GENERATION OF BIOLUMINESCENT STREPTOCOCCUS-MUTANS AND ITS USAGE IN RAPID ANALYSIS OF THE EFFICACY OF ANTIMICROBIAL COMPOUNDS

The oral bacterium Streptococcus mutans was transformed by electropora tion with a shuttle vector (pCSS945) containing insect luciferase gene from a click beetle (Pyrophorus plagiophthalamus) resulting in a biol uminescent phenotype, This S, mutans strain was used in experiments in which light emission was used as a rapid and, compared to conventiona l CFU counting, more convenient means of estimating the effects of var ious antimicrobial treatments. The basic parameters affecting in vivo light production by the strain were studied. It was found that pH 6.0 was optimal for incorporation of the substrate D-luciferin for the luc iferase reaction. The optimum concentration of D-luciferin was approxi mately 150 mu M at room temperature. Under optimum conditions the ligh t emission in vivo increased rapidly to a constant level and thereafte r had a decay of 0.6%/min when logarithmic growth-phase cells were use d. The light emission closely paralleled the numbers of CFU, giving a detectable signal from 30,000 cells and having a dynamic measurement r ange over 4 log CFU/relative light unit. The cells were treated with v arious antimicrobial agents, and the emitted bioluminescence was measu red. With the bioluminescent measurements, the results were obtained w ithin hours compared to the days required for agar plates, and also, t he kinetics of the antibacterial actions could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time in dicator of the bacteriostatic actions of the antimicrobial agents test ed.