Esse estudo comparou diferentes métodos fenotípicos e biomoleculares para avaliação da resistência antimicrobiana de 107 cepas de Salmonella spp. Isoladas de carcaças e couro de bovinos e de produtos cárneos. Os antimicrobianos testados foram ampicilina, cefotaxima, cloranfenicol, ciprofloxacina, gentamicina, imipenem, tetraciclina e trimetoprima. Os testes fenotípicos empregados foram: difusão do disco em ágar, utilizando discos produzidos por duas empresas diferentes e determinação da Concentração Inibitória Mínima (CIM), realizada em placas de microtitulação e empregando tiras comerciais de antibióticos. O método biomolecular (PCR) objetivou a pesquisa de genes de resistência a antimicrobianos (blaTEM, blaOXA-1, blaPSE-1, cat1, cat2, floR, cmlA, cmlB, aac(3)IVa, aadB, aadD, aphA1, tetA, tetB, tetC, tetD, tetE, tetG e dhfrI) , localizados no cromossomo, plasmídeos e integrons classe 1, e foi aplicado para as cepas que apresentaram resistência a pelo menos um dos antibióticos testados por algum dos testes fenotípicos e para as cepas que apresentaram resultados contraditórios nos testes fenotípicos. Todos os métodos foram executados de acordo com as normas do ClinicaI and Laboratory Standards Institute. Foi detectada resistência a pelo menos um antibiótico em 26 cepas (24%), sendo que 20 (77%) dessas foram multirresistentes. A correlação de resultados obtidos pelo método de difusão de discos empregando os discos de duas marcas diferentes foi de 99,7%, enquanto a correlação dos resultados obtidos pelos dois métodos de determinação da CIM foi de 98,6%. Para os resultados obtidos pelos métodos de difusão de discos e determinação da CIM a correlação foi de 98,4%. Não foi observada uma boa correlação entre a presença dos genes detectados e o resultado apresentado pelos métodos fenotípicos. / This study compared different phenotypic and molecular methods for the evaluation of antimicrobial resistance of 107 strains of Salmonella spp. isolated from hides and carcasses of cattle and meat products. The antimicrobials tested were ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, imipenem, tetracycline and trimethoprim. Phenotypic tests included agar disk diffusion, using disks produced by two different companies and determination of the Minimum Inhibitory Concentration (MIC) conducted in microtiter plates and commercial antibiotic strips. The biomolecular method (PCR) focused antimicrobial resistance genes (blaTEM, blaOXA-1, blaPSE-1, cat1, cat2, floR, cmlA, cmlB, aac(3)IVa, aadB, aadD, aphA1, tetA, tetB, tetC, tetD, tetE, tetG e dhfrI) located on chromosomes, plasmids and class 1 integrons, and was applied to the strains that were resistant to at least one of the antibiotics tested as detected by the phenotypic tests and to strains that showed contradictory results in the phenotypic tests. All methods were performed according to the standards of the Clinical and Laboratory Standards Institute. Resistance to at least one antibiotic was detected in 26 strains (24%), and 20 (77%) of these were multidrug-resistant. The correlation between results obtained by disk diffusion method using discs of two different brands was 99.7%, while the correlation of the results obtained by the two methods of determining the MIC was 98.6 %. For the results of the methods of disc diffusion and MIC determination the correlation was 98.4%. There was no good correlation between the presence of genes detected and the result presented by phenotypic methods.

The ecological niche of the vagina contains a large number of different microbes that are constantly interacting with each other and the host. Culture methods have not been sufficient in order to resolve the complexity of the normal vaginal flora. Further, the methods for delineating normal flora from not normal flora are not easily handled and are traditionally not based on culture but on microscopy of elements of the vaginal fluid. In the work presented in this thesis, an international collaboration was established that pin-pointed some of the difficulties in classifying vaginal floras, including staining, sampling, and discordance when lactobacilli are few in number, and that emphasized the importance of the size of the vision field in microscopes. As lactobacilli are prominent members of the normal vaginal flora they need to be carefully classified if further work towards more robust scoring tools is to be achieved. Phenotypic methods have not been able to separate the closely related Lactobacillus species of the vagina. Progress in molecular biology has provided possibilities to characterize these lactobacilli, which are mainly from the Lactobacillus acidophilus group. In this work a large number of strains collected by true random sampling were subjected to RAPD-PCR, TTGE and multiplex PCR for species identification. The major species found were L. crispatus, L. gasseri and L. jensenii and the recently described L. iners. The presence of L. iners has not been detected in previous studies due to its special nutrient requirements. Development of pyrosequencing technology also made it possible to match signatures of the two variable regions V1 and V3 of the 16S rRNA gene of the vaginal lactobacilli and identify them to the species level in a high throughput manner. The study confirmed that the dominating flora in women with normal vaginal flora comprises the four species mentioned previously. Repetitive sampling during IVF-treatment with highly varying oestrogen levels demonstrates changes that possibly occur during changes in the natural life cycle. Furthermore, L. iners was found to be the first species to be established after spontaneously resolved or treated Bacterial Vaginosis. These findings can be of help in developing new strategies for regaining and retaining the normal vaginal flora.

Esse estudo comparou diferentes métodos fenotípicos e biomoleculares para avaliação da resistência antimicrobiana de 107 cepas de Salmonella spp. Isoladas de carcaças e couro de bovinos e de produtos cárneos. Os antimicrobianos testados foram ampicilina, cefotaxima, cloranfenicol, ciprofloxacina, gentamicina, imipenem, tetraciclina e trimetoprima. Os testes fenotípicos empregados foram: difusão do disco em ágar, utilizando discos produzidos por duas empresas diferentes e determinação da Concentração Inibitória Mínima (CIM), realizada em placas de microtitulação e empregando tiras comerciais de antibióticos. O método biomolecular (PCR) objetivou a pesquisa de genes de resistência a antimicrobianos (blaTEM, blaOXA-1, blaPSE-1, cat1, cat2, floR, cmlA, cmlB, aac(3)IVa, aadB, aadD, aphA1, tetA, tetB, tetC, tetD, tetE, tetG e dhfrI) , localizados no cromossomo, plasmídeos e integrons classe 1, e foi aplicado para as cepas que apresentaram resistência a pelo menos um dos antibióticos testados por algum dos testes fenotípicos e para as cepas que apresentaram resultados contraditórios nos testes fenotípicos. Todos os métodos foram executados de acordo com as normas do ClinicaI and Laboratory Standards Institute. Foi detectada resistência a pelo menos um antibiótico em 26 cepas (24%), sendo que 20 (77%) dessas foram multirresistentes. A correlação de resultados obtidos pelo método de difusão de discos empregando os discos de duas marcas diferentes foi de 99,7%, enquanto a correlação dos resultados obtidos pelos dois métodos de determinação da CIM foi de 98,6%. Para os resultados obtidos pelos métodos de difusão de discos e determinação da CIM a correlação foi de 98,4%. Não foi observada uma boa correlação entre a presença dos genes detectados e o resultado apresentado pelos métodos fenotípicos. / This study compared different phenotypic and molecular methods for the evaluation of antimicrobial resistance of 107 strains of Salmonella spp. isolated from hides and carcasses of cattle and meat products. The antimicrobials tested were ampicillin, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, imipenem, tetracycline and trimethoprim. Phenotypic tests included agar disk diffusion, using disks produced by two different companies and determination of the Minimum Inhibitory Concentration (MIC) conducted in microtiter plates and commercial antibiotic strips. The biomolecular method (PCR) focused antimicrobial resistance genes (blaTEM, blaOXA-1, blaPSE-1, cat1, cat2, floR, cmlA, cmlB, aac(3)IVa, aadB, aadD, aphA1, tetA, tetB, tetC, tetD, tetE, tetG e dhfrI) located on chromosomes, plasmids and class 1 integrons, and was applied to the strains that were resistant to at least one of the antibiotics tested as detected by the phenotypic tests and to strains that showed contradictory results in the phenotypic tests. All methods were performed according to the standards of the Clinical and Laboratory Standards Institute. Resistance to at least one antibiotic was detected in 26 strains (24%), and 20 (77%) of these were multidrug-resistant. The correlation between results obtained by disk diffusion method using discs of two different brands was 99.7%, while the correlation of the results obtained by the two methods of determining the MIC was 98.6 %. For the results of the methods of disc diffusion and MIC determination the correlation was 98.4%. There was no good correlation between the presence of genes detected and the result presented by phenotypic methods.