Introduction: Colonization of the anogenital tract of pregnant women by Group B Streptococcus (GBS) is the main risk factor for early-onset neonatal sepsis. Identification of maternal colonization allows antimicrobial prophylaxis and prevention of vertical transmission. Objectives: To identify the genetic diversity and phenotypic characteristics of GBS using molecular biology techniques and culture in specific medium, and the epidemiological aspects of pregnant women colonized by this bacterium. Methods: It was performed a cross-sectional study, anogenital samples of 316 pregnant women were collected between 35 and 37 weeks and submited to culture in specific medium, antimicrobial susceptibility test, multiplex PCR, multi locus sequence typing (MLST) and multi locus variable number of tandem repeat analysis (MLVA). The epidemiological aspects of colonized and non-colonized pregnant women were also investigated. Results: It was obtained a prevalence of 36.4% by culture and 38.6% by PCR. Multiplex PCR had sensitivity of 100%, specificity of 96.5%, positive predictive value of 94.3% and negative predictive value of 100%. The most common serotypes were Ia, V, II and III. Resistance genes were identified in 34 samples. Sensitivity to penicillin was universal, 24.3% presented resistance to erythromycin and 14.8% to clindamycin. Evaluating the epidemiological variables, there were no differences between the colonized and non-colonized pregnant women. Diversity index and number of genotypes found by MLST was 0.608 and 6, and by MLVA was 0.840 and 15, respectively. Conclusion: We found a high prevalence of maternal GBS colonization, with serotype distribution similar to the Americas region. Multiplex PCR was more accurate than culture, and maternal epidemiological variables showed no difference when evaluating presence or absence of bacterial colonization, its serotypes and antimicrobial resistance. MLVA showed a higher discrimination capacity among the unrelated strains than MLST. / Submitted by Otto Henrique May Feuerschuette (otto.feurschuette@unisul.br) on 2018-04-26T00:54:22Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) / Approved for entry into archive by Silvane Cauz (silvane.cauz@unisul.br) on 2018-04-26T12:14:53Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) / Made available in DSpace on 2018-04-26T12:14:53Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) OTTO TESE CORRIGIDA PÓS BANCA (1).pdf FINAL.pdf: 3081595 bytes, checksum: afad5beba2687558aafe2f1705d36e01 (MD5) Previous issue date: 2018 / Introdução: A colonização do trato anogenital de gestantes pelo Estreptococo do grupo B (EGB) é o fator de risco primário para a sepse neonatal precoce. A identificação da colonização materna permite a profilaxia antimicrobiana e prevenção da transmissão vertical. Objetivos: Identificar a diversidade genética e as características fenotípicas do EGB utilizando-se técnicas de biologia molecular e cultura em meio específico, e avaliar os aspectos epidemiológicos de gestantes colonizadas por essa bactéria Métodos: Foi realizado um estudo transversal com 316 amostras anogenitais de gestantes entre 35 e 37 semanas para realização de cultura em meio específico, teste de sensibilidade aos antimicrobianos, PCR multiplex, multi locus sequence typing (MLST) e multi locus variable number of tandem repeat analysis (MLVA). Pesquisou-se também os aspectos epidemiológicos dessas gestantes. Resultados: Foi encontrada prevalência de 36,4% pela cultura e 38,6% pela PCR. A PCR multiplex apresentou sensibilidade de 100%, especificidade de 96,5%, valor preditivo positivo de 94,3% e valor preditivo negativo de 100%. Os sorotipos mais encontrados foram Ia, V, II e III. Os genes de resistência foram identificados em 34 amostras. A sensibilidade à penicilina foi universal, 24,3% apresentaram resistência à eritromicina e 14,8% à clindamicina. Avaliando-se as variáveis epidemiológicas, não se identificaram diferenças entre as gestantes colonizadas e não colonizadas. O índice de diversidade e o número de genogrupos encontrados pelo MLST foi 0,608 e 6, e pela MLVA foi 0,840 e 15, respectivamente Conclusão: Constatou-se uma alta prevalência de colonização materna, com distribuição dos sorotipos semelhante à região das Américas. A PCR multiplex foi mais acurada que a cultura, e as variáveis epidemiológicas maternas não apresentaram diferença significativa ao avaliar-se a presença ou não de colonização bacteriana, seus sorotipos e a resistência aos antimicrobianos. A MLVA apresentou uma capacidade de discriminação entre as cepas não relacionadas maior do que a MLST

Die Untersuchung der antimikrobiellen Fähigkeiten von Bodenisolaten ist von großer Bedeutung, um neue antibiotische Substanzen zu entdecken. Im Rahmen eines mikrobiologischen Praktikums wurden von Studierenden Bodenproben gesammelt und daraus 32 Streptomyces-Isolate gewonnen. Diese Isolate wurden auf ihre antimikrobiellen Leistungen untersucht und taxonomisch eingeordnet. Mittels Multi-Locus-Sequenz-Typisierung (MLST) wurden die 32 Bodenisolate der Gattung Streptomyces zugeordnet. Es gelang, alle Isolate erfolgreich dieser Gattung zuzuordnen. Allerdings konnte nicht für jedes Isolat eine eindeutige Spezieszugehörigkeit durch MLST festgestellt werden. Der Fokus lag insbesondere auf der detaillierten Analyse und Charakterisierung der Bodenisolate #4I1 und #12I2. Mittels Baumrekonstruktionen, basierend auf 16S-rRNA, MLST- und Gesamtgenomanalysen, wurden diese Stämme der Spezies Streptomyces silvae For3T zugeordnet. Das Genom des endophytischen Streptomyces sp. M3, welches aus der NCBI-Datenbank entnommen wurde, konnte ebenfalls dieser Spezies zugeordnet werden. Weiterhin wurde eine vergleichende Genomanalyse durchgeführt, um die genetische Struktur und die metabolischen Eigenschaften der S. silvae-Stämme zu erforschen. Diese umfassenden genetischen Untersuchungen trugen erheblich zum Verständnis der innerartlichen Variabilität und der adaptiven Merkmale der untersuchten Streptomyces-Stämme bei. Die physiologischen Profile der S. silvae-Stämme wurden mittels verschiedener Tests auf ihre antimikrobiellen Eigenschaften und ihre Anpassungsfähigkeit an verschiedene Umweltbedingungen untersucht. Besondere Aufmerksamkeit wurde dem Stamm S. silvae #4I1 gewidmet. Dieser wurde ausführlichen physiologischen Tests unterzogen, um eine präzise taxonomische Beschreibung als Repräsentant seiner Spezies zu ermöglichen. Die Ergebnisse der Studie zeigten, dass die S. silvae-Stämme ein breites Spektrum antimikrobieller Aktivitäten aufweisen. Die Ergebnisse der vergleichenden Genomanalyse verdeutlichten eine enge evolutionäre Verwandtschaft zwischen den S. silvae-Stämmen und offenbarten das Potenzial dieser Stämme zur Produktion von Sekundärmetaboliten, die zur Bekämpfung von pathogenen Mikroorganismen beitragen könnten.:Inhaltsverzeichnis___________________________________________________ I Abkürzungsverzeichnis______________________________________________ VI Zusammenfassung________________________________________________ VIII Summary_________________________________________________________ X Einleitung________________________________________________________ 1 Antibiotika________________________________________________________ 1 Innovative Lösungen zur Bekämpfung von Antibiotikaresistenzen_____________ 3 Identifizierung von Antibiotikaklassen mittels Ganzzell-Biosensoren____________ 4 Gattung Streptomyces______________________________________________ 6 1.4.1 Merkmale und Lebensweise von Streptomyces spp.___________________ 8 1.4.2 Genomstruktur von Streptomyces: Charakteristika und Besonderheiten___ 13 1.4.3 Genomische Organisation der Gene für Sekundärmetaboliten in Streptomyces spp. ____ 15 Zielstellung______________________________________________________ 17 2 Material und Methoden___________________________________________ 18 Verwendete Laborgeräte____________________________________________ 18 Materialien______________________________________________________ 19 2.2.1 Verbrauchsmaterialien_________________________________________ 19 2.2.2 Chemikalien_________________________________________________ 19 2.2.3 Medien_____________________________________________________ 21 2.2.4 Verwendete Bakterienstämme und Oligonukleotide___________________ 29 Physiogische Methoden____________________________________________ 31 2.3.1 Isolierung und Herstellung der Sporensuspensionen von Streptomyces spp. aus Bodenproben 31 2.3.2 Kultivierung der Streptomyces-Stämme____________________________ 32 2.3.3 Kultivierung der Biosensoren und Testorganismen___________________ 33 Methodische Herangehensweisen zur Evaluierung der antimikrobiellen Aktivitäten von Streptomyces spp. 34 2.4.1 Evaluierung des Hemmpotenzials von Streptomyces-Stämmen gegenüber ausgewählten Testorganismen 34 2.4.2 Biosensor-basierte Identifikation antibiotischer Substanzklassen_________ 36 Vergleichende Morphologie der S. silvae-Stämme 37 Physiologische Charakterisierung von S. silvae #4I1______________________ 37 2.6.1 Morphologie und Bildung melanoider Pigmente______________________ 37 2.6.2 Bestimmung der Nutzung verschiedener Kohlenstoffquellen sowie der Natriumchloridtoleranz und Lysozymresistenz_ 37 2.6.3 Evaluierung der pH-Präferenzen_________________________________ 38 2.6.4 Bestimmung der Hämolyseaktivität________________________________ 39 2.6.5 Bestimmung des Temperaturoptimums_____________________________ 39 2.6.6 Analyse der Mikromorphologie mittels Elektronenmikroskopie___________ 39 Molekularbiologische und genetische Methoden__ 40 2.7.1 Isolation genomischer DNA aus Bodenisolaten______________________ 40 2.7.2 Messung der Konzentration genomischer DNA______________________ 40 2.7.3 Gesamt-Genom-Sequenzierung__________________________________ 40 2.7.4 MLST (Multi-Locus-Sequenz-Typisierung)__________________________ 41 2.7.4.1 PCR (Polymerasekettenreaktion)_______________________________ 41 2.7.4.2 Gelektrophorese____________________________________________ 43 2.7.4.3 Extraktion und Aufreinigung der PCR-Produkte____________________ 43 2.7.4.4 Sequenzierung der PCR-Produkte______________________________ 43 Bioinformatische Methoden__________________________________________ 44 2.8.1 Anwendung der MLST-Methode bei den Bodenisolaten________________ 44 2.8.2 Bioinformatische Methoden zur vergleichenden Genomik der vier S. silvae-Stämme ___ 45 2.8.3 Bioinformatische Strategien zum Genomassembling__________________ 46 2.8.3.1 Optimierung der Sequenzdaten aus der Gesamtgenomsequenzierung__ 47 2.8.3.2 de-novo-Genomassembling___________________________________ 48 2.8.3.3 Mapping__________________________________________________ 48 2.8.3.4 Extraktion der Genomsequenz_________________________________ 50 2.8.3.5 Auswahl der Referenzsequenzen für die Contig-Verschmelzung (Scaffolding) _ 50 2.8.3.6 Genomrekonstruktion durch Scaffolding__________________________ 51 2.8.4 Bioinformatische Ansätze zur Ermittlung genomischer Kennziffern________ 52 2.8.4.1 Berechnung des ANI-Wertes___________________________________ 53 2.8.4.2 Berechnung des genomischen GC (Guanin-Cytosin)- und AT (Adenin-Thymin)-Gehaltes 53 2.8.4.3 Berechnung des GC-Versatzes_________________________________ 54 2.8.5 16S-rRNA-Analyse____________________________________________ 55 2.8.5.1 16S-rRNA-Sequenzextraktion__________________________________ 55 2.8.5.2 16S-rRNA-Prozessierung und Alignment__________________________ 56 2.8.5.3 Generierung des 16S-rRNA-Baums_____________________________ 57 2.8.6 AutoMLST_______________________________________________________ 59 2.8.7 Gesamtgenom-basierte Konstruktion eines phylogenetischen Baumes____ 60 2.8.8 Genomische Charakterisierung und Identifizierung relevanter Gencluster in den Genomen von S.silvae__ 61 2.8.8.1 Genannotation___________________________________________________ 61 2.8.8.2 Identifikation von BGCs_______________________________________ 62 2.8.8.3 Identifikation von Antibiotikaresistenzgenen_______________________ 63 2.8.9 Visualisierung____________________________________________________ 63 2.8.9.1 Erstellung zirkulärer Genomkarten______________________________ 64 2.8.9.2 MAUVE-Alignement__________________________________________ 64 3 Ergebnisse_____________________________________________________ 66 Systematische Taxonomie von Streptomyces-Bodenisolaten mittels MLST__ 66 Vielfalt und Spezifität von Streptomyces-Isolaten: Inhibitorische Kapazitäten und biosensorische Charakterisierungen_ 69 3.2.1 Bestimmung der inhibitorischen Kapazität von Streptomyces-Isolaten gegenüber ausgewählten Testorganismen__70 3.2.2 Systematische Evaluierung der biosensorischen Profile von 36 Streptomyces-Stämmen _ 73 Komparative Genomanalyse von S. silvae______ 78 3.3.1 Optimierung der Genomassemblierung der Streptomyces-Isolate #12I2 und #4I1 ______ 79 3.3.2 Phylogenetische Einordnung von S. silvae-Stämmen mittels 16S-rRNA-Analyse und MLST 81 3.3.3 GBDP-gestützte Analyse zur Aufklärung der Phylogenie von S. silvae-Stämmen_______ 82 3.3.4 Komparative Genomkartografie zur Visualisierung der genetischen Diversität in der S. silvae-Klade_ 84 3.3.5 Identifikation homologer Genombereiche___________________________ 88 3.3.6 Vergleichende Analyse der BGCs für Sekundärmetabolite in den vier S. silvae-Genomen 91 3.3.7 Komparative Analyse der Antibiotikaresistenz-assoziierten Gentypen in den S. silvae-Stämmen__ 95 Vergleichende physiologische Untersuchungen von S. silvae-Stämmen________ 99 3.4.1 Differenzierte Analyse antimikrobieller Aktivitäten von S. silvae-Stämmen in Abhängigkeit vom Nährmedium__ 99 3.4.2 Klassifikation der antimikrobiellen Sekundärmetaboliten von S. silvae-Stämmen unter Einsatz lumineszierender Biosensoren_101 3.4.3 Vergleichende Analyse der Makromorphologie von S. silvae-Stämmen auf diversen Nährmedien_ 103 Physiologische Charakterisierung von S. silvae #4I1_____________________ 106 3.5.1 REM-basierte Untersuchung der Mikromorphologie von S. silvae #4I1___ 107 3.5.2 Morphologische Analyse und Melanoidpigmentsynthese von S. silvae #4I1 auf ISP-Medien__ 108 3.5.3 Analyse des Verwertungsspektrums von Kohlenstoffquellen durch S. silvae #4I1 _____ 110 3.5.4 Bestimmung der Salztoleranz von S. silvae #4I1_____________________ 112 3.5.5 Einfluss des pH-Wertes auf das Wachstum von S. silvae #4I1__________ 113 3.5.6 Bestimmung der Lysozymresistenz bei S. silvae #4I1_________________ 114 3.5.7 Untersuchung der hämolytischen Aktivität von S. silvae #4I1___________ 116 3.5.8 Bestimmung der Temperaturpräferenz von S. silvae #4I1______________ 117 4 Diskussion____________________________________________________ 118 Genetische und Phänotypische Diversität der Streptomyces-Isolate__________ 119 4.1.1 MLST-basierte Einordnung und genetische Diversität der Streptomyces-Isolate _______ 119 4.1.2 Kultivierbarkeit der Streptomyces-Isolate__________________________ 119 4.1.3 Hemmeffekte von Streptomyces-Isolaten__________________________ 120 4.1.4 Biosensorische Profile und antimikrobielle Fähigkeiten von Streptomyces-Bodensolaten 123 Fokussierte Untersuchungen der S. silvae-Stämme_______ 125 4.2.1 Signifikanz der 16S-rRNA-Sequenzidentität in S. silvae_______________ 125 4.2.2 Etablierung von S. silvae als monophyletischen Gruppierung__________ 126 4.2.3 Sekundärmetabolismus und der Einfluss variabler Kulturbedingungen auf die antimikrobielle Aktivität und Biosensor-Induktion der S. silvae-Stämme__130 5 Ausblick______________________________________________________ 139 Literaturverzeichnis_______________________________________________ 142 Anhang________________________________________________________ 173

Long term longitudinal surveys have the advantage to enable several sampling of the studied phenomena and then, with the repeated measures obtained, find a confirmed tendency. However, these long term surveys generate large epidemiological datasets including more sources of noise than normal datasets (e.g. one single measure per observation unit) and potential correlation in the measured values. Here, we studied data from a long-term epidemiological and genetic survey of malaria disease in two family-based cohorts in Senegal, followed for 19 years (1990-2008) in Dielmo and for 16 years (1993-2008) in Ndiop. The main objectives of this work were to take into account familial relationships, repeated measures as well as effect of covariates to measure both environmental and host genetic (heritability) impacts on the outcome of infection with the malaria parasite Plasmodium falciparum, and then use findings from such analyses for linkage and association studies. The outcome of interest was the occurrence of a P. falciparum malaria attack during each trimester (PFA). The two villages were studied independently; epidemiological analyses, estimation of heritability and individual effects were then performed in each village separately. Linkage and association analyses used family-based methods (based on the original Transmission Disequilibrium Test) known to be immune from population stratification problems. Then to increase sample size for linkage and association analyses, data from the two villages were used together.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

Orientador: Suzete Aparecida Lanza Destéfano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T14:37:32Z (GMT). No. of bitstreams: 1 Tonin_MarianaFerreira_D.pdf: 2371085 bytes, checksum: d7c915d664efeec24397b855164c65b9 (MD5) Previous issue date: 2012 / Resumo: Espécies pertencentes ao gênero Xanthomonas são responsáveis por doenças que podem causar grandes perdas econômicas em diversas culturas. Esses fitopatógenos têm sido objeto de diversos estudos taxonômicos resultando em significativas alterações na classificação em nível inter e infraespecífico. O presente estudo teve por objetivo caracterizar taxonomicamente bactérias do gênero envolvendo: (1) diferenciação e análises filogenéticas de espécies do gênero Xanthomonas; (2) esclarecimento da posição taxonômica de linhagens classificadas como Xanthomonas sp.; (3) diferenciação de patovares da espécie X. campestris; (4) desenvolvimento de primers específicos para X. campestris, X. translucens, X. cucurbitae e X. melonis. O par de primers rpoB2F/rpoB3R, desenhado a partir de sequências do gene rpoB, foi empregado em experimentos de amplificas;ao utilizando-se DNAs de 26 espécies do gênero Xanthomonas e os produtos de amplificas;ao (800 pb) foram digeridos com diversas endonucleases. Os perfis de restrição obtidos com a utilização da enzima Hae III permitiram a diferenciação da maioria das espécies, incluindo os patógenos de mesmo hospedeiro como X. albilineans e X. sacchari (patogênicas à cana-de-açúcar); X. cucurbitae . e X. melonis (patogênicas ao melão); X. vesicatoria, X. gardneri e X. euvesicatoria/X. perforans (patogênicas ao tomateiro). Ainda, os produtos de amplificação do gene rpoB foram seqüenciados e a árvore filogenética construída a partir destas sequências também possibilitou a diferenciação das espécies do gênero, indicando que o gene rpoB pode ser considerado um marcador molecular eficiente para o estudo das relações filogenéticas de espécies do gênero Xanthomonas. Os DNAs de linhagens classificadas como Xanthomonas sp. também foram analisados por meio de experimentos de amplificação com primers específicos para a espécie X. axonopodis, de hibridizas;ao DNA-DNA, e analise de multilocus utilizando-se sequências dos genes rpoB, rpoA, atpA, recA e região espaçadora 16S-23S DNAr. Os resultados mostraram que as linhagens de X. sp. pv. viticola, X. sp. pv. betae e X. sp. pv. paulliniae pertencem a espécie X. axonopodis, entretanto, não foi possível definir a posição taxonômica das linhagens de X. sp. pv. arracaciae, X. sp. pv. esculenti e X. sp. pv. eucalypti, embora várias ferramentas tenham sido utilizadas. Assim, somente estudos complementares deverão ser conduzidos visando esclarecer a classificação dessas linhagens. Nesse estudo também foram analisadas as espécies X. campestris, X. translucens, X. cucurbitae e X. melonis. Visando a diferenciação dos patovares da espécie X. campestris, os DNAs destas linhagens foram submetidos a experimentos de PCR-RFLP do gene rpoB e as digestões duplas utilizando-se Cfo I/Mbo I. Os resultados permitiram a diferenciação dos patovares X.c. pv. raphani, X.c. pv. barbariae, X.c. pv. incanae, X.c. pv. armoraciae e X.c. pv. campestris/ X.c. pv. aberrans. Ainda, os dados obtidos nas amilises do gene rpoB permitiram o desenvolvimento de primers específicos para as espécies X. campestris, patogênicas as crucíferas (rpoB2F/xcamR) e X. translucens, patogênicas a gramíneas e cereais (trans1F/trans2R). Alem disso, amilises de sequências da região espaçadora 16S-23S DNAr possibilitaram o desenho do par de primers mecF/mecR, especifico para X. cucurbitae e X. melonis, espécies patogênicas ao melão. A especificidade destes primers foi confirmada em experimentos de amplificação utilizando-se DNAs de algumas bactérias de diferentes gêneros isoladas de mesma espécie de plantas hospedeiras. O nível de sensibilidade da técnica de PCR utilizando-se os primers desenvolvidos foi de 0,1 pg para rpoB/xcam, de 0,01 ng para trans1F/trans2R e de 1 pg para mecF/mecR / Abstract: Xanthomonas species are responsible for diseases causing economic losses in many crops. These phytopathogens have been subject of several taxonomic studies resulting in significant changes at interespecific or infraspecific level. This study aimed the taxonomic characterization of Xanthomonas species including: (1) differentiation and phylogenetic analysis of Xanthomonas species; (2) clarification of taxonomic position of strains classified as Xanthomonas sp.; (3) differentiation of the pathovars of X. campestris; ( 4) development of specific primers for X. campestris, X. translucens, X. cucurbitae and X. melonis. The rpoB2F/rpoB3R primers, designed from rpoB gene sequences, were employed in amplification experiments using DNAs from 26 species of Xanthomonas genus and the products (800 bp) were digested with different restriction enzymes. Profiles using Hae III allowed to differentiate the most species of the genus, including pathogens the affect the same plant host as X. albilineans e X. sacchari (pathogenic to sugarcane); X. cucurbitae e X. melonis (pathogenic to melon); X. vesicatoria, X. gardneri and X. euvesicatoria/X. perforans (pathogenic to tomato). Amplification products of the rpoB gene were sequenced and the phylpgenetic tree constructed from these sequences also allowed the differentiation of the Xanthomonas species, indicating that the rpoB gene can be used as an efficient molecular marker for phylogenetic relationships studies within the Xanthomonas genus. DNA of the strains classified as Xanthomonas sp. were also analysed by the amplification experiments using specific primers for X. axonopodis species, DNA-DNA hybridization and multilocus sequence analysis of the genes rpoB, rpoA, atpA, recA and intergenic spacer region 16S-23S rDNA. Results showed that the strains of X. sp. pv. viticola, X. sp. pv. betae and X. sp. pv. paulliniae belong to X. axonopodis species, however, it was not possible to define the taxonomic position of X. sp. pv. arracaciae, X. sp. pv. esculenti and X. sp. pv. eucalypti, although different approaches have been used. Thus, further studies should be conducted in order to clarify their classification. In this study X. campestris, X. translucens, X. cucurbitae and X. melonis were also analyzed. In order to differentiate the pathovars of the X. campestris specie, the DNAs of these strains were submitted to PCR-RFLP analysis of the rpoB gene and the double digestions using Cfo I/Mbo I allowed the differentiation of the pathovars X. c. pv. raphani, X.c. pv. barbariae, X.c. pv. incanae, X.c. pv. armoraciae and X.c. pv. campestris/ X.c. pv. aberrans. Also, the data obtained in the rpoB gene analysis allowed the development of specific primers for the species X. campestris, pathogenic to crucifers (rpoB2F/xcamR) and X. translucens, pathogenic to grasses and cereals (trans1F/trans2R). Furthermore, intergenic spacer 16S-23S rDNA sequences analysis enabled the development of the mecF/mecR primers, specific for X cucurbitae and X melonis, both pathogenic to melon. The specificity of these primers was confirmed by amplification experiments using DNAs from bacteria belonging to different genera pathogenic to the same plant hosts. Sensitivity level of the PCR technique using the primers developed was 0,1 pg for rpoB/xcam, 0,01 ng for trans1F/trans2R and 1 pg for mecF/mecR / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

The increasing number of Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli (E. coli) associated with sepsis in China is the reason for designing the current study. During 2014-2016, thirty hospitals representing 10 different provinces in China was involved in collecting E. coli isolates causing blood stream infections. Early treatment with suitable antibiotics have been found to be of lifesaving importance in the case of care for septic patients. Thorough understanding of the pathogens involved is therefore crucial. Using antimicrobial susceptibility testing, PCR and Multi Locus Sequence Typing (MLST), the molecular characteristics of ESBL producing E. coli isolates could be determined. This study can report that the most common ESBL producing genes found were CTX-M-14 (51 isolates, 45,5%), CTX-M-55 (23 isolates, 20,5%) CTX-M-15 (22 isolates, 19,6%). In addition, 2 isolates (1,8%) were found to be SHV-11 positive which is another ESBL producing gene. As a side finding, 5 isolates harbored Metallo-beta-lactamase (MBL) encoding genes such as NDM-5 and NDM-1 which were found to coexist with CTX-M-55 and CTX-M-14 respectively. An MLST analysis resulted in the finding of 25 different and 17 previously unknown (16,2 %) sequence types. The most common sequence types were ST131 (18 isolates, 17,1 %) as reported previously.  No significant differences in antimicrobial susceptibility were identified whether ESBL producing genes such as SHV and CTX-M was present or not. This study indicates that there could be novel resistance mechanisms present among those isolates not encoding the genes of interest. However, this finding requires further research before it can be confirmed.

Avian pathogenic Escherichia coli (APEC) causes a wide range of diseases in chickens called colibacillosis, resulting a substantial economic loss to the poultry industry. This dissertation aims at addressing this disease by exploring key virulence-associated genes (VAGs), swarming motility (SM), and multi-locus sequence types (MLST) of E. coli isolates obtained from colibacillosis-infected or asymptomatic commercial hens. Secondly, by classifying the E. coli isolates into different virulence categories based on the presence of five key VAGs [iroN, ompT, hlyF, iutA, and iss]. Finally, by performing embryo lethality assays and experimental infection studies to establish the effect of these VAGs. Our findings showed a significantly higher proportion of E. coli isolates obtained from colibacillosis lesions possessed the ompT gene compared to the isolates of asymptomatic commercial hens. A trend of a higher occurrence of the iutA gene was also observed in the isolates of colibacillosis cases. Based on the presence of all five VAGs, we categorized 87.5% of the isolates obtained from colibacillosis lesions into the virulent category and 64.71% of the isolates obtained from asymptomatic commercial hens into the avirulent category. During the embryo lethality assay, we found an interaction effect of virulence categories and SM on embryo mortality. Motile and hyper-motile isolates of virulent and moderately virulent categories caused significantly higher embryo mortality than the non-motile isolates of the same categories. Isolates of the avirulent category significantly reduced the relative embryo weight of the remaining live embryos. The MLST of the isolates did not have any influence on embryo lethality, or relative embryo weight. Yolk sac retention of the remaining live embryo was unaffected by virulence category, MLST, and SM of the isolates. During the experimental infection studies, we observed higher mortality and lesion scores in layer chicks inoculated intratracheally by virulent isolates compared to avirulent isolates. These two classes of isolates also had a different colonization pattern in the extra-intestinal tissues. The avirulent isolates preferably colonized deeper respiratory tracts such as airsacs whereas, the virulent isolates colonized systemic organs such as the liver. Overall, we expect this dissertation will establish the contribution of five key VAGs on embryo and chick mortality, lesion development, and colonization pattern of E. coli isolates. These findings will facilitate the selection of VAGs for field diagnosis of APEC.

No description available.

Les études longitudinales sur une longue période permettent d’échantillonner plusieurs fois le phénomène étudié et ainsi, avec des mesures répétées, dégager une tendance confirmée. Mais, dès lors, elles produisent de très larges bases de données épidémiologiques accompagnées de plus de sources de bruit par rapport aux études à observation unique ; et souvent, contiennent de la corrélation dans les mesures. Ici, nous avons présenté à travers cette thèse une étude de long terme des facteurs épidémiologiques et génétiques du paludisme menée dans deux cohortes familiales du Sénégal, l’une dans le village de Dielmo suivi pendant 19 années consécutives (1990 – 2008) et l’autre dans le village de Ndiop suivi pendant 16 années consécutives (1993 – 2008). L’objectif de ce travail de thèse a été de développer des méthodes d’analyse statistique pour identifier des gênes de susceptibilité / résistance au paludisme prenant en compte les relations familiales, les mesures répétées et des potentielles interactions génotypes – environnement dans l’évaluation des phénotypes. Par la suite, de tels phénotypes corrigés des facteurs identifiés comme potentielles sources de confusion et/ou de bruit ont été alors utilisés pour les tests de liaison et d’association génétique. Le phénotype principal étudié chez chaque volontaire a été la survenue ou non d’accès palustre, attribué à une infection au parasite Plasmodium falciparum, durant chaque trimestre de présence (PFA). Les études ont été menées de manière indépendante dans chacun des deux villages, de même que les analyses descriptives, l’estimation de la contribution génétique humaine et des effets individuels. Les tests de liaison et d’association génétique ont été réalisés par des méthodes familiales basées sur l’analyse de la transmission d’allèles des parents aux enfants (Transmission Disequilibrium Test). Ces méthodes sont connues pour être robustes par rapport au problème de la stratification de population et donc nous permettent d’augmenter la taille de notre échantillon dans les études de liaison et d’association génétique en analysant les deux villages en même temps. / Long term longitudinal surveys have the advantage to enable several sampling of the studied phenomena and then, with the repeated measures obtained, find a confirmed tendency. However, these long term surveys generate large epidemiological datasets including more sources of noise than normal datasets (e.g. one single measure per observation unit) and potential correlation in the measured values. Here, we studied data from a long-term epidemiological and genetic survey of malaria disease in two family-based cohorts in Senegal, followed for 19 years (1990–2008) in Dielmo and for 16 years (1993–2008) in Ndiop. The main objectives of this work were to take into account familial relationships, repeated measures as well as effect of covariates to measure both environmental and host genetic (heritability) impacts on the outcome of infection with the malaria parasite Plasmodium falciparum, and then use findings from such analyses for linkage and association studies. The outcome of interest was the occurrence of a P. falciparum malaria attack during each trimester (PFA). The two villages were studied independently; epidemiological analyses, estimation of heritability and individual effects were then performed in each village separately. Linkage and association analyses used family-based methods (based on the original Transmission Disequilibrium Test) known to be immune from population stratification problems. Then to increase sample size for linkage and association analyses, data from the two villages were used together.

Dans la plupart des questions écologiques, les phénomènes aléatoires d'intérêt sont spatialement structurés et issus de l'effet combiné de multiples variables aléatoires, observées ou non, et inter-agissant à diverses échelles. En pratique, dès lors que les données de terrain ne peuvent être directement traitées avec des structures spatiales standards, les observations sont généralement considérées indépendantes. Par ailleurs, les modèles utilisés sont souvent basés sur des hypothèses simplificatrices trop fortes par rapport à la complexité des phénomènes étudiés. Dans ce travail, la démarche de modélisation hiérarchique est combinée à certains outils de la statistique spatiale afin de construire des structures aléatoires fonctionnelles "sur-mesure" permettant de représenter des phénomènes spatiaux complexes en écologie des populations. L'inférence de ces différents modèles est menée dans le cadre bayésien avec des algorithmes MCMC. Dans un premier temps, un modèle hiérarchique spatial (Geneclust) est développé pour identifier des populations génétiquement homogènes quand la diversité génétique varie continûment dans l'espace. Un champ de Markov caché, qui modélise la structure spatiale de la diversité génétique, est couplé à un modèle bivarié d'occurrence de génotypes permettant de tenir compte de l'existence d'unions consanguines chez certaines populations naturelles. Dans un deuxième temps, un processus de Poisson composé particulier,appelé loi des fuites, est présenté sous l'angle de vue hiérarchique pour décrire le processus d'échantillonnage d'organismes vivants. Il permet de traiter le délicat problème de données continues présentant une forte proportion de zéros et issues d'échantillonnages à efforts variables. Ce modèle est également couplé à différents modèles sur grille (spatiaux, régionalisés) afin d'introduire des dépendances spatiales entre unités géographiques voisines puis, à un champ géostatistique bivarié construit par convolution sur grille discrète afin de modéliser la répartition spatiale conjointe de deux espèces. Les capacités d'ajustement et de prédiction des différents modèles hiérarchiques proposés sont comparées aux modèles traditionnellement utilisés à partir de simulations et de jeux de données réelles (ours bruns de Suède, invertébrés épibenthiques du Golfe-du-Saint-Laurent (Canada)).