TAXONOMIC STUDY OF CORYNEBACTERIUM GROUP ANF-1 STRAINS - PROPOSAL OF CORYNEBACTERIUM-AFERMENTANS SP-NOV CONTAINING THE SUBSPECIES C-AFERMENTANS SUBSP AFERMENTANS SUBSP NOV AND C-AFERMENTANS SUBSP LIPOPHILUM SUBSP NOV

We have determined the cell wall composition, guanine-plus-cytosine (G+C) contents of the DNA, rRNA gene restriction patterns, and the levels of DNA-DNA relatedness of 11 strains identified biochemically as Centers for Disease Control (CDC) Corynebacterium group absolute nonfermenter 1 (Corynebacterium group ANF-1). For seven of these strains, growth is abundant on 5% sheep blood agar, which differentiates them from the four other strains, whose growth requires a lipid supplement such as Tween 80. Two of the lipid-requiring strains produced mucoid colonies on 1% Tween 80-supplemented sheep blood agar. All strains possess cell wall component type IV, short-chain mycolic acids, and (G + C contents of DNA of 66 to 68 mol% as determined by reverse-phase high-performance liquid chromatography. DNA-relatedness experiments by an S1 nuclease procedure showed that nine of these strains, including two of the lipid-requiring strains, constitute a new genomic species less than 40% related to Corynebacterium species and other coryneform groups. The lipid-requiring strain T18502 exhibited 98% DNA relatedness with another lipid-requiring strain, T88593 (difference in thermal denaturation midpoint [DELTAT(m)] = 2-degrees-C) and 71 to 77% similarity with the nonlipophilic strains (DELTAT(m) range of from 2 to 5-degrees-C). Conversely, the DNA relatedness between strain LCDC 88199 and the six other nonlipophilic strains ranged from 86 to 100% (DELTAT(m) range of from 1 to 3-degrees-C) and was only 73 and 76% with the lipophilic strains T18502 and T88593, respectively (DELTAT(m), 3 and 4-degrees-C). These results indicated that these two cultural types of bacteria constitute two subspecies within the new genomic species. These subspecies can be identified within the genus Corynebacterium by their phenotypic characteristics and rRNA gene restriction patterns by PvuII and EcoRI endonuclease digestion. The two mucoid strains were not related to other Corynebacterium group ANF-1 strains or Corynebacterium species reference strains. Further studies should allow the determination of the taxonomic status of these mucoid strains. Therefore, we propose a new species, Corynebacterium afermentans sp. nov., which contains two subspecies: C. afermentans subsp. afermentans subsp. nov. (type strain, LCDC 88199 = CIP 103499) for nonlipophilic Corynebacterium group ANF-1 strains and C. afermentans subsp. lipophilum subsp. nov. (type strain, T18502 = CIP 103500) for two lipid-requiring Corynebacterium group ANF-1 strains.