Merritt Justin, Kreth Jens, Qi Fengxia, Sullivan Richard, Shi Wenyuan
UCLA, School of Dentistry, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90025, United States.
J Microbiol Methods. 2005 May;61(2):161-70. doi: 10.1016/j.mimet.2004.11.012. Epub 2004 Dec 18.
Streptococcus mutans is one of a small number of recognized pathogens that lives among the hundreds of other bacterial species which comprise the oral flora. The virulence of this organism is intimately associated with its ability to live as an attached biofilm community on the tooth surface, and consequently, there is a great interest in its biofilm lifestyle. Currently, there are no established protocols that facilitate drug screening against this organism while it is entrenched in the biofilm. Furthermore, greater complications arise when attempting to perform these experiments in a multi-species setting. In an effort to circumvent these problems, we developed a quick, real-time, and non-disruptive method to probe the metabolic status of S. mutans growing as either a planktonic culture or a biofilm community. This assay takes advantage of the proven utility of luciferase measurements for drug screening. We placed the luciferase gene under the control of the S. mutans lactate dehydrogenase promoter (ldh) and integrated the construct onto its native position on the chromosome. We found this construct to be both highly expressed (<10000 cells easily detectable) and insensitive to many different growth parameters. When testing this reporter in both planktonic and biofilm cultures receiving either bacteriostatic or bactericidal antibiotics, we found the ldh-luc reporter to be a very accurate measurement of cell viability. Furthermore, we also demonstrated that this assay can generate useful information about the characteristics of intoxication caused by antibiotic activity. In addition, we modified the biofilm assay into the 96-well format and demonstrated the feasibility of high throughput drug screening of biofilm embedded S. mutans.
变形链球菌是少数已知的病原体之一，它生活在构成口腔菌群的数百种其他细菌物种之中。这种微生物的毒力与其在牙齿表面以附着生物膜群落形式生存的能力密切相关，因此，人们对其生物膜生活方式极为关注。目前，尚无既定方案可在变形链球菌处于生物膜状态时方便地对其进行药物筛选。此外，在多物种环境中进行这些实验时会出现更多复杂情况。为了规避这些问题，我们开发了一种快速、实时且非破坏性的方法，用于探究作为浮游培养物或生物膜群落生长的变形链球菌的代谢状态。该检测方法利用了荧光素酶测量在药物筛选方面已被证实的效用。我们将荧光素酶基因置于变形链球菌乳酸脱氢酶启动子（ldh）的控制之下，并将构建体整合到其染色体上的天然位置。我们发现该构建体表达水平高（易于检测到<10000个细胞）且对许多不同的生长参数不敏感。在对接受抑菌或杀菌抗生素的浮游和生物膜培养物中的该报告基因进行测试时，我们发现ldh-luc报告基因是细胞活力的非常准确的测量指标。此外，我们还证明该检测方法可以生成有关抗生素活性所致中毒特征的有用信息。此外，我们将生物膜检测方法改进为96孔板形式，并证明了对包埋在生物膜中的变形链球菌进行高通量药物筛选的可行性。
