Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

Abstract

ABSTRACT 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 Tn SC189 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.