UCLA

The mechanisms by which pathogenic Leptospira survive in the environment are poorly understood, in part due to limitations in current methodologies available to assess bacterial viability. The importance of environmental reservoirs in the transmission dynamics of leptospirosis has been difficult to establish without a mechanistic understanding of persistence. In this paper, a new method for assessing leptospiral viability is presented, and then applied to determine the salinities at which Leptospira can survive in the environment. Viable and heat-killed Leptospira were enumerated using a common dye agent, propidium monoazide (PMA), that is known for its effectiveness at removing dead bacterial DNA from qPCR amplification. Characterization of the toxicity and removal efficiency of one treatment cycle of 50 μM PMA showed moderately toxic effects on samples at 25°C compared to untreated samples, and equivalent removal of dead bacterial DNA at 4°C, 25°C, 30°C, and 37°C. Two treatment cycles at 50 μM PMA resulted in the detection of as few as 100 viable cells in 1.0 × 10⁵ dead cells in a 490 μL sample. Comparing PMA-qPCR to traditional qPCR, PMA-qPCR detected 5 orders of magnitude fewer dead cells in samples of heat-killed cells. Motivated by leptospirosis epizootics in marine species, PMA-qPCR was applied to study the persistence of Leptospira in fresh and brackish water microcosms. An initial concentration of 9.93 × 10⁶ cells/ml of model species Leptospira licerasiae serovar Varillal spiked into freshwater (0 ppt), low brackish (10 ppt), and high brackish (18 ppt) microcosms showed abundance of over 1.0 × 10⁴ cells/ml at 22 days. The abundance seen in the brackish microcosms may be due to biofilm formation in Leptospira, or to the expression of genes shared by Leptospira licerasiae serovar Varillal with either of its related pathogenic or saprophytic cousins. PMA-qPCR provides a rapid, inexpensive, and efficient method for leptospiral investigation that will assist in answering critical questions about persistence and transmission of the pathogen within the environment.