RESUMO
An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods.
Assuntos
Técnicas Bacteriológicas/métodos , Enterococcus/crescimento & desenvolvimento , Enterococcus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Microbiologia da Água , Enterococcus/genética , Sensibilidade e EspecificidadeRESUMO
rRNA gene sequences were used for identification and target adequacy controls in a DNA probe assay to identify isolates as Staphylococcus and, more specifically, as S. aureus within 1 hour. mecA status was simultaneously determined using a specific DNA probe. The target adequacy control guarded against false-negative mecA results.
Assuntos
Proteínas de Bactérias/genética , Coagulase/metabolismo , Sondas de DNA , Genes de RNAr , Resistência a Meticilina/genética , Staphylococcus aureus/classificação , Técnicas de Tipagem Bacteriana , Humanos , Proteínas de Ligação às Penicilinas , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Fatores de TempoRESUMO
One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at -70 degrees C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).