A emergência de resistência à vancomicina no mundo iniciou-se no final dos anos 80, sendo primeiro documentada na parte ocidental da Europa e posteriormente nos EUA. Depois disso, o isolamento de enterococos resistentes à vancomicina (VRE - do inglês, vancomycin-resistant enterococci) tem sido continuamente reportado em diversas localizações geográficas, inclusive no Brasil. As infecções nosocomiais causadas por enterococos são grandes desafios para os médicos devido à ocorrência de isolados resistentes a múltiplos antibióticos. Diversos métodos de tipagem molecular têm sido utilizados para estudar a epidemiologia de VRE. Neste trabalho, foi realizada a caracterização genética da resistência à vancomicina e epidemiologia molecular de VREs isolados de pacientes de dois hospitais de Ribeirão Preto, o Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP-USP) e a Santa Casa de Misericórdia de Ribeirão Preto (SCMRP), no período de setembro de 2008 a setembro de 2010. Foram estudados também os primeiros VREs isolados no HCFMRP-USP, em meados de 2005/2006. Foram determinadas as espécies e os genótipos de 53 VREs, pela reação da polimerase em cadeia (PCR), e todos eram E. faecium e apresentavam o gene vanA. Todos E. faecium isolados dos 2 hospitais em estudo apresentaram CIM para vancomicina >256?g/mL pelo Etest®, já daptomicina mostrou valores de CIM dentro do limite de sensibilidade para todos os enterococos analisados. Com relação aos fatores de virulência, foi evidente a predominância dos genes acm e esp nos E. faecium estudados. Os primeiros VREfm (do inglês, vancomycin-resistant E. faecium) isolados em meados de 2005/2006 de pacientes do HCFMRP-USP apresentaram o transposon Tn1546 intacto, já todos os E. faecium isolados do HCFMRP-USP e da SCMRP, no período de setembro de 2008 a setembro de 2010, apresentaram produto de amplificação maior do que os 4,4kb esperados para grupamento gênico vanRSHAX. Resultados de overlapping PCR e sequenciamento da região do transposon que amplificou fragmento de DNA com tamanho maior que o esperado utilizando primers P11-P12, mostraram que 81% (43 VREfm) isolados nos dois hospitais em estudo, no período de setembro de 2008 a setembro de 2010, apresentaram deleção da extremidade esquerda do Tn1546 e insersão (IS1251), entre genes vanS e vanH. A análise da PFGE dos VREfm em estudo mostrou que havia ocorrido disseminações clonais com determinados perfis de PFGE em períodos específicos. O fato dos primeiros VREfm terem apresentado perfil de PFGE diferente da maioria dos isolados, não permitiu que se determinasse o ancestral comum por PFGE, mas resultados de MLST mostraram que VREfm isolados, no período de 2008-2010, podem ser descendentes diretos destes cinco primeiros VREfm ou podem ter evoluído de um mesmo ancestral comum. A análise da tipagem por MLST de 31 linhagens de VREfm selecionadas a partir dos resultados de PFGE, mostrou que havia 9 STs diferentes, dentre estes, sendo 5 STs novos (656, 657, 658, 659 e 660). Os STs predominantes foram STs 412 e 478. Todos STs identificados pertenciam ao complexo clonal 17 (CC17), com exceção do ST658 que era um singleton. O ST78 que vem se disseminando mundialmente, foi identificado pela primeira vez no Brasil em linhagens do presente estudo. E, por fim, a tipagem por MLST comprovou o que já havia sido determinado pelos resultados de PFGE, que existe relação clonal entre linhagens isoladas nos dois hospitais estudados de Ribeirão Preto. / World reports on vancomycin resistance emerged in the late 1980s and first documented in Western Europe and later in the United States. Since then, isolation of vancomycin-resistant enterococci (VRE) has been continuously reported in several geographic locations, including Brazil. The widespread occurrence of strains resistant to multiple antibiotics present a challenge to clinicians when treating enterococci caused nosocomial infections. Several molecular typing methods have been used to study the epidemiology of VRE. In this study, genetic characterization of vancomycin resistance and molecular epidemiology were performed in VREs isolated from patients in two hospitals in Ribeirão Preto, Sao Paulo/Brazil, the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (HCFMRP-USP) and the Santa Casa de Misericórdia de Ribeirão Preto (SCMRP), from September 2008 to September 2010. The first five VREs isolated in the HCFMRP-USP, in mid 2005/2006 were also included in this study. Species and genotypes of 53 VREs determined by the polymerase chain reaction (PCR) were all E. faecium and had the vanA gene. MICs for vancomycin determined by Etest® were >256?g/mL for all E. faecium isolated in the two hospitals, whereas daptomycin showed MIC values within the limit of susceptibility for all the enterococci analyzed. acm and esp genes predominated as virulence factors. The first five vancomycin resistant E. faecium (VREfm) isolated in mid 2005/2006 showed an intact Tn1546 transposon, whereas all E. faecium isolated later, September 2008 to September 2010, gave a larger amplified DNA fragment than the expected 4,4kb gene cluster vanRSHAX. Results of overlapping PCR and sequencing (primers P11-P12) of the transposon region that amplified the larger than expected DNA fragment showed that 81% (n=43) of the these VREfm had a deletion of the left end of Tn1546 and also a IS1251, between the vanS and vanH genes. The analysis of VREfm PFGEs indicated the occurrence of clonal disseminations with some PFGE profiles at specific times. The different PFGE profiles of the first VREfm (2005-2006) in relation to the latter isolates showed that the common ancestor could not be determined by PFGE. However, MLST results indicated that VREfm isolated, in the period from 2008 to 2010, could be direct descendants of the first five VREfm or could have evolved from a common ancestor. MLST analysis of 31 VREfm isolates selected according to the PFGE results, showed that there were nine different STs, among these five new STs (656, 657, 658, 659, 660). The predominant STs were ST412 and 478. All STs identified belonged to clonal complex 17 (CC17), except for ST658, a singleton. The ST78 that has spread worldwide is being identified in Brazil for first time in this study. MLST confirmed PFGE results showing that there is a clonal link between strains isolated in the two hospitals of Ribeirao Preto, Sao Paulo, Brazil.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus pneumoniae é um importante agente etiológico de infecções invasivas e não invasivas, incluindo meningite, pneumonia e otite média. A cápsula polissacarídica é o principal fator de virulência desse microrganismo, sendo também considerada um importante marcador em estudos epidemiológicos. Dentre os mais de 90 tipos capsulares conhecidos, o sorotipo 14 se destaca pela prevalência elevada em várias regiões, inclusive no Brasil. A avaliação da diversidade genética desse microrganismo também inclui a aplicação de métodos moleculares, como PFGE e MLST. Entretanto, essas metodologias são relativamente onerosas, consomem muito tempo e os resultados obtidos com a técnica de PFGE são de difícil comparação entre diferentes laboratórios. A técnica de análise do polimorfismo numérico de segmentos repetitivos em múltiplos loci [MLVA, do inglês Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis] se apresenta como uma alternativa, embora ainda necessite de padronização e avaliação mais ampla para a espécie em questão. No presente estudo, 60 amostras de Streptococcus pneumoniae pertencentes ao sorotipo 14, isoladas de diversas fontes clínicas, em diferentes locais e períodos de tempo, foram caracterizadas pelas técnicas de MLVA (baseada na análise de 18 loci distintos), MLST, PFGE e tipagem do gene pspA. O gene pspA2 predominou entre as amostras analisadas, seguido pelo gene pspA1. Os tipos de MLVA, perfis de PFGE, e STs encontrados apresentaram resultados, em geral, concordantes, indicando o elevado poder discriminatório da versão da técnica de MLVA empregada. Cinco complexos clonais (CC) de MLVA e cinco singletons puderam ser definidos. O CC de MLVA denominado de L7 foi o predominante, compreendendo 36,7% da amostragem estudada. O CC L7 mostrou-se relacionado com genes pspA da família 2, com o CC1 de MLST, com o CC Pen14-H de PFGE, e com a não susceptibilidade à penicilina, Entre os complexos clonais de MLST, o CC1 foi o prevalente e incluiu predominantemente o ST156, pertencente ao clone internacional Spain9V-3. O CC L3 e o singleton L17 de MLVA apresentaram-se associados ao CC de PFGE Eri14-A, a família 1 de PspA e ao CC2 de MLST, que por sua vez também estava relacionado com o clone internacional England14-9. O CC L15 de MLVA esteve associado ao CC de PFGE Pen14-A, ao gene pspA2, aos CC3 e CC4 de MLST e ao clone internacional do PMEN Tennessee14-18. A técnica de MLVA revelou-se significativamente mais discriminatória que as técnicas de PFGE e MLST, conforme exemplificado pela detecção de 21 perfis de MLVA, 13 perfis de PFGE e cinco STs, entre as 22 amostras pertencentes ao CC de MLVA L7. Uma versão de MLVA, compreendendo um painel com os oito loci de maior poder discriminatório, pôde ser proposta a partir da análise dos resultados obtidos. Estes aspectos, aliados ao menor tempo e custo de execução, indicam que a técnica de MLVA constitui uma alternativa importante e satisfatória para uso em estudos sobre a diversidade genética de S. pneumoniae. / Streptococcus pneumoniae is a major pathogen causing invasive and non-invasive diseases in humans, including meningitis, pneumonia and otitis media. The polysaccharide capsule of this microorganism is considered a major virulence factor and an important marker for epidemiological studies. More than 90 pneumococcal capsular serotypes are recognized, and serotype 14 is highly prevalent in many regions, including Brazil. Genotyping methods, such as PFGE and MLST, are essential to evaluate genetic diversity of this bacterium. However, these methods are expensive, time-consuming and results from different laboratories are difficult to compare. Multiple Loci VTNR (Variable-Number Tandem Repeat) Analysis (MLVA) appears as an alternative, despite the fact that standardization and wide evaluation for application to this species is still required. In the present study, a total of 60 S. pneumoniae isolates belonging to serotype 14, isolated from different sources, regions and periods of time, were analyzed by MLVA (based on the analysis of 18 distinct loci), MLST, PFGE and pspA typing methods. Gene pspA2 was the predominant, followed by pspA1. Overall, the results of PFGE, MLST and MLVA typing were congruent, and indicated the discriminatory power of the MLVA method used. Five clonal complexes (CC) and five singletons were identified by MLVA. CC L7 was the predominant MLVA CC, comprising 36.7% of all the isolates. L7 was associated with pspA2 gene and non-susceptibility to penicillin, and it was related to MLST CC1, and to PFGE Pen14-H. CC1 was the prevalent MLST CC and included mostly ST156 that belongs to international clone (IC) Spain9V-3. Another MLVA CC, named L3, and the singleton L17 were related to PFGE CC Eri14-A, MLST CC2, and IC England14-9. MLVA CC L15 was related to PFGE Pen14-A, MLST CC3 and CC4, and IC Tennessee14-18. MLVA was found to be more discriminatory than PFGE and MLST, as exemplified by detection of 21 MLVA types, 13 PFGE profiles and 5 STs among the 22 strains belonging to L7, the predominant MLVA CC. A modified version of the MLVA method, based on the analysis of 8 loci only, is proposed. This aspect, in conjunction with reducing time and costs, indicate that MLVA represents an important and satisfactory alternative method to evaluate the genetic diversity of S. pneumoniae.

To understand transmissible human diseases, disciplines such as epidemiology and the surveillance of affected cases are as essential as the knowledge about the pathogenesis and the course of a disease. Epidemiologists categorize and estimate factors for public health risks by taking metadata into account including geographic aspects, health and social states to study a disease transmission and prevent further cases. In addition, a focus on the causative agents itself is necessary in order to understand their ecology and hence their virulence traits. The causative agents for a severe pneumonia named Legionnaires’ disease (LD) are bacteria of the genus Legionella. The putative sources of LD infection are any aerosol-generating natural or man-made fresh water systems. Due to this ubiquitous distribution of legionellae, it is difficult to find the source of infection. Therefore, it is necessary to isolate the bacterium from the suffering patients to further characterize it in the laboratory and to compare the clinical isolates with isolates obtained from probable environmental sources. The predominant species isolated from LD patients is Legionella pneumophila serogroup (Sg) 1. Intensive genotyping of L. pneumophila Sg1 isolates by using the current gold standard method, the sequence-based typing scheme (SBT), revealed limitations in the discrimination of several sequence types (ST) which could not be compensated for by additional phenotypic typing scheme. In practical terms, this means that several clones or STs are disproportional frequently found in both, patients and water systems, and cannot be distinguished by current methods. Therefore, a distorted picture of endemic and globally-spread clones is generated and current typing methods cannot add substantial information during the identification of the infectious source. The aim of this thesis is to develop and implement new typing methods for L. pneumophila isolates with a higher resolution than the gold standard methods. A DNA-DNA hybridization based microarray was designed and equipped with probes that target specifically L. pneumophila virulence factors and genes that are involved in the biosynthesis of lipopolysaccharide structures. Legionellae can be subgrouped on the basis of their lipopolysaccharide structures. Here, the usually phenotypic characterization of L. pneumophila Sg1 is successfully transmitted to a DNA-based genotypic method. Furthermore, the detailed validation of the DNA-microarray revealed a higher discriminatory power in comparison to the gold standard methods. It enables previously indistinguishable clones to be subdivided, providing valuable information about probable sources of infection. The second new tool for typing of L. pneumophila is based on the core genome of the bacteria. An extended SBT-scheme was extracted from the core genome and accordingly named core genome multilocus sequence typing (cgMLST). This genome wide gene-by-gene typing approach allows a high genomic resolution of L. pneumophila isolates by retaining epidemiological concordance. A major advantage of this genome-based method is the detection of large recombination events within the analysed genomes, which is, so far, reserved for whole genome sequencing. The population structure of legionellae is largely driven by recombination and horizontal gene transfer rather than by spontaneous mutations. Therefore, the detection of recombination events is essential for typing of L. pneumophila isolates. In addition, the cgMLST-scheme assigns a core genome sequence type to the analysed isolate and allows backwards compatibility with the current SBT-scheme. Both methods proved to be fast, reliable and robust typing methods through their application during outbreak investigations. Furthermore, both systems are particularly suited as routine molecular typing tools for the surveillance of single cases. The raw data are verified and translated into uniform portable codes, which enables the easy transfer and comparison of results. The standardized and portable quality of the results of both methods enables the establishment of a curated global database. This qualifies both methods as potential new gold standard methods for the genotyping of L. pneumophila isolates.

S. pneumoniae coloniza a nasofaringe e é um dos principais agente de otite média, pneumonia, bacteremia e meningite com altas taxas de morbidade e mortalidade. Estima-se que 1,6 milhões de pessoas morram de doença pneumocócica por ano, a maioria crianças menores de cinco anos de idade, principalmente em países em desenvolvimento. A cápsula polissarídica antifagocitária é o principal fator de virulência deste microrganismo e determina os 93 sorotipos conhecidos, sendo o alvo de vacinas pneumocócicas. No presente trabalho foi padronizada a tipagem molecular por Multiplex PCR de S. pneumoniae, que compreende 30 pares de iniciadores agrupados em seis reações sequenciais. Foram tipadas 270 cepas de pneumococo isoladas entre janeiro de 2005 a setembro de 2011, proveniente de líquor (13%), sangue (76%) e líquido pleural (11%) de 232 pacientes atendidos no Hospital Universitário da USP. Além disso, a caraterização dessas amostras quanto ao perfil de sensibilidade aos antimicrobianos e à diversidade foi realizada, segundo o CLSI 2011 e a genotipagem molecular pelas técnicas de Multilocus Sequencie Typing Scheme (MLST) e Pulsed Field Eletrophoresis Gel (PFGE), respectivamente. A tipagem por Multiplex PCR detectou 24 sorotipos/sorogrupos diferentes, que foram: 14 (22%), 5 (12%), 12F/A (11%), 6A/B/C (10%), 7F/A (5%), 1 (4%), 3 (4%), 10A (4%), 19A (4%), 18 A/B/C/F (3%), 4 (3%), 8 (3%), 23F (3%), 19F (3%) e outros (9%) (9V/A, 9N/L, 15A, 22F, 11A/D, 31, 38, 34, 16F, 17F e não tipável). Este método apresentou 100% de especificidade e 98% de sensibilidade para determinação de sorogrupos e 66% para sorotipos. Os sorotipos 14, 6B, 5 e 19F foram significativamente mais comuns em criança até dois anos, já entre adultos, os sorotipos 5 e 12F foram os predominantes. O perfil de sensibilidade em infecções não meníngeas foi de 99% de sensibilidade e 1% de resistência intermediária para penicilina e ceftriaxona. Para infecções meníngeas os resultados mostraram 73% de sensibilidade e 27% de resistência para penicilina e 88% de sensibilidade e 12% de resistência intermediária para ceftriaxona. A resistência aos beta-lactâmicos está ligada principalmente ao sorotipo 14 que foi o sorotipo mais isolado com 52 cepas e dessas foram realizados MLST e PFGE. No MLST encontramos 51 cepas pertencentes ao clone Spain9V-3 (ST 156) que é predominante na região sul e sudeste do Brasil e uma cepa com um tipo de sequência ainda não depositada. Pela técnica de PFGE foram detectados três clusters e quatro amostras não relacionadas, o cluster A foi predominante com 41(79%) cepas com 81,7% de similaridade entre elas. A técnica de Multiplex PCR demonstrou ser excelente ferramenta para a detecção dos sorotipos/sorogrupos de S. pneumoniae. Não foi detectada resistência plena à penicilina e ceftriaxona em infecções não meníngeas consolidando a importância do uso da penicilina no tratamento da doença pneumocócica não meníngea. Houve grande similaridade genética entre cepas de S. pneumoniae sorotipo 14. / S.pneumoniae colonizes the nasopharynx and is a major agent of otitis media, pneumonia, bacteremia and meningitis with high morbidity and mortality. It is estimated that 1.6 million people die of pneumococcal disease every year, mostly children under five years old, mainly in developing countries. The antiphagocytic polissarídica capsule is the main virulence factor of this organism and determine the 93 serotypes known for being the target of pneumococcal vaccines. In the present study was standardized molecular typing by Multiplex PCR molecular typing, which comprises 30 primer pairs grouped into six sequential reactions. We performed antimicrobial susceptibility profile, according to the CLSI 2011 and the most frequent serotype was made by molecular genotyping techniques Multilocus Sequence Typing (MLST) and pulsed-field gel Eletrophoresis (PFGE). We studied 270 pneumococcal strains isolated from 2005 to September 2011, from CSF (13%), blood (76%) and pleural fluid (11%) of 232 patients attended at University Hospital of USP. Typing by Multiplex PCR detected 24 serotypes / serogroups different, which were: 14 (22%), 5 (12%), 12F / A (11%), 6A/B/C (10%), 7F / A (5 %), 1 (4%), 3 (4%), 10A (4%), 19A (4%), 18 A / B / C / F (3%), 4 (3%), 8 (3% ), 23F (3%), 19F (3%) and others (9%) (9V / A, 9N / L, 15A, 22F, 11A / D, 31, 38, 34, 16F, 17F and nontypable). This method showed 100% specificity and 98% sensitivity for the determination of 66% for serogroups and serotypes. Serotypes significantly more common in children under two years were: 14, 6B, 5 and 19F among adults serotypes 5 e12F were predominant. The sensitivity profile in non-meningeal infections was 99% sensitivity and 1% penicillin intermediate resistance to ceftriaxone. For meningeal infections the results showed 73% sensitivity and 27% resistance to penicillin and 88% sensitivity and 12% intermediate resistance to ceftriaxone. Resistance to beta-lactams is linked mainly to serotype 14 was the serotype most isolated, and of these 52 strains were performed MLST and PFGE. MLST found in 51 strains belonging to clone Spain9V-3 (ST 156) which is prevalent in south and southeastern Brazil and a strain with a type of sequence is not deposited. The technique of PFGE found three clusters and four non-related samples, cluster A predominated with 41 (79%) strains with 81.7% similarity between them. Multiplex PCR technique proved to be an excellent tool for the detection of serotypes/serogroups of S. pneumoniae. We did not detect full resistance to penicillin and ceftriaxone in non-meningeal infections showing the importance of use of penicillin in the treatment of pneumococcal non-meningeal disease. There was great genetic similarity among strains of S. pneumoniae serotype 14.

Haemophilus parasuis é o agente etiológico da Doença de Glässer, que causa artrite, pneumonia, meningite e poliserosite em suínos e tem assumido grande importância na suinocultura moderna, uma vez que sua ocorrência tem aumentado significativamente nos últimos anos em rebanhos afetados pelo circovirus suíno tipo 2. No presente estudo foram avaliadas 117 amostras de H. parasuis isoladas dentre os anos de 2009 a 2014, isoladas de suínos de diferentes estados do da região Centro-Sul do Brasil. As estirpes foram submetidas à sorotipificação, confirmação do gênero/espécie pela PCR, o perfil de resistência a antimicrobianos foi avaliado através da determinação da concentração inibitória mínima (CIM), foi realizada a caracterização genotípica das amostras por eletroforese em gel de campo pulsado (PFGE) e por sequenciamento de múltiplos sítios (multilocus sequence typing - MLST) e a presença de genes de virulência vtaA foi analisada. Os sorotipos mais frequentes foram: 4 (21,3%), seguido do 5 (12,9%), do 13 (9,4%), do 14 (7,7%) e do sorotipo 1 (1,7%), e em alguns casos mais de um sorotipo foi identificado na mesma granja e até no mesmo animal, resultado este parecido ao encontrado no restante do mundo. Em todas as amostras o gene vtaA estava presente, para alguns antibióticos os índices de resistência foram elevados, como para tilosina (98,29%), danofloxacina (95,72%), sulfadimetoxina (88,03%), penicilina (77,7%) e a multirrestencia atingiu o índice alarmente de 93,16% das estirpes. Foram identificados 67 perfis diferentes no PFGE e das 9 amostras analisadas pelo MLST foram identificados novos STs, até então, não descritos mundialmente. Quando os novos STs foram comparados com os previamente descritos, estas se dispersaram entre as descritas em diferentes países. Neste estudo foi possível observar que as estirpes de H. parasuis brasileiras possuem alta variabilidade, tanto nos sorotipos, perfis de resistência, análises genômicas de PFGE e MLST / Haemophilus parasuis is the etiological agent of Glässer disease that causes arthritis, pneumonia, meningitis and polyserositis in pigs and has assumed great importance in modern swine production, since its occurrence has increased significantly in recent years in herds affected by porcine circovirus type 2. In the present study 117 strains of H. parasuis isolated between 2009 to 2014 were utilized, isolated from pigs of south center of Brazil. The strains were serotyping, confirmed genus/species by PCR, the antimicrobial resistance profile was evaluated determining the minimum inhibitory concentration (MIC) and strains were genotypically characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and presence of vtA virulence gene. The major serotypes identified were 4 (21.3%), 5 (12.9%), 13 (9.4%), 14 (7.7%) and at last the 1 (1.7%), In some cases more than one serotype was identified in the same farm and in the same animal, this results were identified in others parts of the world. All samples had vtA gene. The resistance for some antibiotics was high for tylosin (98.29%), danofloxacin (95.72%) sulfadimetoxin (88.03%), and penicillin (77.7%). Multidrug resistance rates reached 93.16% of the samples. A total of 67 different profiles were identified in PFGE and nine samples were analyzed by MLST. All nine strains tested were identified as new STs. When these strains were compared with MLST database, they were dispersed among the strains from other countries. In this study, it was clear that the Brazilian H. parasuis strains are highly variable considering serotypes, resistance profiles, genomic analysis of PFGE and MLST

Enterococos são ubíquos no ambiente e fazem parte da microbiota do trato gastrintestinal de humanos e animais. A importância dessas bactérias tem sido associada com infecções hospitalares e resistência a múltiplas drogas, principalmente à vancomicina. O objetivo do presente estudo foi realizar a caracterização molecular de cepas de Enterococcus spp. resistentes à vancomicina (VRE) isoladas a partir de amostras coletadas de pacientes hospitalizados, água superficial de rios urbanos e carne de frango comercializada no Brasil. A presença do gene vanA foi confirmada em 20 cepas multirresitentes isoladas durante 1997-2011. Dentre os isolados VRE, 12 cepas foram identificadas como E. faecium e oito como E. faecalis. Cepas de E. faecium isoladas de amostras clínicas e águas foram classificadas como clonalmente relacionadas pelo PFGE, com perfil virulência predominante (acm+, esp+). Adicionalmente, enquanto cepas de E. faecium isoladas dos rios pertenceram aos ST203, ST412 e ST478 (previamente caracterizados como endêmicos em hospitais brasileiros), novos STs foram identificados entre as cepas de E. faecalis (ST614, ST615 e ST616) e E. faecium (ST953 e ST954) isoladas de alimentos. Sequências completas do transposon Tn1546 das cepas clínicas VREfm 320/07 (ST478) e ambiental VREfm 11 (ST412) mostraram Tn1546-like element de ~12800 pb, com um ponto de mutação no gene vanA na posição 7.698 (substituição do nucleotídeo T pelo C) e uma no gene vanX na posição 8.234 (G pelo T). Além disso, uma deleção na extremidade esquerda do Tn1546, e as sequências IS1251 e IS1216E na região intergênica vanHS e vanYX, respectivamente, também foram detectados. A este respeito, a IS1216E na região intergênica vanXY constitui um conjunto de genes previamente relatado em cepas clínicas de VREfm no Brasil, denotando uma característica regional. IS1216E tem sido associada com os genes tcrB e aadE que conferem resistência ao cobre e aminoglicosídeos, em E. faecium e Streptococcus agalactiae, respectivamente. Portanto, essa IS pode contribuir para a rápida aquisição de resistência antimicrobiana entre as espécies de cocos Gram-positivos clinicamente importantes. Os tipos de Tn1546 indistiguíveis que foram identificados no atual estudo isolados de humano e ambientes aquáticos sugerem uma comum partilha de um pool de genes de resistência à vancomicina. / Enterococci are ubiquitous in the environment and in the intestinal tract of humans and animals. The importance of these bacteria has been associated with nosocomial infection and multiple resistance to antimicrobial agents, mainly vancomycin. The aim of the present study was to perform molecular characterization of vancomycin-resistant Enterococcus spp. strains (VRE) isolated from hospitalized patients, surface water of urban rivers and retail chicken meat in Brazil. The presence of the vanA gene was confirmed in 20 multidrug-resistant strains isolated in 1997-2011. Among these VRE isolates, (n = 12) were identified as E. faecium and (n = 8) as E. faecalis. E. faecium strains isolated from water and clinical samples were classified as clonally related by PFGE, the predominant virulence profile being (acm+, esp+). Additionally, while E. faecium strains isolated from rivers belonging to ST203, ST412 and ST478 (previously characterized as endemic in Brazilian hospitals), new STs were identified among strains of E. faecalis (ST614, ST615 and ST616) and E. faecium (ST953 and ST954) isolated from food. Complete sequences of transposon Tn1546 from VREfm clinical strain 320/07 (ST478) and environmental strain VREfm 11 (ST412) showed a Tn1546-like element of ~12800 bp, with T7698C vanA and G8234T vanX mutations. Moreover, deletion of the Tn1546 left extremity, and the IS1251 and IS1216E sequence inside the vanHS and vanYX intergenic region, respectively, were also detected. In this regard, the IS1216E sequence inside the vanXY intergenic region constitutes a gene array previously reported for Brazilian VREfm clinical strains alone, denoting a regional characteristic. IS1216E has been associated with tcrB and aadE genes, which confer resistance to copper and aminoglycosides, in E. faecium and Streptococcus agalactiae, respectively. Therefore, IS1216E should contribute to rapid acquisition of antimicrobial resistance among species of the clinically important Gram-positive cocci. On the other hand, Tn1546-like elements were identical among clinical and environmental VREfm isolates, suggesting sharing of a common vancomycin resistance gene pool.

Oenococcus oeni est l’agent principalement responsable de la fermentation malolactique durant la vinification. Son adaptation au milieu vin est une étape clé dans la réussite de la FML, et donc dans la qualité des vins finis. Cependant, au sein de l’espèce, il existe une variabilité phénotypique importante, et de nombreuses souches ne sont ainsi pas aptes à réaliser la FML. La sélection des meilleures souches œnologiques passe ainsi par l’analyse de la diversité d’O. oeni. Cette étude a été abordée dans cette thèse, selon trois grands axes de recherche. Premièrement, la diversité génétique a été étudiée par des approches MLST, REA-PFGE et présence de marqueurs, et a montré une structuration de l’espèce en deux groupes phylogénétiques et plusieurs sous-groupes, reliés à des souches d’origine géographique précise. Ensuite, l’étude de la diversité phénotypique a montré la grande variabilité d’adaptation au vin des souches étudiées et un meilleur comportement de celles du groupe phylogénétique A durant la vinification. Enfin, une étude transcriptomique a mis en lumière certains mécanismes moléculaires éventuellement impliqués dans la réponse au stress vin chez O. oeni. / Oenococcus oeni is the main agent responsible for the malolactic fermentation, during the wine making process. Its adaptation to wine environment is a key step for the success of the MLF, and then for wines quality. However, there is a high phenotypic variability among the species and several strains are unable to perform MLF. The selection of the best enological strains implies starting by analyzing the diversity of O. oeni. This study has been divided in three main themes of research. Firstly, the genetic diversity has been analyzed using several approaches, MLST, REA-PFGE and presence of genetic markers. That study proved the structuration of the species in two phylogenetic groups and several subgroups, related to geographical areas. Secondly, the study of the phenotypic diversity showed that all the studied strains present a high variability and the best behavior in wine making conditions is found in those from the phylogenetic group A. Finally, a transcriptional analysis has revealed some molecular mechanisms possibly implicated in stress response in O. oeni

Les bactéries pathogènes opportunistes (BPO) sont responsables d'une grande part de la pathologie infectieuse bactérienne. Les BPO d'origine environnementale doivent subir des changements profonds de mode de vie pour s'adapter et coloniser l'homme. Comprendre les conditions de cette adaptation permettra de préciser la notion d'opportunisme infectieux et le rôle des BPO environnementales dans l'émergence des pathogènes.Les genres Ochrobactrum et Agrobacterium regroupent des bactéries présentant une grande variété de modes de vie et établissant différentes relations avec la cellule eucaryote. Ces bactéries connues pour vivre dans l'environnement sont par ailleurs des pathogènes opportunistes de l'homme principalement responsables d'infections chez les individus immunodéprimés. Dans le cadre de ce travail nous avons entrepris une étude populationnelle par une approche de génétique multilocus sur des collections de souches cliniques et environnementales de différentes origines géographiques. Les structures de population obtenues ont été confrontées à divers caractères phénotypiques reliés à la virulence et/ou l'adaptation chez l'homme, la température de croissance, la formation de biofilm et la virulence vis-à-vis des modèles Caenorhabditis elegans et macrophages humains.Ochrobactrum anthropi et Ochrobactrum intermedium sont les deux principales espèces d'intérêt médical du genre Ochrobactrum. La population d'O. anthropi est de type épidémique qui s'organise en deux complexes clonaux (CCs). Si le CC1 regroupe à la fois des souches de diverses origines, le CC4 ne contient que des souches cliniques. Cette sous-population apparait associée à l'homme même si les caractères phénotypiques étudiés ne révèlent pas de différences entre ces deux sous populations. De la même façon, ces deux CCs ne se distinguent pas par leur comportement en modèle macrophage ou par leur diversité génomique. O. intermedium, tout comme O. anthropi, présente une forte diversité génétique toutefois, aucun regroupement des souches en fonction de leur origine n'est mis en évidence pour cette espèce. La diversité des souches cliniques apparait aussi importante que celle de l'ensemble de la population. Plusieurs arguments suggèrent une niche étroite pour cette espèce, notamment une faible diversité génomique. Par ailleurs, le faible nombre de souches environnementales associé à une meilleure croissance planctonique à 37°C qu'à 25°C et 30°C suggèrent que l'homme pourrait constituer cette niche. L'étude de la virulence d'O. intermedium en modèle macrophage ou C. elegans met en évidence différents comportements, pour autant ceux-ci ne semblent pas liés à la structure de population. Certaines souches sont capables de se multiplier dans le modèle macrophage.L'étude du genre Agrobacterium par une approche multilocus sur une collection représentative des différents modes de vie de ces bactéries met en évidence, tout comme pour O. anthropi, une sous population clinique qui regroupe près de 80% des souches de cette origine. D'autres arguments tels que la croissance à 42°C confirment que le génovar A7 peut correspondre à une sous-population associée à l'homme. Les données obtenues seront confrontées aux connaissances sur d'autres bactéries pathogènes opportunistes d'origine environnementale comme Pseudomonas aeruginosa, Stenotrophomonas maltophilia et les bactéries du complexe Burkholderia cepacia qui présentent également des sous populations associées à l'homme et/ou à certaines pathologies humaines. L'existence de ces sous populations suggère une spécialisation qui sera discutée dans le contexte de la spéciation des bactéries pathogènes afin de revisiter le concept d'opportunisme infectieux. / The opportunistic bacterial pathogens (OBP) cause the main part of bacterial infectious diseases. Environmental-borne OBP should encounter dramatic changes in lifestyle in order to colonize human beings. The conditions of this adaptation should precise concepts about OBP and emerging pathogens.The genera Ochrobactrum and Agrobacterium groups bacteria with versatile lifestyles that establish diverse relationships with the eukaryotic cells. These environmental-borne OBP caused diverse infectious diseases in immune-compromised patients. In this study, we undertook an approach of multilocus genetic on large population of environmental and clinical strains of Ochrobactrum and Agrobacterium. The population structures were compared to phenotypic traits related to adaptation and virulence in man, such as growth temperature, biofilm formation and virulence tested in Caenorhabditis elegans and human macrophages models.Ochrobactrum anthropi and Ochrobactrum intermedium are the two main Ochrobactrum species to be involved in human diseases. O. anthropi displays an epidemic population structure organized in two large clonal complexes (CCs). CC4 groups only human associated strains whereas CC1 contain environmental and clinical strains. Population genetics suggested that CC4 is a human-associated clone although phenotypic, genomic and virulence traits do not differ between CC1 and CC4 strains.As O. anthropi, O. intermedium displays a high genetic diversity without correlation between the genetic structure and the origin of strains. The level of genetic diversity among clinical strains appears as high as observed in the whole population. Several data such as a low level of genomic diversity suggested that O. intermedium is associated to a narrow ecological niche. The low number of environmental strains described for this species as well as an optimal growth at 37°C suggested that human beings could be the main niche for O. intermedium. Virulence in macrophage and C. elegans models showed diverse behaviour whereas some strains are able to survive and multiply in macrophages model.Multilocus genetics in a population of Agrobacterium spp. that displays diverse lifestyles, revealed a human associated population as observed for O. anthropi. The clinical genovar A7 groups 80% of the clinical strains included in the study, this strains growing at 42°C. Data obtained in this study will be confronted to the knowledge about other environmental-borne OBP such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia and bacteria belonging to the species complex Burkholderia cepacia. All these bacteria displayed sub-populations associated to man or to a particular human disease. These sub-populations suggest a specialization process that will be described in the context of the speciation of bacterial pathogen in order to revisite the concept of « opportunisme infectieux ».

L'utilisation de la bactérie C. maltaromaticum dans l'industrie alimentaire n'est pas autorisée en raison de faible connaissance et des pathologies qu'il peut provoquer chez le poisson. Un des objectifs de ce travail est de renforcer la connaissance par la séquence génomique d'une souche fromagère et d'évaluer le devenir de la bactérie après ingestion par le consommateur. L'autre objectif est de mieux connaitre la taxonomie de la bactérie par MLST. La séquence complète de C. maltaromaticum LMA 28 a révélé une taille génomique de 3,8 Mpb qui est atypique dans le genre Carnobacterium. L'analyse génomique indique qu'il contiendrait des gènes d'adaptation à l'environnement intestinal. Il a été montré que la bactérie est capable de survivre le transit gastro-intestinal chez la souris et d'adhérer à des cellules intestinales humaines qui présenteraient des propriétés neutres voire anti-inflammatoires. Le résultat suggère qu'elle est capable de survivre le transit GIT et d'interagir avec l'hôte. Il a été montré que la MLST chez C. maltaromaticum permet d'atteindre un haut niveau de résolution. MLST suggèrent que le lait et les fromages à pâte molle sont peu sélectifs pour C. maltaromaticum. De plus, deux CC majoritaires, principalement représentés par des souches laitières, ont été identifiés. Leur existence suggère qu'une lignée de l'espèce est particulièrement bien adaptée à l'environnement laitier ou qu'un phénomène de domestication est en cours. Un grand nombre de singletons ont été identifiés suggérant que la diversité chez cette espèce est sous-estimée et qu'elle reste à explorer / The bacterium Carnobacterium maltaromaticum is not used in industry due to limited knowledge about this organism and its virulence in fish. One objective of this thesis was to strengthen the body of knowledge by determining the complete genome sequence of the cheese strain and to evaluate the fate of this bacterium after ingestion by the consumer. Another objective was to improve the taxonomic knowledge within the species C. maltaromaticum through the development of a MLST scheme. The complete sequence of the strain C. maltaromaticum LMA 28 revealed a genome size of approximately 3.8 Mbp, which is unusually high in the genus Carnobacterium. The genome analysis of this strain indicates the presence of genes conferring the adaptation to the intestinal environment. The bacterium is able to survive during the gastro-intestinal transit in mice. Moreover, this strain is able to adhere to human intestinal epithelial cell lines and would have neutral or anti-inflammatory properties. These data suggest that C. maltaromaticum LMA 28 is adapted to the digestive tract of mammals. At the taxonomical level, it was shown that MLST is highly discriminatory for the species C. maltaromaticum. In addition, the MLST results suggest that milk and soft cheeses are poorly selective for strains of this species. In addition, two major clonal complexes suggest that a sub-population within this species is well adapted to the dairy environment or that a sub-population is submitted to a domestication process. A high proportion of singletons was obtained suggesting that the diversity was under-estimated and remains to be explored

This thesis considers a novel problem of obtaining global channel state information (CSI) at every node in an ad-hoc wireless network. A class of protocols for dissemination and estimation are developed which attempt to minimize the staleness of the estimates throughout the network. This thesis also provides an optimal protocol for CSI dissemination in networks with complete graph topology and a near optimal protocol in networks having incomplete graph topology. In networks with complete graph topology, the protocol for CSI dissemination is shown to have a resemblance to finding Eulerian tours in complete graphs. For networks having incomplete graph topology, a lower bound on maximum staleness is given and a near optimal algorithm based on finding minimum connected dominating sets and proper scheduling is described in this thesis.