We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 102 CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.
Zusammenfassung: Die Enzyme Glucokinase, Pyruvatkinase und Fructosediphosphat-Aldolase im 100 000 ^-Überstand eines Rattenleber-Homogenates werden durch ein lysosomales Kathepsin inaktiviert. Diese Erscheinung wurde an teilgereinigten Glucokinase-Präparaten näher untersucht. Die Inaktivierung verläuft im schwach sauren Bereich schneller als bei pH 7, wird durch ß-Mercaptoäthanol beschleunigt und durch die Substrate der Glucokinase, Glucose und ATP, sowie durch Kalium-und Ammonium-Ionen gehemmt.Auftrennung eines Lysosomen-Extraktes über Sephadex G-100 ergibt für Kathepsin B zwei Maxima, von denen das kleinere mit der enzym-inaktivierenden Wirkung identisch ist und ein Molekulargewicht von ca. 25000 anzeigt. Im Lysosomen-Extrakt verliert das Enzym während mehrmonatiger Lagerung bei 0°C nur langsam an Aktivität, dagegen ist Summary: The enzymes glucokinase, pyruvate kinase and fructose diphosphate aldolase, in the 100,000 x^ supernatant of a rat liver homogenate, are inactivated by a lysosomal cathepsin. This phenomenon was further investigated with a partly purified glucokinase preparation. The inactivation is more rapid at a weakly acid pH than at pH 7; it is accelerated by ß-mercaptoethanol, and it is inhibited by the substrates of glucokinase (glucose and ATP) and by potassium and ammonium ions.
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