Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages.

The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.

Green fluorescent protein as a reporter in rapid screening of antituberculosis compounds in vitro and in macrophages.

The development of new drugs against Mycobacterium tuberculosis is impeded by slow growth and highly infectious nature of the organism that warrants the need to work under highly stringent biosafety conditions. These problems can be overcome by use of reporter genes and surrogate strains. A strain of rapidly growing M. aurum has been recommended as test organism to screen inhibitors of mycobacteria to preselect compounds for progression into testing against M. tuberculosis. We have investigated the application of recombinant M. aurum expressing green fluorescent protein in rapid screening of antituberculosis compounds in vitro and in infected macrophages. Recombinant M. aurum[pGFM-11] expressing green fluorescent protein was constructed. The assay is based on measurement of fluorescent intensity at 509 nm. A good correlation was found between fluorescence and growth. Fluorescence of recombinant M. aurum was inhibited in vitro within 8 to 24 h by frontline antimycobacterial drugs at their reported MICs whereas inhibition in infected macrophages was observed in 72 h. Therefore green fluorescent reporter system provides a convenient screen to test antimycobacterial compounds that are active in vitro and within infected macrophages.

Diagnosis of tuberculous meningitis by detection of antigen and antibodies in CSF and sera.

OBJECTIVE: To evaluate diagnostic potential of three immunological tests, namely, detection of H37Rv antigen of M. Tuberculosis in CSF, detection of antibodies (IgG) against H37Rv in CSF and detection of antibodies (IgG) against H37Rv in serum for diagnosis of tuberculous meningitis in children. SUBJECTS: 50 children diagnosed as patients of tuberculous meningitis were included as cases and 48 children with CNS diseases of nontubercular etiology [pyogenic meningitis (n = 31), encephalitis (n = 10), seizure disorder of unknown etiology (n = 5), brain tumor (n = 2)] served as controls. METHODS: H37Rv antigen of M. tuberculosis was detected in CSF by Dot ELISA, and antibodies (IgG) against H37Rv in CSF and serum were detected by Plate ELISA. RESULTS: Detection of H37Rv antigen in CSF was the most sensitive (90%) and specific (95.83%) with positive and negative predictive values of 95.74% and 90.19%, respectively, followed by detection of antibodies in CSF (sensitivity-74%, specificity-89.58%, positive predictive value-88.10%, negative predictive value-76.78%). Detection of antibodies in serum had low sensitivity (50%), specificity (91.67%), positive predictive value (86.21%) and negative predictive value (63.76%). CONCLUSIONS: Detection of antigen in CSF is a rapid, sensitive and specific test for diagnosis of tuberculous meningitis in children. Detection of antibody in CSF may be useful in some cases but needs further evaluation. Detection of antibody in serum does not appear to be useful for diagnosis of tuberculous meningitis.

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: evidence for a new repair function.

The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between arg B and pur D loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and lambda-rays and incorporates little or no 3H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of 3H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42 degrees C before their transfer to 30 degrees C results in higher survival as compared to their incubation at 30 degrees C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.