Animals ; Female ; Macrophages ; microbiology ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Mycobacteriophages ; physiology ; Mycobacterium tuberculosis ; virology ; Virus Replication

OBJECTIVETo establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility.

METHODWe used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method.

RESULTSThe sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide.

CONCLUSIONSPhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.

Anti-Bacterial Agents ; pharmacology ; Biological Assay ; methods ; Humans ; Microbial Sensitivity Tests ; methods ; Mycobacteriophages ; physiology ; Mycobacterium tuberculosis ; drug effects ; Nitrate Reductase ; metabolism ; Rifampin ; pharmacology ; Sensitivity and Specificity

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Mycobacteriophages ; enzymology ; Mycobacterium tuberculosis ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics