Bacillus sporothermodurans, a new species producing highly heat-resistant endospores

Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahigh-temperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16S rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. megaterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16S rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. megaterium group, notably, B. firmus (30%), B. benzoevorans (28%), and B. circulans (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermodurans for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).