Flowering plants display diverse reproductive systems, including a variety of gender strategies and mechanisms of clonal propagation. Here, I investigate gender strategies, sex-ratio evolution, and sexual dimorphism in the North American clonal aquatic, Sagittaria latifolia (Alismataceae), which exhibits three sex phenotypes (hermaphrodites, females, males) and two modal sexual systems (monoecy, dioecy). This provides an outstanding opportunity to examine the costs and benefits of combined versus separate sexes. My research focused on the northern range limit in eastern N. America, and on disjunct populations in western N. America. I developed microsatellite (SSR) markers to investigate population genetic structure at several spatial scales, including the clonal structure of local populations to continental patterns. These analyses provided insights on the roles of historical, ecological and reproductive factors in the evolution and maintenance of sexual system diversity. Phenotypic sex ratios varied near continuously from monoecy through subdioecy (three sex phenotypes) to dioecy. A comparison of phenotypic and genotypic sex ratios in dioecious populations demonstrated close correspondence. The northern range limit was characterized by a decline in female frequency and an increased incidence of subdioecy. I evaluated two hypotheses to explain this pattern: (1) increased sex inconstancy in dioecious populations; (2) hybridization between monoecious and dioecious populations. I found support for both hypotheses, although hybridization appears to be the more common pathway to subdioecy. I parameterized a model predicting female frequency and hermaphrodite sex allocation; observed and predicted values were correlated suggesting that subdioecious populations are closer to equilibrium than expected for a clonal perennial. A comparison of eastern and western populations indicated genetic differentiation between monoecy and dioecy in the east, but in the west, due to habitat isolation, geography plays a more important role in genetic differentiation. Evidence from cpDNA haplotype variation indicated that the western range was established following long-distance colonization from the east involving a genetic bottleneck. The discovery of gynodioecious populations of S. latifolia in the west, and the absence of ecological and genetic differentiation between monoecious and dioecious populations, raise the possibility that dioecy may have evolved autochthonously in the west, and more recently than in the eastern range.

Flowering plants display diverse reproductive systems, including a variety of gender strategies and mechanisms of clonal propagation. Here, I investigate gender strategies, sex-ratio evolution, and sexual dimorphism in the North American clonal aquatic, Sagittaria latifolia (Alismataceae), which exhibits three sex phenotypes (hermaphrodites, females, males) and two modal sexual systems (monoecy, dioecy). This provides an outstanding opportunity to examine the costs and benefits of combined versus separate sexes. My research focused on the northern range limit in eastern N. America, and on disjunct populations in western N. America. I developed microsatellite (SSR) markers to investigate population genetic structure at several spatial scales, including the clonal structure of local populations to continental patterns. These analyses provided insights on the roles of historical, ecological and reproductive factors in the evolution and maintenance of sexual system diversity. Phenotypic sex ratios varied near continuously from monoecy through subdioecy (three sex phenotypes) to dioecy. A comparison of phenotypic and genotypic sex ratios in dioecious populations demonstrated close correspondence. The northern range limit was characterized by a decline in female frequency and an increased incidence of subdioecy. I evaluated two hypotheses to explain this pattern: (1) increased sex inconstancy in dioecious populations; (2) hybridization between monoecious and dioecious populations. I found support for both hypotheses, although hybridization appears to be the more common pathway to subdioecy. I parameterized a model predicting female frequency and hermaphrodite sex allocation; observed and predicted values were correlated suggesting that subdioecious populations are closer to equilibrium than expected for a clonal perennial. A comparison of eastern and western populations indicated genetic differentiation between monoecy and dioecy in the east, but in the west, due to habitat isolation, geography plays a more important role in genetic differentiation. Evidence from cpDNA haplotype variation indicated that the western range was established following long-distance colonization from the east involving a genetic bottleneck. The discovery of gynodioecious populations of S. latifolia in the west, and the absence of ecological and genetic differentiation between monoecious and dioecious populations, raise the possibility that dioecy may have evolved autochthonously in the west, and more recently than in the eastern range.

Depuis le Néolithique, les activités anthropiques ont fortement impacté la persistance et l’évolution des espèces méditerranéennes, avec des effets variés sur la biodiversité, notamment son érosion et son homogénéisation. Composé seulement de trois à cinq espèces, le genre méditerranéen et Sud-asiatique Arundo L. (Poaceae) illustre bien les interactions Homme-Biodiversité. Tout d’abord, l’imprécision des statuts taxonomiques pour la plupart de ces espèces dessert autant la conservation des populations françaises protégées d’A. plinii Turra s.l. que la lutte contre l’invasive mondiale A. donax L. Suite à la révision systématique du complexe circumméditerranéen A. plinii Turra s.l., trois taxons ont été distingués, élevant les populations françaises au rang d’endémique ligure, A. donaciformis (Loisel.) Hardion et al. Cette dernière s’est différenciée en marge de son espèce-sœur, l’Italo- Balkanique A. plinii s.s., sous les effets conjugués de la polyploïdie et des oscillations climatiques du Pléistocène. La forte clonalité du genre et la formation récente d’A. donaciformis ont réduit son potentiel adaptatif. Malgré une persistance ancienne au sein d’un paysage agricole, son actuelle position urbaine sur la Côte d’Azur la menace fortement. Enfin, l’origine perse d’A. donax et l’hypothèse de son statut d’archéophyte en Méditerranée illustre l’intérêt primordial de ce genre lors du développement des civilisations antiques. / Human activities have scrambled biodiversity persistence and evolution of Mediterranean species since the Neolithic, inducing various impacts on biodiversity, from reduction to homogenisation. With only three to five species, the Mediterranean and South- Asian genus Arundo L. (Poaceae) well illustrate these Human-Biodiversity interactions. First, the taxonomic mess for almost all Arundo taxa harms the conservation of protected French populations of A. plinii Turra s.l. as much as the biological control of the worldwide invasive A. donax L. The systematic revision of the circum-Mediterranean A. plinii complex in three distinct taxa has distinguished French populations as a Ligurian endemic species, A. donaciformis (Loisel.) Hardion et al. This species was differentiated from its nearest relative, the italo-balkan A. plinii s.s., under the coupled effects of polyploidy and Pleistocene climatic variations. The dominating clonality of the genus and the recent speciation of A. donaciformis have reduced its adaptive potential. Despite its ancient persistence under farmlands, its current urban position on the French Riviera threatens this protected species. Finally, the Persian origin of A. donax and its hypothetic status of archeophyte in the Mediterranean show the essential usefulness of this genus during the development of antic civilisations.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-21T17:43:23Z No. of bitstreams: 1 thiagocesarnascimento.pdf: 3494952 bytes, checksum: 3356415ad599be16280e8e70cee1950d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-01-25T18:47:50Z (GMT) No. of bitstreams: 1 thiagocesarnascimento.pdf: 3494952 bytes, checksum: 3356415ad599be16280e8e70cee1950d (MD5) / Made available in DSpace on 2016-01-25T18:47:50Z (GMT). No. of bitstreams: 1 thiagocesarnascimento.pdf: 3494952 bytes, checksum: 3356415ad599be16280e8e70cee1950d (MD5) Previous issue date: 2014-04-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Staphylococcus aureus é uma das principais causas de infecções associadas à saúde em todo o mundo. O objetivo deste trabalho foi avaliar as características epidemiológicas, fisiológicas e moleculares de S. aureus resistente à oxacilina (ORSA), isolados de infecções em um hospital terciário universitário e avaliar sua susceptibilidade a novas moléculas síntéticas. Foram avaliadas um total de 103 amostras de ORSA quanto a dados clínicos e epidemiológicos relacionados aos pacientes, perfil de susceptibilidade a drogas antimicrobianas, avaliação da produção de biofilme e da capacidade hemolítica e confirmação da identidade genética, detecção do gene mecA e caracterização do SCCmec, por PCR. Para 45 amostras oriundas do CTI, também foram realizadas a análise do perfil de fragmentação do DNA cromossômico por PFGE e caracterização do CC, por PCR. Para 21 amostras foi avaliada a susceptibilidade a aminas aromáticas alquiladas (aminoálcoois). Considerando-se as amostras clínicas, a maioria das amostras foi isolada no sexo masculino (71%) a partir de secreção traqueal (26,2%) e sangue (23,3%) seguido de swab de sítio cirúrgico e ponta do cateter (15,5%), exsudados (14,6%) e urina (4,9%), associados à infecção do sistema respiratório (34%) e bacteremia (20,4%), em unidade de terapia intensiva (43,7%). No geral, uma alta frequência de resistência foi observada contra clindamicina (100%), eritromicina (100%), azitromicina (99%), levofloxacina (93,2%), gentamicina (84,5%), sulfametoxazol-trimetoprim (75,7%), tetraciclina (77,6%), cloranfenicol (59,3%) e rifampicina (50,5%). Os aminoálcoois também apresentaram atividade antibacteriana contra a maioria dos isolados de ORSA. Tipos de SCCmec III (66,7%) , II (17,8%) , IV (4,4% ), I (2,2%) foram encontrados. A maioria (66,7%) dos isolados foram relacionados ao clone epidemico brasileiro (CEB)/CC8/SCCmec III , que prevaleceu entre 2005 e 2008 , enquanto que a linhagem USA100/CC5/SCCmec II surgiu em 2007 e foi mais frequente em 2009 e 2010, na UTI. Os isolados que transportam o tipo de SCCmec IV (USA400/CC1 e USA800/CC5 linhagens) e I (USA500/CC5) também foram detectadas. Nossos dados são altamente relevantes para os sistemas de vigilância permitiu mapear em maior escala a circulação dinâmica de ORSA e levantar discussões sobre estratégias de contenção e uso racional da quimioterapia empírica. / Staphylococcus aureus is a major cause of health care associated infections worldwide. The aim of this work was to evaluate epidemiological, physiological and molecular characteristics of aureus oxacillin resistant S. aureus (ORSA) isolated from infections in a tertiary university hospital and evaluated their susceptibility to new synthetic molecules. A total of 103 samples of ORSA for clinical and epidemiological data related to patients susceptibility profile to antimicrobial drugs, assessment of biofilm production and hemolytic capacity and confirmation of genetic identity, detection of the mecA gene and characterization of SCCmec were evaluated, PCR. For 45 samples from CTI, also the profile of DNA analysis by PFGE and characterization of the CC, PCR were performed. For 21 samples susceptibility to alkylated aromatic amines was evaluated. Considering the clinical samples, most samples contained in males (71%) from tracheal secretion (26.2%) and blood (23.3%) followed by surgical site and swab tip of the catheter (15.5%), exudates (14.6 %) and urine (4.9%), associated with infection of the respiratory system (34%) and bacteremia (20.4%) in the intensive care unit (43.7%). Overall, a high frequency of resistance was observed against clindamycin and erythromycin (100%), azithromycin (99%), levofloxacin (93.2%), gentamicin (84.5%), trimethoprim-sulfamethoxazole (75.7%), tetracycline (77.6%), chloramphenicol (59.3%) and rifampicin (50.5%). The amino alcohols also showed antibacterial activity against most isolates of ORSA. SCCmec type III (66.7%), II (17.8%), IV (4.4%) and I (2.2%) were found. The majority (66.7%) isolates were related to the brazilian epidemic clone (CEB)/CC8/SCCmec III, which prevailed between 2005 and 2008, while the USA100/CC5/SCCmec lineage II emerged in 2007 and was more frequent in 2009 and 2010 in the ICU. The strains carrying the SCCmec type IV (USA400/CC1 and USA800/CC5 lineages) and I (USA500/CC5) were also detected. Our data are highly relevant to surveillance systems enabled map on a larger scale the dynamic movement of ORSA and raise discussions on containment strategies and rational use of empirical chemotherapy.

Les leucémies/lymphomes à cellules T de l’adulte (Adult T-cell leukemia/Lymphoma, ATL) sont des hémopathies lymphoïdes T CD4+ malignes matures rares, induite par le rétrovirus HTLV-1 (Human T lymphotropic virus type 1). Nous avons étudié différents aspects moléculaires de la lymphomagenèse HTLV-1 induite sur une série rétrospective de patients pris en charge pour un ATL.Nous avons dans un premier temps étudié la marque épigénétique hydroxymethylation (5hmc) de l’ADN, ainsi que les enzymes la régulant sur des cellules primaires d’ATL. Nous avons observé une diminution du taux de 5hmc dans les formes agressives comparativement aux formes indolentes qui corrélait avec une diminution de l’expression de la dioxygénase TET2 et avec la survie des malades. Nous avons également mis en évidence la présence de mutations somatiques du gène TET2 chez moins de 10% des patients et identifié un polymorphisme surreprésenté dans le locus de TET2 chez les patients atteints ATL comparé à des patients chroniquement infectés sains ethniquement appariés.Dans un deuxième temps, nous avons exploité une technique de PCR « ligation médiée » suivie d’un séquençage à haut débit afin d’étudier l’architecture de l’intégration virale comme outil de maladie résiduelle. Nous avons retrouvé une meilleure sensibilité de cette technique pour définir la réponse au traitement par rapport aux critères de réponse actuels ouvrant la voie de son utilisation pour le suivi des patients.Enfin, nous avons étudié le paysage des altérations génomiques sur une cohorte de 60 patients par une approche globale. Nous avons observé des mutations sur 3 voies principales : TCR/NF-KB, trafic cellulaire T et échappement au système immunitaire corroborant les résultats d’une récente étude sur une population de patients japonais. L’analyse par RNAseq a révélé la perturbation systématique de l’expression génique cellulaire induite par l’intégration virale soit par le biais d’un transcrit chimérique partant du LTR 3’ viral soit par un arrêt de la transcription d’un gène cellulaireLe suivi de patients progressant d’une forme indolente à une forme agressive a révélé l’acquisition de mutations activatrices principalement sur la voie de signalisation TCR/NF-KB. Le suivi de patients ayant rechuté après une période de rémission a également révélé l’acquisition de nouvelles mutations.Ces résultats soulignent la spécificité de la lymphomagenèse HTLV-1, qui procède d’une part d’évènements secondaires à l’intégration virale au sein du génome cellulaire, induisant la perturbation de l’expression de gènes cellulaires (premier évènement oncogénique) et d’autre part à l’accumulation d’altérations génomiques secondaires, communs aux autres hémopathies lymphoïdes T et B matures, responsables de la transformation. / Adult T cell leukemia/lymphoma (ATL) is a rare and mature T cell malignancy induced by the retrovirus HTLV-1 (Human T-lymphotropic virus type 1) which bears a dismal prognosis. We have studied several molecular aspects of HTLV-1 induced lymphomagenesis on a retrospective cohort of ATL patients.First, we analyzed the global level of the DNA epigenetic mark hydroxymethylation (5hmc) as well as of enzymes implicated in its regulation in primary ATL cells. We observed a reduction of the 5hmc level in aggressive ATL compared to indolent forms with a positive correlation between the reduction of the 5hmc level, the decrease of the TET2 dioxygenase transcript and the patient overall survival. We found that somatic mutations in TET2 were present with a frequency of less than 10% but identified a SNP in TET2 locus whose frequency in ATL patients was higher compared to that of an ethnically matched control population.In a second part, we took advantage of a new technique of ligated mediated PCR followed with high throughput sequencing to analyze the viral integration architecture as a means of minimal residual disease detection. We demonstrate that this technique allows a better definition of the treatment response compared to actual consensus response criteria.Finally, we performed an integrated genomic analysis of a retrospective cohort of 60 ATL patients. We identified alterations targeting the TCR/NFKB signaling pathway, T cell trafficking and immune escape mechanisms, consistent with previous findings described in a Japanese ATL cohort. RNAseq analysis revealed the systematic perturbation of host gene expression secondary to viral integration and proceeding via the viral antisense leading to the production of a virus-host chimeric transcript production or the direct transcription termination of a host gene. Analysis of matched sequential samples of patients progressing from an indolent to an aggressive form revealed in most of the cases the acquisition of mutations affecting the TCR/NF-KB pathway. Analysis of sequential samples from patients who relapsed after a remission period also showed the acquisition of additional genetic alterations.These results underscore the specific nature of HTLV-1 induced lymphomagenesis, which proceeds on the one hand through mechanisms induced by the viral integration in the host genome, and consequent host-gene expression perturbation (viral first oncogenic hit) and on the other hand through secondary oncogenic mutations in various pathways, common to other mature B and T cell lymphoid malignancies.

<p>A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. </p><p>In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. </p><p>In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.</p>

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.

Nothofagus moorei is a long-lived, Gondwana relict cool temperate rainforest tree. Nothofagus-dominated rainforests were widespread across much of eastern Australia during the mid-Tertiary but today, N. moorei occurs only as a series of disjunct, isolated populations in south-east Queensland and northern New South Wales. Clonal regeneration via coppicing is reported to be a common feature of most N. moorei populations, while successful sexual regeneration is believed to be rare, occurring largely only in niches with high light levels and limited competition. While clonal propagation enables population persistence and individual longevity, it cannot generate novel genotypes. Isolated populations, potentially high levels of clonality, low-potential for successful sexual regeneration, long-lived individuals and predicted global warming effects make N. moorei vulnerable to local, if not total, population extinction. The current study aimed to assess the relative conservation status of extant N. moorei populations in order to develop appropriate conservation management strategies for long-term population persistence. Levels of genetic diversity and population structure were examined across the remaining natural distribution of N. moorei using nuclear amplified fragment length polymorphism (AFLP), microsatellite and chloroplast DNA markers. In total 607 individuals were sampled from 20 populations and 5 geographical regions: Lamington/Border Ranges, Ballow, Dorrigo/New England, Werrikimbe and Barrington. Genetic results were then analysed to assess conservation status of each population and geographical region. Microsatellite and AFLP data identified comparatively high levels of genetic diversity in all remnant populations sampled. The prevalence of coppicing in the northern Lamington/Border Ranges populations appears to have had little impact on relative levels of genetic diversity, heterozygosity or population structure. Population differentiation was limited, with the majority of genetic variation retained within populations, no regional structuring and high levels of admixture. Analysis of cpDNA variation showed that the three Dorrigo/New England populations were divergent from all other populations, suggesting an ancient divergence in N. moorei prior to Pleistocene glaciations. While levels of genetic diversity were essentially the same across all populations, Bayesian analysis of genetic structure did identify four populations with differing gene pool proportions which would be important to include in conservation efforts in addition to individuals from other populations. Similarly, individuals from four significantly differentiated groups identified using traditional F-statistics suggests individuals from each of these four groups should be included in future conservation plans. In order to maintain ancient chloroplast lineages, populations from the Dorrigo/New England region should also be assigned special conservation value. Populations of N. moorei appear to have retained significant levels of genetic diversity and show little population divergence in spite of marked reductions in the natural distribution since the Early Miocene. Sampling of these ancient trees however, suggests current levels of diversity in N. moorei actually reflect past diversity and differentiation, and that there have been insufficient generations since the historical contraction in distribution for genetic diversity to be adversely affected and regional differentiation to evolve. Long-term persistence of N. moorei is still threatened by future accelerated climate change and the limited preferred habitat that remains where N. moorei can expand its range. While the ability to regenerate clonally may enable long-term persistence of N. moorei, populations are still likely to continue to decline as climatic conditions will increasingly favour sub-tropical and warm temperate species across much of N. moorei's northern distribution. Southern populations of N. moorei, in contrast, could expand their ranges into eucalypt woodlands as predicted climate becomes warmer and wetter. However, this will ultimately be determined by the frequency of fires, with increased fire frequencies favouring the expansion of eucalypts and contraction and possible local population extinction of N. moorei dominated cool temperate rainforests.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-17T18:26:18Z No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) / Made available in DSpace on 2014-02-17T18:26:18Z (GMT). No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A infecção do trato urinário (ITU) é considerada a segunda infecção mais comum em humanos, estima-se que ocorram cerca de 150 milhões de casos de ITUs por ano no mundo. O aumento das taxas de resistência aos antimicrobianos entre os uropatógenos tem tornado mais difícil o tratamento das ITUs. Objetivo: Determinar a distribuição clonal das cepas de E. coli isoladas em pacientes com ITU adquirida na comunidade de acordo com o perfil de susceptibilidade aos antimicrobianos e avaliar o papel dos grupos clonais na disseminação e persistência da resistência nestas infecções. Métodos: Foram isoladas 874 cepas de E. coli em pacientes com ITU, procedentes de unidades ambulatoriais em três hospitais na cidade de Salvador–Ba, no período de 2001 a 2009. O perfil de susceptibilidade foi determinado por microdiluição em placa (Microscan-Siemens®). Nas amostras selecionadas para genotipagem (n=275), a identificação dos grupos clonais foi realizada pela comparação dos padrões de PFGE, utilizando os critérios de Tenover (1995). Em todas as etapas do estudo foi utilizada como controle de qualidade a cepa ATCC E. coli 25922. Resultado: Entre os antibióticos testados, a maior prevalência de resistência foi encontrada para ampicilina (AMP) (49%), cefalotina (12-33%) e sulfametoxazol-trimetropin (SXT) (36-42%). A taxa de resistência à ciprofloxacina (CIP) variou de 9 a 14%. Na análise da distribuição clonal, segundo os fenótipos de resistência aos antimicrobianos, encontramos maior predomínio de um grupo clonal CgA (63%) entre as cepas consideradas multidroga resistentes. Diferentemente das amostras com algum grau de resistência ou multi-sensíveis, nas quais observamos diversidade clonal. Conclusão: A alta prevalência de resistência a SXT, AMP e cefalotina contraindica o uso destes antimicrobianos no tratamento empírico das ITU adquiridas na comunidade. A taxa de resistência à CIP relativamente alta, alerta para o aumento e disseminação de resistência a este antimicrobiano na comunidade. Isto irá dificultar e onerar o tratamento destas infecções. Observamos a surgimento de um grupo clonal (CgA) no período final do estudo (2008 a 2009) associado às cepas multidroga resistentes. Este achado sugere que a expansão de determinados clones pode ter um papel importante na disseminação de resistência bacteriana em ITUs adquiridas na comunidade. / Urinary tract infection (UTI) is considered the second most common infection in humans. It is estimated that there are about 150 million cases of UTIs per year worldwide. Increasing rates of antimicrobial resistance among uropathogens challenges UTI treatments. Objective: To determine the distribution of clonal strains of E. coli isolated from patients with community-acquired UTI according to the profile of antimicrobial susceptibility; and to evaluate the role of clonal groups in the spread and persistence of resistance in these infections. Methods: Eight hundred seventy four strains of E. coli were isolated from patients with UTI, coming from outpatient clinics in three hospitals in the city of Salvador - Bahia, from 2001 to 2009. The susceptibility profile was determined by broth microdilution method (Siemens - Microscan ®). The samples selected for genotyping (n = 275) were identified for clonal groups by comparing the patterns of PFGE, using the criteria of Tenover (1995). All study stages were quality control by strain E. coli ATCC 25922. Results: Among the antibiotics tested, the highest prevalence of resistance was for ampicillin (AMP) (49%) followed by trimethoprim - sulfamethoxazole (SXT) (36-42%) and for cephalothin (12-33%). The rate of resistance to ciprofloxacin (CIP) ranged between 9-14 %. In the Clonal Analysis distribution, performed according to antimicrobial resistance phenotypes, we found a higher prevalence of a clonal group CgA (63%) among multidrug resistant strains. This result differs from samples with some degree of resistance or multi-sensitive in which we observed clonal diversity. Conclusion: The high prevalence of resistance to SXT, AMP, and cephalothin contraindicate the use of these antibiotics in the empirical treatment of community-acquired UTI. The relatively high rate of resistance to CIP, raises attention to the increase and spread of antimicrobial resistance in this community and potentially complicate and encumber the treatment of these infections. We observe the emergence of a clonal group (CgA) in the final period of the study (2008-2009) associated with multidrug resistant strains. This finding suggests that the expansion of particular clones may have an important role in the spread of bacterial resistance in community-acquired UTI.

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-04-30Bitstream added on 2014-06-13T20:23:21Z : No. of bitstreams: 1 ciampi_mb_dr_jabo.pdf: 501699 bytes, checksum: 6171bb1c109f747a0aeff63805c59aaf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O basidiomiceto Rhizoctonia solani AG-1 IA é um dos principais patógenos da soja no Brasil, onde as perdas estimadas com a doença podem atingir 30 a 60%. 232 isolados de R. solani AG-1 IA foram coletados de campos comerciais de soja nas principais regiões produtoras do país e genotipados usando dez locos polimórficos de microssatélites. As baixas diversidades genotípicas, os desvios do equilíbrio de Hardy-Weinberg (EHW), o desequilíbrio gamético e o elevado grau de subdivisão populacional encontrados nessas populações são consistentes com predominância de reprodução assexuada e dispersão de propágulos vegetativos a curtas distâncias. Os níveis de subdivisão observados poderiam ser explicados pela migração histórica assimétrica entre as populações, indicando a população do Tocantins como a provável fundadora. As evidências de fluxo gênico restrito e modo reprodutivo misto enquadrariam o fungo na categoria de médio risco para potencial evolutivo de patógenos, sugerindo precaução quanto à aplicação de fungicidas ou melhoramento para genes de resistência. Também foi desenvolvido um método para detecção de SNPs em múltiplos locos por PCR, através da conversão de sondas de RFLP em seis marcadores co-dominantes de seqüenciamento, altamente informativos e polimórficos. Detectou-se de um a múltiplos alelos em cada isolado, para cada região analisada, indicando a condição heterocariótica do fungo. O maior número de polimorfismos SNPs foi detectado para o marcador R68L, com 18 mutações em 303 pares de bases. O conjunto de novos marcadores desenvolvido mostrou-se um sistema de genotipagem viável, possibilitando discriminação alélica precisa, com potencial de complementar os métodos existentes para estudo da biologia populacional de R. solani AG-1 IA e viabilizar estudos de caráter evolutivo. / The Basidiomycete fungus Rhizoctonia solani AG-1 IA is a major pathogen of soybean in Brazil, where the average yield losses have reached 30 to 60%. 232 isolates of R. solani AG1 IA were collected from soybean fields in the most important soybean production areas in the country. These isolates were genotyped using ten microsatellite loci. Low genotypic diversity, departures from Hardy-Weinberg equilibrium (HWE), gametic disequilibrium and high degree of population subdivision found in these populations are consistent with predominantly asexual reproduction, short-distance dispersal of vegetative propagules, and limited long-distance dispersal. The observed levels of subdivision could be explained by asymmetric historical migration among the soybean-infecting populations, denoting TO06 as the founder population. Evidences of restricted gene flow and a mixed reproductive mode would fit the fungus into the medium-high risk category for pathogen evolutionary potential, suggesting the need for caution when applying fungicides or breeding for major-gene resistance. We also developed a method to detect SNPs in multiple loci by PCR, converting RFLP probes in six highly informative and polymorphic co-dominants sequencing markers. We have identified single and multiple alleles per isolate in each analyzed region, indicating the fungus heterokaryotic condition. The highest number of SNPs was detected at the R68L marker, with 18 mutations along 303 base pairs. The developed set of new markers proved to be a viable genotyping system, allowing precise allelic discrimination, with the potential to complement the methods already described to study the R. solani AG-1 IA population biology and making evolutionary studies feasible.