Department of Microbiology, Monash University, Melbourne, Victoria 3800, Australia.
J Clin Microbiol. 2010 Jun;48(6):2037-42. doi: 10.1128/JCM.02541-09. Epub 2010 Apr 7.
Rapid and reliable in vitro methods for the detection of pathogenic leptospires, such as Leptospira interrogans, are lacking. The present study investigated the use of luminescence to replace the existing enumeration techniques. Transposon TnSC189 was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens and was used to construct luminescent Leptospira spp. There was a linear relationship between luminescence and cell number, with the theoretical detection limit being less than 10(4) leptospires. A comparison of enumeration by a standard method (counting by dark-field microscopy) and enumeration by luminescence was conducted with luminescent L. interrogans. There was a good correlation between the two methods of enumeration (R(2) = 0.766), although variation in the luminescence early and late in growth phase reduced the degree of correlation. To demonstrate the utility of luminescence as a viability and cell number reporter, in vitro assays, including MIC determination, an extracellular matrix binding experiment, and a complement killing experiment, were conducted. In each case, the results obtained by luminescence matched those obtained by traditional means with high correlations (binding assay R(2) = 0.916, complement killing assay R(2) = 0.988). A strain expressing the luxCDABE transposon retained virulence in the hamster model of infection. Despite some variation in luminescence as a result of the growth phase or the particular assay conditions, enumeration by luminescence was found to be a quick, reliable, and highly sensitive method for the in vitro detection of leptospires that has the potential to replace more time-consuming methods of enumeration.
目前缺乏快速可靠的体外方法来检测致病性钩端螺旋体，如问号钩端螺旋体。本研究探讨了利用发光来替代现有的计数技术。转座子 TnSC189 经过修饰后，整合了发光杆菌的 luxCDABE 盒，并用于构建发光钩端螺旋体属。发光与细胞数量之间存在线性关系，理论检测下限小于 10(4)个钩端螺旋体。通过标准方法（暗场显微镜计数）和发光计数对发光的问号钩端螺旋体进行了计数比较。两种计数方法之间存在良好的相关性（R(2) = 0.766），尽管在生长早期和晚期发光的变化降低了相关性的程度。为了证明发光作为活力和细胞数量报告器的实用性，进行了体外测定，包括 MIC 测定、细胞外基质结合实验和补体杀伤实验。在每种情况下，发光获得的结果与传统方法高度相关（结合实验 R(2) = 0.916，补体杀伤实验 R(2) = 0.988）。表达 luxCDABE 转座子的菌株在仓鼠感染模型中保持了毒力。尽管由于生长阶段或特定测定条件的不同，发光存在一些变化，但发光计数被发现是一种快速、可靠和高度敏感的体外检测钩端螺旋体的方法，有可能替代更耗时的计数方法。
