Pectobacterium carotovorum subsp. brasiliense (Pcb) is a plant pathogenic bacterium that causes blackleg and tuber soft rot disease of potato worldwide. Pectobacterium spp. are characterized by the secretion of large quantities of plant cell wall degrading enzymes. As the name indicates, Pectobacteria are pectinolytic pathogens, producing enzymes such as pectate lyase, polygalacturonase, and many others that are used to catalyse the breakdown of pectin, the main plant cell wall component. Consequently, virulence of Pectobacteria is highly reliant upon the production and secretion of macerating enzymes. Hence these bacteria are also referred to as “brute-force” pathogens. Infection and disease symptoms on plants commonly result in the development of blackleg disease, a characteristic black-like decay extending on the stems of infected potato plants. Furthermore, the infection of tubers results in the development of soft rot disease. Pcb is of particular interest in that among Pectobacterium spp. such as Pectobacterium atrosepticum (Pa), P. carotovorum subsp. carotovorum (Pcc), and P. wasabiae, Pcb strains are reported to be the most aggressive and virulent pathogens causing blackleg and soft rot disease of potato in many growing regions across the world. The fact that strains of Pcb were recently reported and isolated in South Africa has necessitated that this work be undertaken in order to characterise this newly described important pathogen of potato in regard to its phenotypic, genetic diversity, virulence and host range traits. Therefore in this work Pcb strains were subjected to multilocus phylogenetic analyses (MLSA) in order to investigate and determine whether there is any interspecies and intraspecies genetic diversity among the South African Pcb isolates. It was thus established that there is a significant genetic diversity that exists both on an interspecies and intraspecies level among Pcb isolates. As a result we sought to investigate further if the level of genetic diversity observed can be reflected in terms of the pathogen’s virulence, biochemical, phenotypic as well as host range characteristics. The results of virulence assays on potato tubers and stems indicated that Pcb strains are significantly much more virulent on potato compared to closely related Pectobacterium spp. such as Pa and Pcc. Moreover, the level of intraspecies diversity observed through phylogeny was also evident and reflected on the phenotypic, virulence and host range characteristics of the pathogen. This study also focused on investigating virulence factors employed by Pectobacterium spp. during infection. Such factors include the ability to produce and secrete of various extracellular macerating enzymes, as well as screening for the presence of virulence associated effectors and phytotoxin genes. It was of interest to observe that Pcb strains have the ability to grow and produce substrate-degrading enzymes much more rapidly compared to Pa and Pcc. This phenomenon was also observed in virulence assays where Pcb strains were noted to cause more rapid and most severe maceration symptoms on potato tubers and stems. Thus in agreement with other studies, our results suggests that Pcb is a uniquely sophisticated but diverse plant pathogen which can be considered to be one of the most aggressive causal agents of blackleg and soft rot disease of potato in South Africa. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / Unrestricted

Genetic typing of Klebsiella pneumoniae is used for epidemiological referencing. In the clinical setting it can be useful in outbreak investigations, understanding transmission and managing hospital infections. Multi-drug resistant bacteria exist and proliferate either due to natural selection of clonal lineages or the transfer of mobile genetic elements, sometimes in response to antibiotic-use selective pressure. Pulsed-field gel electrophoresis (PFGE) is highly discriminatory and the gold standard typing method for the characterisation of K. pneumoniae isolates. The aim of the study was to genetically characterise K. pneumoniae isolates by PFGE and multilocus sequence typing (MLST). One hundred unrepeated ESBL-producing K. pneumoniae isolates were collected from the National Health Laboratory Service (NHLS). The PFGE was performed on a Rotaphor VI system (Biometra, Germany). Clonal representatives were further characterised by MLST. All the strains were typeable by PFGE using XbaI, which discerned multiple pulsotypes and MLST identified 10 different STs including a novel sequence type, ST1632. The diverse pulsotypes of K. pneumoniae isolates are not suggestive of clonal spread of particular strains. The MLST results further confirmed the variability among isolates tested and elucidated several STs, some of which have been identified internationally and often associated with carbapenem-resistance. Data on K. pneumoniae STs is still limited in the South African clinical setting, although the close monitoring of resistance profiles and characterisation of isolates is imperative for outbreak analysis, identification of prominent STs in clinical settings as compared to international counterparts and surveillance of expanding resistance. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2015. / Medical Microbiology / MSc (Medical Microbiology) / Unrestricted

Beluga whales in Canadian waters are subdivided into at least six genetically distinct stocks maintained by geographic separation and philopatry to estuaries in summer. Belugas in eastern and western Hudson Bay have previously been shown to be compose genetically distinct populations using mitochondrial restriction analysis. It is not known whether these stocks are further subdivided on the basis of specific estuarine use. Mitochondrial DNA control region sequences were used to investigate variation among belugas sampled at several sites along eastern Hudson Bay, Hudson Strait and Ungava Bay. 320 bp were sequenced, including the highly variable 5' region of control region, in 126 belugas. 17 variable sites and 17 haplotypes, which clustered into 2 related groups, were detected among the whales sequenced. Haplotypes of group A were found mostly in eastern Hudson Bay sites, while B group haplotypes were predominant in northern populations. Significant differences in frequencies of haplotype groups were found between eastern Hudson Bay and Southern Hudson Strait/Ungava Bay populations, indicating they are genetically distinct populations. Haplotype distribution patterns also suggested possible differences between belugas using different estuaries along eastern Hudson Bay. The presence of both groups in each population indicated some exchange of individuals between populations, and/or between eastern and western Hudson Bay. Multilocus DNA fingerprinting was used to investigate the extent of gene flow between eastern and western Hudson Bay belugas via interbreeding on common wintering grounds in Hudson Strait. Belugas from St. Lawrence estuary and the Mackenzie Delta were also analyzed to measure their genetic relatedness to Hudson Bay whales as well as for purposes of comparison to earlier fingerprinting analyses. While results supported lower genetic diversity within the St. Lawrence population, the range of bandsharing within and between populations was otherwise low (0.09 -0.17 for Jeffreys 33.15 and 0.12-0.22 for Jeffreys 33.6). Mantel tests showed differences among St. Lawrence, Hudson Bay, and Mackenzie Delta populations, but not within Hudson Bay. The conflicting nature of the data did not allow conclusions regarding gene flow. Therefore, DNA fingerprinting was not considered to have provided sufficient resolution in addressing this issue. / Thesis / Master of Science (MS)

Yes / Background: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. Methods: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. Results: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44–0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29–0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11–0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. Conclusions: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future. / Supported by medical and health science and technology projects of Health and Family Planning Commission of Guangzhou Municipality (grant number 20151A010034) and Guangdong provincial science and technology planning projects (grant number 2014A020212520).

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

Bisherige Methoden der Haplotyp-Block-Definition zielen entweder auf abwesende Rekombinationsereignisse oder eine effiziente Beschreibung genomischer Variation. Die vorliegende Arbeit definiert Blöcke von Single Nucleotide Polymorphisms (SNP) als Gebiete erhöhten Kopplungsungleichgewichtes (LD). Für dieses Ziel wird ein neues, entropie-basiertes Maß für LD zwischen multiplen Markern/Loci (Normalized Entropy Difference) entwickelt und als eine Multilocus-Erweiterung des paarweisen Maßes r2 charakterisiert. Ein zugehöriger Algorithmus für die Block-Definition wird vorgeschlagen. Seine Evaluierung an einem Datensatz des menschlichen Chromosoms 12 vom Internationalen Haplotype Map Projekt zeigt die Nützlichkeit der abgeleiteten Blöcke in Hinblick auf verschiedene Eigenschaften, einschließlich ihrer chromosomalen Coverage und der Anzahl sowie des Anteils der häufigen Block-Haplotypen. Der wesentliche Einfluß der SNP-Dichte auf die zu entdeckenden LD- und Blockstrukturen wird demonstriert. Der Erfolg von Assoziationsstudien in komplexen Erkrankungen mit Block-Haplotypen als multiallelischen Markern wird davon abhängen, ob die Common Variants/Common Diseases (CV/CD) Hypothese für solche Erkrankungen erfüllt ist. / Current approaches to haplotype block definition target either absent recombination events or the efficient description of genomic variation. This thesis aims to define blocks of single nucleotide polymorphisms (SNP) as areas of elevated linkage disequilibrium (LD). To this end, a new entropy-based measure for LD between multiple markers/loci, the Normalized Entropy Difference, is developed and is characterized as a multilocus extension of the pairwise measure r2. A corresponding algorithm for the block definition is proposed. Its evaluation on a data set of human chromosome 12 from the International Haplotype Map project proves the usefulness of the derived blocks with respect to several features, including their chromosomal coverage and the number and portion of common block haplotypes. The critical role of the SNP density for detectable LD and block structure is demonstrated. The success of association studies in common diseases with block haplotypes serving as multi-allelic markers will depend on whether the Common Variants/Common Diseases (CV/CD) hypothesis holds true for those diseases.

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

This thesis focuses on multilocus methods designed to detect single nucleotide polymorphisms (SNPs) that are associated with disease using case-control data. I study multilocus methods that allow for interaction in the regression model because epistasis is thought to be pervasive in the etiology of common human diseases. In contrast, the single-SNP models widely used in genome wide association studies (GWAS) are thought to oversimplify the underlying biology. I consider both pairwise interactions between individual SNPs and modular interactions between sets of biologically similar SNPs. Modular epistasis may be more representative of disease processes and its incorporation into regression analyses yields more parsimonious models. My methodological work focuses on strategies to increase power to detect susceptibility SNPs in the presence of genetic interaction. I emphasize the effect of gene-gene independence constraints and explore methods to relax them. I review several existing methods for interaction analyses and present their first empirical evaluation in a GWAS setting. I introduce the innovative retrospective Tukey score test (RTS) that investigates modular epistasis. Simulation studies suggest it offers a more powerful alternative to existing methods. I present diverse applications of these methods, using data from a multi-stage GWAS on prostate cancer (PRCA). My applied work is designed to generate hypotheses about the functionality of established susceptibility regions for PRCA by identifying SNPs that affect disease risk through interactions with them. Comparison of results across methods illustrates the impact of incorporating different forms of epistasis on inference about disease association. The top findings from these analyses are well supported by molecular studies. The results unite several susceptibility regions through overlapping biological pathways known to be disrupted in PRCA, motivating replication study.

Gobiidae is one of the largest families of teleost fishes with nearly 2000 species currently recognized. They have a worldwide distribution with exception of Arctic and Antarctic areas, inhabiting marine, brackish and freshwaters. Mostly, they are small-sized and live inconspicuously on the bottom. Their phylogeny has been studied only partially. In Europe, three independent lineages of gobies exist (Gobius-, Aphia- and Pomatoschistus-lineage), most of the species of these lineages are marine. In this work, I performed a multilocus study of the Gobius-lineage encompassing the majority of the species. Mitochondrial (cytochrome b and cytochrome c oxidase I) as well as nuclear (rhodopsin and recombination activating gene) markers were used. 480 individuals of 30 species were analyzed in the laboratory and sequences of further 25 - 32 species were downloaded from the Genbank and added to a dataset of each marker according to availability. Mitochondrial markers were more informative than the nuclear ones. The usefulness of cytochrome c oxidase I for studying phylogenetic relationships of gobies was compared with cytochrome b. Cytochrome c oxidase I showed to be useful for identification of the species, but has some limitations in resolving deeper phylogenetic relationships in gobies. Cytochrome b showed...

<p>Streptococcus agalactiae, also designated group B streptococcus (GBS), is a Gram-positive coccus, and it is an important pathogen that causes invasive disease in neonates, pregnant adults, and non-pregnant adults with predisposing conditions. The group II intron GBSi1 is one of the major mobile genetic elements identified in S. agalactiae. The aim of this thesis was to characterize the GBSi1 distribution pattern, the population structure, and the influence of serotype- and clone-specific properties on the invasive capacity among clinical invasive and non-invasive isolates of S. agalactiae.</p><p>Two additional copies of GBSi1 were identified at sites different from the primarily identified scpB-lmb locus. The distribution of GBSi1 was uneven among different serotypes. Three intron copies were only found in isolates of serotype III, and these targeted all the three identified gene loci. In contrast, a single copy of GBSi1 was found in isolates of serotype II and V and only located at the scpB-lmb locus. Furthermore, at the 5′ flanking region of the scpB-lmb gene locus, a novel 2.1 kb DNA fragment with plasmid features was identified only in intron carrying isolates. This may suggest that GBSi1 once was brought into the S. agalactiae genome by an integrated plasmid.</p><p>Multilocus sequence typing was used to characterize totally 314 invasive and non-invasive S. agalactiae isolates collected in Northern and Western Sweden from the years 1988 to 2004. Five major genetic lineages (clonal complexes) were identified among both invasive and non-invasive isolates, including serotype Ia, Ib, and II to V, indicating a clonal population structure of S. agalactiae isolates. A number of genetically highly related isolates were found to express different capsular types, suggesting that capsule switching occurs rather frequently between isolates. Furthermore, non-invasive isolates belonging to the same clonal complexes displayed more heterogeneity in capsule expression as well as in the distribution patterns of mobile genetic elements than invasive isolates. This indicates that less variability is allowed in a highly selective environment such as the blood. All major clonal complexes and serotypes caused invasive disease, although their ability to do so varied greatly. CC17 was significantly associated with neonatal invasive disease; whereas CC19 was equally common among isolates from adult and neonatal disease, despite that both CC17 and CC19 expressed capsular type III. This striking difference seen between CC17 and CC19 suggests that clonal complex associated properties, in addition to capsular type, play important roles in the virulence of S. agalactiae. CC1, a new emerging clone since early 1990s, has caused substantial amount of disease among adults. In addition, mutually exclusive distribution of mobile elements GBSi1 and IS1548 was seen, and they were shown to constitute genetic markers for serotype III CC17 and CC19 isolates, respectively.</p>