The gene encoding resistance to β-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated Staphylococcal Chromosome Cassette mec or SCCmec. In addition to the resistance gene, mecA, SCCmec also contains site specific recombinase genes, ccrAB, that are capable of catalyzing the chromosomal excision and integration of SCCmec. The increasing prevalence of methicillin-resistant Staphylococcus aureus infections may be due, in part, to the transfer of SCCmec into successful methicillin-sensitive S. aureus lineages. In this work we attempt to better characterize the factors associated with SCCmec transfer, beginning with CcrAB-mediated SCCmec excision in a collection of MRSA containing type IV SCCmec. CcrAB-mediated excision of type IV SCCmec was not demonstrated for all strains tested, as a subset of S. aureus strains with type IV SCCmed did not excise their element. These strains are all highly related and represent a lineage of successful community associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the presence of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately outside of SCCmec on the chromosome. Staphylococcus aureus strain USA300 contains SCCmec type IV and a genomic island containing an arginine deiminase pathway, known as ACME, inserted adjacent to one another in the SCCmec chromosomal attachment site. Each element was site-specifically excised from the chromosome by CcrAB, resulting in two independent, extra-chromosomal, circularized elements. Therefore the presence of ACME did not disrupt SCCmec excision.Next, we describe three MRSA strains that become resistant to vancomyein during passage on increasing concentrations of the drug. In each case, mecA was lost during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec while the other two strains (3130VR and VP32) deleted portions of their SCCmec elements in a manner that appears to involve IS431. Conversion to vancomycin resistance caused a decrease in growth rate that was partially compensated for by deletion of mecA. In mixed culture competition experiements, vancomycin resistant strains that lacked mecA readily out-competed their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.Finally, we examined the genetic structure surrounding the SCCmec attachment site in a diverse collection of methicillin-sensitive S. aureus isolates. This region of the chromosome varies greatly from strain to strain and appaers to be prone to recomination. Open reading frames found in this region were homologous to enterotoxins, restriction-modification enzymes, and transposases. Several open reading frames that have not been previously reported in staphylococci were also present in this region. 28 out of the 42 isolates examined did not contain the attachment site sequence found in S. aureus isolates known to be capable of CcrAB-mediated SCCmec integration or excision. This may indicate that these strains do not contain a functional attachment site and therefore may not have the potential to acquire SCCmec by CcrAB-mediated recombination.

A mastite bovina é considerada a enfermidade de maior impacto na pecuária leiteira mundial. Apesar da adoção de práticas de manejo para minimizar a ocorrência desta, os resultados não são satisfatórios para o controle de Staphylococcus aureus, o principal agente causador. Além da importância veterinária, possui implicações na saúde humana devido ao uso extensivo de antibióticos no tratamento e controle da doença e devido ao potencial de disseminação zoonótica de S. aureus. A principal preocupação em relação à resistência aos antimicrobianos é referente à meticilina, mediada pelo gene mecA. Estudos moleculares são importantes para o conhecimento da diversidade genética dos agentes causadores de mastite. A tipagem por sequenciamento de um único locus, do gene da região X da proteína A de S. aureus (spa Typing), tem sido utilizada com sucesso para investigar estrutura populacional, sendo adequada para estudos epidemiológicos. O presente projeto teve como objetivo pesquisar o perfil de resistência aos antimicrobianos e a presença de genes de resistência à meticilina e de S. aureus isolados de mastite bovina, além de compreender a diversidade genética, identificar possíveis novos perfis e conhecer a distribuição destes isolados. Os resultados encontrados foram a ausência de resistência à meticilina no antibiograma, confirmada pela ausência do gene mecA, e a baixa prevalência de multirresistência aos antimicrobianos testados. Apesar de mais de 58,74% dos isolados (242/412) serem resistentes a pelo menos um antibiótico, 53,64% (221/412) correspondem a resistência à ampicilina. Foram determinados 44 spa tipos diferentes, sendo os três mais frequentes t605 (57,45%), t002 (8,4%) e t127 (8,13%). Não houve associação entre a distribuição dos spa tipos e as variáveis analisadas de ano, estado, município e fazenda, e perfil de resitência aos antibicrobianos. Este é o primeiro estudo que utiliza o spa typing para avaliar uma coleção temporalmente diversa e, portanto, representativa da região estudada ao longo de 22 anos. Logo, espera-se que a descrição dos tipos de S. aureus obtidos contribua para a compreensão dos aspectos epidemiológicos envolvidos na transmissão do patógeno e do papel dos hospedeiros como reservatórios para linhagens resistentes. Tal compreensão é imprescindível para a prevenção, controle e tratamento da mastite bovina. / Bovine mastitis is considered the disease of greatest impact in global dairy industry. Despite the adoption of management practices to minimize its occurrence, the results are not satisfactory for the control of Staphylococcus aureus, the major causative agent. Besides veterinary importance, there are also implications for human health due to the extensive use of antibiotics in treatment and control of the disease and due to the potential for zoonotic spread of S. aureus. The main concern regarding antimicrobial resistance is methicillin resistance mediated by the mecA gene. Molecular epidemiology studies are important for the understanding of the genetic diversity of the causative agents of mastitis. Typing by sequencing of a single locus of the X region gene of the S. aureus protein A (spa Typing) has been successfully used to investigate population structure. This project aims to investigate the antimicrobial susceptibility profile and the presence of methicillin resistance genes in S. aureus isolated from bovine mastitis, in addition to understanding the genetic diversity, identifying possible new profiles and knowing the distribution of these isolates. The results obtained to the present were the absence of methicillin resistance in the antibiogram, confirmed by the absence of mecA gene, and the low prevalence of multidrug resistance to antimicrobials. Even though resistance to at least one antibiotic was present in more than 58,74% of the isolates (242/412), ampicillin resistance corresponds to 53.64% (221/412). Different spa types were determined, the three most frequent being t605 (57.45%), t002 (8.4%) and t127 (8.13%). There was no association between the spa type distribution and the variables of year, state, municipality, farm, and antibiotic resistance profile analyzed. This is the first study that uses spa Typing to evaluate a collection as temporarily diverse and representative of the region studied in 22 years. Therefore, it is expected that the results of this study, by allowing comparison of the profile of S. aureus from different sources (animals and property), will contribute to the understanding of the epidemiological aspects involved in the transmission of the pathogen and the role of hosts as reservoirs for pathogenic strains. Such understanding is essential for the prevention, control and treatment of bovine mastitis

Made available in DSpace on 2016-04-28T20:17:24Z (GMT). No. of bitstreams: 1 2007- Lidiane de Castro Soares.pdf: 1336181 bytes, checksum: 47680a74331a705e5ecd649c1639aa3f (MD5) Previous issue date: 2007-02-28 / Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Coagulasenegative staphylococci (SCN) take part of normal microbiota. Although it has been considered saprophytic, nowadays there is a concern about its pathogenic potential.Nevertheless, all advance in SCN identification assays, these microorganisms are continually neglected in laboratorial routine of infectious diseases, because of the wide range of species. In spite of the difficulties, it is necessary to make an appropriated species identification in order to differentiate the potential pathogenic agents and to determine its antimicrobial susceptibility pattern. Resistance in SCN is related to the presence of mecA gene, which expresses a heterogeneous pattern that can be detected through diverse phenotypics tests. In this study, 72 SCN isolates obtained from external ear conducts of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal and S. cohnii subsp.urealyticus in human sample. The antimicrobial resistance patterns were evaluated through disc diffusion test, and a high level of resistance to penicillin and ampicillin were detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association ampicillin+sulbactam. Oxacillin resistance was phenotypically detected by modified disc diffusion test, agar screen, broth microdilution and agar dilution. Presence of mecA gene where detected in 5,55% of the isolates by Polimerase Chain Reaction (PCR). Correlation with the detection of mecA gene was used as gold standard for evaluation of sensitivity and specificity of phenotypical assays. The low number of mecA positives isolates did not allowed the association between cefoxitin and oxacillin resistance as a test to detect the presence this gene. The broth microdilution and agar dilution tests presented the best accuracy in phenotypic detection of the oxacillin resistance. / Os estafilococos coagulasenegativos (ECN) fazem parte da microbiota normal da pele e apesar de terem sido considerados sapr?fitas por muito tempo, o seu significado cl?nico como agente etiol?gico tem aumentado com o passar dos anos. No entanto, apesar de todo avan?o nas t?cnicas de identifica??o dos ECN e do conhecimento destes como agentes etiol?gicos em diversos processos infecciosos, estes microrganismos muitas vezes s?o negligenciados na rotina laboratorial, devido a enorme diversidade de esp?cies encontradas. A identifica??o das esp?cies de ECN, embora de dif?cil realiza??o para a maioria dos laborat?rios cl?nicos, ? necess?ria para diferenciar o potencial patog?nico e o perfil de resist?ncia de cada isolado. A resist?ncia ? oxacilina em ECN ? mediada pelo gene mecA e usualmente heterog?nea, sendo detectada por v?rios m?todos fenot?picos. Neste estudo, foram avaliados 72 isolados de ECN provenientes de amostras do conduto auditivo de c?es da ra?a Beagles, de mastite bovina e de infec??es humanas. Staphylococcus xylosus foi o microrganismo mais isolado, nas amostras animais, e S. cohnii subsp.urealyticus em humanos, dentro de uma ampla gama de esp?cies identificadas. O perfil de resist?ncia aos antimicrobianos em isolados de animais e humanas foi avaliado atrav?s da t?cnica de difus?o em disco, na qual detectouse um elevado n?vel de resist?ncia ? penicilina e ampicilina. A gentamicina, vancomicina e associa??o ampicilina+sulbactam foram eficientes frente aos isolados testados. A avalia??o da resist?ncia ? oxacilina foi realizada atrav?s da difus?o em disco modificada, ?gar screen, microdilui??o em caldo e em ?gar. A presen?a do gene mecA foi determinada pelo m?todo da Rea??o em Cadeia de Polimerase (PCR), sendo 5,55% dos isolados mecA positivos. Os resultados fenot?picos de resist?ncia a cefoxitina e a oxacilina foram correlacionados com a detec??o do gene mecA, que foi utilizado como padr?o ouro para avalia??o da sensibilidade e especificidade das t?cnicas utilizadas. O reduzido n?mero de isolados mecA + n?o permitiu estabelecer o grau de correla??o entre a cefoxitina e a oxacilina como m?todo de predi??o do gene, embora ambas tenha apresentado o mesmo percentual de resist?ncia. O teste fenot?pico que apresentou melhor acur?cia na detec??o da resist?ncia ? oxacilina foi a microdilui??o em caldo.

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

A mastite bovina é considerada a enfermidade de maior impacto na pecuária leiteira mundial. Apesar da adoção de práticas de manejo para minimizar a ocorrência desta, os resultados não são satisfatórios para o controle de Staphylococcus aureus, o principal agente causador. Além da importância veterinária, possui implicações na saúde humana devido ao uso extensivo de antibióticos no tratamento e controle da doença e devido ao potencial de disseminação zoonótica de S. aureus. A principal preocupação em relação à resistência aos antimicrobianos é referente à meticilina, mediada pelo gene mecA. Estudos moleculares são importantes para o conhecimento da diversidade genética dos agentes causadores de mastite. A tipagem por sequenciamento de um único locus, do gene da região X da proteína A de S. aureus (spa Typing), tem sido utilizada com sucesso para investigar estrutura populacional, sendo adequada para estudos epidemiológicos. O presente projeto teve como objetivo pesquisar o perfil de resistência aos antimicrobianos e a presença de genes de resistência à meticilina e de S. aureus isolados de mastite bovina, além de compreender a diversidade genética, identificar possíveis novos perfis e conhecer a distribuição destes isolados. Os resultados encontrados foram a ausência de resistência à meticilina no antibiograma, confirmada pela ausência do gene mecA, e a baixa prevalência de multirresistência aos antimicrobianos testados. Apesar de mais de 58,74% dos isolados (242/412) serem resistentes a pelo menos um antibiótico, 53,64% (221/412) correspondem a resistência à ampicilina. Foram determinados 44 spa tipos diferentes, sendo os três mais frequentes t605 (57,45%), t002 (8,4%) e t127 (8,13%). Não houve associação entre a distribuição dos spa tipos e as variáveis analisadas de ano, estado, município e fazenda, e perfil de resitência aos antibicrobianos. Este é o primeiro estudo que utiliza o spa typing para avaliar uma coleção temporalmente diversa e, portanto, representativa da região estudada ao longo de 22 anos. Logo, espera-se que a descrição dos tipos de S. aureus obtidos contribua para a compreensão dos aspectos epidemiológicos envolvidos na transmissão do patógeno e do papel dos hospedeiros como reservatórios para linhagens resistentes. Tal compreensão é imprescindível para a prevenção, controle e tratamento da mastite bovina. / Bovine mastitis is considered the disease of greatest impact in global dairy industry. Despite the adoption of management practices to minimize its occurrence, the results are not satisfactory for the control of Staphylococcus aureus, the major causative agent. Besides veterinary importance, there are also implications for human health due to the extensive use of antibiotics in treatment and control of the disease and due to the potential for zoonotic spread of S. aureus. The main concern regarding antimicrobial resistance is methicillin resistance mediated by the mecA gene. Molecular epidemiology studies are important for the understanding of the genetic diversity of the causative agents of mastitis. Typing by sequencing of a single locus of the X region gene of the S. aureus protein A (spa Typing) has been successfully used to investigate population structure. This project aims to investigate the antimicrobial susceptibility profile and the presence of methicillin resistance genes in S. aureus isolated from bovine mastitis, in addition to understanding the genetic diversity, identifying possible new profiles and knowing the distribution of these isolates. The results obtained to the present were the absence of methicillin resistance in the antibiogram, confirmed by the absence of mecA gene, and the low prevalence of multidrug resistance to antimicrobials. Even though resistance to at least one antibiotic was present in more than 58,74% of the isolates (242/412), ampicillin resistance corresponds to 53.64% (221/412). Different spa types were determined, the three most frequent being t605 (57.45%), t002 (8.4%) and t127 (8.13%). There was no association between the spa type distribution and the variables of year, state, municipality, farm, and antibiotic resistance profile analyzed. This is the first study that uses spa Typing to evaluate a collection as temporarily diverse and representative of the region studied in 22 years. Therefore, it is expected that the results of this study, by allowing comparison of the profile of S. aureus from different sources (animals and property), will contribute to the understanding of the epidemiological aspects involved in the transmission of the pathogen and the role of hosts as reservoirs for pathogenic strains. Such understanding is essential for the prevention, control and treatment of bovine mastitis

The presented thesis was created in cooperation with Siemens, s.r.o. and deals with the analysis of the bending of aluminium sheets. These products are used as conductors of electrical energy. The problem happened when the bending angle was 90 ° when there was a violation outside of the bend. The aim of the thesis is to verify whether the bend is correctly designed using analytical and empirical relations and the finite element method. Because of the requirement for running numerical simulation on one of the freely available software the Salome Meca program environment was used. The results of numerical simulation did not confirm that the violation should occur. In deeper analysis appeared doubts whether it is possible to correctly evaluated result only by data from tensile test. In the final part, there are recommended some experiments which would be necessary for the correct calibration of the ductil fracture criteria.

The focus of this thesis is the confirmation of the presence of the methicillin-resistant Staphylococcus aureus on a selected dairy bovine farm and the following typing of its isolates while using common genotyping and phenotyping methods. After the isolation of this bacteria from the samples, they were tested for the presence of mecA gene and the genes responsible for the production of enterotoxins. It was tested whether the isolates belong to the 398 sequence type and their type of spa gene sequence was determined. Then the resistance against antimicrobial agents was tested. Results of the typing were compared to those of other related articles. Finally, this thesis observes the efficiency of the sanitizing agent used at this farm and suggests further methods to reduce the occurrence of MRSA in this herd.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that has spread into the community since the 90s. In general, the community-associated (CA- MRSA) strains are more virulent, but less resistant to antimicrobials than the hospital- acquired strains (HA-MRSA). Some lineages of MRSA such as sequence type 8 have been transmitted more successfully around the world than others and there are situations where a dominant lineage has been replaced by a new one. The factors that are crucial for the selection of dominant lineage are often not clear. As part of this thesis, a longitudinal study of MRSA epidemiology in the Motol University Hospital during the period of 2018-2020 was performed and the occurrence of MRSA clonal complexes was characterized. A multicenter study of the epidemiological situation of MRSA in Slovakia during the period of January - April 2020 was also performed concomitantly. Moreover, several isolates from dominant lineages were further characterized by their phenotype. In the Motol University Hospital study, it was found that the HA-MRSA of the clonal complex 5 dominates and is represented mainly by spa-type t586; a finding that concurs with recent studies from the Czech Republic. In Slovakia, similar to the Czech Republic, MRSA lineages from clonal...