Simple SummaryLeptospirosis is one of the most neglected zoonotic diseases in the world and humans can easily acquire the pathogen after exposure to water, soil, or mud contaminated with urine from infected animals. In September 2018, a leptospirosis outbreak caused by a pathogenicLeptospiragenospeciesinterroganswas identified in one farm in North Sardinia, Italy. In addition, culture and isolation of a saprophyticLeptospiragenospeciesmontravelensisfrom two urine samples (from one pig and one donkey) was reported for the first time, indicating that co-infection with more than one strain ofLeptospirain the same reservoir host is possible. These results provide new information on the epidemiology of leptospirosis and on the genotypes circulating in Sardinia, emphasizing the conducting of active surveillance of leptospirosis in humans and animals.AbstractSaprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genusLeptospira,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 ofLeptospira interrogansserovar 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 ofLeptospira. Sera of all tested animals resulted positive for antibodies toLeptospirausing a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive forLeptospiraDNA after qPCR. Results obtained after MLST analysis and sequencing ofrrs,rpoB, andsecYgenes, performed on sixLeptospirastrains isolated in culture, revealed the presence of the genomospeciesL. interrogansserovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophyticL. montravelensisin 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.