Staphylococcus epidermidis has emerged in recent years as an important nosocomial pathogen, especially in infections associated with implanted foreign body materials (e.g., prosthetic joints and heart valves) and in individuals with a compromised immune system (e.g., cancer patients and neonates). Although rare, implant infections are long lasting and cause severe suffering for the patient that includes pain and disability and even increased mortality. One aim of the present thesis was to develop and evaluate a genetic method for species identification and simultaneous detection of rifampicin resistance in staphylococci. A second aim was to examine S. epidermidis isolated from prosthetic joint infections (PJIs) and from wrists and nares of healthy individuals regarding their antibiotic susceptibility, biofilm production, virulence factors, and epidemiology. Comparison with phenotypic diagnostics revealed that 8 (16%) of 49 isolates differed in their species identification in favour of the genetic method. In addition, mutations associated with rifampicin resistance, including two not previously reported, were possible to detect in all isolates resistant to rifampicin. Antibiotic susceptibility testing of 61 PJI isolates showed multi-drug resistance in 91%. Furthermore, the results of the synergy testing revealed that no antibiotic combination was significantly better than the others. Hence, the effects that were possible to detect were isolate dependent. To find a method for discriminating between invasive (n=61) and commensal (n=24) isolates of S. epidermidis genotypic and phenotypic characterisations of biofilm production (including the ica and aap genes), antibiotic susceptibility, virulence-related genes (such as agr and ACME) and epidemiology were performed (using multilocus sequence typing [MLST], typing of the staphylococcal chromosome cassette mec [SCCmec] and PhenePlate). Significant differences were found in antibiotic susceptibility, i.e. there was more resistance among invasive isolates. MLST sequence types (ST) ST2 and ST215 dominated the invasive isolates.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Bacterial contamination of platelet concentrates (PCs) represents the highest post-transfusion infectious risk. The skin flora bacterium Staphylococcus epidermidis has been reported to be the predominant aerobic contaminant of PCs. The Ramirez' group has shown that S. epidermidis can form surface-attached bacterial aggregates known as biofilms, and can outcompete other coagulase-negative staphylococci, such as Staphylococcus capitis, in PCs. The ability of S. epidermidis to form biofilms has been linked to increased pathogenicity and missed detection during PC screening with an automated culture system (BacT/ALERT). This thesis aimed at investigating the proliferative advantage and resistance mechanisms displayed by S. epidermidis in the PC milieu. Furthermore, in an effort to enhance PC safety for transfusion patients, I studied the anti-biofilm properties of essential oils and antimicrobial peptides (AMPs). My studies aimed at improving PC safety by focussing on both the point of introduction of bacterial contaminants (blood collection), and the stage at which bacterial contaminants can form biofilms and proliferate (PC storage). S. epidermidis can be found in the skin of blood donors as biofilms, which are resistant to the blood donor skin disinfectant currently used by Canadian Blood Services, chlorhexidine-gluconate and isopropyl alcohol (CHG-IPA). Here, several plant-extracted essential oils were evaluated for their ability to enhance the anti-biofilm activity of CHG-IPA. Data revealed that the Lavandula multifida oil and its main component (linalool) greatly enhanced the activity of CHG-IPA against S. epidermidis biofilms. Furthermore, the ability of a combination of three synthetic AMPs to inhibit S. epidermidis biofilm formation during PC storage was assessed These results showed that the combination of AMPs could inhibit biofilm formation but was ineffective against pre-formed S. epidermidis biofilms. The accumulation associated protein (Aap) encoded by the aap gene, found in most S. epidermidis strains and absent in S. capitis, plays a role in biofilm formation. When S. epidermidis aap is transformed into S. capitis, this bacterium displayed increased biofilm formation and proliferated to higher concentrations compared to untransformed S. capitis and to a S. epidermidis aap deletion mutant. Based on these results, aap appears to play a role in providing S. epidermidis a proliferative advantage in PCs by enhancing biofilm formation. Lastly, the GraRS system and SepA were studied for their role in S. epidermidis resistance to platelet-derived AMPs using the synthetic AMP PD4 as a model molecule. Results indicate that the GraS mechanism is involved in resistance towards PD4. The work presented in my thesis provides further insights into why S. epidermidis has a proliferative advantage in the PC storage environment and allows for the proposal of alternative methods to enhance PC safety for transfusion patients.

Made available in DSpace on 2014-07-28T18:09:54Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 103.pdf: 652449 bytes, checksum: ea164ab2e7463c3fab8f472fadef32d6 (MD5) Previous issue date: 2008 / Made available in DSpace on 2014-12-22T16:56:35Z (GMT). No. of bitstreams: 2 103.pdf: 652449 bytes, checksum: ea164ab2e7463c3fab8f472fadef32d6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Staphylococus aureus e Staphylococcus Coagulase negative(CNS) são reconhecidos como causadores de infecções hospitalares e comunitárias em todas as regiões do mundo. Nós estudamos a produção de biofilme em 43 cepas de Staphylococus aureus meticilina resistente (MRSA) e em 21 isolados de Staphylococus epidermidis meticilina resistente (MRSE) e a susceptibilidade a antibióticos destas cepas que foram obtidas de pacientes infectados do Hospital Universitário Antônio Pedro (HUAP), localizado na cidade de Niterói no estado do Rio de Janeiro. Foi realizado também a Eletroforese em campo pulsado (PFGE) de fragmentos de macrorestrição de SmaI do DNA genômico, bem como a tipificação do cassete cromossômico estafilocócico mec (SCCmec).Os isolados que carreaiam mecA foram usados para estudar a distribuição do tipo clonal entre 43 cepas de MRSA colhidas no HUAP de 2003 a 2006. Nossos resultados demonstraram que a maioria das cepas de MRSA (83.7 por cento) e MRSE (52,4 por cento) produziram biofilme,e altas taxas de resistência de MRSA e MRSE à eritromicina, à clindamicina e ao ciprofloxacino foram observadas. Dois tipos clonais predominantes (A e B) foram identificados por PFGE, tendo ambos compreendendo a maioria das cepas (32,6 por cento cada um). De acordo com a tipificação de SCCmec, o SCCmec tipo IV foi o que prevaleceu (51,2 por cento). Porém, o mais interessante em nosso estudo foi a mudança observada no background genético dos MRSA isolados do HUAP, pois o Clone Epidêmico Brasileiro(CEB) que era o clone predominante neste hospital, está sendo substituído pelo USA-400, um clone que a princípio foi denominado de clone comunitário Staphylococcus aureus meticilina resistente (CA-MRSA). / Staphylococus aureus and coagulase-negative staphylococci (CNS) are recognized as causing nosocomial and community-acquired infection in every region of the world. We study biofilm production in 43 isolates of methicillin-resistant Staphylococcus aureus (MRSA) and 21 isolates methicillin-resistant Staphylococcus epidermidis (MRSE) and the antibiotics susceptibilities of these strains that were obtained from infected patients at Antônio Pedro University Hospital (HUAP) located in Niterói city, Rio de Janeiro. In addition, Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA as well as staphylococcal cassette chromosome mec (SCCmec) typing for mecA-carrying isolates were used to study the distribution of clonal types among 43 MRSA recovered in HUAP between 2003 and 2006. Our results demonstrated that the majority strains of MRSA (83.7%) and MRSE (52,4%) produced biofilm and high resistance of MRSA and MRSE to erytromicin, clindamicin and ciprofloxacin were observed. The two major clones types (A and B) were identified by PFGE, with both them comprising the majority of strains (32,6% each). According to SCCmec typing, SCCmec type IV were the most prevalent type, showing 51,2% . But the most interesting in our study is a changed observed in the genetic background of MRSA isolates from HUAP, because of Brazilian clone, which was the major clone in this hospital, was replaced by the USA-400, one type of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA).

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

Made available in DSpace on 2016-06-02T19:02:42Z (GMT). No. of bitstreams: 1 5562.pdf: 2010923 bytes, checksum: 99b0ace4e740c99eb2fc0708b19a70fd (MD5) Previous issue date: 2013-05-10 / Financiadora de Estudos e Projetos / The staphylococci belong a diverse group of bacteria that cause diseases ranging from minor skin infections to death-threatening bacteraemia. The two major opportunistic pathogens of this genus, Staphylococcus epidermidis and S. aureus are the most frequent causes of nosocomial infections and infections associated with the use of medical devices. Staphylococci, is the leading cause of infections associated with biofilm formation. Biofilmrelated infections are challenging to treat with conventional antimicrobial agents, limiting the efficacy of antibiotic therapy and becoming a crucial problem for treatment of chronic infections. Therefore, the main aim of this thesis was to study the susceptibility of staphylococcal biofilm cells against antimicrobial agents. For that, it was evaluated in vitro activities of N-acetylcysteine (NAC), rifampicin, linezolid, daptomycin and vancomycin alone and in combination on biofilm cells of Staphylococcus epidermidis and S. aureus. The activities of antimicrobial agents were evaluated in concentrations CIM, 10xCIM and peak serum. The biofilms susceptibility to agents studied was assessed through, colony-forming units (CFU/ml), staining with crystal violet (CV) that is the measure total biofilm biomass and cellular activity using XTT reduction assay. The results of viable cells (expressed as log10 CFU/ml) to N-acetylcysteine alone, in the concentration 10xCIM on both the biofilms of staphylococcal evaluated showed a greater effect compared with other antimicrobial agents evaluated, with reductions of approximately 4-5 log10. The combination NAC (10xCIM) - vancomycin (independent of concentration evaluated) showed a greater reduction (p<0.05) on viable cells of S. epidermidis and S. aureus, compared with other combinations evaluated. This combination presented a reduction about of 5-6 log10 CFU/ml. The results of CV showed loss the total biofilm biomass and were observed decrease in the metabolic activity measured by the XTT, these results are in very good agreement with those obtained in terms of cell viability. In conclusion, the results obtained in the studies of this thesis constitutes a promising therapeutic strategy in the treatment of infections associated with biofilm formation by S. epidermidis and S. aureus. In addition, the use of antimicrobial agents in combinations may be an alternative for monotherapy, thus avoiding the development of resistance. / Os estafilococos pertencem a um grupo diversificado de bactérias que causam doenças que vão desde infecções de pele, a risco de morte como bacteriemia. Os dois principais patógenos oportunistas deste gênero, Staphylococcus epidermidis e S. aureus são as causas mais frequentes de infecções nosocomiais e infecções associadas ao uso de dispositivos médicos. Estafilococos é a principal causa de infecções associadas com a formação de biofilme. As infecções relacionadas à formação de biofilme são difíceis de tratar com agentes antimicrobianos convencionais, limitando a eficácia da terapia com antibióticos e tornando um problema crucial para o tratamento de infecções crônicas. Portanto, o objetivo principal desta tese foi estudar a susceptibilidade das células de estafilococos em biofilme contra agentes antimicrobianos. Para isso, foram avaliadas as atividades in vitro de N-acetilcisteína (NAC), rifampicina, linezolida, daptomicina e vancomicina isoladamente e em combinação contra as células em biofilme de Staphylococcus epidermidis e S. aureus. As atividades dos agentes antimicrobianos foram avaliadas nas concentrações CIM, 10xCIM e a concentração máxima no soro. A susceptibilidade dos biofilmes para os agentes estudados foi avaliada através de unidades formadoras de colônia (UFC/ml), a coloração com cristal violeta (CV), que é a medida da biomassa total do biofilme e, a atividade celular usando ensaio de redução de XTT. Os resultados das células viáveis (expressos em log10 UFC/ml) para N-acetilcisteína sozinho, na concentração 10xCIM para ambos os biofilmes de estafilococos avaliados mostraram um maior efeito em comparação com outros agentes antimicrobianos avaliados, com redução de cerca de 4-5 log10. A combinação de NAC (10xCIM) - vancomicina (independente da concentração avaliada) mostrou uma maior redução (p<0,05) em células viáveis de S. epidermidis e S. aureus, em comparação com as outras combinações avaliadas. Esta combinação apresentou uma redução de cerca de 5-6 log10 CFU/ml. Os resultados de CV mostraram perda da biomassa total do biofilme e, foram observados diminuição da atividade metabólica, medida pelo ensaio de XTT, este resultados estão em boa concordância com os resultados obtidos em termos de viabilidade celular. Em conclusão, os resultados obtidos nos estudos desta tese constituem uma estratégia terapêutica promissora para o tratamento de infecções associadas a formação de biofilme por S. epidermidis e S. aureus. Além disso, a utilização de agentes antimicrobianos em combinação pode ser uma alternativa para a monoterapia, evitando assim o desenvolvimento de resistência.

O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

Orientador: Maria Luiza Moretti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T09:55:21Z (GMT). No. of bitstreams: 1 Bratfich_OrlandoJose_D.pdf: 1128343 bytes, checksum: 5a4d981849f245636703326e9af0c8ad (MD5) Previous issue date: 2005 / Resumo: Isolados clínicos de Staphylococcus epidermidis provenientes de infecções de corrente sangüínea do hospital de clínicas HC ¿ UNICAMP, Campinas foram analisados. Os isolados foram caracterizados por análises fenotípicas e genotípicas. Os testes de susceptibilidade aos antimicrobianos foram realizados para comparar os métodos de disco difusão, concentração inibitória mínima e diluição em Agar, para determinar a relevância clínica da resistência aos antimicrobianos. Foram analisados a presença do gene mecA por PCR, produção de biofilme, genotipagem por PFGE com enzima SmaI. Dados epidemiológicos dos pacientes foram analisados para detectar a importância clínica dessas cepas. Para oxacilina 82 isolados foram resistentes, sendo que 78 (95,12%) apresentaram reação de PCR positiva para o gene mecA, dois isolados apresentaram-se como hiperprodutores de ß-lactamases. Para teicoplanina os testes de CIM apresentaram 52 isolados sensíveis, 35 isolados intermediários e 13 isolados resistentes e, quando comparado com os testes de disco difusão foram observadas discordâncias em 43% dos isolados, onde o teste de disco difusão não foi capaz de identificar isolados intermediários e resistentes (P < 0,001). Para vancomicina um único isolado apresentou resultado intermediário com CIM de 8,0 µg/mL. Isolados vancomicina resistentes não foram observados. Analises dos dados epidemiológicos, através de regressão logística, as variáveis estatisticamente significantes foram neutropenia (P = 0,002) e quimioterapia (P = 0,015). As análises genotípicas por PFGE demonstraram a disseminação clonal de S. epidermidis no ambiente hospitalar e verificou-se que testes dilucionais são necessários quando se estudam glicopeptideos. A análise filogenética demonstrou grande estabilidade e adaptabilidade genética do gene mecA responsável pela resistência a oxacilina / Abstract: Clinical strains of Staphylococcus epidermidis taken from bloodstream infection from UNICAMP (teaching hospital in Campinas São Paulo State, Brazil) were analyzed. The strains were characterized by phenotypic and genotypic analysis. The antimicrobial susceptibility tests were made to compare the disk diffusion tests, MIC, and agar-salt screening-test to determine the relevant clinical antimicrobial resistance. There were also made PCR assays for mecA detection, biofilm production, genotypic by SmaI-digested and pulsed-field gel electrophoresis. Patients epidemiological data were employed to determine the major clinical importance of these strains. For oxacillin 82 strains were resistant, and 78 (95.12%) had positive PCR assay for mecA gene, and two strains presented characteristics of ß-lactamases hyperproduction. In the MIC tests for teicoplanin, 52 strains were susceptible; 35 strains intermediate and 13 strains were resistant, and when compared to the results obtained by disk diffusion tests, MIC, were observed discordant results in 43% of the strains where the disk diffusion test was not able to identify strains intermediate and resistant (P < 0.001). MIC test had only one intermediate of 8.0 µg/mL for vancomycin. Strains vancomycin-resistant were not observed. The variable data statistically significant in the logistic regression analysis related to the treatment of the BSI by S. epidermidis were: neutropenia (P = 0.002) and chemotherapy (P = 0.015). The genotyping analysis of isolates demonstrated a clonal distribution of S. epidermidis in the hospital environment and it was checked that dilution tests are necessary when glycopeptides are being studied.. The phylogenetic analysis showed great genetic stability and adaptability of the mecA gene responsible for the oxacillin resistance / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-03T17:25:23Z No. of bitstreams: 1 Oliveira, Hanna Lara da Cruz Dinéas de [Dissertação, 2013].pdf: 1766254 bytes, checksum: 30cf8e6b52cfd32b623ecf94d3cee01b (MD5) / Made available in DSpace on 2017-04-03T17:25:23Z (GMT). No. of bitstreams: 1 Oliveira, Hanna Lara da Cruz Dinéas de [Dissertação, 2013].pdf: 1766254 bytes, checksum: 30cf8e6b52cfd32b623ecf94d3cee01b (MD5) / Os estafilococos coagulase negativos emergem como importantes patógenos nosocomiais, frequentemente isolados em bacteremias humanas, fato intimamente relacionado com o aumento do uso de dispositivos médicos ao longo dos últimos anos. Sua capacidade de aderir a superfícies poliméricas e produzir biofilmes constitui o principal fator de virulência associado a estes dispositivos. A fim de minimizar esta possibilidade, antissépticos são largamente utilizados para desinfecção de pele e mucosas na rotina hospitalar, no entanto, tem-se observado uma diminuição da susceptibilidade bacteriana a estes agentes. A diminuição da susceptibilidade dos microrganismos a antissépticos, associados a sua capacidade de formar biofilmes tem elevado a morbidade, mortalidade, período de internação e os custos relativos ao cuidado com a saúde. No estudo aqui apresentado, foram analisadas 81 cepas de Staphylococcus epidermidis resistentes a meticilina (MRSE) e 55 cepas de Staphylococcus haemolyticus resistentes a meticilina (MRSHa), todas resistentes a meticilina, isoladas de pacientes internados em hospital Universitário localizado na cidade de Niterói-RJ nos anos de 2008 e 2010. A determinação do perfil de susceptibilidade aos biocidas triclosan, clorexidina e cloreto de benzalcônio mostrou que frente ao triclosan, as amostras apresentaram as menores taxas de susceptibilidade bacteriana. Papaína não apresentou atividade antibacteriana. Entretanto, foi capaz de reduzir expressivamente a formação de biofilme (p<0,06) em ambas as espécies, mostrando-se a mais eficiente entre os produtos analisados. Foi capaz também de desintegrar biofilmes maduros formados por Staphylococcus epidermidis. Os biocidas não levaram à redução significante de biofilmes, exceto a clorexidina, que foi capaz de reduzir a formação do biofilme pelas MRSE. Foi possível verificar que cepas de MRSHa tratadas com cloreto de benzalcônio têm 40% menos probabilidade de formar biofilme se comparada às tratadas com triclosan e clorexidina. A análise estatística nos mostrou que a expressão do gene qacA/B influenciou significativamente a formação de biofilme e confirmou a relação existente entre os valores de concentração mínima inibitória (CMI) e a formação de biofilme (p< 0,001) / Coagulase negative staphylococci have emerged as important nosocomial pathogens, commonly isolated in human bacteremia, a fact closely related to the increased use of medical devices over the last few years. Its ability to adhere to polymer surfaces and produce biofilms is the main virulence factor associated with these devices. To minimize this possibility, antiseptics are widely used for disinfection of skin and mucosa in routine hospital, however, there has been an increase in bacterial resistance to these agents. Decreased susceptibility of microorganisms to antiseptics, associated with its ability to form biofilms has high morbidity, mortality, length of stay and costs related to health care. In the study presented here, we analyzed 81 strains of methicillin-resistant Staphylococcus epidermidis (MRSE) and 55 strains of methicillin-resistant Staphylococcus haemolyticus (MRSHa), all meticillin- resistant, isolated from patients hospitalized at University Hospital in the city of Niterói, Rio de Janeiro in 2008 and 2010. The determination of the susceptibility profile of biocides triclosan, chlorhexidine and benzalkonium chloride demonstrated that front of triclosan, the strains have the biggest rates of bacterial susceptibility. Papain showed no antibacterial activity. However, it was able to significantly reduce biofilm formation (p <0.06) in both species, being the most efficient among the analyzed products. It was also able to degrade established biofilms formed by Staphylococcus epidermidis. Biocides showed no significant reduction of biofilms, except chlorhexidine, which was able to reduce biofilm formation by MRSE. We noticed that strains MRSHa treated with benzalkonium chloride are 40% less likely to form biofilm compared to those treated with chlorhexidine and triclosan. Statistical analysis showed that the gene expression qacA/B was significantly influential to biofilm formation and confirmed positive relationship between the values of MIC and biofilm formation (p <0.001)

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk with skin flora, such as Staphylococcus epidermidis and Staphylococcus capitis, being the predominant contaminants. These bacteria are able to form surface-attached aggregates or biofilms, which are present in the skin of healthy blood donors and can subsequently be isolated from contaminated PCs. Disinfection of the venipuncture area before donation with a combination of 2% chlorhexidine-gluconate and 70% isopropanol is used at Canadian Blood Services. However, not all bacteria are eliminated during skin disinfection since contaminated PCs are still captured during routine PC screening. In this thesis, the ability of biofilm-forming S. epidermidis and S. capitis to resist the currently used disinfectants was explored. It was demonstrated that although a combination of chlorhexidine and isopropanol has a bactericidal effect, it is unable to completely eradicate skin flora biofilms. Several countries have implemented Pathogen Inactivation Technologies (PITs) as a measure to help control transfusing bacterially-contaminated PCs by exposing PC units to ultra violet light. However, no investigations have been done to evaluate the ability of PITs against bacterial biofilms, which was one of the objectives of this thesis. Data revealed that the efficacy of a currently used PIT, the Mirasol® system, is similar for S. epidermidis present in PCs produced from whole blood inoculated with biofilm or non-biofilm cells. However, treatment effectiveness was strain dependent. In conclusion, further investigation to improve donor skin disinfection and PITs should be considered. Surveillance at Canadian Blood Services shows that contamination rates in single-donor apheresis PCs (Aph-PCs) is generally higher than in four-donor buffy coat platelet pools (BC-PCs). This study investigated whether the BC-PC production method contributes to this observation as BC-PCs are derived from WB that is left to rest overnight while Aph-PCs are collected directly from the donor. Data showed that WB hold during BC-PC production does not have a broad-spectrum bactericidal effect and therefore other factors contribute to low rates of contamination in BC-PCs. The work presented in this thesis provides an insight to bacterial residence and persistence during blood product manufacturing and makes suggestions for PC safety improvements.