Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction.

INTRODUCTION: Staphylococcus spp. - both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) - are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. MATERIAL AND METHODS: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. RESULTS: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. CONCLUSIONS: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

Do alterations in gene expressions influence tumorigenesis in the transmissible venereal tumor in dogs? / Alterações nas expressões gênicas influenciam na tumorigênese no tumor venéreo transmissível em cães?

Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.(AU)

O tumor venéreo transmissível canino (TVTC) se trata de uma neoplasia transmissível, que se propaga naturalmente entre os cães pela transferência halogênica de células tumorais, principalmente, durante o coito. É a mais antiga linhagem tumoral conhecida na natureza e relatos sobre mutações gênicas vêm sendo ampliadas. Além disso, este tumor compartilha uma série de mutações genéticas com alguns cânceres em seres humanos, dentre eles, carcinomas de pulmão, melanoma, próstata, mama, entre outros tipos de câncer. Assim, quantificou-se a expressão de genes supressores de tumores, como TP53, P21 e genes relacionados à apoptose, como BAX, BCL-2 e BCL-xL, tanto in vivo quanto in vitro (cultura celular primária). No presente estudo, na comparação das expressões gênicas, o gene TP53 se mostrou elevado na maioria dos casos em tecidos (65%) e em cultura celular primária (100%), enquanto BCL-2, BCL-xL e BAX apresentaram-se variáveis entre os animais analisados. Ademais, nessas situações os resultados sugerem que não ocorreu regulação apoptótica desses genes pelo TP53. O gene P21 mostrou-se, em sua maioria, normal (70%), embora a ausência (6%) e subexpressões (24%) também tenham sido observadas. A análise estatística do gene BCL-xL demonstrou diferenças significativas entre os tecidos dos animais, quando comparadas às culturas celulares, entretanto, para os demais genes, não foi observada diferença estatística entre os grupos. Preliminarmente, os resultados sugerem a presença de alterações nas expressões gênicas dos genes TP53, P21, BAX, BCL-2 e BCL-xL, levando a perda de função desses genes, os quais afetam a tumorigênese do CTVT.(AU)

Detection of the mecA gene and identification of Staphylococcus directly from blood culture bottles by multiplex polymerase chain reaction

ABSTRACT Introduction: Staphylococcus spp. - both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) - are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. Results: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. Conclusions: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

Comparação de diferentes temperaturas de armazenamento de sêmen refrigerado de garanhões da raça Mangalarga Marchador e Quarto de Milha / Comparison of differences storage temperatures in cooling semen of Mangalarga Marchador and Quarter Horses stallions / Comparación de diferentes temperaturas de almacenamiento de semen refrigerado de caballos Mangalarga Marchador y Cuarto de Milla

Pesquisas têm sido realizadas com o objetivo de prolongar a longevidade espermática por meio da refrigeração ou criopreservação do sêmen equino e com isso melhorar as taxas de fertilidade. Muitas dessas pesquisas têm como objetivo desenvolver diluidores, aditivos, curvas e protocolos de refrigeração, assim como tempos e temperaturas de estocagem que garantam a motilidade e integridade do sêmen equino submetido à refrigeração e com isso índices de fertilidade melhores e menos variáveis. Nos últimos anos, houve um aumento considerável na utilização de sêmen equino refrigerado, principalmente devido à aceitação de grande parte das associações de criadores de cavalos. A raça Mangalarga Marchador, de origem nacional, tem se mostrado extremamente sensível ao processo de manipulação, refrigeração e transporte de sêmen. Como em outras espécies de produção, o sucesso da inseminação artificial nos equinos depende da manutenção da viabilidade e fertilidade do sêmen durante o armazenamento. Além disso, quanto maior o tempo ao qual se mantem o potencial fertilizante do sêmen refrigerado, maior flexibilidade terá o médico veterinário para coletar e enviar o sêmen aos haras em que as éguas se encontram, resultando num maior êxito da técnica. O presente experimento teve por objetivo comparar as raças Mangalarga Marchador (MM) e Quarto de Milha (QM) quanto à resistência à refrigeração em duas temperaturas de armazenamento de sêmen refrigerado (5°C e 15°C). Os resultados deste trabalho demonstraram que não houve diferença entre as temperaturas de armazenamento (5ºC e 15°C), mostrando que ambas são eficientes na manutenção da viabilidade espermática pelo período de 24 horas para ambas as raças estudadas. Além disso, foi observada uma característica espermática superior dos garanhões da raça Quarto de Milha quando comparada aos da raça Mangalarga Marchador, tanto nos parâmetros de velocidade espermática, avaliado pelo CASA, revelando uma maior sensibilidade dos animais da raça Mangalarga ao processo de refrigeração do sêmen.

Studies have been conducted in order to prolong sperm longevity using cooling or frozen-thawed equine semen and in that way improve fertility rates. Many of these studies have focused on developing extenders, additives, cooling curves and protocols, as well as storage times and temperatures to ensure the integrity and motility of equine semen exposed to cooling. Recent years have increased the use of cooled equine semen, mainly due to the acceptance of the horse breeders associations. The Brazilian breed called "Mangalarga Marchador" has proven to be extremely sensitive to the cooling semen. As in other species, the success of artificial insemination in horses depends on maintaining the viability and fertility of semen during storage. Moreover, the longer you keep the cooling semen fertilizing potential, more flexibility will have the veterinarian to collect and send the semen samples, resulting in higher technique success. The aim of this study was to compare breeds Mangalarga Marchador (MM) and Quarter Horses (QM) for resistance to cooling in two semen storage systems (5°C and 15°C). The results showed no difference between the storage temperatures (5°C and 15°C), showing that both are effective in maintaining sperm viability for a period of 24 hours for both breeds studied. In addition, we observed superior sperm characteristics for Quarter Horse stallions when compared to the Mangalarga Marchador, evaluated by CASA, revealing a higher sensitivity for the cooling process by Mangalarga Marchador stallions.

Últimamente, varios estudios se han realizado con el fin de prolongar la longevidad de los espermatozoides mediante refrigeración o criopreservación del semen equino y de esa manera mejorar las tasas de fecundidad. Muchos de estos estudios se han centrado en los diluyentes, aditivos, curvas de enfriamiento y protocolos, así como los tiempos y temperaturas de almacenamiento para garantizar la integridad y la motilidad del semen equino expuestos al enfriamiento. En los últimos años han aumentado el uso de semen refrigerado equino, principalmente debido a la aceptación de las asociaciones de criadores de caballos. La raza brasileña "Mangalarga Marchador" ha demostrado ser extremadamente sensible a la refrigeración del semen. Como en otras especies, el éxito de la inseminación artificial en los caballos depende del mantenimiento de la viabilidad y fertilidad del semen durante el almacenamiento. Por otra parte, cuanto más tiempo se mantenga el potencial fertilizante, más flexibilidad tendrá el veterinario para colectar y enviar las muestras de semen, lo que resulta en mejores resultados de la técnica. El objetivo de este estudio fue comparar las razas Mangalarga Marchador (MM) y Caballos Cuarto de Milla (CM) para la resistencia a la refrigeración en dos temperaturas de almacenamiento de semen (5°C y 15°C). Los resultados no mostraron diferencia entre las temperaturas de almacenamiento (5°C y 15°C), mostrando que ambos son efectivos en el mantenimiento de la viabilidad espermática por un período de 24 horas para ambas las razas estudiadas. Además, se observó características espermáticas superiores de los sementales Cuarto de Milla en comparación con los Mangalargas Marchadores, evaluada por CASA, indicando una mayor sensibilidad para el proceso de refrigeración para la raza Mangalarga Marchador.