Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

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 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.

Expanding the Genetic Toolbox for Leptospira Species by Generation of Fluorescent Bacteria

“…In a recent study, the luxCDABE cassette from Photorhabdus luminescens was transferred into Leptospira spp. to generate luminescent bacteria (26). In this work, we constructed a set of plasmid vectors which contain either gfp or mRFP1 alleles.…”

High-Temperature Protein G Is an Essential Virulence Factor of Leptospira interrogans

“…Two key defenses against pathogens are macrophages and serum complement. L. interrogans is resistant to killing by serum complement (39,59), whereas the saprophytic species L. biflexa is sensitive. To assess whether the disruption of htpG in M1233 affected resistance to human complement, wildtype and M1233 cells were incubated with 90% pooled normal human serum at 37°C for 60 min; the viability of the cells was then determined by direct microscopic counts.…”

“…Nonmotile cells are considered nonviable (60), with direct microscopic counts employed as a measure of viability in several studies (9,59,61,62), including assessment of sensitivity to complement (59,61). Additionally, studies with luminescent leptospires have shown motility to be an accurate measure of cell viability (39). Leptospires tend to aggregate in culture and have diffuse colony morphologies (60) that make viable plate counts problematic and highly inaccurate.…”

“…Complement resistance. The resistance of the wild type and M1233 to complement was determined by incubating 4 ϫ 10 6 cells with 90% pooled human serum (39). The cells were incubated at 37°C for 60 min before survival was determined by dark-field microscopy comparing the proportion of motile cells to that of nonmotile cells (see Results and Discussion).…”

Identification of Genes Essential for Leptospirosis

“…Various modifi ed transposons have been used, including those carrying different selection markers [ 5 , 17 ], an alternative origin of replication to allow purification of higher plasmid quantities [ 18 ], or addition of markers such as green fl uorescent protein [ 19 ] and the Photorhabdus luminescens luxCDABE luminescence genes [ 20 ]. Transposon insertions are stable, as the transposase is encoded outside of the transposon; mutants have retained the transposon during passage through animals, and over 100 generations in the absence of selection [ 18 ].…”