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On the basis of phenotypic and DNA relatedness data, three novel species of the genus Bosea are proposed, Bosea massiliensis (63287T =CIP 106336T =CCUG 43117T), Bosea vestrisii (34635T =CIP 106340T =CCUG 43114T) and Bosea eneae (34614T =CIP 106338T =CCUG 43111T). The original description of the genus Bosea included thiosulphate oxidation as a phenotypic feature, when the sole and type species of the genus, Bosea thiooxidans, was proposed. The three novel species described herein were not able to oxidize thiosulphate; thus, it is proposed that this characteristic be removed from the description of the genus and considered as specific for B. thiooxidans. The novel species of the genus Bosea proposed here form a well-separated cluster in the Bradyrhizobium group of the α-2 subclass of the Proteobacteria, on the basis of 16S rDNA gene sequence analysis. However, 16S rDNA gene sequence analysis was not sufficient to delineate the species; hence, DNA–DNA relatedness and phenotypic data were also required. All of the novel species described in this study are fastidious bacteria isolated from a hospital water supply, using co-cultivation with amoebae. This group of bacteria are hypothesized to be a potential cause of nosocomial infections. For treatment of infections caused by these novel bacteria, doxycycline appears to be the sole antibacterial compound with a consistently low MIC value.