Loimaranta V, Tenovuo J, Koivisto L, Karp M
Institute of Dentistry and Turku Immunology Centre, University of Turku, FIN-20520 Turku, Finland.
Antimicrob Agents Chemother. 1998 Aug;42(8):1906-10. doi: 10.1128/AAC.42.8.1906.
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. This S. 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 of D-luciferin was approximately 150 microM 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.
口腔细菌变形链球菌通过电穿孔法用一种穿梭载体（pCSS945）进行转化，该载体含有来自叩头虫（Pyrophorus plagiophthalamus）的昆虫荧光素酶基因，从而产生了生物发光表型。这种变形链球菌菌株被用于实验中，在实验中，与传统的菌落形成单位（CFU）计数相比，发光被用作一种快速且更便捷的手段来评估各种抗菌治疗的效果。研究了影响该菌株体内发光的基本参数。发现pH 6.0最适合荧光素酶反应底物D - 荧光素的掺入。在室温下，D - 荧光素的最佳浓度约为150微摩尔。在最佳条件下，当使用对数生长期细胞时，体内发光迅速增加到恒定水平，此后以0.6%/分钟的速率衰减。发光与CFU数量密切平行，从30,000个细胞可检测到信号，动态测量范围超过4个对数CFU/相对光单位。用各种抗菌剂处理细胞，并测量发出的生物发光。通过生物发光测量，与琼脂平板培养所需的天数相比，数小时内即可获得结果，而且还可以跟踪抗菌作用的动力学。因此，发现发光是所测试抗菌剂抑菌作用的可靠、灵敏且实时的指标。
