Optimization of Culture Protocols to Isolate spp. from Environmental Water, Field Investigation, and Identification of Factors Associated with the Presence of spp. in the Environment.

The successful culture of spp. from the environment is challenging. Here, we optimized the isolation of spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of subclade P1 were culture positive within 2 weeks. For subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 10 colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with spp. Cultures of spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.