Environmental Biofilms from an Urban Community in Salvador, Brazil, Shelter Previously Uncharacterized Saprophytic Leptospira

Biofilms are complex microecosystems with valuable ecological roles that can shelter a variety of microorganisms. Spirochetes from the genus Leptospira have been observed to form biofilms in vitro, in rural environments, and in the kidneys of reservoir rats. The genus Leptospira is composed of pathogenic and non-pathogenic species, and the description of new species is ongoing due to the advent of whole genome sequencing. Leptospires have increasingly been isolated from water and soil samples. To investigate the presence of Leptospira in environmental biofilms, we collected three distinct samples of biofilms formed in an urban setting with poor sanitation: Pau da Lima, in Salvador, Bahia, Brazil. All biofilm samples were negative for the presence of pathogenic leptospires via conventional PCR, but cultures containing saprophytic Leptospira were identified. Whole genomes were generated and analyzed for twenty isolates obtained from these biofilms. For species identification, we used digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis. The obtained isolates were classified into seven presumptive species from the saprophytic S1 clade. ANI and dDDH analysis suggest that three of those seven species were new. Classical phenotypic tests confirmed the novel isolated bacteria as saprophytic Leptospira . The isolates presented typical morphology and ultrastructure according to scanning electron microscopy and formed biofilms under in vitro conditions. Our data indicate that a diversity of saprophytic Leptospira species survive in the Brazilian poorly sanitized urban environment, in a biofilm lifestyle. We believe our results contribute to a better understanding of Leptospira biology and ecology, considering biofilms as natural environmental reservoirs for leptospires.