Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“

<p>The aim was to characterize 100 coagulase positive staphylococci isolates originating from pig carcasses from Swedish slaughterhouses by biotyping, antibiotic susceptibility testing, typing with pulsed field gel electrophoresis (PFGE) and real-time PCR-screening of the enterotoxin genes sea, sec, seg and sei in order to evaluate the impact on human health. The biotyping classified 56 as non host specific (NHS), 29 as human biotype, five as poultry, one as ovine, one as bovine biotype and eight were unclassified (UCF). Susceptibility testing to 16 antibiotics revealed that 49% of the isolates were resistant to penicillin, which the biotype human dominated among these isolates. The results from the PFGE showed correlation between the biotypes and the pulsotypes obtained with several groups with identical strains. The results from the 47 isolates tested for enterotoxins were that the combination of seg and sei was the most common but sea and sec were also detected. There were slaughterhouses that had certain biotypes and penicillin resistance linked to them.</p>

The aim was to characterize 100 coagulase positive staphylococci isolates originating from pig carcasses from Swedish slaughterhouses by biotyping, antibiotic susceptibility testing, typing with pulsed field gel electrophoresis (PFGE) and real-time PCR-screening of the enterotoxin genes sea, sec, seg and sei in order to evaluate the impact on human health. The biotyping classified 56 as non host specific (NHS), 29 as human biotype, five as poultry, one as ovine, one as bovine biotype and eight were unclassified (UCF). Susceptibility testing to 16 antibiotics revealed that 49% of the isolates were resistant to penicillin, which the biotype human dominated among these isolates. The results from the PFGE showed correlation between the biotypes and the pulsotypes obtained with several groups with identical strains. The results from the 47 isolates tested for enterotoxins were that the combination of seg and sei was the most common but sea and sec were also detected. There were slaughterhouses that had certain biotypes and penicillin resistance linked to them.

Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.

<p>Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women,&nbsp / Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa / therefore, we embarked on a study to determine the&nbsp / prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics&nbsp / units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history,&nbsp / including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG)&nbsp / and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV&nbsp / status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p &lt / 0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history&nbsp / of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV.&nbsp / </p>

<p>Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women,&nbsp / Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa / therefore, we embarked on a study to determine the&nbsp / prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics&nbsp / units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history,&nbsp / including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG)&nbsp / and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV&nbsp / status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p &lt / 0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history&nbsp / of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV.&nbsp / </p>

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:42Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:59Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Made available in DSpace on 2015-01-13T10:47:59Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) Previous issue date: 2013-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Poultry products are recognized as sources of contamination by several pathogens. In the last decades the genus Campylobacter, particularly thermophilic species, stands out in developed countries, where it is considered the primary agent responsible for Foodborne Illnesses (DVA's). Due to the importance of this pathogen in public health and economics, researches are conducted in order to identify and characterize the isolated, as well as develop and improve techniques for the identification of the genus Campylobacter spp. with attention focused mainly to important thermophilic species related to food, allowing the determination of the prevalence of this pathogen in main placement sources, such as chicken. The objective of this study was to verify the occurrence of Campylobacter spp. and identify isolated thermophilic species from chicken carcasses in slaughterhouses in the state of Goiás submitted to the Federal Inspection Service which had proper authorization to internal and external trade. A total of 200 samples have been evaluated using the immunoenzymatic screening assay, with subsequent bacterial samples isolation and identification through biotyping. In this study it was observed that the employed methods allowed the isolation of Campylobacter spp. in 24% (48/200) of the assessed samples. Among these samples, 21 (10.5%) were demonstrably positive for genre, having as basis the set of analytical screening method, sequential bacterial isolation for positive samples and evidence of enzymatic and biochemical assimilation and antimicrobial profile. C.coli was the most frequently isolated species (58.6%), followed by C.jejuni ssp. jejuni (17.2%), C.lari (13.8%) and Campylobacter jejuni ssp. doylei (3.4%). By using a screening method, conventional isolation system and numerical biotyping it was immediately revealed the characterization of the isolated Campylobacter. It was observed that some suggestive isolated, due to its positivity in the screening test, growth on selective sources, incubation temperature, motility and morphology, characteristics of the genus assessed, have not been identified by numerical system and thus were considered negative. From the results, it can be concluded that the association of screening method to conventional isolation and biotyping numerical profile allowed a greater understanding of the isolated. It can also be concluded that Campylobacter spp. is present in chicken meat for human consumption and three thermophilic species and two subspecies have been identified. The pathogen presence attests public health risk. / Os produtos de origem avícola podem veicular diversos microrganismos patogênicos. Nas últimas décadas, o gênero Campylobacter, em especial as espécies termofílicas, destaca-se como um dos principais agentes responsáveis por Doenças Veiculadas por Alimentos (DVA’s). Em decorrência da importância deste patógeno na saúde pública e na economia, pesquisas são realizadas com o objetivo de identificar e caracterizar os isolados, bem como desenvolver e aprimorar as técnicas para a identificação do gênero, permitindo a determinação da prevalência deste patógeno nas principais fontes de veiculação, como a carne de frango. O presente estudo teve como objetivo verificar a ocorrência de Campylobacter spp. e identificar as espécies termofílicas isoladas de carcaças de frango de abatedouros do estado de Goiás submetidos ao Serviço de Inspeção Federal e que possuíam autorização para o comércio interno e externo. Um total de 200 amostras foi avaliado utilizando o ensaio imunoenzimático como triagem, com posterior isolamento bacteriano e identificação por meio da biotipificação. Observou-se que os métodos empregados permitiram a identificação de Campylobacter spp. em 24% (48/200) das amostras avaliadas. Destas, 21 (10,5%) foram comprovadamente positivas para o gênero, tendo por fundamentação o isolamento bacteriano de amostras positivas à triagem e testes de assimilação enzimática, bioquímicas e de perfil antimicrobiano. Campylobacter coli foi a espécie mais isolada (58,6%), seguida por Campylobacter jejuni ssp. jejuni (17,2%), Campylobacter lari (13,8%) e Campylobacter jejuni ssp. doylei (3,4%). Observou-se que alguns isolados sugestivos, por apresentar positividade no teste de triagem, crescimento em meios seletivos, temperatura de incubação, morfologia e motilidade características do gênero em questão, não foram identificados pelo sistema numérico empregado neste estudo e, portanto, foram considerados negativos. Pelos resultados, pode-se concluir que a associação do método de triagem ao isolamento convencional e perfil numérico para biotipificação permitiram melhor identificação dos isolados. Assim, é possível afirmar que Campylobacter spp. foi identificada em carne de frango destinada ao consumo, havendo a descrição de três espécies termofílicas e duas subespécies. A presença deste patógeno confere risco à saúde pública por ingestão de alimentos contaminados e decorrência de doenças gastroentéricas e extraintestinais e em casos mais graves, porém não raros, a síndromes reumatológicas, oftalmológicas e neurológicas. Especificamente as espécies Campylobacter coli e Campylobacter jejuni descritas como de maior interesse em alimentos foram as de maior frequência e relacionam-se à maioria dos casos de campilobacteriose de origem alimentos.

>Magister Scientiae - MSc / Risk factors such as intrauterine and vaginal infection put pregnant women at risk for delivering preterm. Bacterial vaginosis (BV) is a polymicrobial clinical syndrome commonly diagnosed in women of reproductive age, with women of African descent with low socioeconomic status and previous preterm delivery at high risk. Although frequently isolated from healthy women, Gardnerella vaginalis has been most frequently associated with BV. There is limited data available on the prevalence of BV in Southern Africa; therefore, we embarked on a study to determine the prevalence of BV and G. vaginalis in predominantly black communities in the Western Cape, in order to establish the role of G. vaginalis in BV. Women attending various Maternity and Obstetrics units (MOU) in the Cape Peninsula with and without a history of pre-term delivery (PTD) were invited to participate in the study. Several factors were statistically associated with pregnancy history, including location of study population, parity, smoking and presence of clinical symptoms. The presence of G. vaginalis was determined by culture in 51.7% of the preterm delivery group (PTDG) and 44% of the full-term delivery group (FTDG) women. BV was detected in 31.13% of PTDG and 23.67% of FTDG by Gram stained analysis according to Nugent scoring criteria, with age and HIV status posing as risk factors. When comparing PTDG and FTDG for an association between the presence of G. vaginalis and BV, a stronger association was observed in the PTDG but it was not statistically significant. In both PTDG and FTDG, G. vaginalis was isolated significantly more often in women diagnosed with BV at 24.5% (p < 0.05). Antibiogram studies revealed both Metronidazole and Clindamycin resistant strains of G. vaginalis. G. vaginalis Biotype 7 is specifically associated with BV, while Biotype 2 appears to be associated with BV in women with a history of PTD. Accuracy of diagnostic tools were tested and it was determined that Nugent scoring is more sensitive in diagnosing BV (76.04%), but culture for G. vaginalis is more specific (83.21%). Although this study was limited in that we were unable to follow-up pregnancy outcomes, we were able to confirm the perceived role of G. vaginalis in BV. / South Africa

In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.