Surveys for entomopathogenic bacteria and their associated nematode hosts were conducted locally (University of Bath campus) and across southern England. Sampling involved trialing a novel Android app. (Epicollect) to manage sample collection data. Galleria larvae were used to bait UK soil samples. Insects which became infected were placed on White traps to collect any emerging nematodes, from which bacteria were isolated. Bacteria were also isolated from the haemolymph of any infected larvae. Bacterial isolates were classified on the basis of 16s rDNA and recA gene sequences. Serratia proteamaculans-like strains dominated the samples, and Multilocus sequence analysis (MLSA) was developed for the characterization of these Serratia isolates. We determined the sequences of (350-450-bp) fragments from five housekeeping genes of 84 isolates of Serratia proteamaculans. MLSA was shown to be effective for distinguishing closely related strains found in the insects’ haemolymph and from different nematodes. goeBURST was used to visualize the relationships between the STs, and the data showed a high level of discrimination, resolving 69 STs from the 84 isolates. In addition, the data derived from this study were represented in a phylogenetic network using the Splits Tree-network methods, to show the rate of recombination within and between the genes. From a total of 256 infected Galleria 23.04% were nematode positive. The nematodes were identified based on 18S rDNA 19 isolates were close relatives of the species Pristionchus entomophaga and Diplogasteriodes magnus (Diplogastridae). A further 16 isolates were more closely related to Steinernema glaseri (Steinernematidae). All three nematode types were isolated from diverse habitats and soil types, but were isolated more frequently in cold seasonal conditions. The bacterial sequence data suggest that the nematode- associated strains of bacteria belong to specific clades, distinct from the free living infective strains, which hints at ecological diversity within the S. proteamaculans population. Two of the Serratia proteamaculans-like strains had been chromosomally labeled with GFP to confirm the specifics of their association with the nematode hosts. The associated S. proteamaculans-like isolates isolated from Bath and Chepstow soils were examined further for their pathogenicity to Galleria mellonella and Manduca sexta larvae. Serratia Bath isolates, isolated from Pristionchus were more virulent toward both insect hosts than the Serratia from the Chepstow isolates associated with Steinernema nematodes. This suggests that host specificity may play important role in the virulence of the strain.

Nous avons étudié le rôle des éléments génétiques mobiles (EGM) dans l’évolution et la virulence de Streptococcus agalactiae, bactérie pathogène opportuniste responsable d'infections chez l’homme. La structure génétique de la population a été analysée par multilocus sequence typing à partir d’un souchier représentatif de la diversité de l’espèce. La distribution de 11 EGM (sept séquences d’insertion, trois transposases, un intron) a été confrontée à la structure de la population. Cette confrontation a permis i) de démontrer que l’acquisition des EGM corrélait avec l’évolution de l’espèce, ii) de proposer une hypothèse de la chronologie d’acquisition des EGM, iii) d’identifier certains EGM comme marqueurs de l’écosystème d’origine des souches, et iv) de conforter l’hypothèse de l’émergence du clone humain invasif CC17 à partir d’un ancêtre bovin. Nous avons également cartographié les copies des EGM présentes sur le génome de S. agalactiae, puis nous avons identifié huit nouveaux sites d’insertion, dont deux sont significativement associés à l’origine écologique de la souche. / We studied the role of mobile genetic elements (MGE) on the evolution and the virulence of Streptococcus agalactiae, an opportunistic bacterium responsible for human infections. The genetic population structure was analyzed by multilocus sequence typing using a collection representative of the species diversity. The distribution of 11 MGE (seven insertion sequences, three transposases, one intron) was compared to the population structure. We demonstrated that the MGE prevalence strongly correlates with the genetic lineages. We proposed an evolutionary scheme for the acquisition of the MGE. Several MGE appeared to be markers of the origin of the strains. MGE analysis brought evidence for a bovine origin of the human virulent clone CC17. We also identified the position of the MGE copies on the S. agalactiae genome and we identified eight new MGE insertion sites, from which two were significantly associated with the ecological origin of the isolates.

O gênero Stenotrophomonas inclui doze espécies de bactérias Gram- negativas, não fermentadoras, que podem ser encontradas no ambiente. Até o presente, S. maltophilia é a única espécie com linhagens que são patógenos oportunistas em seres humanos. Essa espécie tem ganhado importância clínica por estar envolvida em um crescente número de infecções em pacientes imunodeprimidos e em UTI neonatais, apresentando altas taxas de morbimortalidade. Isolados ambientais e clínicos de S. maltophilia compartilham 85% de homologia genômica, podendo ser a diferença uma consequência da adaptação da bactéria aos diferentes nichos ecológicos em que é encontrada. Embora infecções e/ou colonizações por S. maltophilia aconteçam principalmente em ambiente nosocomial, diversos estudos têm mostrado um aumento do número de infecções de origem comunitária. Neste estudo, isolados nosocomiais e linhagens clínicas de S. maltophilia foram fenotipados e comparados quanto ao seu perfil filogenético por Multilocus Sequence Typing (MLST). Na análise por MLST utilizaram-se os genes atpD, gapA, guaA, nuoD, ppsA, recA e rpoA, por serem genes constitutivos. O perfil filogenético mostrou alta variabilidade clonal, provavelmente refletindo o processo de adaptação de S. maltophilia ambientais a outros habitats. Verificou-se que dois subgrupos de isolados clínicos de S. maltophilia com grande homogeneidade filogenética apresentam recombinação intergrupos, indicando alta permissividade à transferência horizontal de informação genética, envolvida na resistência a antibióticos e na expressão de fatores de virulência. Mais ainda, para a maioria das amostras clínicas aqui estudadas, inferências filogenéticas podem ser feitas apenas com o uso do gene ppsA. Portanto, o sequenciamento de apenas um fragmento específico para esse gene seria suficiente, em muitos casos, para determinar se a infecção por S. maltophilia foi causada por cepa já presente no ambiente nosocomial ou por bactérias de origem comunitária introduzidas nesse ambiente pela circulação de pessoas e materiais. Finalmente, foi possível mostrar que até mesmo isolados e linhagens clínicas filogeneticamente próximos não compartilham similaridade de perfil metabólico, o que indica sua origem comunitária / The genus Stenotrophomonas comprises twelve species of Gram- negative and non-fermentative bacteria, which can be found in the environment. The species S. maltophilia is the only one, until the present, that includes opportunistic pathogenic strains in humans. S. maltophilia gained clinical importance by being involved in an increasing number of infections in immunocompromissed patients and in NICUs, with high rates of morbidity and mortality. Environmental and clinical isolates of S. maltophilia share around 85% of genomic homology and this difference may be a consequence of adaptation to the different ecological niches where S. maltophilia can be found. Although infection and/or colonization by S. maltophilia occur mainly in the nosocomial environment, several studies have shown a growing number of community-acquired infections. In this study, nosocomial isolates and clinical strains of S. maltophilia were phenotyped and their phylogenetic profiles compared by using Multilocus Sequence Typing (MLST). In order to accomplish the MLST analysis the constitutive genes atpD, gapA, guaA, nuoD, ppsA, recA and rpoA were used. The resultant global phylogenetic profile showed high clonal variability, what correlates with the adaptability process of environmental S. maltophilia to other habitats. It was found that two clinical isolates subgroups of S. maltophilia with great phylogenetic homogeneity present intergroup recombination indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. Moreover, for most of the clinical samples studied here, phylogenetic inferences can be made with the use of the gene ppsA only. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, to determine whether the infection with S. maltophilia ? ? was caused by a strain already present in the nosocomial environment, or by bacteria introduced from the community in this environmental through the movement of people and materials. Finally, phenotyping data showed that even closely phylogenetic related nosocomial isolates and clinical strains do not share the same metabolic profile, thus indicating their community- acquired origin

O gênero Stenotrophomonas inclui doze espécies de bactérias Gram- negativas, não fermentadoras, que podem ser encontradas no ambiente. Até o presente, S. maltophilia é a única espécie com linhagens que são patógenos oportunistas em seres humanos. Essa espécie tem ganhado importância clínica por estar envolvida em um crescente número de infecções em pacientes imunodeprimidos e em UTI neonatais, apresentando altas taxas de morbimortalidade. Isolados ambientais e clínicos de S. maltophilia compartilham 85% de homologia genômica, podendo ser a diferença uma consequência da adaptação da bactéria aos diferentes nichos ecológicos em que é encontrada. Embora infecções e/ou colonizações por S. maltophilia aconteçam principalmente em ambiente nosocomial, diversos estudos têm mostrado um aumento do número de infecções de origem comunitária. Neste estudo, isolados nosocomiais e linhagens clínicas de S. maltophilia foram fenotipados e comparados quanto ao seu perfil filogenético por Multilocus Sequence Typing (MLST). Na análise por MLST utilizaram-se os genes atpD, gapA, guaA, nuoD, ppsA, recA e rpoA, por serem genes constitutivos. O perfil filogenético mostrou alta variabilidade clonal, provavelmente refletindo o processo de adaptação de S. maltophilia ambientais a outros habitats. Verificou-se que dois subgrupos de isolados clínicos de S. maltophilia com grande homogeneidade filogenética apresentam recombinação intergrupos, indicando alta permissividade à transferência horizontal de informação genética, envolvida na resistência a antibióticos e na expressão de fatores de virulência. Mais ainda, para a maioria das amostras clínicas aqui estudadas, inferências filogenéticas podem ser feitas apenas com o uso do gene ppsA. Portanto, o sequenciamento de apenas um fragmento específico para esse gene seria suficiente, em muitos casos, para determinar se a infecção por S. maltophilia foi causada por cepa já presente no ambiente nosocomial ou por bactérias de origem comunitária introduzidas nesse ambiente pela circulação de pessoas e materiais. Finalmente, foi possível mostrar que até mesmo isolados e linhagens clínicas filogeneticamente próximos não compartilham similaridade de perfil metabólico, o que indica sua origem comunitária / The genus Stenotrophomonas comprises twelve species of Gram- negative and non-fermentative bacteria, which can be found in the environment. The species S. maltophilia is the only one, until the present, that includes opportunistic pathogenic strains in humans. S. maltophilia gained clinical importance by being involved in an increasing number of infections in immunocompromissed patients and in NICUs, with high rates of morbidity and mortality. Environmental and clinical isolates of S. maltophilia share around 85% of genomic homology and this difference may be a consequence of adaptation to the different ecological niches where S. maltophilia can be found. Although infection and/or colonization by S. maltophilia occur mainly in the nosocomial environment, several studies have shown a growing number of community-acquired infections. In this study, nosocomial isolates and clinical strains of S. maltophilia were phenotyped and their phylogenetic profiles compared by using Multilocus Sequence Typing (MLST). In order to accomplish the MLST analysis the constitutive genes atpD, gapA, guaA, nuoD, ppsA, recA and rpoA were used. The resultant global phylogenetic profile showed high clonal variability, what correlates with the adaptability process of environmental S. maltophilia to other habitats. It was found that two clinical isolates subgroups of S. maltophilia with great phylogenetic homogeneity present intergroup recombination indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. Moreover, for most of the clinical samples studied here, phylogenetic inferences can be made with the use of the gene ppsA only. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, to determine whether the infection with S. maltophilia ? ? was caused by a strain already present in the nosocomial environment, or by bacteria introduced from the community in this environmental through the movement of people and materials. Finally, phenotyping data showed that even closely phylogenetic related nosocomial isolates and clinical strains do not share the same metabolic profile, thus indicating their community- acquired origin

<p>Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.</p>

Made available in DSpace on 2015-07-22T20:49:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A metodologia do “Multilocus Sequence Typing (MLST)” tornou-se uma ferramenta importante para tipagem molecular para C. albicans porque esta metodologia pode caracterizar grande número de isolados rapidamente e está isenta da interpretação subjetiva de padrões de bandas em géis de eletroforese. Este método é muito útil no entendimento da filogenia e epidemiologia de cepas de C. albicans recuperadas de infecções fúngicas invasivas. Objetivos: 1) aplicar as metodologias de MLST e Tipagem ABC para isolados de C. albicans recuperados de infecções de corrente sanguínea em hospitais terciários no Brasil e 2) determinar se cepas indistinguíveis ou diferentes foram responsáveis pelos episódios de candidemia persistente ou candidemia recorrente em isolados sequenciais de mesmo paciente. Material e Métodos: Nós aplicamos a metodologia do MLST e Tipagem ABC em isolados de C. albicans de 61 pacientes com candidemia coletados durante um estudo multicêntrico realizado em 11 hospitais públicos terciários de 9 cidades brasileiras. Também foram avaliados os isolados sequenciais de 8 pacientes com candemia persistente ou recorrente. Candidemia persistente foi definido como um episódio de fungemia com duas ou mais culturas positivas para C. albicans, em 2 ou mais dias diferentes, a despeito da contínua terapia antifúngica adotada. Candidemia recorrente foi definida como um episódio de candidemia ocorrendo ao menos 1 mês após o episódio incidente e a negativação de duas hemoculturas sequenciais após introdução da terapia antifúngica, envolvendo a mesma espécie de Candida. Resultados: Um total de 48 únicos “diploid sequence types (DSTs)” foram observados, incluindo 10 novos genótipos e 32 novos DSTs. DST 69 foi o mais comum entre os nossos isolados. Isolados clado 1 responderam a 56% da nossa coleção. O clado 3 e clado 8 foram os clados com maior número de isolados depois de clado 1, ambos respondendo por 10% das amostras. O clado 9 e clado 17 foram responsáveis por 6,5% dos isolados cada um. Isolados clado 12 responderam por 5%. Foi isolada uma única cepa (1,5%) do clado 2, clado 4, clado 16 e um isolado categorizado como “solitário”. Para Tipagem ABC, 82% dos isolados foram classificados como tipo A, seguido por tipo B com 16,5% e tipo C com 1,5%. Quanto aos pacientes com candidemia persistente ou recorrente, para todos os pacientes exceto um, verificou-se a permanência dos mesmos DSTs encontrados entre a primeira e última amostra coletada. Um único paciente com coletas sequenciais pelo período de 10 dias apresentou 3 cepas distintas discriminadas pelo MLST. Uma destas 3 cepas foi a única representante do clado 2 em nosso estudo. Conclusão: Mais de 50% dos isolados deste estudo apresentaram novos DSTs, predominando o clado 1 em 56% das amostras. Para a Tipagem ABC, 82% dos isolados foram do tipo A. Este é o primeiro estudo de nosso conhecimento a descrever infecção de corrente sanguínea por 3 cepas distintas de C. albicans documentadas no período de 10 dias. / The DNA sequence-based genotyping technique multilocus sequence typing (MLST) has emerged as an alternative typing tool for C. albicans because can characterize large numbers of isolates rapidly, and does not require the subjective interpretation of banding patterns. This methodology is a very useful tool in understanding the phylogenetics and epidemiology of C. albicans strains from invasive candidiasis. Objective: Our goal was 1) to apply MLST and ABC typing to C. albicans strains recovered from bloodstream infection from public tertiary care hospitals in Brazil and 2) determine whether indistinguishable or different strains were responsible for persistent or recurrent fungemia by performing MLST and ABC typing on sequential C. albicans isolates from the same patient. Methods: We applied MLST and ABC typing, which is based on the presence or absence of an intron in the 25S rDNA region, to C. albicans strains from 61 patients with candidemia collected during a multicenter surveillance study in 11 public tertiary care hospitals, representative of the public health system of 9 of the largest cities in Brazil. We also analyzed C. albicans strains from 8 patients with persistent or recurrent candidemia. Persistent candidemia was defined as two or more blood cultures positive for C. albicans on 2 or more separate days. Recurrent candidemia was defined as an episode of candidemia occurring at least 1 month after the apparent complete resolution of an infectious episode caused by the same Candida species. Results: A total of 48 unique profiles or diploid sequence types (DST) were observed, with 10 new sequence types (STs) and 32 new DSTs. DST 69 was the most common DST isolated. C. albicans clade 1 accounted for 56% of the collection, clade 3 and clade 8 for 10% each, clades 9 and 17 for 6.5% each, and clade 12 for 5%. Clade 2, clade 4, clade 16 and a singleton strain had 1 isolate each (1.5%). For ABC typing, 82% of the isolates were classified as type A, followed for 16.5% type B and 1.5% type C. All the patients’ strains related to persistent or recurrent candidemia but one showed the same MLST diploid sequence type (DST), ABC type and susceptibility profile to antifungals in the first and second samples. One patient with 7 samples collected sequentially over 10 days showed 3 distinct strains, well discriminated by MLST. One of the 3 strains recovered from this patient showed a single C. albicans isolate found in our total collection classified as clade 2, although clade 2 is commonly found worldwide. Conclusion: More than 50% of isolates from this study form a unique set of DSTs and clade 1 was responsible for 56% of the isolates. For ABC typing, 82% of the isolates were type A. To the best of our knowledge, this is the first study describing a blood stream infection with 3 distinct C. albicans strains in the same patient within a short period of time. / CNPq: GM/GD 142025-2005-4 / CNPq: SWE 200669/2007-9 / TEDE / BV UNIFESP: Teses e dissertações

Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.

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

Nas últimas décadas observou-se um número crescente de relatos de infecções invasivas por Trichosporon em ambientes hospitalares, devido ao aumento da população suscetível e a melhoria dos métodos diagnósticos. Leveduras do gênero Trichosporon, depois de Candida, são as mais relacionadas à infecção fúngica invasiva em pacientes hematológicos, sendo Trichosporon asahii responsável por 90% dos casos. A identificação de espécies de Trichosporon é realizada através do sequenciamento da região IGS1 do DNA ribossomal, técnica considerada padrão-ouro. Através do estudo dos polimorfismos da região IGS1 do DNA ribossomal, diversos genótipos de T. asahii têm sido descritos, entretanto sem relação com a distribuição geográfica, perfil de suscetibilidade aos antifúngicos ou patogenicidade. O presente estudo teve como objetivo padronizar um método de análise por sequenciamento multilocus para a espécie T. asahii, definindo novos genes (loci) para melhor descrever a filogenia da espécie. Foram analisadas 21 cepas de T. asahii de diferentes origens (Brasil, Europa, Ásia) e genótipos (1,3,4,5,6,7). As sequências de genes estruturais (housekeeping genes) dos genomas de T. asahii (CBS2479 e CBS8904) disponíveis no GenBank foram alinhadas e analisadas in silico para o delineamento e avaliação dos novos primers. Após as reações de PCR e análise das sequências de DNA, quatro novos loci foram selecionados para a análise filogenética multilocus: phosphate carrier protein, topoisomerase 1 (TOP1), beta-1-tubulin, copper-exporting ATPase. As árvores filogenéticas demonstraram dois clados bem distintos, com altos valores de bootstraps. Além disso, os genótipos 1 e 3 foram alocados em clados diferentes. Nossos resultados sugerem uma reclassificação genética para a espécie T. asahii. Novos estudos, incluindo um maior número de cepas e outros marcadores genéticos, são necessários para melhor abordar a filogenia atual de T. asahii / In the last decades there has been a significant increase of the reported cases of invasive fungal infections by Trichosporon in hospital settings, related to the increase of the susceptible population and to the improvement of diagnostic methods. Trichosporon are the most frequent yeast related to invasive fungal infection in hematological patients after Candida, with Trichosporon asahii accounting for 90% of the cases. The gold standard method for Trichosporon species identification is the sequence analysis of the intergenic spacer region 1 (IGS1) from the ribosomal DNA. Based on the polymorphisms of the IGS region of ribosomal DNA, several T. asahii genotypes have been described, without relation with geographical distribution, antifungal susceptibility profile or pathogenicity. The objective of the study was to evaluate a multilocus sequencing method for the T. asahii species, defining new loci to better describe the phylogeny of the species. Twenty-one strains of T. asahii from different origins (Brazil, Europe, Asia) and genotypes (1,3,4,5,6,7) were analyzed. Housekeeping genes from T. asahii genomes (CBS2479 and CBS8904) available in GenBank were aligned and in silico analyses were carried out to design and evaluate the new primers. After PCR reactions and DNA sequence analysis, four new loci were selected for the multilocus plylogenetic analysis along with the IGS1 region from the rDNA: topoisomerase 1 (TOP1), phosphate carrier protein, beta-1-tubulin, copper-exporting ATPase. Phylogenetic trees revealed two well-distinct clades, with high bootstraps values. Moreover, IGS genotypes 1 and 3 strains were split into the different clades. Our results suggest a different genetic background for the species T. asahii. Further studies including more T. asahii strains and other genetic markers are necessary to better address the current phylogeny of T. asahii

<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>