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. 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. 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.

The possibility of identifying single nucleotide polymorphisms (SNPs) that would be useful for rapid bacterial typing was investigated. Neisseria meningitidis was the organism chosen for modelling the approach since informative SNPs could be found amongst the sequence data available for multi-locus sequence typing (MLST) at http://www.mlst.net. The hypothesis tested was that a small number of SNPs located within the seven gene fragments sequenced for MLST provide information equivalent to MLST. Preliminary investigations revealed that a small number of SNPs could be utilised to highly discriminate sequence types (STs) of clinical interest. Laboratory procedures demonstrated that SNP fingerprinting of N. meningitidis isolates is achievable. Further tests showed that laboratory identification of a defining SNP in the genome of isolates was to be a practical method of obtaining relevant typing information. Identification of the most discriminating SNPs amongst the ever-increasing amount of MLST sequence data summoned the need for computer-based assistance. Two methods of SNP selection devised by the author of this thesis were translated into computer-based algorithms by contributing team members. Software for two computer programs was produced. The algorithms facilitate the optimal selection of SNPs useful for (1) distinguishing specific STs and (2) differentiating non-specific STs. Current input information can be obtained from the MLST database and consequently the programs can be applied to any bacterial species for which MLST data have been entered. The two algorithms for the selection of SNPs were designed to serve contrasting purposes. The first of these was to determine the ST identity of isolates from an outbreak of disease. In this case, isolates would be tested for their membership to any of the STs known to be associated with disease. It was shown that one SNP per ST could distinguish each of four hyperinvasive STs of N. meningitidis from between 92.5% and 97.5% of all other STs. With two SNPs per ST, between 96.7% and 99.0% discrimination is achieved. The SNPs were selected from MLST loci with the assistance of the first algorithm which scores SNPs according to the number of base mismatches in a sequence alignment between an allele of an ST of interest and alleles belonging to all other STs at a specified locus. The second purpose was to determine whether or not isolates from different sources belong to the same ST, regardless of their actual ST identity. It was shown that with seven SNPs, four sample STs of N. meningitidis could, on average, be discriminated from 97.1% of all other STs. The SNPs were selected with the aid of the second algorithm which scores SNPs at MLST loci for the relative frequency of each nucleotide base in a sequence alignment as a measure of the extent of their polymorphism. A third algorithm for selecting SNPs has been discussed. By altering the method of scoring SNPs, it is possible to overcome the limitations inherent in the two algorithms that were utilised for finding SNPs. In addition, the third approach caters for finding SNPs that distinguish members of a complex from non-members.

This research addressed the effects of cultural management practices, cultivar selection, and applications of seasonal fungicides on ultradwarf bermudagrass health and playability and occurrence of foliar diseases. Additionally, novel ectotrophic root-infecting fungi were isolated from ultradwarf bermudagrass roots exhibiting symptoms of summer decline, identified via multilocus phylogenetic analyses, and characterized by morphological assessments and pathogenicity evaluations.

No description available.

Echinococcus multilocularis est un parasite nécessitant pour survivre un passage successif entre les carnivores, comme le renard et les micro-mammifères. Le parasite est responsable chez l'homme de l'Echinococcose Alvéolaire, une maladie mortelle si elle n'est pas prise en charge. Uniquement décrit dans l'hémisphère nord (Chine, Japon, Europe et Amérique du Nord), la distribution spatiale du parasite semble connaître une évolution récente, notamment en Europe, où l'Arc alpin est décrit comme le foyer historique d'E. multilocularis dans cette région. Le génotypage a été choisi pour étudier la diffusion du parasite en Europe. Cependant, le manque d'outils de détection du polymorphisme du parasite nécessitait la recherche et la caractérisation de marqueurs possédant un haut pouvoir discriminant. Après caractérisation et validation de la cible EmsB multilocus répétée en tandem, la diversité génétique du parasite en Europe a été étudiée à différentes échelles spatiales d'analyse. A l'échelle micro-locale (rongeurs parasités d'une même pâture), les isolats présentaient une faible diversité génétique entre eux, évoquant une contamination des rongeurs par une même source infectieuse (e.g. les fèces d'un même renard parasité). A l'échelle locale (900 km²), 140 parasites de 25 renards ont été étudiés. Les parasites présentaient une diversité génétique permettant de distinguer 6 profils EmsB. La présence simultanée de différents profils chez le renard a été décrite de manière fréquente, évoquant des infestations répétées des renards. Un faible taux d'hétérozygotie a été trouvé chez le parasite, ce qui pourrait être expliqué par un mode de reproduction principalement clonal. A l'échelle continentale (9 sous-régions européennes de la zone endémique historique et de la périphérie de celle-ci) la diversité génétique et la structure spatiale du polymorphisme ont été étudiées à partir de 653 isolats (596 vers adultes isolés de 129 renards, 57 lésions opérés chez des patients et des animaux vivant en captivité). Une grande diversité génétique a été observée en Europe, avec la description de 54 profils EmsB. Des profils transversaux ont été trouvés de part et d'autre de la zone d'étude alors que d'autres plus endémiques étaient limités spatialement. L'étude de la composition génétique au sein des sous-régions européennes a permis de mettre en évidence une plus grande diversité génétique dans le foyer historique par rapport à sa zone périphérique, où quelques profils représentaient la majorité des parasites. Cette distribution évoque une dispersion du parasite à partir de la zone centrale vers la zone périphérique dans un système de transmission « continent-île ». Chez l'homme et l'animal en captivité des profils EmsB décrits comme endémiques ont été trouvés sur plusieurs années, montrant une contamination de manière locale par une même souche. Cette étude constitue la première application d'un marqueur microsatellite multilocus pour l'étude de la circulation d'un helminthe à l'échelle continentale.

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

Methicillin-resistenta Staphylococcus aureus (MRSA) som bär på genen mecA, har förekommit och spridit sig över hela världen, främst i sjukhusmiljö, och orsakat utbrott av vårdrelaterade (så kallade nosokomiala) infektioner. Dessa infektioner kan inte behandlas med stafylokock-penicilliner och MRSA-bakterierna är ofta resistenta även mot flera andra grupper av antibiotika vilket medför att infektionerna ofta är påtagligt svårbehandlade. Under senare år har emellertid allt fler fall beskrivits av samhällsförvärvad MRSA infektion, det vill säga uppträdande av MRSA hos personer som tidigare ej har haft kontakt med sjukhusvård eller behandlats med antibiotika. Det har länge varit oklart om de samhällsförvärvade MRSA [community-acquired (CA-MRSA)] representerar spridning av bakterier från sjukhusmiljön ut till samhället eller om dessa MRSA är spontant uppträdande. Många av dessa stammar har dessutom visat sig bära på sjukdomsrelaterade gener som vanligen inte återfinns hos S. aureus, t.ex. Panton Valentine leukocidin (PVL) som associeras med hudinfektioner och allvarlig lunginflammation med hög dödlighet hos unga och annars friska individer. Denna avhandling beskriver den molekylära epidemiologin hos MRSA med fokus på samhällsförvärvade MRSA som utgjorde mer än hälften av samtliga fall av MRSA i Örebro län och som dessutom ofta producerade PVL toxinet, vars funktion vidare analyserades i detalj. Undersökning av ursprung och släktskap hos samtliga MRSA som isolerats i Örebro län, samt karaktärisering av det genetiskt element som kallas staphylococcal cassette chromosome mec (SCCmec) vilket innehåller genen mecA och ibland även andra resistensgener, visade att CA-MRSA inte är relaterade till de nosokomiala MRSA, och att dessa har uppstått oberoende av varandra. Flertalet MRSA visade sig dessutom bära på SCCmec, och resistensmekanismer, som tidigare inte beskrivits. Troligen har dessa MRSA uppstått genom ett genetiskt utbyte av SCCmec mellan methicillin-resistenta koagulas-negativa stafylokocker (MR-KNS), som utgör huvudparten av normalfloran på huden, och methicillin-känsliga S. aureus som därvid erhåller genen mecA och resistensmekanismer mot samtliga stafylokockantibiotika. I den här avhandlingen framläggs bevis för att ett sådant genetiskt utbyte har skett på Barnkliniken på Universitetssjukhuset i Örebro i slutet på 1990-talet, vilket resulterade i uppkomsten av en ny klon av MRSA som därefter orsakade ett allvarligt utbrott. Kartläggning av DNA-sekvensen hos flertalet unika SCCmec från svenska MRSA gav dessutom en bättre förståelse för hur resistens uppkommer och sprider sig, samt mekanismerna bakom detta. Dessa nya kunskaper kan bidra till en förbättrad diagnostik av MRSA. Detta är framför allt av stor betydelse eftersom nya effektiva kloner av MRSA verkar kunna uppstå ute i samhället med potential att orsaka svårbehandlade infektioner men även att sprida sig bland den friska befolkningen. / Material and methods - During a period of 14 years, around 2000 patients with head injuries were admitted to the emergency ward at Lindesberg County Hospital and Örebro Medical Centre Hospital. Six hundred subjects suffered from skull fracture and/or brain contusion and diagnosis was established using a computed tomography scan (CT). The degree of initial brain injury was estimated using the Swedish Reaction Level Scale (RLS). Sixty-six subjects were investigated with pure tone audiometry in close proximity to the trauma, and this gave an opportunity to study the issue of progress. The investigation took place two to 14 years after trauma, and the results were compared to matched control groups. A battery of different audiological methods was used to investigate peripheral and central auditory function, and a specially designed acoustic environmental room was also utilized. Cognition was investigated using a computer-based test-battery, text information process system (TIPS). Self-assessed hearing, cognition and quality of life were explored using different questionnaires. Results - A high percentage of peripheral and central auditory impairments and also cognitive shortcomings were demonstrated. Progress of SNHL was a common finding, and fracture, high age at trauma and large initial hearing loss predicted progress. Antibody-mediated autoimmunity as a mechanism behind posttraumatic progress of SNHL or clear evidence for sympathetic cochleolabyrinthitis could not be demonstrated. Binaural auditory deficits could be demonstrated when tested in a realistic acoustic environment. Tinnitus, vertigo and memory shortcomings proved to be common sequelae, even in a long-term perspectiveCognitive shortcomings were found in several of these well-rehabilitated subjects.On a group level, there was a good correlation between self-assessments and audiometric results, even if some individuals had a tendency to over- or underestimate their abilities. Conclusion - Auditory and cognitive long-term sequelae of CHI are a common finding even in well-rehabilitated and socially well-functioning subjects, as are vertigo and tinnitus. Vertigo and tinnitus are also common sequelae after CHI, therefore a basic audiovestibular investigation after CHI is recommended, at least in selected cases.Early awareness of the risk for hearing and cognitive sequelae after CHI could lead to measurements taken to prevent tension-related symptoms.Early detection of HI offers an opportunity to try immunosuppressive treatment in cases with a large initial SNHL.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-12-03T17:48:36Z No. of bitstreams: 1 frederico_m_r_silva_ioc_bcm_0032_2005.pdf: 983631 bytes, checksum: c53725cd06ca616bc0df79ce5088aa9f (MD5) / Made available in DSpace on 2012-12-03T17:48:36Z (GMT). No. of bitstreams: 1 frederico_m_r_silva_ioc_bcm_0032_2005.pdf: 983631 bytes, checksum: c53725cd06ca616bc0df79ce5088aa9f (MD5) Previous issue date: 2005 / Universidade Estácio de Sá. Campus Arcos da Lapa. Rio de Janeiro, RJ, Brasil / No Brasil, apenas uma cepa disseminada, o clone epidêmico brasileiro (BEC) de MRSA, foi amplamente caracterizada, apresentando o cassete estafilocócico de resistência à meticilina (SCCmec) IIIA de 67 Kb e multirresistência antibiótica. Entretanto, novos clones de MRSA não-multirresistentes com alta virulência têm sido descritos em infecções comunitárias e hospitalares em vários países. Estes clones abrigam SCCmec curtos, com cerca de 24 Kb, portanto mais eficiente na transferência, replicação e tradução. Este estudo objetiva descrever o background genético dos clones de S. aureus envolvidos em infecções no Rio de Janeiro/RJ. Foram coletadas 139 amostras de S. aureus (54 MRSA), isoladas de pacientes de sete hospitais e de um laboratório de patologia clínica. As amostras foram submetidas a análises moleculares por diversos métodos. Com a análise por Multilocus Restriction Fragment Typing (MLRFT), caracterização do tipo de SCCmec e a presença de genes para leucocidina Panton-Valentine (PVL), juntamente com o perfil de resistência antimicrobiana, definiu-se 37 cepas representativas que foram posteriormente submetidos ao sequenciamento por Multilocus Sequence Typing (MLST) e caracterizados por Pulsed Field Gel Electrophoresis (PFGE). A combinação de todas as abordagens permitiu o agrupamento dos isolados em 22 grupos clonais. O genótipo prevalente correspondeu à cepa nosocomial MRSA/BEC e foi caracterizada pelo RTF-BAAACAC / Sequence Type (ST) 239/SCCmec IIIA / Pulsotipo A / PVL negativo / multirresistência antibiótica. Outros 5 grupos clonais apresentaram cepas de MSSA e MRSA não-MDR com SCCmecs curtos. Quinze RFTs apresentaram somente cepas de MSSA com grande diversidade genotípica entre si, sendo 27,4% dessas PVL-positivas. Algumas cepas MRSA não-MDR PVL-positivas foram agrupadas em dois genótipos: RFT-AAAAAAA (ST-1) e RFT–BBBBBAB (ST-30), com SCCmec IVa e IV, respectivamente. Essas são características de clones comunitários disseminados pelo mundo, porém foram encontradas em MRSA hospitalares. De forma interessante, amostras pertencentes ao ST-30 foram identificadas como de origem hospitalar. Entretanto, um isolado foi derivado de infecção comunitária, demonstrando compartilhamento de cepas entre comunidade e hospital. A presença de SCCmecs curtos em cepas MRSA não-MDR direciona à possível emergência de novos clones resistentes. Os perfis heterogêneos de MRSA encontrados no Brasil reforçam a dinâmica da estrutura genética populacional deste patógeno e a importância de procedimentos de tipagem molecular para definir as relações entre isolados nosocomiais e comunitários. / In Brazil, a solely strain has been found disseminated, the Brazilian Epidemic Clone (BEC) of MRSA, was detected, characterized by the presence of a 67Kb staphylococcal chromosomal cassette of methicillin resistance (SCCmec) IIIA and antibiotic multiresistance. However, new non-multiresistant highly virulent MRSA strains have been described in community and nosocomial infections in several countries. These clones harbor a smaller SCCmec of around 24Kb and therefore more efficient in transferring, replication and translation. This study aim to describe the genetic background of S. aureus clones associated to infections in Rio de Janeiro/RJ. One hundred and thirty nine S. aureus samples were collected (54 MRSA isolates) from patients from seven hospitals and from a clinical pathology laboratory. These samples were submitted to molecular analyses by several methods. Multilocus Restriction Fragment Typing (MLRFT), SCCmec typing and the presence of the Panton-Valentine Leucocidin (PVL) genes, in association with the antimicrobial resistance profile, defined 37 representative isolates that were further submitted to DNA sequencing by Multilocus Sequence Typing (MLST) and also characterized by Pulsed Field Gel Electrophoresis (PFGE). The combination of all approaches allowed the clustering of the isolates into 22 clonal groups. The most prevalent genotype corresponded to MRSA/BEC nosocomial strain and was characterized as RFT-BAAACAC, Sequence Type (ST) 239, SCCmec IIIA, Pulsotype A, PVL-negative and antibiotic multiresistance. The other 5 clonal groups presented MSSA and non-MDR MRSA strains with smaller SCCmecs. Fifteen RFTs presented only MSSA strains with large genotypic diversity being 27,4% of the MSSA samples PVL-positives. Some PVL-positive non-MDR MRSA strains were clustered into two genotypes: RFT-AAAAAAA (ST-1) and RFT–BBBBBAB (ST-30), with SCCmec IVa and IV, respectively. These are characteristics of community isolates worldwide disseminated, however they are found in nosocomial MRSA strains. Notably, in this study, samples characterized as ST-30 was identified, in its majority, from hospital origin. However, an isolate was derived from community infection, depicting sharing among community and hospital settings. The presence of shorter SCCmecs in non-MDR MRSA strains leads to the posible emergence of new resistant clones. The heterogeneous MRSA profiles found in Brazil reinforce the dynamics of the genetic population structure of this pathogen and the importance of molecular typing procedures to define the relationship among nosocomial and community strains.