Generation of BioluminescentStreptococcus mutansand Its Usage in Rapid Analysis of the Efficacy of Antimicrobial Compounds

Abstract

The oral bacterium Streptococcus mutans was transformed by electroporation with a shuttle vector (pCSS945) containing insect luciferase gene from a click beetle (Pyrophorus plagiophthalamus) resulting in a bioluminescent phenotype. ThisS. mutans strain was used in experiments in which light emission was used as a rapid and, compared to conventional CFU counting, more convenient means of estimating the effects of various 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 luciferase reaction. The optimum concentration ofd-luciferin was approximately 150 μM at room temperature. Under optimum conditions the light emission in vivo increased rapidly to a constant level and thereafter had a decay of 0.6%/min when logarithmic-growth-phase cells were used. The light emission closely paralleled the numbers of CFU, giving a detectable signal from 30,000 cells and having a dynamic measurement range over 4 log CFU/relative light unit. The cells were treated with various antimicrobial agents, and the emitted bioluminescence was measured. With the bioluminescent measurements, the results were obtained within hours compared to the days required for agar plates, and also, the kinetics of the antibacterial actions could be followed. Thus, the light emission was found to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents tested.

Optimisation of Bioluminescent Reporters for Use with Mycobacteria

“…Finally, the signal obtained is also affected by the metabolic state of the bacteria since the luciferase reaction relies on bacterial metabolites, mainly ATP and FMNH 2 for FFluc and Lux respectively. This is exemplified by the decrease in the luminescence observed when M. smegmatis enters the stationary phase and has also been previously described for other bacteria [67], [68], [69], [70], [71]. Although this could represent a handicap for the study of, for example, dormant M. tuberculosis it could be a powerful tool to gain insights into the metabolic state of M. tuberculosis during infection, to study entrance into dormancy, as well as for the rapid detection of drugs acting on bacterial metabolism.…”

Real-time assessment of Streptococcus mutans biofilm metabolism on resin composite

“…Our results are supported by previous findings [24, 25] that demonstrated a linear and directly proportional relationship between the cells’ metabolic status and their viability. Robrish et al [31] highlighted that bacteria present in oral biofilms have uniform cytoplasmic volumes, which allows the intracellular ATP content of any given organism to be used as a good indicator of biomass.…”

“…In 1998, Loimaranta et al [24] introduced a bioluminescent strain of S. mutans that was used to screen the in vitro efficacy of antimicrobial agents. The luc gene was placed under the control of a phage promoter and introduced into S. mutans via shuttle plasmid.…”

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

“…To explore this phenomenon, we measured the intracellular ATP concentration in the presence of D-luciferin via light emission by the bioluminescent strain MIM945, which is a derivative of S. thermophilus DSM20617 T [16]. The light emission of the MIM945 EdC was significantly higher when the bacteria were supplemented with lactose and urea or lactose and ammonia, compared to lactose alone (Figure 1D).…”

“…The bioluminescent derivatives mutants of S. thermophilus DSM 20617 T , the urease-negative mutant A16( ΔureC3 ) [2] and Lactococcus lactis IL1403, were created by electrotransformation with the shuttle vector pCSS945, which contains the luciferase gene from Pyrophorus plagiophtalamus [16].…”