Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi 
sensu lato (sl) complex transmitted to humans by Ixodes ticks. B. burgdorferi sl 
complex, currently comprising at least 19 genospecies, includes the main 
pathogenic species responsible for human disease in Europe: B. burgdorferi sensu 
stricto (ss), B. afzelii, and B. garinii. In this study, for the first time, 
MALDI-TOF MS was applied to Borrelia spp., supplementing the existing database, 
limited to the species B. burgdorferi ss, B . spielmanii and B. garinii, with 
the species B. afzelii, in order to enable the identification of all the species 
potentially implicated in LB in Europe. Moreover, we supplemented the database 
also with B. hermsii, which is the primary cause of tick-borne relapsing fever 
in western North America, B. japonica, circulating in Asia, and another 
reference strain of B. burgdorferi ss (B31 strain). The dendrogram obtained by 
analyzing the protein profiles of the different Borrelia species reflected 
Borrelia taxonomy, showing that all the species included in the Borrelia sl 
complex clustered in a unique branch, while Borrelia hermsii clustered 
separately. In conclusion, in this study MALDI-TOF MS proved a useful tool 
suitable for identification of Borrelia spp. both for diagnostic purpose and 
epidemiological surveillance.