yes / More than 2 million people/year suffer a bone fracture in the UK1. Reconstruction of bone defects represents a major clinical challenge and is addressed using a number of medical devices. Although medical device compositions and applications may differ widely, all attract microorganisms and represent niches for medical device associated infections. For open fractures, the risk of infection can be 55%2. These infections are often resistant to many of the currently available antibiotics and represent a huge and growing financial and healthcare burden. The aim of this study was a fundamental understanding of how the presence of host defence peptides (HDPs)3 and/or RGD can influence the outcome of cell vs. bacterial viability and proliferation. / Presented at the conference: eCM XVI - Bone and Implant Infection June 24-26, 2015, Convention Centre, Davos Platz, Switzerland.

Made available in DSpace on 2016-01-26T18:56:03Z (GMT). No. of bitstreams: 1 Deigilam Cestari Esteves.pdf: 2776358 bytes, checksum: 7e1a46b5b264cbf8e0c0d96e68b501e2 (MD5) Previous issue date: 2014-12-18 / The environmental and hospital infections, caused by bacteria which are resistant to a wide variety of antibiotics, have shown increasing records in the last years, manifesting themselves in high mortality and lethality. Recent researches report that the bacteria presents a survival profile on dry surfaces in order to maintain their virulence when exposed to biological fluids such as urine, saliva and blood. The objective of this study was to document - through laboratorial analysis the survival capacity of the main bacteria of medical interest on abiotic surfaces. The adopted procedures were completely conducted in the microbiology laboratory at Unoeste, in Presidente Prudente SP. Standard ATCC strains of Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC35218), Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa (ATCC27583) were used, to which biological fluids and water were added. The surfaces were tile, synthetic fabric, mattress and cotton fabric. The surfaces were contaminated with suspension composed by the ATCC strains, biological fluids and water in addition to the control containing only the ATCC strains, which were stored in petri dishes, kept in room temperature. Every seven days the surfaces were dipped in trypticase soy broth and kept in the oven for 24h at 37ºC. They were spread from the contaminated brothin Mueller Hinton agar and kept for 24h in the oven at 37ºC. The viability analysis was done through the colony-forming unity (CFU) counting. For the statistical analysis, the Software R was used, through the Friedman and Kruskal Wallis tests for multiple comparisons, analyzing within each factor (solution or surface) which differ between one another (Friedman s test) and also which treatments interactions are different (Kruskal-Wallis). Through this analysis it was possible to observe that in the current work that Staphylococcus aureus kept its viability for a longer period than other microorganisms under all the tested conditions, presenting significant differences between the fluids and surfaces, with a particular colony growth in blood and cotton. The Klebsiella pneumoniae showed differences between fluids, with saliva containing the highest colony number. With regards to the other tested bacteria, there was no isolated significance. For the multiple comparisons analysis, only the Pseudomonas aeruginosa presented no significant difference between any pair of treatments, whereas the other bacteria presented significant differences between the correlations. The need to analyze the environmental impact of these microorganisms persistence in environments, which are vulnerable to human beings, guides the creation of measures in order to control the spread of pathogenic microorganisms. / As infecções ambientais e hospitalares causadas por bactérias resistentes a um amplo espectro de antibióticos têm índices crescentes nos últimos anos, manifestando-se com alta morbidade e letalidade. Pesquisas recentes evidenciam que as bactérias demonstram um perfil de sobrevivência, em superfícies secas de modo a manter sua virulência quando expostas a fluidos biológicos como urina, saliva e sangue. O objetivo desse estudo foi documentar através de análises laboratoriais a capacidade de sobrevivência das principais bactérias de interesse médico em superfícies abióticas. Os procedimentos foram totalmente realizados no laboratório de microbiologia da Unoeste em Presidente Prudente - SP., utilizando cepas padrão ATCC de Staphylococcus aureus(ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli(ATCC35218), Klebsiella pneumoniae(ATCC700603) e Pseudomonas aeruginosa(ATCC27583), ao qual foram adicionados fluídos biológicos e água. As superfícies utilizadas foram piso, tecido sintético, colchão e tecido algodão. As superfícies foram contaminadas com suspensão composta pelas cepas ATCC, fluídos biológicos e água, além do controle contendo somente as cepas ATCC, armazenadas em placas de petri e mantidas em temperatura ambiente. A cada sete dias as superfíciesarmazenadas foram mergulhadas em caldo Tripscaseína de Soja (TSB), colocadas na estufa por 24h a 37ºC. Foram semeados do caldo contaminado em ágar Mueller Hinton e mantidos por 24h em aquecimento a 37º em Estufa.A análise da viabilidadefoi realizada através da contagem de unidade formadora de colônias (U.F.C.). Para a análise estatística utilizou o software R, realizando os testes de Friedman e de Kruskal Wallis para comparações multiplas, analisando dentro de cada fator (solução ou superfície) quais diferem entre si (Teste de Friedman) e, também, quais interações de tratamentos são diferentes (Kruskal-Wallis). Através dessas análises foi possível observar no presente trabalho que Staphylococcus aureus manteve a viabilidade por tempo maior que os outros microrganismos em todas as condições testadas, apresentando diferença significativa entre os fluídos e as superfícies, tendo sangue e tecido algodão crescimento de maior número de colônias. A Klebsiella pneumoniae apresentou diferença entre os fluídos, sendo asaliva com maior número de colônias. Para as outras bactérias testadas não houve significância isoladamente. Para as análises de comparações múltiplas somente para a Pseudomonas aeruginosanão houve diferença significativa entre algum par de tratamentos, as outras bactérias apresentaram diferenças significativas entre as correlações. A necessidade de analisar o impacto ambiental da persistência desses microrganismos em ambientes vulneráveis ao ser humano, norteia o delineamento de medidas para o controle na disseminação de microrganismos patogênicos.

Made available in DSpace on 2016-07-18T17:46:20Z (GMT). No. of bitstreams: 1 Deigilam Cestari Esteves.pdf: 2776358 bytes, checksum: 7e1a46b5b264cbf8e0c0d96e68b501e2 (MD5) Previous issue date: 2014-12-18 / The environmental and hospital infections, caused by bacteria which are resistant to a wide variety of antibiotics, have shown increasing records in the last years, manifesting themselves in high mortality and lethality. Recent researches report that the bacteria presents a survival profile on dry surfaces in order to maintain their virulence when exposed to biological fluids such as urine, saliva and blood. The objective of this study was to document - through laboratorial analysis the survival capacity of the main bacteria of medical interest on abiotic surfaces. The adopted procedures were completely conducted in the microbiology laboratory at Unoeste, in Presidente Prudente SP. Standard ATCC strains of Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC35218), Klebsiella pneumoniae (ATCC700603) and Pseudomonas aeruginosa (ATCC27583) were used, to which biological fluids and water were added. The surfaces were tile, synthetic fabric, mattress and cotton fabric. The surfaces were contaminated with suspension composed by the ATCC strains, biological fluids and water in addition to the control containing only the ATCC strains, which were stored in petri dishes, kept in room temperature. Every seven days the surfaces were dipped in trypticase soy broth and kept in the oven for 24h at 37ºC. They were spread from the contaminated brothin Mueller Hinton agar and kept for 24h in the oven at 37ºC. The viability analysis was done through the colony-forming unity (CFU) counting. For the statistical analysis, the Software R was used, through the Friedman and Kruskal Wallis tests for multiple comparisons, analyzing within each factor (solution or surface) which differ between one another (Friedman s test) and also which treatments interactions are different (Kruskal-Wallis). Through this analysis it was possible to observe that in the current work that Staphylococcus aureus kept its viability for a longer period than other microorganisms under all the tested conditions, presenting significant differences between the fluids and surfaces, with a particular colony growth in blood and cotton. The Klebsiella pneumoniae showed differences between fluids, with saliva containing the highest colony number. With regards to the other tested bacteria, there was no isolated significance. For the multiple comparisons analysis, only the Pseudomonas aeruginosa presented no significant difference between any pair of treatments, whereas the other bacteria presented significant differences between the correlations. The need to analyze the environmental impact of these microorganisms persistence in environments, which are vulnerable to human beings, guides the creation of measures in order to control the spread of pathogenic microorganisms. / As infecções ambientais e hospitalares causadas por bactérias resistentes a um amplo espectro de antibióticos têm índices crescentes nos últimos anos, manifestando-se com alta morbidade e letalidade. Pesquisas recentes evidenciam que as bactérias demonstram um perfil de sobrevivência, em superfícies secas de modo a manter sua virulência quando expostas a fluidos biológicos como urina, saliva e sangue. O objetivo desse estudo foi documentar através de análises laboratoriais a capacidade de sobrevivência das principais bactérias de interesse médico em superfícies abióticas. Os procedimentos foram totalmente realizados no laboratório de microbiologia da Unoeste em Presidente Prudente - SP., utilizando cepas padrão ATCC de Staphylococcus aureus(ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli(ATCC35218), Klebsiella pneumoniae(ATCC700603) e Pseudomonas aeruginosa(ATCC27583), ao qual foram adicionados fluídos biológicos e água. As superfícies utilizadas foram piso, tecido sintético, colchão e tecido algodão. As superfícies foram contaminadas com suspensão composta pelas cepas ATCC, fluídos biológicos e água, além do controle contendo somente as cepas ATCC, armazenadas em placas de petri e mantidas em temperatura ambiente. A cada sete dias as superfíciesarmazenadas foram mergulhadas em caldo Tripscaseína de Soja (TSB), colocadas na estufa por 24h a 37ºC. Foram semeados do caldo contaminado em ágar Mueller Hinton e mantidos por 24h em aquecimento a 37º em Estufa.A análise da viabilidadefoi realizada através da contagem de unidade formadora de colônias (U.F.C.). Para a análise estatística utilizou o software R, realizando os testes de Friedman e de Kruskal Wallis para comparações multiplas, analisando dentro de cada fator (solução ou superfície) quais diferem entre si (Teste de Friedman) e, também, quais interações de tratamentos são diferentes (Kruskal-Wallis). Através dessas análises foi possível observar no presente trabalho que Staphylococcus aureus manteve a viabilidade por tempo maior que os outros microrganismos em todas as condições testadas, apresentando diferença significativa entre os fluídos e as superfícies, tendo sangue e tecido algodão crescimento de maior número de colônias. A Klebsiella pneumoniae apresentou diferença entre os fluídos, sendo asaliva com maior número de colônias. Para as outras bactérias testadas não houve significância isoladamente. Para as análises de comparações múltiplas somente para a Pseudomonas aeruginosanão houve diferença significativa entre algum par de tratamentos, as outras bactérias apresentaram diferenças significativas entre as correlações. A necessidade de analisar o impacto ambiental da persistência desses microrganismos em ambientes vulneráveis ao ser humano, norteia o delineamento de medidas para o controle na disseminação de microrganismos patogênicos.

Le microbiote intestinal est constitué d’un écosystème complexe de microorganismes appartenant à différents règnes. Cependant, la majorité de ces microorganismes sont d’origine bactérienne. Par conséquent, de nombreuses études, y compris la présente, se concentrent sur l’étude des communautés bactériennes. Les microorganismes ont développé une relation mutualiste avec le corps humain et agissent de plusieurs manières sur sa santé. Une perturbation du microbiote intestinal, nommée dysbiose, est reliée au développement d’une multitude de problèmes de santé chez diverses espèces animales. La transplantation de microbiote fécal suscite l’intérêt dans le domaine de la médecine vétérinaire. La préparation et l’entreposage affectent la composition et la viabilité bactérienne des fèces destinées à la transplantation de microbiote fécal (TMF). Jusqu’à présent, il demeure l’absence d’un protocole vétérinaire pour effectuer la préparation et l’entreposage des transplants fécaux canins. Par conséquent, l’objectif de cette étude était de comparer la viabilité bactérienne d’échantillons fécaux en présence et en absence d’oxygène et d’effectuer la congélation à l’aide de deux cryoprotecteurs différents. Les hypothèses de ce projet étaient les suivantes : la préparation des échantillons en absence d’oxygène préservera la viabilité bactérienne, l’utilisation d’un cryoprotecteur contenant des antioxydants pour la congélation générera le meilleur taux de viabilité, et le microbiote de chaque individu n’aura pas la même capacité à résister aux effets de la préparation et de l’entreposage. Les fèces de 10 chiens en santé ont été collectées et immédiatement transférées à l’intérieur d’une chambre anaérobique. Des aliquotes de 1,8 g ont été diluées dans 7,2 ml d’un cryoprotecteur contenant du glycérol à 10% (Gly) ou des antioxydants (Cryo). Les échantillons ont été homogénéisés et filtrés en condition aérobique (Ae) et en condition anaérobique (An) à l’intérieur d’une chambre anaérobique, simulant la préparation de la TMF. Les échantillons ont été congelés à -20 °C durant 90 jours (F) pour l’évaluation des effets de l’entreposage. La viabilité bactérienne des échantillons a été déterminée à l’aide de la cytométrie de flux. L’analyse de la composition bactérienne chez les 10 donneurs de matières fécales a été réalisée par le séquençage de la région V4 du gène de l’ARNr 16S à l’aide de la plateforme Illumina MiSeq. Les échantillons non congelés, préparés en absence d’oxygène et dilués avec Cryo présentaient les plus grands taux de viabilité (66,78 %) par rapport aux autres groupes (p < 0,05). Les échantillons exposés à l’oxygène avaient une viabilité bactérienne inférieure (p < 0,01). Toutefois, les échantillons dilués avec Cryo présentaient une viabilité plus élevée (65,26 %) que les échantillons dilués dans Gly (55,20 % ; p < 0,001) en présence d’oxygène. La viabilité bactérienne a diminué en raison de la congélation des échantillons (p < 0,001). L’ensemble des échantillons frais avaient une viabilité médiane de 62,23 % et, à la suite de la congélation, elle était de 22,68 %. Cependant, les échantillons congelés à l’aide de glycérol avaient une viabilité plus élevée (30,61 % ; p < 0,001). Le genre Prevotella était fortement corrélé à la viabilité (R = 0,731 ; p < 0,05, R = 0,756 ; p < 0,05, R = 0,834 ; p < 0,01, R = 0,752 ; p < 0,05). Ces résultats indiquent que la viabilité bactérienne est optimale lors de l’utilisation de matières fécales en absence d’oxygène et lors d’une dilution à l’aide d’un cryoprotecteur contenant des antioxydants. La congélation a significativement réduit la viabilité bactérienne, mais le glycérol semble mieux préserver les bactéries. La présence de certaines espèces plus résistantes et l’impact de la composition du microbiote sur l’efficacité de la TMF nécessitent une enquête plus approfondie. / The intestinal microbiota is made up of a complex ecosystem of microorganisms belonging to different kingdoms. However, bacterial cells are much more numerous. Therefore, many studies, including the present one, focus on the study of bacterial communities. Microorganisms have developed a mutualistic relationship with the animal body and act in several ways on its health. A disturbance of the intestinal microbiota, called dysbiosis, is linked to the development of a multitude of health problems in various animal species. There is an emerging interest in the transplantation of fecal microbiota in veterinary medicine. Preparation and storage affect the quality of transplants intended for faecal microbiota transplantation (FMT). Considering the absence of a protocol in veterinary medicine, the objective of this study was to optimize bacterial viability during the preparation and storage of canine fecal transplants. The hypotheses of this project were that the preparation of samples in the absence of oxygen will preserve bacterial viability, that the use of a cryoprotectant containing antioxidants for freezing will yield the best viability rate and the microbiota of individuals does not have the same ability to withstand the effects of preparation and storage. Feces from ten healthy dogs were collected, and immediately transferred inside an anaerobic chamber. Aliquots of 1.8 g were diluted in 7.2 ml of a cryoprotectant containing glycerol (Gly) or antioxidants (Cryo). The samples were homogenized and filtered, simulating the TMF preparation. To evaluate the impact of oxygen on bacterial viability, the procedures were performed outside (Ae) and inside (An) the anaerobic chamber. Samples were frozen at -20°C for 90 days (F) to evaluate effect of freezing. The bacterial viability of samples was determined using flow cytometry. Analysis of the bacterial composition was performed by sequencing the V4 region of the 16S rRNA gene, using the Illumina MiSeq platform. Fresh samples prepared under anaerobiosis and diluted with Cryo had the highest viability (66.78%) compared to the other groups (p < 0.05). Bacterial viability was affected by oxygen (p < 0.01) but solutions prepared with Cryo had higher viability (65.26%) than samples diluted in Gly (55.20%; p < 0.001). Freezing decreased bacterial viability from 62.23% to 22.68% (p < 0.001). However, samples frozen using glycerol showed higher viability (30.61%; p < 0.001). The genus Prevotella was strongly correlated with viability (R = 0.731; p < 0.05, R = 0.756; p < 0.05, R = 0.834; p < 0.01, R = 0.752; p < 0.05). These results show that bacterial viability is optimal when preparing feces under anaerobic conditions and using a cryoprotectant containing antioxidants. If freezing is necessary, glycerol seems to preserve the bacteria better. The presence of some more resilient species and the impact of microbiota composition on the efficacy of TMF requires further investigation.