Doctor of Philosophy / Department of Biology / Lynn E. Hancock / Enterococci are gram-positive bacteria typically found as commensals in the gastro-intestinal tracts of most mammals. Enterococci, most notably Enterococcus faecalis and Enterococcus faecium, have become problematic causative agents of several nosocomially acquired infections including urinary tract infections, bacteremia, surgical sight infections, and endocarditis. These bacteria must first overcome the innate immune response in order to establish infection. Many bacteria produce capsular polysaccharides that contribute to pathogenesis by helping the microbe evade the host innate immune response. The capsular polysaccharide produced by E. faecalis has been shown to play a role in pathogenesis; however the mechanisms of innate immune avoidance were unknown. Moreover, the number of capsule serotypes produced by E. faecalis and the genetic differences that contribute to capsule serospecificity were in doubt. In the current study it is made clear that only two capsule serotypes are produced by E. faecalis and that both capsule serotypes contribute to evasion of the host innate immune system. This work shows two mechanisms by which the capsule of E. faecalis contributes to immune evasion. First, the presence of capsule inhibited complement mediated phagocytosis through limiting the detection of opsonic complement protein C3 on the surface of the bacteria. Secondly, the presence of capsule altered cytokine signaling of macrophages by shielding bacterial components from detection. Many pathogenic strains of E. faecalis also produce an extracellular protease known as gelatinase (GelE). This work also shows a novel mechanism involving GelE in innate immune evasion through the degradation of the anaphylatoxin C5a. Degradation of C5a by GelE resulted in decreased neutrophil recruitment in vitro. A rabbit model of endocarditis was employed to assess the effect of GelE production on disease development and progression. Rabbits infected with GelE producing strains had increased bacterial burdens in the heart compared to rabbits infected with strains that were GelE negative. Reduced phagocyte infiltration at primary and secondary infection sites was also observed in rabbits infected with GelE producing strains compared to GelE negative strains. The work presented here demonstrates that both the capsular polysaccharide and GelE play roles in E. faecalis evasion of innate immune responses. Moreover, these pathogenic determinants would be suitable targets for developing alternative therapeutics used to treat E. faecalis infections.

En los últimos años los casos de infecciones causadas por enterococos resistente a vancomicina (ERV) han ido cobrando importancia debido a su capacidad innata para almacenar información e inclusive transferirla a otras cepas. Los ERV han sido aislados de Unidades de Cuidados Intensivos (UCI), de infecciones de tracto urinario, bacteriemias, endocarditis, etc. Las dos especies clínicamente importantes son Enterococcus faecium y Enterococcus faecalis, las cuales portan genes de resistencia a vancomicina. El objetivo de esta investigación fue analizar genéticamente las cepas ERV del Hospital Nacional Guillermo Almenara Irigoyen (HNGAI). Para ello se recolectaron cepas ERV y se realizó la evaluación molecular en el Laboratorio de Microbiología y Biotecnología Molecular de la Universidad Nacional Mayor de San Marcos – Lima. Se seleccionaron un total de 28 cepas, 82% (n = 23) correspondieron a la especie E. faecium y 18% (n = 5) a E. faecalis. Se determinó el nivel de resistencia a vancomicina con la Concentración Mínima Inhibitoria (CMI) mediante el E-test, 23 cepas de Enterococcus faecium obtuvieron una CMI ≥ 256µg/mL; 5 cepas de Enterococcus faecalis mostraron subpoblaciones heterorresistentes a vancomicina dentro del halo de inhibición. Para determinar la ubicación de la resistencia se utilizó la prueba de curación de plásmidos resultando que el 96% (n = 22) de las cepas de E. faecium mantuvo la resistencia después del curado y el 100% de Enterococcus faecalis mostraron sensibilidad y transferibilidad, siendo la frecuencia de transferencia más alta de 5 x10-3 para la cepa EC0507. Mediante PCR múltiplex se determinó la presencia de un sólo genotipo vanA, en todas las cepas Enterococcus faecium y Enterococcus faecalis. De esta manera se concluye que existe un solo genotipo vanA en los ambientes intrahospitalarios del HNGAI y que estos pueden ser transferibles.

El propósito de este estudio fue determinar la efectividad de la asociación de Cetrimida -Clorhexidina (15% / 0.15%) frente a Clorhexidina 2%; en la erradicación de biofilms de Enterococcus faecalis in vitro. La metodología usada para este fin fue la creación de biofilms, en láminas portaobjeto de 48 horas de incubación a 37 ºC en condiciones aerobias a partir de la cepa Enterococcus faecalis ATCC 29212 en caldo BHI, las cuales fueron sometidas a los irrigantes testados durante los periodos de tiempo de 1, 3 y 5 minutos, respectivamente. Se llevaron a cabo dos grupos de experimentos independientes, más un control negativo (suero fisiológico); en tres repeticiones para cada irrigante y tiempo evaluado. Los irrigantes evaluados fueron: Cetrimida-Clorhexidina (15%/0.15%) “SAFEBLON”, Clorhexidina (2%), y Suero Fisiológico. Luego del contacto con los irrigantes, los biofilms fueron sembrados en Agar Tripticasa Soya (TSA) e incubados durante 24 horas a 37 ºC para la cuantificación final de UFC/mL y así determinar el porcentaje de inhibición bacteriana y consecuentemente la efectividad de los irrigantes. El resultado obtenido muestra que, el porcentaje de inhibición bacteriana de Biofilms de Enterococcus faecalis con Cetrimida-Clorhexidina (15%/0.15%) fue 99,99%; con Clorhexidina (2%), fue 100% y con Suero Fisiológico(control negativo) fluctuó entre 24% y 31%; durante los tiempos evaluados de 1,3 y 5 minutos. Por lo tanto, se concluye que no existe diferencia significativa en la Erradicación de biofilms de Enterococcus faecalis; mediante la aplicación de la asociación Cetrimida-Clorhexidina (15%/1.5%) y Clorhexidina 2% a cualquiera de los tres tiempos experimentados; permitiéndose utilizar indistintamente menor concentración de Clorhexidina de 2% al 0.15% , mediante el uso coadyuvante de Cetrimida 15%. Palabras Clave: Cetrimida, Surfactante, biofilms, Enterococcus faecalis, tensión superficial. / The purpose of this study was to determine the effectiveness of the association of Cetrimide-Chlorhexidine 15%/0.15% with Chlorhexidine 2% in the eradication of biofilms of Enterococcus faecalis in vitro. The used methodology for this aim was the creation of biofilms in glass slides for 48 hours of incubation at 37 ºC, in aerobic conditions from Enterococcus faecalis ATCC 29212 strains in BHI infusion. They were subjected to the tested irrigants during the periods of 1, 3 and 5 minutes, respectively. There were two groups of independent experiments and a negative control with saline solution in three repetitions for each irrigant and time tested. The tested irrigants were Cetrimide/ Chlorhexidine 15%/015%, (SAFEBLON) , Chlorhexidine 2% and physiological saline. After the contact with the irrigants , biofilms were cultivated in TSA agar and incubated during 24 hours at 37 ºC for the quantification of the UFC/mL and determine the percentage of bacterial inhibition and consequently the percentage of the irrigants effectiveness. The results obtained show that the obtained percentage of bacterial inhibition of Enterococcus faecalis biofilms with Cetrimide - Chlorhexidine (15%/0.15%) was 99,99% ; with Chlorhexidine (2%), was at 100% and with Physiological Saline (negative control) ranged between 24% and 31%; during three tested times : 1,3 and 5 minutes. Therefore, It concluded that there is no significant difference in the eradication of Enterococcus faecalis biofilms; by the application of the association Cetrimide-Chlorhexidine (15%/ 0.15%) and Chlorhexidine 2% at any of the three tested times; allowing to use indistinctly less concentration of Chlorhexidine from 2% to 0.15% by the adjuvant use of Cetrimide 15%. Keywords: Cetrimide, surfactant, biofilms, Enterococcus faecalis, surface tension

El presente trabajo in vitro evaluará la capacidad antibacteriana del yoduro de potasio yodado al 2% como un coadyuvante para la erradicación de la bacteria Enterococcus faecalis de los conductos radiculares posterior al tratamiento quimiomecánico, bajo un modelo de evaluación y control microbiológico de las paredes del conducto radicular, reproduciendo características clínicas tales como la anatomía de los conductos radiculares y el efecto mecánico de una técnica de instrumentación rotatoria.

Orientador: Carla Raquel Fontana Mendonça / Resumo: O presente estudo tem como objetivo avaliar a fototoxicidade do azul de metileno (AM), clorina-e6 (Ce-6) e curcumina (CUR) associados a irradiações com diferentes intensidades de luz para a terapia fotodinâmica (TFD) contra cepas de Enterococcus faecalis e Cutibacterium acnes, em suspensão e em biofilme. As fontes de luz utilizadas na TFD foram LEDs no comprimento de onda azul (450 nm) e vermelho (660 nm) de acordo com cada fotossensibilizador testado. Como resultado observamos que, em E. faecalis a TFDa mediada por AM e Ce6 foi mais eficiente quando exposta a intensidade de luz de 153 mW/cm², porém com a CUR os melhores resultados foram obtidos com a intensidade de luz de 103 mW/cm². Para C. acnes em suspensão, a Ce6 e a CUR submetidas às diferentes intensidades de luz, causaram reduções bacterianas similares. Em biofilme, a Ce6 associada a intensidade de 122 mW/cm² de luz foi mais eficiente, no entanto, a CUR não proporcionou morte de biofilmes de C. acnes independente das intensidades testadas. O AM, independente da fase, apresentou os melhores resultados com a intensidade de luz de 153 mW/cm². Considerando a quantificação de espécies reativas de oxigênio, a concentração de AM foi determinante na produção de EROs. Na concentração de 100 μg/mL de AM na dose de 120 J/cm2 radicais hidroxila foram mais evidentes em relação a quantidade de oxigênio singleto formado. Na concentração de 50 μg/mL, a intensidade de 61 mW/cm² gerou similarmente as espécies testadas, quanto que a int... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Orientador: Cristiane Duque / Coorientador: Eloi Dezan Júnior / Banca: Rogério de Castilho Jacinto / Banca: Denise Madalena Palomari Spolidorio / Resumo: Na busca por fontes alternativas de antimicrobianos, diversos autores tem explorado o uso de plantas medicinais para o tratamento de várias doenças. A atividade antimicrobiana de várias espécies de plantas como, Cordia verbenacea DC, Mikania laevigata Schultz Bip. Ex Baker e Psidium cattleianum, popularmente chamadas de erva-baleeira, guaco e de araçá, respectivamente, tem sido relatada contra diversos microrganismos orais. O objetivo do presente estudo foi avaliar o efeito antimicrobiano dos extratos hidroetanólico bruto e as suas respectivas frações aquosa, butanol, hexano e acetato de etila de Cordia verbenacea (CV) e Mikania laevigata (ML) e extratos aquoso e hidroetanólico de Psidium cattleianum (PC) contra Enterococcus faecalis, Pseudomonas aeruginosa, Actinomyces israelii e Candida albicans, sob condições planctônicas e de biofilme. Os microrganismos foram analisados em condições planctônicas por meio de ensaios para a determinação da concentração inibitória mínima (CIM) e concentração letal mínima (CLM), pelo método de microdiluição em caldo. Posteriormente, foram selecionados somente os extratos capazes de eliminar completamente cada microrganismo (CLM 100%), para a análise da atividade antimicrobiana em ensaios de biofilme. Os resultados mostraram que os valores de CIM e CLM variaram de 250 à 4000μg/ml. O biofilme formado por E. faecalis foi eliminado frente à ação dos extratos aquoso e hidroetanólico de PC e à fração acetato de etila de ML. A mesma fração de ML e o extrato hidroetanólico de PC também foram capazes de eliminar biofilme de P. aeruginosa. O mesmo ocorreu com as frações hexânicas de CV e ML e o extrato hidroetanólico de ML frente ao biofilme de A. israelii. Nenhum extrato/fração foi capaz de eliminar biofilme de C. albicans. Este estudo demonstrou o potencial antimicrobiano de extratos ou frações de Cordia verbenacea, Mikania laevigata... / Abstract: Seeking for alternative sources of the antimicrobials, several authors have explored the use of herbal medicines for the treatment of diseases. The antimicrobial activity of various species of plants, such as Cordia verbenacea DC, Mikania laevigata Schultz Bip. Ex Baker and Psidium cattleianum, known as erva-baleeira, guaco and araçá, respectively, has been reported against several oral microorganisms. The present study aimed to evaluate the antimicrobial effect of Cordia verbenacea (CV) and Mikania laevigata (ML) hydroethanolic crude extracts and their fractions aqueous, butanol, hexane and ethyl acetate and aqueous and hydroethanolic extracts of Psidium cattleianum (PC) against Enterococcus faecalis, Pseudomonas aeruginosa, Actinomyces israelii and Candida albicans, under planktonic and biofilm conditions. The microorganisms in planktonic conditions were analyzed by determination of minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) assays using the microdilution broth method. Posteriorly, only the extracts that were able to totally eliminate each microorganism (MLC 100%) were selected for the antimicrobial activity analysis in the biofilm assay. The results showed that the MIC and MLC values ranged between 250 and 4000μg/ml. E. faecalis biofilm was eliminated by PC aqueous and hydroethanolic extracts and the ML ethyl acetate fraction. The same ML fraction and PC hydroethanolic extract were also able to eliminate the P. aeruginosa biofilm. The same occurred with the CV and ML hexane fractions and ML hydroethanolic extract against the A. israelii biofilm. None of the extracts/fractions were able to eliminate the C. albicans biofilm. This study confirmed the antimicrobial activity potential of Cordia verbenacea, Mikania laevigata and Psidium cattleianum, suggesting their use for endodontic purposes due to their effectiveness against pathogens associated with persistent or se... / Mestre

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-04-14T14:53:02Z No. of bitstreams: 1 PDF - Amaro Lafayette Nobre Formiga Filho.pdf: 1284130 bytes, checksum: caeaf28bc01f345af720116bf8d65ead (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:00:45Z (GMT) No. of bitstreams: 1 PDF - Amaro Lafayette Nobre Formiga Filho.pdf: 1284130 bytes, checksum: caeaf28bc01f345af720116bf8d65ead (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:00:53Z (GMT) No. of bitstreams: 1 PDF - Amaro Lafayette Nobre Formiga Filho.pdf: 1284130 bytes, checksum: caeaf28bc01f345af720116bf8d65ead (MD5) / Made available in DSpace on 2016-07-22T15:00:53Z (GMT). No. of bitstreams: 1 PDF - Amaro Lafayette Nobre Formiga Filho.pdf: 1284130 bytes, checksum: caeaf28bc01f345af720116bf8d65ead (MD5) Previous issue date: 2012-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this study was to evaluate in vitro experimental drugs made from plants of the Brazilian semiarid region, Schinopsis brasiliensis Engl. and Ximenia americana L., comparing its effectiveness with commercial medications. Hydroalcoholic extracts were prepared from six medicinal plants (Schinopsis brasiliensis Engl., Ximenia americana L., Cereus jamacaru DC., Croton campestres ST. Hill, Spondias mombin, Zornia reticulata SM.), which were subjected to an Enterococcus faecalis microbiological screening by agar dilution method, cylinder technique. The two groups of greater efficiency, Schinopsis brasiliensis Engl. and Ximenia americana L., were nebulized and potency was evaluated by the same method. In vitro contamination with E. faecalis was performed in teeth for 60 days with bacterial growth evaluated by scanning electron microscopy, optical microscopy and microbiological tests. After the inoculation period, the teeth were irrigated, dried and filled with different drugs: 1 - experimental S. brasiliensis paste, 2 - experimental X. americana paste, 3 - Calen ® paste and 4 – CTZ paste. The pastes removal and analysis of the results were performed after 8 and 28 days after their application. The results showed that the X. americana paste had greater efficacy and was able to inhibit the growth of E. faecalis after 28 days of treatment, compared with the other studied pastes, with CTZ exception, that presented the best results during the entire treatment period. / O objetivo deste estudo foi avaliar in vitro medicações experimentais formuladas a partir de plantas do semiárido brasileiro, Schinopsis brasiliensis Engl. e Ximenia americana L., comparando sua eficácia com medicações comerciais. Foram preparados inicialmente extratos hidroalcoólicos de seis plantas medicinais (Schinopsis brasiliensis Engl., Ximenia americana L., Cereus jamacaru DC., Croton campestres ST. Hill, Spondias mombin, Zornia reticulata SM.), os quais foram submetidos a um screening microbiológico frente à Enterococcus faecalis pelo método de diluição em ágar, técnica do cilindro. Os dois extratos de maior eficácia, Schinopsis brasiliensis Engl. e Ximenia americana L., foram nebulizados e sua potência foi avaliada através do mesmo método. Em seguida foi realizada a contaminação in vitro dos elementos dentários com E. faecalis por 60 dias, com o crescimento bacteriano comprovado através da Microscopia Eletrônica de Varredura, Microscopia Óptica e de testes microbiológicos. Decorrido o período de inoculação, os elementos dentários foram irrigados, secos e preenchidos com diferentes medicamentos: 1 – pasta experimental de S. brasiliensis, 2 – pasta experimental de X. americana, 3 – pasta comercial Calen ® e 4 – pasta CTZ. A remoção das pastas e a leitura dos resultados foram realizadas após 8 e 28 dias da sua aplicação. Os resultados obtidos mostraram que a pasta formulada a base da X. americana apresentou maior eficácia e foi capaz de inibir o crescimento de E. faecalis após 28 dias de tratamento, quando comparado com as outras pastas testadas, com exceção da CTZ que apresentou os melhores resultados durante todo o período de tratamento.

Los enterococos, considerados patógenos emergentes, presentan una gran versatilidad genética actuando en el medio ambiente como reservorios genéticos de virulencia y resistencia. Se determinó comparativamente en 33 cepas de enterococos clínicos y 34 cepas de origen marino los perfiles de resistencia antimicrobiana, perfil fenotípico y la presencia de los marcadores de virulencia gelE, cylA, esp y hyl, relacionados respectivamente con: la gelatinasa, hemolisina, la proteína de superficie enterocócica y una putativa glicosil-hidrolasa; además se realizaron ensayos de mortalidad en el modelo experimental Galleria mellonella (Orden: Lepidoptera). Se determinó en cepas marinas sólo la presencia de gelE y esp, con una expresión fenotípica fuerte de biopelículas en casi todas las cepas, aunque no se demostró asociación con estos marcadores. Sólo el 14.3% de enterococos gelE+ presentó actividad gelatinasa y ninguna cepa resultó ser betahemolítica ni portar cylA (Activador de la hemolisina). En el grupo clínico se observó múltiples antibiotipos y perfiles de virulencia en relación directa a la especie y al origen de la infección. El marcador hyl estaba presente sólo en cepas resistentes a vancomicina (EVR) y portadoras de esp, además se detectó en este grupo cepas betahemolíticas (12.1%) y portadoras de cylA (15.2%) mientras que el 48.5% presentaron gelE y la actividad gelatinasa fue detectada en un 30.3% de la cepas. Las cepas clínicas con actividad gelatinasa y hemolisina positivas desarrollaron una alta mortandad en G. mellonella, en tanto una cepa de E. faecalis de origen marino (gelatinasa positiva) presentó una mortandad similar al grupo clínico. Se determinó que los enterococos marinos, principalmente E. faecalis, conservan ciertas características de virulencia y de resistencia antimicrobiana por lo que su persistencia en el ambiente marino representa una amenaza potencial a la salud pública / --- Enterococci are emerging pathogens, retaining virulence and antimicrobial resistance genes in marine environmental. antimicrobial resistance profiles, phenotype profile and presence of virulence markers were compared in 33 clinical isolates and 34 marine strains. Virulence markets gelE, CylA, esp and hyl, these respectively are gelatinase, hemolysin, enterococcal surface protein, and putative glycosyl-hydrolase; besides mortality trials were conducted in the experimental model Galleria mellonella (Order: Lepidoptera). Was found only in marine enterococci the presence of gelE and esp, with strong biofilms in almost all strains, no association showed with these markers. Only 14.3% of enterococci gelE+ and gelatinase activity showed no strain proved beta hemolytic or carry cylA (hemolysin activator). In the clinical group multiple virulence antibiotypes and profiles related to the species and origin of infection was observed, hyl marker was present only in strains vancomycin resistance (VRE) and esp-positives, also detected in this group, beta-hemolytic (12.1%) and strains carrier cylA (15.2%), in addition 48.5% of clinical enterococci are gelE and gelatinase activity was detected in 30.3% of the strains. Gelatinase positive isolates and hemolysin developed a high mortality in G. mellonella, a strain of E. faecalis of marine origin (positive gelatinase) with similar clinical group mortality. It was determined that marine enterococci, mainly E. faecalis, retain some virulence characteristics and antimicrobial resistance so its persistence in the marine environment is a threat to public health. Keywords: Virulence, enterococci, multiresistance, pathogenicity, G. mellonella. / Tesis

No description available.

Thesis (M.D.Sc.) - University of Queensland, 2003. / 2 Pts. ; abstract for each. Includes bibliography.