Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-22T13:39:31Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Mestrado fase final defendido 3 versão digital.pdf: 1373305 bytes, checksum: 2ed41bad53b29893bffe574008593b64 (MD5) / Made available in DSpace on 2016-07-22T13:39:31Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Mestrado fase final defendido 3 versão digital.pdf: 1373305 bytes, checksum: 2ed41bad53b29893bffe574008593b64 (MD5) Previous issue date: 2016-02-29 / CAPEs / Staphylococcus aureus é um dos microrganismos mais comuns em infecções patogênicas no mundo, tornando-se de grande importância hospitalar e comunitária. Pessoas vivendo com HIV/aids (PVHA) são mais susceptíveis de serem colonizados por Staphylococcus aureus resistente á meticilina (MRSA). S. aureus pode adquirir resistência a antimicrobianos, devido à presença de genes de vários tipos contidos no cassete cromossômico estafilocócico - mec (SCCmec) conferindo resistência a diversos antibióticos. A investigação da colonização por MRSA foi realizada através do isolamento de amostras oriundas de secreção nasal e posteriormente screening de oxacilina combinado a reação de PCR convencional para investigação do gene mecA. Foram entrevistados no estudo 500 PVHA ambulatoriais do hospital terciário. Aproximadamente 95% fazia uso de terapia antirretroviral, sendo que 89,3% destes apresentavam contagem de células CD4 >200 e 73,4% com carga viral ≤100 cópias. A maioria foi do sexo masculino (64,4%), com média etária de 41,5 anos e se declararam de cor parda (54,7%). Exposição a antimicrobianos nos últimos 12 meses foi encontrado em 27,4% dos indivíduos e 25,1% relataram uso de drogas ilícitas ao menos uma vez na vida. Colonização nasal por S. aureus foi encontrada em 31,4% (157/500) da totalidade dos indivíduos estudados, nos quais, 14% (22/157) foram MRSA. A colonização foi maior nos indivíduos acima de 40 anos, entre os que relataram uso de drogas ilícitas ao menos uma vez na vida, nos que não havia registro de exposição prévia a antimicrobianos nos últimos 12 meses, porém, não foi encontrada nenhuma associação de MRSA com as variáveis estudadas. A colonização de MRSA, embora alta, não foi associado com as variáveis estudadas para fator de risco em PVHA. O perfil antimicrobiano mostra alta resistência aos antibióticos mais utilizados para profilaxia e tratamento por infecções bacterianas. Esse estudo pode contribuir para orientar na vigilância e conduta terapêutica entre as PVHA. / Staphylococcus aureus is one of the most common pathogenic microorganisms in infections in the world, making it of great importance hospital and community. People living with HIV/aids (PVHA) are more likely to be colonized with Staphylococcus aureus resistant to methicillin (MRSA). S. aureus may acquire resistance to antibiotics due to the presence of various types of genes contained in the chromosomal staphylococcal cassette - mec (SCCmec) conferring resistance to various antibiotics. The investigation of MRSA colonization was carried out by isolating samples from nasal discharge and subsequently screening oxacillin combined with conventional PCR to investigate the mecA gene. They were interviewed in the study 500 outpatient PVHA tertiary hospital. Approximately 95% made use of antiretroviral therapy, and 89.3% of them had CD4 cell counts> 200 and 73.4% with viral load ≤100 copies. Most were male (64.4%) with a mean age of 41.5 years and declared mulatto (54.7%). antimicrobial exposure in the last 12 months was found in 27.4% of patients and 25.1% reported using illicit drugs at least once in life. nasal colonization by S. aureus was found in 31.4% (157/500) of all subjects studied, in which, 14% (22/157) were MRSA. The colonization was higher in individuals over 40 years among those who reported using illicit drugs at least once in life, in which there was no antimicrobial previous exposure record in the last 12 months, however, it found no MRSA association with the variables studied. Colonization of MRSA, although high, was not associated with the variables for risk factor for PVHA. The antimicrobial profile shows high resistance to antibiotics most commonly used for treatment and prevention of bacterial infections. This study may help guide surveillance and therapeutic management among PVHA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pandemic human pathogen accounting for most of health-care associated infections throughout the world. However, in recent years, a more virulent strain of MRSA has emerged in the community defined as community-associated MRSA (CA-MRSA). These emerging strains of CA-MRSA are described to have different antibiotic susceptibility profiles, possess the SCCmec type IV element and usually produce the Panton-Valentine leukocidin (PVL) toxin. The majority of these CA-MRSA strains are associated with skin and soft tissue infections and necrotising pneumonia, with a 34% mortality rate. Identification and characterisation of MRSA isolates is mainly performed using phenotypic methods, which are time consuming. Little information exists on the prevalence and characteristics of MRSA isolates including antibiotic susceptibility patterns, PVL-producing CAMRSA strains, the SCCmec types and genotypes that might be circulating in the Steve Biko Academic Hospital. Identification and characterisation of MRSA isolates based on these criteria are important in controlling possible outbreaks in the clinical setting. In this study, 97 clinical MRSA isolates from the Steve Biko Academic Hospital, South Africa were collected between April 2006 to February 2007. These isolates were analysed and characterised using multiplex PCR (M-PCR), real-time PCR as well as staphylococcal protein A (spa) and hyper-variable region (HVR) typing. The aim of this study was to determine the antibiotic profiles, prevalence of MRSA isolates, the SCCmec types and the genotypes. Antibiotic susceptibility determination was performed using the disk diffusion susceptibility method as guidelined by the CLSI. Six distinct antibiotypes were identified with a total of 73%, 71%, 70% and 7% of MRSA isolates resistant to clindamycin, erythromycin, gentamicin and fusidic acid, respectively. The presence of Staphylococcus aureus specific 16S rRNA, the mecA and PVL genes was determined using a modified M-PCR assay. A total of 4% of the MRSA isolates possessed the PVL gene. Real-time PCR analysis also showed a 100% prevalence of the PVL gene in the same 4% MRSA isolates confirming the results of the first M-PCR assay. The second M-PCR was used to determine the SCCmec type prevalence and to distinguish between health-care associated MRSA (HA-MRSA) and CA-MRSA. SCCmec typing showed 67% of the isolates belonged to SCCmec type II and 14.4% SCCmec type III, both types belonging to HA-MRSA. A total of 4% of the MRSA isolates were CA-MRSA belonging to SCCmec type IVd. Genotyping results showed three distinct spa clusters whilst HVR showed six distinct clusters. Molecular-based assays proved to be useful tools to determine the prevalence and monitoring of MRSA outbreaks as well as to identify the SCCmec types, subtypes and genotypes of MRSA strains that might be circulating in the hospital. The determination of the different antibiotypes of MRSA can assist in the monitoring of the antibiotic resistant profile trends in the Steve Biko Academic Hospital, thus assisting with the correct implementation of antibiotic regimens for suspected MRSA infections. In an endeavour to assess the dissemination of MRSA strains especially PVL expressing CA-MRSA strains, it is of paramount importance to continuously monitor the emergence of these strains in clinical settings. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Medical Microbiology / unrestricted

The Gram positive bacterium Staphylococcus aureus is a major cause of human disease in industrialized countries. This multifaceted pathogen is adapted to thrive in a variety of host niches, including the intracellular compartment. S. aureus rapidly develops antibiotic resistance, and infections due to resistant clones pose a global threat, calling for novel therapeutic approaches. The ability to exploit host nutrients and efficiently metabolize these resources for growth is paramount for bacterial pathogenesis. Understanding the nutritional and metabolic determinants that underpin bacterial virulence may lead to the identification of novel antimicrobial targets. This thesis investigates carbon nutrition and metabolism of community-acquired methicillin resistant S. aureus (CA-MRSA) USA300, a widely spread, hyper virulent multi-resistant strain. The dependence of S. aureus on carbohydrates for growth was considered first. In vitro studies in supplemented chemically defined media showed that sugar phosphates, such as hexose phosphates and glycerol phosphates, promote staphylococcal growth more efficiently than glucose. Deletion mutations were introduced to the two putative sugar phosphate transporter genes present in the S. aureus genome, uhpT (hexose phosphate permease) and glpT (glycerol phosphate permease). Phenotypic analysis of USA300 mutants and heterologous expression of the transporters in a previously described Listeria monocytogenes Δhpt mutant, totally unable to use sugar phosphates, confirmed that S. aureus UhpT and GlpT have different substrate specificities. Whilst both can transport glycerol monophosphate (excluding glycerol-2-phosphate) and the organophosphate antibiotic fosfomycin, hexose monophosphates are only imported via UhpT. Since sugar phosphates are only present in significant amounts inside living tissues, particularly the intracellular compartment, the role of S. aureus UhpT and GlpT in pathogenesis was investigated by constructing a double deletion mutant. The ΔuhpTΔglpT USA300 mutant was used to infect several relevant mammalian cell lines. In the conditions tested, it was found that UhpT and GlpT played no role in the intracellular replication of S. aureus. By contrast, Listeria exploits sugar phosphates from the host cell cytosol via the homologous hexose phosphate transporter, Hpt, to maximise replication and enhance virulence. The distinct requirement of sugar phosphates for intracellular proliferation may reflect intrinsic differences in carbon nutrient dependence between the two organisms. It was confirmed that S. aureus can efficiently use other readily available carbon sources for growth, such as amino acids. In contrast, Listeria is strictly dependent upon sugar-derived carbon for growth, due to an incomplete tricarboxylic acid cycle. Whilst the double ∆uhpT∆glpT mutation had no effect in S. aureus, expression of staphylococcal uhpT or glpT restored wild-type intracellular growth in the L. monocytogenes ∆hpt mutant. Taken together, the results illustrate that sugar phosphate permeases have a contextual role in bacterial virulence, where the background in which the genes are expressed determine their contribution as a virulence factor. The intracellular dynamics of S. aureus was also explored using immunofluorescence microscopy. It was observed that, during epithelial cell infection, USA300 remains enclosed in a membrane-bound vacuole. This localisation may form a barrier to cytosolic sugar phosphates and potentially explain the absence of effect of the sugar phosphate permease deletions in intracellular proliferation. Preliminary characterisation of the S. aureus containing vacuole (SACV) was performed and it was found to be positive for the Rab7 late-endosomal GTPase and for trans-Golgi markers. This suggests that SACVs converge at the Golgi apparatus. Interestingly, a USA300 mutant lacking the global regulatory system agr was unable to proliferate intracellularly and did not acquire Rab7 or Golgi markers. Since the Δagr mutation did not cause any impairment in carbon source dependent growth, these preliminary data suggest that modification of the SACV by Agr-regulated effectors may play a key role in modulating cellular processes that control staphylococcal intracellular survival and/or replication. Evidence presented in this thesis provides a platform for further exploration of S. aureus host cell nutrient dependence and the mechanisms that drive replication.

Staphylococcus aureus is a remarkably successful pathogen, accounting for an estimated 95,000 invasive infections annually in the U.S. alone. The burden of MRSA infections on public healthcare continues to rise, particularly with the continued spread of antibiotic resistant strains and the hyper-virulent CA-MRSA strains. The pathogenic nature of S. aureus can be attributed to the cache of virulence factors encoded within the genome of this organism. Typically, these are secreted toxins which directly interact with the host during infection, and facilitate pathogenesis. A previous screen in our laboratory investigating proteases in S. aureus identified a mutant in aminopeptidase Z as being attenuated in disease causation. Classically aminopeptidases function in the bioactivation/inactivation of proteins; and/or the utilization of imported peptides for cellular nutrition. We therefore hypothesize that cells deficient in one of these two processes would have decreased fitness levels, resulting in reduced virulence. We therefore sought to explore the role of PepZ in S. aureus; either in the processing of exogenous oligopeptides for nutrition, or in protein bioactivation/inactivation, and protein stability. We determined that S. aureus strains deficient in PepZ are less viable when cultured under conditions of starvation or while in competition for nutrients with the parent strain, and does not appear to be peptide driven. Using protein analysis approaches we have identified PepZ externalization, suggesting a potential for the aminopeptidase beyond the confines of the cell membrane. Additionally, we have also identified a potential role for PepZ in protein stability in this organism. Lastly, we present the essential role for PepZ in S. aureus virulence.

Methicillin-resistant Staphylococcus aureus (MRSA) is a growing concern in Canada especially in Aboriginal communities in remote regions. The northern and remote communities possess some or many of the risk factors that are identified in previous research to be associated with Community-Associated Methicillin-resistant Staphylococcus aureus (CA-MRSA) infections such as overcrowding, challenges in maintaining personal hygiene and limited access to healthcare. CA-MRSA spreads rapidly in the communities. It is known to not only affect young and healthy individuals, but it is also associated with high morbidity and mortality rates. Furthermore, antibiotic resistance in CA-MRSA is increasing in Canada. It is known that inappropriate and greater use of antibiotics is associated with increased antibiotic resistance. Resistant CA-MRSA infections are more difficult to treat. To reduce and to control the antibiotic resistance, monitoring the prevalence of CA-MRSA and the changing antibiotic susceptibility profiles at the population level, especially in highly affected communities, is crucial. By monitoring this trend, it will enable healthcare practitioners to provide more adequate and appropriate treatment. To our knowledge, there was no community-based study that examined the epidemiology of CA-MRSA skin and soft-tissue infections (SSTIs) in First Nations communities in Canada at large scale, and the knowledge on the risk factors, outcomes and antibiotic susceptibility profiles is still very limited. This study aimed to describe the local epidemiology of SSTIs at the community level in selected First Nations communities as well as to describe the antibiotic use to treat SSTIs and the antibiotic susceptibility patterns of CA-MRSA. A retrospective chart review was conducted in 12 nursing stations in the First Nations communities across 5 provinces in Canada. The charts of individuals over 18 years of age who had received service at the nursing station in the previous 12 months, starting retrospectively from the date of collection, were reviewed in this study. Each antibiotic prescription that was noted in the chart in this period prior to chart review was recorded in the antibiotic tracking case report form. Data collected included demographics, indication for antibiotic use, antibiotic prescription parameters and patient outcomes. In total, of 372 patient charts reviewed, 224 patient charts contained at least one case (an encounter that resulted in an antibiotic prescription during the study period). Of those 224 charts, 459 cases were recorded and, of those, 137 cases had a diagnosis of an SSTI. In the 65 patients accounting for all cases of SSTI patients, more than 80% of the study population were under the age of 50. The prevalence of impaired renal function was low, diabetes was present in 20% of cases, cardiac disease was present in 15% of cases, and reported alcohol misuse was present in 30% of cases. The presence of indwelling devices was very rare. There were 137 cases of SSTIs over 372 charts reviewed in total. The prevalence of SSTI among the selected First Nations communities in 2012-2013 was estimated at almost 37% (137 cases of SSTIs / 372 charts reviewed). In the 137 SSTI cases, 55 cases were identified as MRSA infections either by laboratory test such as wound culture or by history of colonization documented in the chart. The overall prevalence of MRSA in all SSTI cases was estimated at 40.1% (55 confirmed MRSA positive cases / 137 cases of SSTIs). The majority of SSTIs were purulent infections and wounds. We also found that a wound culture and susceptibility test were performed only in 29% of all SSTI cases. An orally administered antibiotic was most frequently used (in 71.5% of treatments). Topical antibiotics were used in 18.3% and IV antibiotics were used in 8.8%. Other than the antibiotic treatment, wound care was performed in 49% of the SSTI cases and incision and drainage (I&D) procedure in 9%. The majority of MRSA isolates in this study were susceptible to clindamycin and co-trimoxazole (90.5% and 95.2%), but only 29% were susceptible to erythromycin. In general, higher rates of SSTIs were seen in communities where overcrowding and poor access to running water are more prevalent. In this study, we found that the diagnostic tests such as wound culture and sensitivity test was not ordered very often and there was also lack of follow-up or lack of documentation of the follow-up. More research is needed to better understand some of the challenges and risk factors associated with CA-MRSA infections in remote communities. Developing a national-level surveillance system that can help with monitoring the epidemiology of SSTIs and the antibiotic susceptibility test results of CA-MRSA at community level would be essential for better prevention, control, and management. Furthermore, adopting other initiatives such as antibiotic stewardship programs at community and healthcare settings as well as addressing the socio-environmental factors such as housing and access to water would be all very important in the steps to curb antibiotic resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health problem nationwide, threatening to develop into an epidemic. Many of these patients are presenting to their primary care clinics with skin and soft tissue infections (SSTIs). The CDC has reported that in 2005, MRSA was responsible for an estimated 94,000 life-threatening infections and 16,650 deaths. The purpose of this study is to estimate the incidence of CA-MRSA within a specific family practice in Florida and to identify epidemiologic factors, classify antibiotic susceptibility patterns, and evaluate patient education in regard to disease management and prevention. This study was a descriptive, epidemiologic, three-year retrospective medical record review of all wound cultured skin and soft tissue infections that presented to a family practice between January 2007 and December 2009. Sixty-two medical records met the inclusion and exclusion criteria for the study. Of these 62 SSTIs, 44 cultures grew one or more bacterial organisms. The incidence of CA-MRSA was 66% (n=29). The mean age of those with CA-MRSA was 40 years old, with a range from 7 to 90 years old. Sixty-two percent (n=18) were male and 38% (n=11) were female; additionally 69% (n=20) lived within a 10 mile radius from the family practice, while 31% (n=9) lived in a surrounding suburb. The most frequent race was Caucasian 83% (n=24), with African American at 10% (n=3) and Hispanics 7% (n=2). Risk factors associated with CA-MRSA was obesity 41% (n=10), diabetes mellitus 24% (n=7), and a previous history of MRSA infection 24% (n=7). Skin and soft tissue infections were diagnosed as either an abscess 62% (n=18), boil 24% (n=7), pustule 10% (n=3), or cellulitis 4% (n=1). CA-MRSA isolates were susceptible to trimethoprim-sulfamethoxazole 100% (n=29), doxycycline 93% (n=27), and rifampin 100% (n=14). Clindamycin susceptibility was 65% (n=15) with resistance at 30% (n=7) and 5% (n=1) intermediate. Both cephalexin and erythromycin were 100% resistant. Documentation in the medical record on wound care was found in 45% (n=13) of the records. The incidence of CA-MRSA SSTI was 66%, which identifies this suburban community at high risk for this bacterial infection. Risk factors associated with CA-MRSA included obesity (BMI > 30), history of previous MRSA infection, and diabetes mellitus. There were no clinical characteristics that helped distinguish MRSA infection from other bacterial SSTIs. Most SSTI were treated with incision and drainage and a susceptible antibiotic. Judicious use of antibiotics not only provides appropriate treatment, but is also critical in prevention of antibiotic resistance. Lastly, patient education in adequate hygiene is essential in preventing the spread of CA-MRSA

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.

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.

Staphylococcus aureus é um patógeno humano comum, causador principalmente de infecções de pele na comunidade e infecções em diversos sítios em ambiente hospitalar. Desde a última década, tornou-se motivo de preocupação devido ao aumento na incidência de infecções por cepas resistentes a meticilina (methicillinresistant S. aureus [MRSA]) na comunidade sem os clássicos fatores de risco associados. O cassete cromossômico SCCmec (staphylococcal cassette chromosome) tipo IV, carreador do gene mecA (responsável pela resistência à meticilina/oxacilina), está associado predominantemente ao MRSA comunitário e ao denominado clone pediátrico do MRSA, causador de infecções hospitalares. Isolados de MRSA comunitário normalmente produzem uma toxina chamada Panton-Valentine leukocidin (PVL), associada à destruição dos leucócitos e à necrose tecidual. MRSA carreadores de SCCmecIV do clone Oceania Southwest Pacific (OSPC), foram relatados no Brasil causando principalmente infecções de pele e tecidos moles em pacientes de Porto Alegre. Entretanto, pouco se conhece sobre as características clínicas dos casos ocorridos no Brasil. Este estudo objetivou descrever a epidemiologia clínica e molecular associada ao MRSA carreador de SCCmecIV em pacientes atendidos em hospital universitário de Porto Alegre, Rio Grande do Sul. De julho de 2006 a junho de 2008 foram selecionados isolados de MRSA isolados de pacientes provenientes do Hospital São Lucas da PUC-RS, que apresentaram resistência à oxacilina e a não mais que três antibióticos não -ß lactâmicos. Foi realizada análise molecular por Reação em Cadeia Polimerase (Polimerase Chain Reaction [PCR]) para detecção dos genes mecA e lukF-pv (que codifica a toxina PVL), teste de restrição modificado (RM) para identificar o complexo clonal a que as amostras pertencem e eletroforese em campo pulsado (Pulse Field Gel Electrophopresis -PFGE) para a identificação dos clones de MRSA isolados. As características clínicas foram revisadas nos prontuários médicos dos pacientes. Vinte um isolados de 13 pacientes preencheram critérios de inclusão. Somente o primeiro foi considerado para análise molecular. Os 13 isolados foram carreadores de SCCmecIV e pertenciam a duas linhagens diferentes: complexo clonal (CC) 30 (relacionado ao clone OSPC, 11 isolados) e CC5 (relacionado ao clone pediátrico, 2 isolados). Seis isolados apresentaram PFGE padrão A1; outros 5 isolados foram relacionados ao clone A1 (A2 ao A4). Outros dois isolados apresentaram PFGE padrão B (os dois do CC5). Todas os isolados CC30 eram produtoras de PVL. Cinco pacientes apresentaram infecção associada a cuidados de saúde (IACS), com início hospitalar; cinco apresentaram IACS com início comunitário; e apenas três associadas à comunidade (CA) sem fatores de risco para MRSA. Este estudo apresentou o perfil genotípico e fenotípico de isolados de MRSA carreadores de SCCmecIV presentes em Porto Alegre e demonstrou que isolados do clone OSPC não causam apenas infecções comunitárias no Brasil, mas também podem causar IACS. / We evaluated clinical outcomes and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmecIV recovered from patients attended at a teaching hospital from Porto Alegre, Brazil. All PVL-producer isolates belonged to clonal complex (CC) 30 (11 isolates, related to Oceania Southwest Pacific clone - OSPC) and the PVL-negative isolates were typed as CC5 (2 isolates, related to the pediatric clone). Five patients had health-care associated infections (HCAI) with hospital onset, five HCAI with community-onset and three community-acquired infections without risks. A high overall mortality (30.8%) was found. This study show that OSPC isolates are not only causing community-associated infections but are also involved in HCAI in our country.

Staphylococcus aureus é um patógeno humano comum, causador principalmente de infecções de pele na comunidade e infecções em diversos sítios em ambiente hospitalar. Desde a última década, tornou-se motivo de preocupação devido ao aumento na incidência de infecções por cepas resistentes a meticilina (methicillinresistant S. aureus [MRSA]) na comunidade sem os clássicos fatores de risco associados. O cassete cromossômico SCCmec (staphylococcal cassette chromosome) tipo IV, carreador do gene mecA (responsável pela resistência à meticilina/oxacilina), está associado predominantemente ao MRSA comunitário e ao denominado clone pediátrico do MRSA, causador de infecções hospitalares. Isolados de MRSA comunitário normalmente produzem uma toxina chamada Panton-Valentine leukocidin (PVL), associada à destruição dos leucócitos e à necrose tecidual. MRSA carreadores de SCCmecIV do clone Oceania Southwest Pacific (OSPC), foram relatados no Brasil causando principalmente infecções de pele e tecidos moles em pacientes de Porto Alegre. Entretanto, pouco se conhece sobre as características clínicas dos casos ocorridos no Brasil. Este estudo objetivou descrever a epidemiologia clínica e molecular associada ao MRSA carreador de SCCmecIV em pacientes atendidos em hospital universitário de Porto Alegre, Rio Grande do Sul. De julho de 2006 a junho de 2008 foram selecionados isolados de MRSA isolados de pacientes provenientes do Hospital São Lucas da PUC-RS, que apresentaram resistência à oxacilina e a não mais que três antibióticos não -ß lactâmicos. Foi realizada análise molecular por Reação em Cadeia Polimerase (Polimerase Chain Reaction [PCR]) para detecção dos genes mecA e lukF-pv (que codifica a toxina PVL), teste de restrição modificado (RM) para identificar o complexo clonal a que as amostras pertencem e eletroforese em campo pulsado (Pulse Field Gel Electrophopresis -PFGE) para a identificação dos clones de MRSA isolados. As características clínicas foram revisadas nos prontuários médicos dos pacientes. Vinte um isolados de 13 pacientes preencheram critérios de inclusão. Somente o primeiro foi considerado para análise molecular. Os 13 isolados foram carreadores de SCCmecIV e pertenciam a duas linhagens diferentes: complexo clonal (CC) 30 (relacionado ao clone OSPC, 11 isolados) e CC5 (relacionado ao clone pediátrico, 2 isolados). Seis isolados apresentaram PFGE padrão A1; outros 5 isolados foram relacionados ao clone A1 (A2 ao A4). Outros dois isolados apresentaram PFGE padrão B (os dois do CC5). Todas os isolados CC30 eram produtoras de PVL. Cinco pacientes apresentaram infecção associada a cuidados de saúde (IACS), com início hospitalar; cinco apresentaram IACS com início comunitário; e apenas três associadas à comunidade (CA) sem fatores de risco para MRSA. Este estudo apresentou o perfil genotípico e fenotípico de isolados de MRSA carreadores de SCCmecIV presentes em Porto Alegre e demonstrou que isolados do clone OSPC não causam apenas infecções comunitárias no Brasil, mas também podem causar IACS. / We evaluated clinical outcomes and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmecIV recovered from patients attended at a teaching hospital from Porto Alegre, Brazil. All PVL-producer isolates belonged to clonal complex (CC) 30 (11 isolates, related to Oceania Southwest Pacific clone - OSPC) and the PVL-negative isolates were typed as CC5 (2 isolates, related to the pediatric clone). Five patients had health-care associated infections (HCAI) with hospital onset, five HCAI with community-onset and three community-acquired infections without risks. A high overall mortality (30.8%) was found. This study show that OSPC isolates are not only causing community-associated infections but are also involved in HCAI in our country.