Torrero MN, Collins LA, Franzblau SG; American Society for Microbiology. General Meeting.

Abstr Gen Meet Am Soc Microbiol. 1997 May 4-8; 97: 555 (abstract no. U69).

GWL Hansen's Disease Center, Baton Rouge, LA.

The ideal drug screening assay for mycobacteria would combine the sensitivity and high-throughput properties of the luciferase reporter assays with the ability to easily monitor the kinetics of inhibition as found in the BACTEC 460 system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule which (in contrast to luciferase) requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher intensity GFP was introduced by electroporation into Mycobacterium tuberculosis H37Ra (H37Ra) on expression vector pFPV2. The plasmid was stably maintained in the absence of kanamycin selection and expressed high levels of fluorescence as observed by epifluorescence microscopy. A microplate fluorescence assay was developed for the recombinant H37Ra-gfp to determine antimicrobial susceptibility to existing anti-TB agents. Fluorometric MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone and capreomycin were within one log2 dilution of those determined using the BACTEC 460 system. Equivalent MICs of anti-TB agents (except for kanamycin to which H37Ra-gfp is resistant) in the BACTEC 460 system for both H37Ra-gfp and the H37Ra parent strain suggested that introduction of pFPV2 did not influence drug susceptibility of H37Ra in general. Preliminary attempts to detect fluorescent H37Ra-gfp within mouse peritoneal macrophage cultures by fluorescence microscopy were successful. GFP provides a unique tool with which to easily and rapidly monitor the dynamic response of Mycobacterium tuberculosis to the antimicrobial effects of existing and potential anti-TB agents as well as to cellular defense mechanisms.

Publication Types:

• Meeting Abstracts

Keywords:

• Animals
• Anti-Bacterial Agents
• Drug Evaluation, Preclinical
• Ethambutol
• Ethionamide
• Gene Expression
• Green Fluorescent Proteins
• Isoniazid
• Kanamycin
• Luciferases
• Mice
• Microbial Sensitivity Tests
• Mycobacterium
• Mycobacterium tuberculosis
• Ofloxacin
• Rifampin
• Streptomycin
• genetics

Other ID:

• 98928825

UI: 102235478

From Meeting Abstracts