Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O Staphylococcus aureus resistente a meticilina (MRSA) foi inicialmente descrito como um patógeno associado a infecções relacionadas à assistência em saúde; porém, um clone de MRSA, o CA-MRSA emergiu na comunidade e está atualmente incrementando nos hospitais. O objetivo desta tese foi descrever aspectos relacionados com a epidemiologia das infecções por cepas CA-MRSA no Hospital Universitário Pedro Ernesto da Universidade do Estado do Rio de Janeiro (HUPE/UERJ), avaliando especificamente fatores de risco relacionado com as infecções por CA-MRSA. Usando informações das bases de dados do laboratório de microbiologia, da farmácia e da Comissão para Controle da Infecção Hospitalar do HUPE/UERJ foi realizado um estudo retrospectivo de infecções/colonizações por cepas de S. aureus (fevereiro 2005 a Julho 2011). Foi realizado um estudo caso e controle, utilizando como casos os pacientes com infecções por cepas CA-MRSA. Na avaliação da susceptibilidade aos antimicrobianos usados em infecções graves por MRSA (vancomicina, teicoplanina, daptomicina e linezolida), foram determinadas as concentrações inibitórias mínimas (CIM) das amostras por diferentes metodologias (testes de difusão em agar, microdiluição em caldo e E-test). Nas analises das tendências temporais da apresentação dos subtipos de MRSA, usando um critério fenotípico para classificação das cepas MRSA, foi observada uma diminuição do número de cepas de MRSA multirresistente (HA-MRSA) (p<0.05). Também foi observada uma tendência ao aumento de cepas não-multirresistentes (CA-MRSA), mas sem alcançar a significância estatística (p = 0.06) igual que os S. aureus sensíveis a meticilina (MSSA) (p = 0.48). Não houve associação entre o subtipo de MRSA e a mortalidade devida à infecção por cepas MRSA. Uma idade acima de 70 anos (OR: 2.46, IC95%: 0.99 - 6.11), a presença de pneumonia adquirida no hospital (OR: 4.94, IC95%: 1.65 -14.8), a doença pulmonar obstrutiva crônica (OR: 6.09, IC95% 1.16 31.98) e a leucemia (OR: 8.2, IC95%: 1.25 54.7) foram fatores de risco associadas à mortalidade nas infecções por cepas de S. aureus. Usando curvas de Kaplan-Meier, foi observada uma tendência ao aumento da mortalidade em infecções causadas por MSSA na primeira semana, porém sem alcançar significância estatística (p = 0.07). Não foram observadas amostras MRSA com susceptibilidade intermediaria a vancomicina, linezolida, daptomicina ou teicoplanina. A dinâmica das infecções por S. aureus no HUPE/UERJ mudou durante o período de estudo, com menor número de episódios infecciosos causados por cepas de MRSA multirresistentes. Existe uma tendência ao aumento das cepas não-multirresistentes de MRSA entanto que a taxa de infecções por MSSA permaneceu estável no período do estudo. O perfil de resistência dos estafilococos não teve associação com a mortalidade / The methicillin-resistant Staphylococcus aureus (MRSA) was described initially like a health-care associated pathogen. However, an MRSA clone called community-adquired S. aureus emerged with success in the community and now has a worring increasing frequency in hospital settings. The aim of this study was to descript issues related to the epidemiology of infections due tu CA-MRSA isolates at the Pedro Ernesto Universitary Hospital (HUPE/UERJ) in Rio de Janeiro, Brazil from february 2005 to june 2011, analyzing risk factors related to these infections. Thus, using databases of the microbiology laboratory, pharmacia department and the infection control committee of the HUPE-UERJ, was realized an restrospective study of S. aureus isolates obtained from infected/colonizated patients hospitalized from February 2005 to July 2011. To evaluate risk factors related to CA-MRSA infections was conduced a case-control study, using patients with true infections due to MRSA like cases and patients with methicillin susceptible S. aureus (MSSA) like controls. To test the antimicrobial susceptibility of the antibiotics used in MRSA severe infections (Vancomycin, teicoplanin, daptomycin and linezolid), were determinated the minimal inhibitory concentration (MIC) of MRSA isolates using differents methods (disk-difusion test, microdilution in broth and E-test strips). The trend analyses of the MRSA types, using a phenotypic criteria to classificate the MRSA isolates, found a decrease in the infections due to multi-resistant MRSA isolates (HA-MRSA) in our hospital (p<0.05). Also was observed and increase in non-multi-resistant MRSA strains (CA-MRSA), but without reach statistic significancy (p = 0.06), similar to MSSA (p = 0.48). There is not association between the MRSA phenotype and the mortality due to S. aureus infection. In the multivariate analysis, were observed that an older age than 70 years (OR: 2.46, IC95%: 0.99 - 6.11), health-care pneumonia (OR: 4.94, IC95%: 1.65 -14.8), chronic obstructive pulmonary disease (OR: 6.09, IC95% 1.16 31.98) and leucaemia (OR: 8.2, IC95%: 1.25 54.7) were risk factors associated with mortality due to S. aureus infections. The Kaplan-Meier analysis, found a trend to high mortality due to MSSA infections in the first week, but without get statistic significancy (p = 0.07). We dont found any MRSA isolated with resistance or intermediary resistance to vancomycin, linezolid, daptomycin or teicoplanin. There is good correlation between both MICs determinations, with broth microdiluiton and E-Test strips metodhology. Its were concluded that the dynamic of the S. aureus infections at the HUPE/UERJ is changing, with less number of infectious episodes due to multi-resistant MRSA isolates. Moreover, there are an increasing number of infections due to non-multi-resistant MRSA isolate. The prevalence of infections due to MSSA dont have change in the time of period study. The kind of the S. aureus phenotype dont has association with all-causes-mortality

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.

Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant bacterium of epidemiologic importance in Canadian healthcare facilities. The contact between MRSA colonized or infected patients with other patients, healthcare workers (HCWs) and/or the healthcare environment can result in MRSA transmission and healthcare-associated MRSA (HA-MRSA) infections in hospitals. These HA-MRSA infections are linked with increased length of hospital stay, economic burden, morbidity and mortality. Although infection prevention and control programs initiated in 2009 in Canada and other developed countries (e.g., UK, France, Belgium, Denmark, etc.) have been relatively successful in reducing the rate of HA-MRSA infections, they continue to pose a threat to patients, especially to the more vulnerable in long term care and geriatric institutions. Historically, MRSA was a problem mainly in hospital settings but after mid-1990s new strains of MRSA have been identified among people without healthcare-related risks and have been classified as community-associated MRSA (CA-MRSA). Furthermore, the distinction between HA-MRSA and CA-MRSA strains is gradually waning due to both the introduction of HA-MRSA in communities, and the emergence of CA-MRSA strains in hospitals. The purpose of this thesis was to explore the feasibility of constructing healthcare networks to evaluate the role of healthcare providers (e.g., physicians) and places (e.g., patient rooms) in the transmission of MRSA in a large tertiary care facility. Method of investigation: a secondary data case-control study, using individual characteristics and network structure measures, conducted at The Ottawa Hospital (TOH) between April 1st, 2013 and March 31th, 2014. Results: It was feasible to build social networks in a large tertiary care facility based on electronic medical records data. The networks' size (represented by the number of vertices and lines) increased during the outbreak period (period 1) compared to the pre-outbreak period (period 0) for both groups and at all three TOH campuses. The calculated median degree centrality showed significant increase in value for both study groups during period 1 compared to period 0 for two of the TOH campuses (Civic and General). There was no significant difference between the median degree centrality calculated for each study group at the Heart Institute when compared for the two reference periods. The median degree centrality of the MRSA case group for period 0 showed no significant difference when compared to the same measure determined for the control group for all three TOH campuses. However, the median degree centrality calculated for period 1 was significantly increased for the control group compared to the MRSA case group for two TOH campuses (Civic and General) but showed no significant difference between the two groups from the Heart Institute. In addition, there was a correlation between the two network measures (degree centrality and eigenvector centrality) calculated to determine the most influential person or place in the MRSA case group networks. However, there was no correlation between the two network’s measures calculated for physicians included in MRSA case group networks. Conclusions: It is feasible to use social network analysis as an epidemiologic analysis tool to characterize the MRSA transmission in a hospital setting. The network's visible changes between the groups and reference periods were reflected by the network measures and supported also by known hospital patient movements after the outbreak onset. Furthermore, we were able to identify potential source cases and places just prior of the outbreak start. Unfortunately, we were not able to show the role of healthcare workers in MRSA transmission in a hospital setting due to limitations in data collection and network measure chosen (eigenvector centrality). Further research is required to confirm these study findings.

The literature directly comparing the epidemiology of MRSA among adult and pediatric hospitalized patients is strikingly minimal. The objective of this thesis was to identify any differences between these two patient groups. The Canadian Nosocomial Infections Surveillance Program MRSA data (1995 to 2007: n=1,262 pediatric and 35,907 adult cases) were used to compare MRSA clinical and molecular characteristics and rates. Hospital characteristics were modeled using repeated measures Poisson regressions. The molecular and epidemiological characteristics of MRSA differed significantly between adults and children. Compared to children, MRSA in adults was more likely to be healthcare-associated, colonization, SCCmec type II, PVL negative, and resistant to most antibiotics. Rates of MRSA in Canada increased in both populations over time but were significantly higher in adults. The hospital characteristics associated with increased MRSA rates differed in adult and pediatric facilities. Implications for infection prevention and control strategies are discussed.

The literature directly comparing the epidemiology of MRSA among adult and pediatric hospitalized patients is strikingly minimal. The objective of this thesis was to identify any differences between these two patient groups. The Canadian Nosocomial Infections Surveillance Program MRSA data (1995 to 2007: n=1,262 pediatric and 35,907 adult cases) were used to compare MRSA clinical and molecular characteristics and rates. Hospital characteristics were modeled using repeated measures Poisson regressions. The molecular and epidemiological characteristics of MRSA differed significantly between adults and children. Compared to children, MRSA in adults was more likely to be healthcare-associated, colonization, SCCmec type II, PVL negative, and resistant to most antibiotics. Rates of MRSA in Canada increased in both populations over time but were significantly higher in adults. The hospital characteristics associated with increased MRSA rates differed in adult and pediatric facilities. Implications for infection prevention and control strategies are discussed.