Staphylococcus aureus belongs to the normal flora. Many healthy people are colonized by the bacterium mainly in the nose but also on the skin and on other mucous membranes without showing symptoms. After damage to the skin, the bacterium can enter the wound and cause infections. Methicillin-resistant S. aureus (MRSA) is resistant to b-lactam antibiotics such as penicillin and methicillin. The gene that gives resistance characteristic of MRSA is the mecA-gene. MRSA strains are spread in both hospitals and in the community, and it is important to identify these bacteria with rapid and sensitive methods. In this study, Taq Man RT-qPCR was compared with SYBR Green RT-qPCR (LightCycler480, Roche) to explore which method had the best sensitivity with the least working hours. In addition, Bullet for automated DNA extraction and CAS 1200 ™ for automated pipetting of the samples were evaluated. Twelve patient isolates and 232 patient samples for MRSA screening were included in the study. The results showed that the primers were of major importance for the outcome of the amplification. It was also shown that the Ct-values were clearly lower when the Bullet, CAS 1200 ™ and LightCycler480 were combined compared with manual DNA extraction, manual pipetting and the Rotor-Gene 6000. In future, the former method will be used by the laboratory when screening patient samples for MRSA.

Clinical S. aureus strains were gathered from four hospitals, two in Turkey (Hacettepe hospital 200 strains and Ankara Hospital 106 strains) and the other two from Libya (Aljalla Hospital 88 strains and Jamahyria Hospital 62 strains). The clinical specimens were collected form different sources including blood, urine, wound, pus, burn, sputum, semen, catheter and aspiration. Patients were aged between 0 to 84 years and from both sexes. Resistance to Methicillin was determined by measuring the Oxacillin MIC / this was done by using the oxacillin E-test, with resistance defined as an MIC of &gt / 2 &micro / g ml. In this study all isolates displayed an Oxacillin MIC of &amp / #8805 / 256&micro / g/ml. The MRSA strains were (56%) in Turkish hospitals, and (59%) in Libyan hospitals. The percentage of the VRSA and VISA in Libyan hospitals was (7%) and (26%) respectively, although the percentage of VRSA in Turkish hospitals was only 2% and there were no intermediately susceptible Staphylococcus aureus (VISA). Besides the MRSA isolates, Coagulase Negative Staphylococcus showing Methicillin resistance was collected from clinical isolates in thirteen patients in Turkish hospitals. In both countries, the majority MRSA isolates were multiresistant to more than five classes of antibiotics including / Ampicillin, Amoxicillin, Tetracycline, Erythromycin and Ciprofloxacin. Most of the MRSA isolates were from blood (68%), wounds (57%) and pus (50%).The results of genetic investigations indicated that the mecA gene was present in the majority of isolates in both countries / the community acquired MRSA type (ccr-BIV) was present in three samples out of thirty in Turkish hospitals and in one case out of twenty in Libyan hospitals / There was no case out of fifty specimens that carry the hospitals acquired MRSA type (ccr-BI, II, III) in both countries. Besides the Methicillin resistance gene, the incidence of Tetracycline resistance gene was quite high (tetM and tetK 50%) in Turkish hospitals isolates, and the prevalence of Panton-Valentine Leukocidin gene was high (PVL 70%) in Libyan hospitals specimens.

This thesis is an investigation of two common skin conditions of pigs: exudative epidermitis (EE) and ear necrosis (EN). The cause of exudative epidermitis and risk factors are well understood, however the study was prompted because of reports of treatment failure. A survey of veterinary practitioners (n=15) and pork producers (n=58) was conducted to determine which treatments are commonly used. Amongst farmer respondents topical treatments were often used and in serious cases injectable penicillin G was administered. Thirty farms with a history of EE were visited and skin samples taken from affected pigs. The antimicrobial resistance pattern for isolates of Staphylococcus hyicus and Staphylococcus aureus revealed that almost all isolates were resistant to penicillin G and ampicillin. In addition, certain isolates of S. hyicus as well as S. aureus were shown to possess the mecA gene which is associated with resistance to methicillin. The presence of widespread resistance to penicillin G among staphylococci isolates suggests a reason for poor treatment response. The presence of the mecA gene in staphylococci other than S. aureus recovered from pigs has not been reported before and is of interest from a public health standpoint. A second study investigated EN. The causative agent(s) and the associated risk factors are not well understood. Eleven case farms were visited and skin biopsies and oral swabs taken from pigs in early, mid and late stages of the disease. Bacteriological culturing was performed for staphylococci and spirochetes as well as histological examination of the biopsy samples. Farm-level risk factors were assessed on 14 case farms and 9 control farms. Staphylococci were generally recovered in abundance from the majority of samples but spirochetes were not cultured and only identified microscopically in a small number of tissue samples. Histology revealed that the disease appeared to occur first as a lesion on the epidermal surface that caused tissue damage and led to subsequent invasion of the dermis. This pathogenesis was consistent with the hypothesis that staphylococci colonize the skin surface and produce exfoliating toxins. Ear biting was noted to be commonly present and may be an important contributing factor. / Ontario Pork Animal Health Strategic Initiative Fund Ontario Ministry of Agriculture, Food and Rural Affairs(OMAFRA) Ontario Veterinary College, University of Guelph

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Multiple antibiotic resistant Staphylococcus aureus represent a big problem in the control of hospital infections. Resistance pattern of isolated S. aureus presented in central vascular catheter of patients interned in the Intensive Therapy Center of the School Hospital of the Universidade Federal do Triângulo Mineiro was evaluated by antimicrobial tests, in which it was possible to detect high level of resistance to penicillin (94.7%) and ampicillin (86.8%), only considering the samples that presented resistance, beyond one strain that presented resistance to vancomycin. The oxacillin resistance evaluation was confirmed by PCR with the presence of the gene mecA. The association of the results obtained in the phenotypic test with the presence of the gene mecA, considered the reference method, was confirmed through the Table of Contingency and the Test of Χ2 with Yates correction. In 49 isolates evaluated, 23 were resistant to oxacillin, being possible to detect the mecA gene in 21 samples. The test of molecular screening by RAPD allowed the separation of the phenotypic groups in two different grouping patterns, the ones that presented resistance to antimicrobials and the sensible ones, with a dissimilarity of 73,3%. There is a higher genetic similarity between groups that present the same type of resistance, thus confirming the phenotypic analyses. Molecular markers for detection of resistance to oxacillin, like the gene mecA, were more sensitive than the phenotypic markers. / Staphylococcus aureus resistentes a múltiplos antibióticos representam um grande problema no controle das infecções hospitalares. O perfil de resistência a antimicrobianos de isolados de S. aureus presentes em cateteres vasculares central de pacientes internados em leitos do centro de terapia intensiva do Hospital Escola da Universidade Federal do Triângulo Mineiro foi avaliado por meio de testes antimicrobianos, pelos quais foi possível detectar um elevado nível de resistência à penicilina (94,7%) e ampicilina (86,8%), considerando-se somente as amostras que possuíam resistência, além de uma cepa resistente à vancomicina. A avaliação da resistência à oxacilina foi confirmada por PCR através da presença do gene mecA. A associação dos resultados obtidos no teste fenotípico com a presença do gene mecA, considerado um método de referência, foi confirmada através da Tabela de Contingência e do Teste de Χ2 com correção de Yates. Em 49 amostras avaliadas, 23 apresentaram resistência à oxacilina, sendo possível detectar a presença do gene de resistência mecA em 21 amostras. O teste de tipagem molecular por RAPD permitiu a separação dos grupos fenotípicos em dois padrões diferentes de agrupamento, os que possuíam resistência e os sensíveis aos antimicrobianos, com uma dissimilaridade de 73,3%. Há maior similaridade genética entre grupos que apresentam o mesmo tipo de resistência, confirmando assim as análises fenotípicas. Marcadores moleculares para detecção de resistência à oxacilina, como o gene mecA, foram mais sensíveis que os marcadores fenotípicos. / Mestre em Genética e Bioquímica

Made available in DSpace on 2014-07-29T15:30:43Z (GMT). No. of bitstreams: 1 DISSERTACAO DE MESTRADO JULIANA ROSA.pdf: 303226 bytes, checksum: 9585889e11363ca2d5297656fdeae6e5 (MD5) Previous issue date: 2008-04-17 / Coagulase negative staphylococci (CNS) is in human and animal microbiota, but may cause with infections with significant morbidity and mortality rates. Healthy care workers may be carriers of many microorganisms and spread resistant ECN in the hospital. This study aimed to identify the CNS species isolated from healthy care workers saliva, establishe the oxacillin resistance pattern and detect the mecA gene in resistant isolates. We evaluated 100 ECN, isolated from the saliva of an institution of professional health of large Riberão Preto in Sao Paulo state. The ECN identification was based on biochemical tests, and 41 were identified as S. epidermidis, 25 S. saprophyticus, 18 S. haemolyticus, 8 S. cohnii, 4 S. lugdunenses, 3 S. capitis, and 1 S. simulans. Thirty-two percent were nonsusseptible to oxacillin, 84.4% to mupirocin, 43.7% to cefoxitin, but all were vancomycin susceptible. The oxacillin nonsusseptible ECN, detected by disk diffusion test were grown in agar screening oxacillin (6 μ g) supplemented with sodium chloride (4.0%) and submited to mecA detection by the PCR. Of the 32 nonsusseptible oxacillin CNS,, 93.7% developed in the oxacillin agar and the mecA gene was detected in 75.0%. This is the first report of mecA gene presence in CNS isolated from the saliva of healthy care workers. Attention must be given to CNS species identification, as well as the characterization of the nonsusceptible microorganisms, since healthy care workers may represent a reservoir of CNS / Estafilococos coagulase negativa (ECN) fazem parte da microbiota autóctone humana e animal, embora possam causar infecções. Profissionais da saúde podem ser portadores de inúmeros microrganismos virulentos entre eles os ECN resistentes a oxacilina que podem ser disseminados por esses profissionais. O estudo teve como objetivo identificar espécies de ECN isolados da saliva de profissionais da saúde, de uma instituição de saúde de grande porte em Riberão Preto no estado de São Paulo, determinar o perfil de resistência à oxacilina e detectar o gene mecA. Foram avaliados 100 ECN, e através de testes bioquímicos 41 estafilococos foram identificados como S. epidermidis, 25 S. saprophyticus, 18 S. haemolyticus, 8 S. cohnii, 4 S. lugdunenses, 3 S. capitis e um S. simulans. Trinta e dois por cento apresentaram resistência in vitro a oxacilina, 84,4% a mupirocina, 43,7% a cefoxitina, e todos suscetíveis a vancomicina. Os ECN resistentes a oxacilina, detectados pelo teste de disco difusão, foram cultivados no agar triagem oxacilina (6µg) suplementado com cloreto de sódio e a detecção do gene mecA foi realizada pela técnica de PCR. Dos 32 ECN resistentes a oxacilina, 93,7% desenvolveram no agar oxacilina e o gene mecA foi detectado em 75,0% das amostras analisadas. Vale ressaltar que este estudo é pioneiro em mostrar a presença do gene mecA em ECN isolados da saliva de profissionais da saúde saudáveis. Uma maior atenção deve ser dada na identificação das espécies de ECN, e na caracterização da resistência desse grupo de microrganismos, uma vez que os profissionais da saúde podem representar um reservatório de ECN resistentes

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: A alta frequência de Estafilococos Coagulase-Negativa (CoNS) na pele de indivíduos saudáveis e em doenças associadas ao sangue, associados à seleção de cepas resistentes devido a uso indiscriminado de antimicrobianos, tornou mais estreitos os limites entre o ambiente hospitalar e o comunitário quanto à distribuição de cepas. Objetivou-se, com este estudo, caracterizar isolados de CoNS de origem hospitalar e comunitária da cidade de Botucatu-SP quanto ao perfil clonal, analisar os aspectos de resistência à oxacilina pela aferição de metodologia de detecção, e investigar os determinantes de heterorresistência à vancomicina nessas cepas. As espécies estudadas incluíram S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans e S. xylosus. O teste de disco-difusão (TDD) com discos de oxacilina e cefoxitina, fitas de Etest impregnadas com oxacilina e pesquisa do gene mecA por PCR em tempo real foram realizadas. A triagem em ágar com 6 e 8 µg/ml de vancomicina, microdiluição em caldo para aferição da Concentração Inibirtória Mínima (MIC), microscopia eletrônica de transmissão para verificar espessamento da parede celular e alterações fenotípicas por testes bioquímicos foram realizadas. O perfil clonal foi determinado por PFGE (Pulsed-Field Gel Eletrophoresis) e para clones de S. epidermidis, o MLST (Multilocus Sequence Typing). S. epidermidis apresentou alta diversidade clonal, mas presença de clusters no ambien... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The high frequency of Coagulase-Negative Staphylococci (CoNS) on the skin of healthy individuals and in bloodstream infections, together with the selection of resistant strains, has narrowed the boundaries between the hospital and the community environment for the distribution of strains. This study aimed to characterize CoNS isolated from clinical and colonization specimens of patients and individuals from Botucatu-SP, to compare their clonal profile, to analyze the determination of oxacillin resistance by the evaluation of the methodology of detection, and to investigate the determinants of reduced susceptibility to vancomycin in those strains. CoNS species included S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans and S. xylosus. The disc diffusion test (DDT) using oxacillin and cefoxitin discs was employed, Etest strips impregnated with oxacillin and mecA gene detection by real-time PCR were used. An agar screening with 6 and 8 µg/ml of vancomycin, the broth microdiluition method for the Minimal Inhibitory Concentration (MIC), the transmission eletronic microscopy for evaluation of cellwall thickening and phenotypic modifications by biochemical tests were performed. Clonal profile was determined by PFGE (Pulsed-Field Gel Eletrophoresis) and, for S. epidermidis clones, MLST (Multilocus Sequence Typing). S. epidermidis presented high clonal diversity, despite some clusters circulating within hospi... (Complete abstract click electronic access below) / Doutor