Escherichia coli enteropatogênica atípica (EPECa) é causadora de diarréia infantil e apresenta alta heterogeneidade quanto aos fatores de virulência. O objetivo deste trabalho foi estudar o comportamento de EPECa na interação com fagócitos profissionais. Duas amostras de EPECa sorotipo O55:H7 mostraram-se capazes de reduzir a fagocitose. Os sobrenadantes dos cultivos foram submetidos a SPE e HPLC e as frações com efeito antifagocítico foram submetidas a espectrometria de massas. A fração capaz de reduzir a fagocitose de bactérias reduziu também a fagocitose de Saccharomyces cerevisiae. Além de mostrar que EPECa é capaz de induzir a antifagocitose, mostrou-se também que o fator antifagocitico é secretado, solúvel em meio aquoso, termoestável, apresenta baixo peso molecular, não é microbicida ou citotóxico e, por último, há indicativos de que possa apresentar uma região glicosídica. Estes achados sugerem que o fator antifagocítico pode, embora não sozinho, exercer um papel importante na adaptabilidade e patogenicidade das EPECa. / Atypical enteropathogenic Escherichia coli (aEPEC) causes diarrhea mainly in children and presents a high heterogeneity of virulence factors. The objective of this work was to study the behavior of aEPEC regarding its interaction with professional phagocytes. Two samples of aEPEC serotype O55:H7 were able to reduce phagocytosis, The culture supernatants were submitted to SPE and HPLC and the active fractions were tested and analyzed by mass spectrometry. The results show that the fraction with bacterial antiphagocytic activity also reduces phagocytosis of Saccharomyces cerevisiae. In addition to demonstrating that aEPEC can induce antiphagocytosis, this work shows that it is due to a secreted antiphagocytic factor that is soluble in aqueous medium, is thermo-stable, has a low molecular weight, is not bactericide or cytotoxic and, finally, possibly presents a glycosidic region. These findings suggest that the antiphagocytic factor may, though maybe not alone, play an important role in the adaptability and pathogenicity of aEPEC.

Gram-negative infection has been linked to hospital water although few studies have examined whether water systems are reservoirs of nosocomial pathogens. This study investigated longitudinal recovery of the opportunistic pathogens Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii from water outlets of a haematology unit and evaluated Point-Of-Use Filtration (POU-F) as a control measure. In a two-year double cross-over trial, water samples and swabs were taken weekly from 39 showers/taps on the unit. Four study phases alternated between non-filtered (Phases 1 & 3), and filtered outlets (Phases 2 & 4) using Pall AquasafeTM 14-day filters. In Phases 1 & 3; 99% of 1396 samples yielded bacterial growth, with colonies generally too numerous to count. Target species were isolated from 22% of water samples (P. aeruginosa 14%; S. maltophilia 10%) and 10% of swabs. P. aeruginosa was particularly associated with handwash stations and S. maltophilia with showers. A. baumannii was not isolated. With POU-F; 22% of 1242 samples yielded bacterial growth (mean CFU/100ml ,4.6). S. maltophilia was isolated only once from water but never from outlet swabs. PCR typing identified clusters of isolates colonizing different outlets over time but no clear association between water and patient isolates was identified. The incidence of Gram negative infections remained low throughout the study. Without POU-F, water from taps/showers represented a source of bacteria including the target species. POU-F substantially reduced the frequency and number of target species from every outlet, and merits further investigation as an intervention to protect immunocompromised patients from opportunistic pathogens.

Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: Seasonality of healthcare-associated infections (HCAIs) has been recently reported, especially involving Gram-negative bacilli (GNB). Factors underlying this phenomenon were not elucidated. It is theoretically conceivable it reflects seasonal variations in traditional risk factors for those infections. With this in mind, we conducted a study to analyze the interplay of season, weather and usual predictors of healthcare-associated bloodstream infections caused by Gram-negative bacilli (GNB-BSI). The study had a retrospective, case-only desing. It was conducted in the teaching hospital from Botucatu School of Medicine (450 beds). The study enrolled 446 patients with GNB-BSI caused by Escherichia coli, Enterobacter spp., Klebsiella spp., Pseudomonas aeruginosa or Acinetobacter baumannii, diagnosed from July 2012 through June 2016. Demographic data, comorbidities, invasive procedures and use of antimicrobials were reviewed in medical charts. The season in which GNB-BSI occurred, as well as weather parameters of the day of diagnosis, were recorded. We analyzed factors associated with occurrence of GNB-BSI in different seasons (with winter as reference category) and caused by different GNB (reference category, E. coli). Univariate and multivariable models of polytomous (multinomial) logistic regressions were used for analysis. In multivariable analysis, GNB-BSI diagnosed in summer were more likely to be caused by Klebsiella spp. (OR, 5.33; 95%CI, 2.04-13.96) or A. baumannii (OR, 2.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Seasonality of healthcare-associated infections (HCAIs) has been recently reported, especially involving Gram-negative bacilli (GNB). Factors underlying this phenomenon were not elucidated. It is theoretically conceivable it reflects seasonal variations in traditional risk factors for those infections. With this in mind, we conducted a study to analyze the interplay of season, weather and usual predictors of healthcare-associated bloodstream infections caused by Gram-negative bacilli (GNB-BSI). The study had a retrospective, case-only desing. It was conducted in the teaching hospital from Botucatu School of Medicine (450 beds). The study enrolled 446 patients with GNB-BSI caused by Escherichia coli, Enterobacter spp., Klebsiella spp., Pseudomonas aeruginosa or Acinetobacter baumannii, diagnosed from July 2012 through June 2016. Demographic data, comorbidities, invasive procedures and use of antimicrobials were reviewed in medical charts. The season in which GNB-BSI occurred, as well as weather parameters of the day of diagnosis, were recorded. We analyzed factors associated with occurrence of GNB-BSI in different seasons (with winter as reference category) and caused by different GNB (reference category, E. coli). Univariate and multivariable models of polytomous (multinomial) logistic regressions were used for analysis. In multivariable analysis, GNB-BSI diagnosed in summer were more likely to be caused by Klebsiella spp. (OR, 5.33; 95%CI, 2.04-13.96) or A. baumannii (OR, 2.... (Complete abstract click electronic access below) / Mestre

Si les antibiotiques ont amélioré le pronostic des maladies infectieuses, l’apparition de résistances importantes et les manipulations génétiques volontaires peuvent faire craindre l’émergence de pathogènes très virulents et résistants aux antibiotiques recommandés. Aussi, la multidrug résistance (MDR) est devenue un problème majeur pour traiter des infections impliquant des bactéries à Gram-négatif. La surexpression des mécanismes d’efflux contribue largement au phénotype de multi-résistance aux antibiotiques et l’inhibition de ces mécanismes intéresse donc de plus en plus le monde de la recherche.Les phénothiazines font partie des molécules les plus prometteuses du 20e siècle. La présence d’un substituant en position C-2 sur le noyau tricyclique et celle d’une chaine alkyl aminée en position N-10 se sont révélées être critiques pour des activités de type neuroleptique, antihistaminique et anthelminthique. Une activité anti-MDR a également été mise en évidence, notamment contre les cancers et la tuberculose. Le mécanisme par lequel ces molécules inhiberaient l’efflux est cependant encore mal connu. Différents dérivés de la phénothiazine ont été synthétisés afin de comprendre ce mécanisme et de dégager les propriétés physico-chimiques mises en jeu chez Burkholderia pseudomallei. Cette bactérie à Gram-négatif, responsable de la mélioïdose, est classée parmi les agents potentiels du bioterrorisme. Elle est en effet extrêmement pathogène et présente une sensibilité très réduite à une majorité de classes d’antibiotiques. L’activité des phénothiazines a été évaluée par la technique Etest® sur Burkholderia thailandensis, modèle d’étude non-pathogène. / If antibiotic drugs improved the prognosis of infectious diseases, the appearance of antimicrobial resistance and deliberate genetic modifications could be followed by the worrying emergence of highly virulent pathogens resistant to usual antibiotics. Thus, Multi-Drug Resistance (MDR) became a major problem to treat infections involving Gram negative bacteria. The overexpression of efflux mechanisms contributes to a great extent to antibiotic resistance and the inhibition of these mechanisms increasingly interest research areas.Phenothiazines are the most promising molecules of the 20th century. The presence of substituent in C-2 position on the tricyclic structure and the one of alkyl amino chain in N-10 position proved their critical activity as neuroleptic, antihistaminic and antihelmintic drugs. An anti-MDR activity has also been put into evidence against cancers and tuberculosis, but the mechanism by which molecules would inhibit efflux is not well known yet. Different phenothiazine derivatives have been synthesized in order to better understand this mechanism and to draw the physicochemical properties involved in Burkholderia pseudomallei. This Gram negative bacterium is responsible of melioidosis and classified as potential bioterrorism infectious agent. This bacterium is indeed extremely pathogenic and has a very low susceptibility to most classes of antibiotics. The activity of phenothiazine derivatives was evaluated using the Etest® method in Burkholderia thailandensis, the non-pathogenic study model.

Submitted by THAÍS ALVES BARBOSA null (thaalvesb@hotmail.com) on 2016-03-20T00:20:03Z No. of bitstreams: 1 Dissertação.pdf: 1818604 bytes, checksum: 7ea9f39f4ad30e976729ba7907b30ff1 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-22T14:21:02Z (GMT) No. of bitstreams: 1 barbosa_ta_me_bot.pdf: 1818604 bytes, checksum: 7ea9f39f4ad30e976729ba7907b30ff1 (MD5) / Made available in DSpace on 2016-03-22T14:21:02Z (GMT). No. of bitstreams: 1 barbosa_ta_me_bot.pdf: 1818604 bytes, checksum: 7ea9f39f4ad30e976729ba7907b30ff1 (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A necessidade de permanência em Unidade de Terapia Intensiva Neonatal (UTIN) simboliza um dos principais fatores desencadeantes de colonização e infecção. Sabe-se que logo após o nascimento, inicia-se a colonização bacteriana do neonato pelo contato com a microbiota materna, dos profissionais de saúde ou a partir da exposição ambiental. Recém-nascidos (RNs), que permanecem em tratamento intensivo, possuem predisposição aumentada para infecção posteriormente à colonização. A maior sobrevida e o prolongamento do período de internação dos neonatos têm proporcionado uma elevação nas taxas de infecções, principalmente em UTINs. Objetivos: Estudar a epidemiologia da colonização e infecção microbiana em uma coorte de neonatos admitidos em uma UTIN com abordagem clínica e molecular. Metodologia: Foram incluídos no estudo todos os neonatos admitidos na UTIN, nascidos no HC da FMB, por um período de um ano, e assim coletadas amostras de aspirado traqueal, como também por meio de swabs estéreis dos sítios nasal e anal e acompanhamento do recém-nascido até o desfecho final (alta da UTI ou óbito). Micro-organismos isolados foram submetidos à identificação e ao teste de sensibilidade às drogas antimicrobianas para a determinação da concentração inibitória mínima (CIM) pela técnica de E-test. Dentre os Staphylococcus spp. que apresentarem resistência à meticilina foi determinado o tipo de cassete cromossômico estafilocócico mec (SCCmec). Para a pesquisa de clones prevalentes na unidade, foi realizada a caracterização dos clusters pela técnica de eletroforese em gel de campo pulsado (PFGE). Resultados: Os resultados revelaram maior incidência de infecção (27,8%) e colonização por Staphylococcus epidermidis principalmente na mucosa nasal (56,4%). Na mucosa anal predominou a colonização por Serratia marcescens (26,8%). Os resultados evidenciaram cepas de S. epidermidis resistentes a sulfametoxazol-trimetoprim, e S.epidermidis e Enterococcus faecalis resistentes a quinopristina/dalfopristina. Das 402 amostras de Staphylococcus spp. estudadas, 204 (50,7%) apresentaram o gene mecA, com uma maior frequência de isolados provenientes da mucosa nasal, sendo detectados 47 isolados albergando o SCCmec tipo I, 12 carreando o SCCmec tipo II, 77 carreando o SCCmectipo III e 43 albergando o SCCmec tipo IV. A tipagem molecular para determinação dos clusters pela técnica de PFGE demonstrou a presença de isolados de diferentes RNs, com perfis idênticos ou alta taxa de similaridade, sugestivo de contaminação cruzada. Além disso, isolados obtidos do mesmo RN, porém, de sítios diferentes também se apresentaram como idênticos, comprovando que o micro-organismo que colonizava o RN no momento da coleta também era o agente infeccioso. A análise estatística revelou que o processo de colonização pode ser considerado fator de risco para infecção, com um risco de três vezes mais quando comparado com RNs não colonizados. Dentre os fatores de risco para colonização a utilização de nutrição parenteral e cateter central de inserção periférica (PICC) aumentaram o risco em seis vezes mais e quatro vezes mais, respectivamente. Conclusão: Os achados deste trabalho podem auxiliar na escolha antimicrobiana adequada visto que o processo de colonização ocorre posteriormente ao nascimento e que a terapia empírica muitas vezes é necessária. O conhecimento do perfil microbiológico da unidade possibilita a criação de protocolos antimicrobianos adequados para prevenção e tratamento de IRAS, objetivando melhoria na qualidade da assistência prestada. Para tanto, salienta-se a importância de implementação de culturas de vigilância, que auxiliam a comissão de controle de IRAS, e a equipe assistencial na elaboração de medidas a serem adotadas. / The need to stay in the Neonatal Intensive Care Unit (NICU) represents one of the main factors of colonization and infection. It is known that, soon after birth, bacterial colonization of the newborn starts from contact with maternal microbiota, health professionals, or from environmental exposure. Newborns (NBs) who remain in intensive care have a greater predisposition to infection after the colonization. The longer survival and prolonged hospitalization of NBs have led to an increase in infection rates, especially in NICUs. Objectives: To study the epidemiology of colonization and microbial infection in a cohort of neonates admitted to a NICU with a clinical and molecular approach. Methodology: All neonates born in the University Hospital of Botucatu Medical School admitted to the NICU for one year were included in the study. Tracheal aspirate samples were collected, as well as samples of the nasal and anal sites by using sterile swabs. The NBs were followed up until the final outcome – ICU discharge or death. Isolated mircroorganisms were submitted to identification and were tested for antimicrobial drug susceptibility in order to determine the minimum inhibitory concentration (MIC) by the E-test. The type of staphylococcal cassette chromosome mec (SCCmec) was determined among the methicillin-resistant Staphylococcus spp. For the research on prevalent clones in the unit, the characterization of clusters was performed by pulsed-field gel electrophoresis (PFGE). Results: The results showed higher incidence of infection (27.8%) and colonization by Staphylococcus epidermidis mainly in the nasal mucosa (56.4%). The colonization by Serratia marcescens was predominant in the anal mucosa (26.8%). The results showed S. epidermidis resistant to trimethoprim-sulfamethoxazole, and S. epidermidis and Enterococcus faecalis resistant to quinopristin-dalfopristin. From the 402 samples of Staphylococcus spp. studied, 204 (50.7%) had the mecA gene, more frequently isolated from the nasal mucosa. There were 47 isolates harboring SCCmec type I, 12 carrying SCCmec type II, 77 carrying SCCmec type III, and 43 harboring SCCmec type IV. Molecular typing to determine the clusters by the PFGE technique demonstrated the presence of isolates of different NBs with either identical or high similarity profiles, which suggests cross-contamination. In addition, isolates from the same NB but of different sites also presented as identical, proving that the microorganism that colonized the NB at the time of collection was also the infectious agent. The statistical analysis revealed that the colonization process can be considered a risk factor for infection with a three times greater risk compared to non-colonized NBs. Among the risk factors for colonization, the use of parenteral nutrition and peripherally-inserted central catheter (PICC) increased the risk six times and four times, respectively. Conclusion: The findings of this study can assist in the appropriate antimicrobial choice as the colonization process occurs after the birth and the empirical therapy is often required. Knowing the microbiological profile of the unit allows to create proper antimicrobial protocols for preventing and treating healthcare-associated infections (HCAIs) aiming to improve the quality of the care provided. To this end, the implementation of surveillance cultures which help the HCAI control commission as well as the care team to develop the measures to be adopted is very important. / FAPESP: 2013/12482-0

Introdução: a infecção hospitalar tem se tornado um problema de saúde pública, no Brasil e na maioria dos países do mundo, sobretudo devido ao aumento gradativo da resistência dos microrganismos aos antimicrobianos. Nos pacientes que apresentam deficiências no sistema imunológigo como os indivíduos que vivem com o vírus da imunodeficiência humana (HIV) ou com a síndrome da imunodeficiência adquirida (aids) o quadro clínico dessas infecções pode se tornar extremamente grave, aumentando a morbimortalidade. Objetivo: determinar a prevalência de microorganismos gram-negativos em indivíduos com HIV/aids internados num hospital escola do interior paulista. Material e Método: trata-se de um estudo de corte transversal, aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto da Universidade de São Paulo. No período de 01 agosto de 2011 a 28 de fevereiro de 2013, foram abordados 365 indivíduos internados em duas unidades especializadas de um hospital escola público do interior paulista, sendo a população do presente estudo composta por 220 sujeitos. Os dados sociodemográficos, clínicos e hábitos de saúde foram obtidos por meio de entrevista individual e consulta aos prontuários. Coletou-se também amostras de saliva e swab nasal nas primeiras 24 horas de internação, as quais foram processadas pelo Laboratório de Microbiologia do referido hospital. Os dados foram inicialmente digitados em planilha do Microsoft Office Excel for Windows 2011, realizada dupla digitação e validação dos dados, a fim de identificar possíveis erros de digitação. Posteriormente, a planilha definitva foi transportada para o programa Statistical Package for the Social Science (SPSS), versão 17.0 for Windows, onde foi estruturado o banco de dados e realizada análise estatística. Resultados: a prevalência de microorganismos gram negativos nos indivíduos com HIV/aids foi de 15,4% independente do sítio onde foi isolado. Pseudomonas aeruginosa foi o micoorganismo mais frequentemente isolado tanto na saliva (50%), quanto no swab nasal (37,5%), seguida por Klebsiella pneumoniae (30,7%) isolada somente na saliva. Em relação aos aspectos clínicos 29,4% dos indivíduos com amostras positivas para microorganismos gram negativos tinham carga viral acima de 1000.000 cópias,ml, CD4 menor que 200 céluas/mm3 (50%), tiveram internações prévias (52,9%), estavam em uso de antimicrobiano (64,7%), não usavam antirretrovirais (52,9%) e tinham algum procedimento invasivo no momento da coleta (67,6%). Nenhum microorganismo apresentou resistência aos antimicrobianos. Conclusão: a prevalência de microorganismos gram-negativos foi maior na saliva (11,8%) que no swab nasal (3,6%), indicando que coletar amostras de mais de um sítio pode favorecer a identificação de indivíduos colonizados e ou infectados / Introduction: the hospital infection has become a public health problem in Brazil and in most countries of the world, mainly due to the gradual increase of resistance of microorganisms to antibiotics. In patients who have deficiencies in the immune system such as individuals living with human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS), the clinical picture of these infections can become very serious, increasing morbidity and mortality. Objective: to determine the prevalence of gram-negative microorganisms in individuals with HIV/AIDS hospitalized at a teaching hospital in the interior of São Paulo state. Material and Method: this is a cross-sectional study, approved by the Ethics Research Committee of the University of São Paulo at Ribeirão Preto College of Nursing. In the period from August 01, 2011 to February 28, 2013, 365 individuals hospitalized in two specialized units of a public teaching hospital in the interior of São Paulo state were approached, and the study population was comprised of 220 subjects. The sociodemographic and clinical data and health habits were obtained through individual interviews and medical records. Saliva samples and nasal swabs were collected in the first 24 hours of admission, which were processed by the Microbiology Laboratory of the hospital. The instrument variables were coded and cataloged in a dictionary (codebook). The data were initially recorded in a Microsoft Office Excel spreadsheet for Windows 2011, performed a double entry and data validation in order to identify possible typing errors. Subsequently, the final worksheet was transported to the Statistical Package for Social Science (SPSS), version 17.0 for Windows, in which database was structured and statistical analysis was performed. Results: the prevalence of gram-negative microorganisms in individuals with HIV/AIDS was 15.4% regardless of where it was isolated. Pseudomonas aeruginosa is the most frequently isolated microorganisms both in saliva (50%) and in nasal swabs (37.5%), followed by Klebsiella pneumoniae (30.7%) isolated only in saliva. In regard to clinical aspects, 29.4% of individuals with positive samples for gram-negative microorganisms had viral load above 1000,000 copies/ml, CD4 less than 200 cells/mm3 (50%), had previous hospitalizations (52.9%), were using antimicrobials (64.7%), did not use antiretroviral drugs (52.9%) and had some invasive procedure at the time of collection (67.6%). No microorganism was resistant to antimicrobials. Conclusion: the prevalence of gram-negative microorganisms was higher in saliva (11.8%) than in nasal swabs (3.6%), indicating that collecting samples from more than one location may facilitate the identification of individuals colonized and/or infected

As Stenotrophomonas são comumente encontradas no trato respiratório de pacientes com doenças pulmonares crônicas e também na rizosfera de plantas. Esse gênero apresenta resistência a diversos antibióticos, promove o crescimento de plantas e algumas espécies apresentam a capacidade de fixar o nitrogênio atmosférico. O Multi Locus Sequence Analysis (MLSA) é uma metodologia baseada em genes constitutivos para definição e alocação taxonômica de novas espécies. O objetivo geral do presente trabalho foi caracterizar taxonomicamente uma coleção ampla de Stenotrophomonas composta por isolados endófitos, linhagens-tipo e de referência. Para tanto, foi estabelecido um sistema de classificação e identificação de Stenotrophomonas por meio de MLSA. Foi possível através da metodologia de MLSA definir 9 novas espécies, detectar a presença de um novo gênero e estabelecer um sistema online de taxonomia para Stenotrophomonas. / The genus Stenotrophomonas is found in the respiratory treatment of patients with chronic pulmonary and also in the rizhosfera of plants. It presents resistance to several antibiotics, promotes the growth of plants and some species present the ability to fix atmospheric nitrogen. The Multi Locus Sequence Analysis (MLSA) is a methodology based on constitutive genes for definition and taxonomic allocation of new species. The general objective of the present work was to characterize a wide collection constituted by Stenotrophomonas from isolated endophytic, type and reference strains. In such a way, a system of classification and identification of Stenotrophomonas by means of MLSA was established. It was possible through the MLSA methodology to define 9 new species, to detect the presence of a new genus and to establish an online system for Stenotrophomonas taxonomy.

Quarenta e nove amostras EPEC típica (tEPEC) e atípica (aEPEC) pertencentes a diferentes sorotipos, isoladas de humanos e animais (cães, gatos, bovinos, ovinos, coelhos e sagüis) foram investigadas quanto ao perfil de virulência pela PCR e similaridade clonal por Multilocus Sequence Typing (MLST) e Pulsed-Field Gel Electrophoresis (PFGE). O objetivo deste estudo foi verificar se animais atuam como reservatório e fonte infecção de aEPEC para humanos. Os marcadores de virulência analisados revelaram que cepas aEPEC isoladas de animais possuem potencial para causar diarréia em humanos. As técnicas MLST e PFGE revelaram que amostras isoladas de animais e humanos compartilham relações clonais próximas ou idênticas. Estes resultados indicam que os animais estudados atuam como reservatório de aEPEC e representam fonte de infecção para humanos. Pelo fato de humanos, também atuarem como reservatório de aEPEC, ciclos de infecção cruzada animal-humano não podem ser descartados, pois a dinâmica de transmissão entre reservatórios de aEPEC não é muito bem compreendida. / Forty-nine typical and atypical EPEC strains belonging to different serotypes, isolated from humans, pets (cats and dogs), farm (bovines, sheep and rabbits) and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. Close clonal relationship between human and animal isolates was found with MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out, since the transmission dynamics between the reservoirs are not yet clearly understood.

Methylobacterium mesophilicum SR1.6/6 é uma bactéria endofítica isolada de ramo de citros previamente esterilizado superficialmente. Esta bactéria possui a capacidade de associar-se com uma ampla variedade de espécies e tecidos de plantas, principalmente nas raízes, mediado pela formação de biofilme e superfícies do hipocótilo de plântulas in vitro. Este trabalho teve como objetivo o desenvolvimento e a aplicação de um modelo para o estudo in vitro da associação entre esta bactéria e plântulas de soja. Metodologias de genômica e transcriptômica foram aplicadas para a obtenção do draft genômico desta bactéria e de um amplo perfil de sequências expressas, obtidas em dois tratamentos distintos; i) células de biofilme células bacterianas aderidas às raízes das plântulas removidas por sonicação, e ii) células planctônicas células bacterianas em suspensão (i.e. interagindo somente com exsudados radiculares). Os dados genômicos obtidos por 454-pirosequenciamento resultaram em uma cobertura de 37 vezes o tamanho do genoma e geraram 242 contigs. Entre estes, 187 contigs grandes representaram 96% do genoma (tamanho estimado de 6.8 Mb), com um conteúdo GC de 69.5%. Considerando a análise da expressão gênica, um procedimento para monitorar a aderência das células bacterianas às raízes das plântulas foi realizado por meio de microscopia eletrônica de varredura de amostras de raízes coletadas durante o experimento (i.e. 24, 48 e 72h após a inoculação). As células bacterianas obtidas em cada tratamento foram inicialmente submetidas à extração de RNA total, seguida de um processo de enriquecimento de mRNA e sequenciamento por meio da tecnologia de 454-pirosequenciamento (RNA-Seq). O amplo perfil de expressão gênica obtido foi mapeado no draft genômico, resultando em um total de 1.930 clusters gênicos. Posteriormente, estes clusters foram filtrados de acordo com sua abundância e ocorrência diferencial em cada tratamento, resultando em 280 genes diferencialmente expressos. Funções relacionadas ao metabolismo de etanol/metanol, divisão celular, resposta ao estresse oxidativo, produção de sideróforos, biossíntese de peptidoglicanos e hopanóides foram induzidas nas células bacterianas aderidas às raízes das plântulas, enquanto que genes relacionados ao metabolismo essencial das células foram observados principalmente nos tratamentos controle e planctônico. Estes dados fornecem uma base para estudos relacionados a mecanismos moduladores da interação bactériaplanta, distinguindo significativamente os tratamentos biofilme e planctônico, mostrando assim que o contato físico é essencial para o sucesso da interação em estudo. Por fim, estas análises permitiram uma ampla visualização de perfis de expressão gênica desta bactéria, utilizando o draft genômico primeiramente obtido como base para o estudo desta interação com a planta hospedeira. Estudos futuros podem ser desenvolvidos visando caracterizar os mecanismos adaptativos desta bactéria, como seu metabolismo metilotrófico e outros metabolismos específicos, os quais podem dar suporte ao comportamento endofítico deste organismo. / Methylobacterium mesophilicum strain SR1.6/6 is an endophytic bacterium, which has originally been isolated from surface-sterilized healthy citrus branch. This bacterium is able to associate with a range of plant species, rather in the roots, mediated by a biofilm structure, and in hypocotyl surfaces of in vitro seedlings. The aim of the present study was the development and application of a model to study in vitro the association between this bacterium and soybean seedlings. Genomic and transcriptomic approaches were applied resulting a draft of this bacterium genome and a broad profile of mapped mRNA sequences obtained in two different treatments; i) biofilm cells root adhered bacterial cells were removed by sonication, and ii) planktonic cells bacteria cells in suspension (i.e. interacting only with root exudates). Genomic data, obtained by 454-pyrosequencing have had an average depth of 37-fold coverage of the genome and yielded 242 contigs. Among these, 187 large contigs represented 96% of the genome sequence (estimate size of 6.8 Mb) with a GC content of 69.5%. Concerning the gene expression survey, the process to monitor the adherence of bacteria cells to the roots was performed by scanning electron microscopy of roots collected along the experiment (i.e. 24, 48 and 72 hours after inoculation). Bacterial cells obtained in each treatment were firstly submitted to RNA extraction, followed by mRNA enrichment and RNA-Seq using 454-pyrosequencing technology. The broad gene expression profile obtained was mapped into the drafted genome, resulting in a total of 1.930 gene clusters. After that, these clusters were filtered according to their abundance and differential occurrence in each treatment resulting in 280 differential expressed genes. Functions related to methanol/etanol metabolism, cell division, oxidative stress response, siderophore production, peptidoglycan and hopanoid biosynthesis were induced in bacterial cells adhered to plant roots, while genes related to essential cell metabolism were observed mostly in control and planktonic treatment. Also, these data provide insights into the mechanisms modulating plant microbe-interaction, significantly distinguishing biofilm and planktonic treatment, showing that the physical contact is a crucial step on plant-microbe interactions. In conclusion, results allowed a strongly supported analysis of gene expression, based on the genome draft of an endophytic bacterium interacting with the host plant. Further studies should focus on the adaptive mechanisms present in this bacterium, like the methylotrophic lifestyle and other specific metabolisms which might support its behavior as an endophytic bacterium.

A bacteria Pectobacterium carotovora causa danos a diferentes hospedeiros por meio da produção de enzimas pectinolíticas que degradam o pectato de cálcio da lamela media próximo a parede celular, causando extravasamento do conteúdo celular, sintomas da podridão mole. Bactérias do gênero Burkholderia tem se mostrado capazes em controlar a podridão mole em orquídeas, no entanto, os aspectos moleculares envolvidos neste controle ainda não foram estudados. Neste trabalho, foram avaliados 602 transformantes quanto a sua habilidade em inibir os sintomas da podridão mole, onde foram observados 16 mutantes defectivos no controle da doença. Destes, foram identificados sete diferentes genes inativados pelo transposon Tn5, e estes genes podem estar envolvidos em processos de síntese de aleloquímicos, competição por nutrientes, adaptação a condições ambientais, e na interação com o hospedeiro e/ou entre microrganismos. No entanto, o envolvimento destes genes na perda da capacidade em controlar a podridão mole deve ser melhor estudado. / The bacterium Pectobacterium carotovora cause damage to different hosts and by production of pectic enzymes that degrade calcium pectate of the middle lamella near of the cell wall, causing overflow of cell content and consequently the soft rot. Burkholderia genus has proven able to control the soft rot in Orchids, however, the molecular aspects involved in the control have not been studied. In this work, 602 transformants were characterized for their ability to inhibit soft rot caused by P. carotovora. We identified 16 mutants showing shifts in inhibition pattern or lost of the ablitity to inhibit soft rot symptoms. Among these mutants, we identified 7 genes related to disease inhibition,and this genes may be involved in process of allelochemicals synthesis, competition for nutrients, adapting to environmental conditions, and interaction between the host and microorganisms. However, the involvement of these genes in loss of ability to control the soft rot disease is being further studied in details.