| Summary: | Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus <i>Leptospira,</i> which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of <i>Leptospira interrogans</i> serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of <i>Leptospira</i>. Sera of all tested animals resulted positive for antibodies to <i>Leptospira</i> using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for <i>Leptospira</i> DNA after qPCR. Results obtained after MLST analysis and sequencing of <i>rrs</i>, <i>rpoB</i>, and <i>secY</i> genes, performed on six <i>Leptospira</i> strains isolated in culture, revealed the presence of the genomospecies <i>L. interrogans</i> serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic <i>L. montravelensis</i> in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications.
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