiluição em caldo. A multirresistência (resistência a 3 ou mais antimicrobianos não ß-lactâmicos) foi observada em 31,5% das amostras, sendo que 53,2% apresentaram resistência ao antimicrobiano clindamicina, uma das opções para o tratamento empírico das infecções de pele/tecidos moles. 86,4% apresentaram concentração inibitória mínima (CIM) para vancomicina ≥ 1,0 g/mL ou seja, elevado percentual de amostras associadas ao fenômeno MIC creep, o qual está associado ao insucesso na terapia antimicrobiana anti-MRSA. Não foi observado até o momento nenhuma amostra com CIM ≥ 4cg/mL para vancomicina, entretanto, já há resistência à linezolida em quatro hospitais do estudo. A tipificação do SCCmec nos permitiu classificar 4,5% das amostras em HA-MRSA e 86,5% em CA-MRSA, nas quais a resistência heterogênea típica à oxacilina foi observada em 57,2%. A toxina de Panton-Valentine (PVL) foi identificada pela metodologia de PCR em 28% das amostras com genótipo CA-MRSA. Os fatores de riscos clássicos, da literatura, relacionados à infecção por HA-MRSA foram também observados nos pacientes com infecção por CA-MRSA portadoras de SCCmec IV e V. No intuito de verificar a existência de similaridades genéticas ou a presença de clone predominante entre as amostras dos cinco hospitais, foi realizada a técnica de eletroforese em gel sob campo pulsado (PFGE) e observou-se diversidade genética assim como a presença de amostras com padrões similares aos clones OSPC (18,5%) e USA400. Não foram encontradas amostras com padrões de eletroforese similares aos clones USA300, USA800 e CEB. É essencial a vigilância da resistência aos antimicrobianos não ß-lactâmicos no CA-MRSA, em especial à vancomicina. A mudança na epidemiologia deste microrganismo vem impactando os padrões característicos dos genótipos limitando os critérios de diferenciação entre eles. Neste contexto, as técnicas moleculares atuam como excelentes ferramentas de caracterização. O conhecimento do patógeno auxilia na elaboração e implementação de medidas preventivas, contribuindo para o controle da doença tanto no ambiente hospitalar quanto na comunidade. / Methicillin-resistant Staphylococcus aureus (MRSA) is a major microrganism involved in healthcare associated infections (HAIs). However, a clone of MRSA, CA-MRSA, has emerged in the community and has been considered agent of HAIs. The goal of this dissertation is to evaluate phenotypically and genotypically 111 samples of methicillin-resistant Staphylococcus aureus susceptible to non ß-lactam antibiotics from patients treated in five hospitals in the city of Rio de Janeiro. Using the Clinical and Laboratory Standards Institute criteria were determined susceptibility to 11 antimicrobials by the disk diffusion method and minimal inhibitory concentration for oxacillin and vancomycin by broth microdilution method. The multidrug resistance (resistance to three or more non ß-lactam antibiotics) was observed in 31.5% of isolates, and 53.2% were resistant to the antimicrobial clindamycin, one of the choices in the empirical treatment of infections of skin / soft tissue. 86.4% showed minimal inhibitory concentration (MIC) for vancomycin ≥ 1.0 mg / mL, representing high percentage of samples associated with the MIC creep phenomenon, which can imply therapeutic failure. The typification of SCCmec enabled us to classify 4,5% of the samples in HA-MRSA and 86.5% in CA-MRSA, among which the typical heterogeneous oxacillin resistance was observed in 57.2%. The Panton-Valentine Leukocidin (PVL) toxin, one of the virulence factors involved in the pathogeneses of MRSA, was present in 28% of samples with genotype CA-MRSA. We performed uptake of demographic and clinical information on patients medical records and verified the presence of classical risk factors for HA-MRSA infection in individuals infected by CA-MRSA carrying SCCmec IV and V. In order to verify the existence of genetic similarities or the presence of predominant clone among the samples of the five hospitals, we applied the technique of pulsed-field gel electrophoresis (PFGE) and observed genetic diversity and the presence of samples with standards similar to OSPC clones (18.5%) and USA400. There were no samples with electrophoresis patterns similar to clone USA300, USA800 and CEB. Surveillance of resistance to non ß-lactam antibiotics is essencial in CA-MRSA, especially vancomycin. The change in the epidemiology of this microrganism has been impacting the characteristic patterns of genotypes limiting criteria of differentiation between them. In this context, molecular techniques serve as excellent characterization tools. Knowledge of pathogen assists in the development and implementation of preventive measures, contributing to disease control both in hospitals and in the community.

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

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais microrganismos envolvidos nas Infecções relacionadas à Assistência à Saúde (IrAS). Porém, um clone de MRSA, o CA-MRSA, emergiu na comunidade e atualmente vem sendo agente de IrAS. O objetivo desta dissertação é avaliar fenotípica e genotipicamente 111 amostras de Staphylococcus aureus resistentes à meticilina e sensíveis a antibióticos não ß-lactâmicos de pacientes atendidos em cinco hospitais no município do Rio de Janeiro. Utilizando os critérios padronizados pelo CLSI 2012, foram determinadas as susceptibilidades a 11 antimicrobianos pelo método de disco difusão em ágar e concentração inibitória mínima para vancomicina e oxacilina pelo método da microdiluição em caldo. A multirresistência (resistência a 3 ou mais antimicrobianos não ß-lactâmicos) foi observada em 31,5% das amostras, sendo que 53,2% apresentaram resistência ao antimicrobiano clindamicina, uma das opções para o tratamento empírico das infecções de pele/tecidos moles. 86,4% apresentaram concentração inibitória mínima (CIM) para vancomicina &#8805; 1,0 g/mL ou seja, elevado percentual de amostras associadas ao fenômeno MIC creep, o qual está associado ao insucesso na terapia antimicrobiana anti-MRSA. Não foi observado até o momento nenhuma amostra com CIM &#8805; 4cg/mL para vancomicina, entretanto, já há resistência à linezolida em quatro hospitais do estudo. A tipificação do SCCmec nos permitiu classificar 4,5% das amostras em HA-MRSA e 86,5% em CA-MRSA, nas quais a resistência heterogênea típica à oxacilina foi observada em 57,2%. A toxina de Panton-Valentine (PVL) foi identificada pela metodologia de PCR em 28% das amostras com genótipo CA-MRSA. Os fatores de riscos clássicos, da literatura, relacionados à infecção por HA-MRSA foram também observados nos pacientes com infecção por CA-MRSA portadoras de SCCmec IV e V. No intuito de verificar a existência de similaridades genéticas ou a presença de clone predominante entre as amostras dos cinco hospitais, foi realizada a técnica de eletroforese em gel sob campo pulsado (PFGE) e observou-se diversidade genética assim como a presença de amostras com padrões similares aos clones OSPC (18,5%) e USA400. Não foram encontradas amostras com padrões de eletroforese similares aos clones USA300, USA800 e CEB. É essencial a vigilância da resistência aos antimicrobianos não ß-lactâmicos no CA-MRSA, em especial à vancomicina. A mudança na epidemiologia deste microrganismo vem impactando os padrões característicos dos genótipos limitando os critérios de diferenciação entre eles. Neste contexto, as técnicas moleculares atuam como excelentes ferramentas de caracterização. O conhecimento do patógeno auxilia na elaboração e implementação de medidas preventivas, contribuindo para o controle da doença tanto no ambiente hospitalar quanto na comunidade. / Methicillin-resistant Staphylococcus aureus (MRSA) is a major microrganism involved in healthcare associated infections (HAIs). However, a clone of MRSA, CA-MRSA, has emerged in the community and has been considered agent of HAIs. The goal of this dissertation is to evaluate phenotypically and genotypically 111 samples of methicillin-resistant Staphylococcus aureus susceptible to non ß-lactam antibiotics from patients treated in five hospitals in the city of Rio de Janeiro. Using the Clinical and Laboratory Standards Institute criteria were determined susceptibility to 11 antimicrobials by the disk diffusion method and minimal inhibitory concentration for oxacillin and vancomycin by broth microdilution method. The multidrug resistance (resistance to three or more non ß-lactam antibiotics) was observed in 31.5% of isolates, and 53.2% were resistant to the antimicrobial clindamycin, one of the choices in the empirical treatment of infections of skin / soft tissue. 86.4% showed minimal inhibitory concentration (MIC) for vancomycin &#8805; 1.0 mg / mL, representing high percentage of samples associated with the MIC creep phenomenon, which can imply therapeutic failure. The typification of SCCmec enabled us to classify 4,5% of the samples in HA-MRSA and 86.5% in CA-MRSA, among which the typical heterogeneous oxacillin resistance was observed in 57.2%. The Panton-Valentine Leukocidin (PVL) toxin, one of the virulence factors involved in the pathogeneses of MRSA, was present in 28% of samples with genotype CA-MRSA. We performed uptake of demographic and clinical information on patients medical records and verified the presence of classical risk factors for HA-MRSA infection in individuals infected by CA-MRSA carrying SCCmec IV and V. In order to verify the existence of genetic similarities or the presence of predominant clone among the samples of the five hospitals, we applied the technique of pulsed-field gel electrophoresis (PFGE) and observed genetic diversity and the presence of samples with standards similar to OSPC clones (18.5%) and USA400. There were no samples with electrophoresis patterns similar to clone USA300, USA800 and CEB. Surveillance of resistance to non ß-lactam antibiotics is essencial in CA-MRSA, especially vancomycin. The change in the epidemiology of this microrganism has been impacting the characteristic patterns of genotypes limiting criteria of differentiation between them. In this context, molecular techniques serve as excellent characterization tools. Knowledge of pathogen assists in the development and implementation of preventive measures, contributing to disease control both in hospitals and in the community.

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

MRSA é um importante agente de colonização e infecção em pacientes hepatopatas e transplantados de fígado. Este estudo tem como objetivo avaliar a clonalidade e a virulência de isolados MRSA de pacientes hepatopatas atendidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. De agosto de 2010 a janeiro de 2012, foram coletados swabs nasais e inguinais de 190 pacientes (126 pré-transplante e 64 de pós-transplante). Isolados de MRSA foram identificados fenotipicamente e foi realizada detecção de genes de virulência, caracterização do tipo de SCCmec, análise de polimorfismo genômico por PFGE e técnica de microarranjo. Além disso, determinou-se a CIM para dez antimicrobianos pelo método de microdiluição em caldo. MRSA foi detectado em 20% dos pacientes pelo método de cultura e em 82% por PCRs. Apenas três pacientes colonizados desenvolveram infecção após o transplante. Entre os 69 isolados de MRSA, 42,0% (29/69) apresentaram SCCmec tipo II, 20,3% (14/69) SCCmec tipo I, 20,3% (14/69) SCCmec tipo III, 13,0% (9/69) SCCmec tipo IVa, 2,9% (2/69) SCCmec tipo IV e 1,5% (1/69) SCCmec tipo V. O gene tst foi detectado em 5,8% (4/69) dos isolados MRSA e todos eles foram definidos como SCCmec tipo I. Outros genes identificados por PCR foram: lukD (89,9%; 62/69), lukE (89,9%; 62/69), clf (91,3%; 63/69) e fnbA (89,9%; 62/69). A análise por PFGE dos 69 isolados mostrou a presença de um clone predominante chamado cluster A em 36,2% (25/69) e este cluster apresentou 84,6% de similaridade com o clone NewYork/Japan (BK2464). O dendrograma demonstrou também a presença de um cluster relacionado com BEC (Clone Endêmico Brasileiro) HSJ216. Atualmente o tipo de SCCmec mais prevalente em nosso hospital é o tipo II. Neste estudo, observou-se a presença de isolados virulentos tanto em pacientes hepatopatas como em pacientes transplantados. Nossos resultados mostraram que o clone predominante (cluster A) apresentou diferentes genes de virulência (genes fnbA, clf e lukD-lukE) e foi resistente a pelo menos seis diferentes drogas, além de ser caracterizado como HA-MRSA SCCmec tipo II. Em conclusão, a técnica de microarranjo permite a genotipagem e detecção de genes estafilocócicos clinicamente relevantes, e pode, na maioria dos casos, ser utilizada como uma importante ferramenta para a triagem da virulência e resistência a antimicrobianos em isolados de MRSA / MRSA is an important agent of colonization and infection in patients with liver disease and liver transplant. This study aims to evaluate clonality and virulence of MRSA isolates from liver diseases patients treated at Hospital of Clinics Faculty of Medicine from University of Sao Paulo. From August 2010 to January 2012, we collected nasal and groin swabs from 190 patients (126 pre-liver and 64 post-liver). MRSA isolates were identified phenotypically and the detection of virulence genes, characterization of SCCmec type, microarray and genomic polymorphism analysis by PFGE were done. In addition, it was determined the MIC for ten antibiotics by broth microdillution method. MRSA was detected in 20% patients by culture method and 82% by PCR. Only three patients colonized developed infection post-transplantation. Among the 69 MRSA isolates, 42.0% (29/69) had type II SCCmec, 20.3% (14/69) SCCmec type I, 20.3% (14/69) SCCmec type III, 13.0% (9/69) SCCmec type IVa, 2.9% (2/69) SCCmec type IV and 1.5% (1/69) SCCmec type V. The tst gene was detected in 5.8% (4/69) of MRSA isolates and all of them were defined as SCCmec type I. Other genes were identified by PCR: lukD (89.9%; 62/69), lukE (89.9%; 62/69), clf (91.3%; 63/69) and fnbA (89.9%; 62/69). The PFGE analysis of 69 isolates showed the presence of a predominant cluster named cluster A in 36.2% (25/69) and this cluster had 84.6% similarity with New York/Japan clone (BK2464). Dendrogram also demonstrated presence of one cluster related with BEC (Brazilian Endemic Clone) HSJ216. Currently the most prevalent SCCmec type in our hospital is type II. In this study, we observed virulent isolates in pre and post-transplantation patients. Our results showed that the predominant clone (cluster A) had different virulence genes (genes fnbA, clf and lukD-lukE) and was resistant to at least six different drugs, in addition to being characterized as HA-MRSA SCCmec type II. In conclusion, microarray profiling allows genotyping and detection of clinically relevant staphylococcal genes, and can, in most cases, be used as an important tool to screening virulence and antibiotic resistance genes in MRSA isolates