Background: Pigment production and acidification of ribose are most frequently used biochemical tests for the differentiation of three enterococcal species carrying vanC genes such as Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. However, pigment production may occasionally be negative in E. casseliflavus, and some of E. casseliflavus may be negative or delayed reaction with ribose fermentation test. So, we performed this study to find out biochemical tests capable of distinguishing the strains possessing vanC genotypes.

Methods: A total of 17 enterococci composed of 14 clinical isolates with motility or pigment positive strains and three ATCC strains (E. gallinarum ATCC 49573, E. casseliflavus ATCC 25788, and E. flavescens ATCC 49997) were tested by multiplex PCR of the vanC genes (vanC-1, vanC-2, and vanC-3) and various biochemical tests.

Results: Among the 17 isolates including three ATCC control strains, four were genotyped as VanC-1, 11 were VanC-2, one were vanC-2/3, and any of vanC genes were not detected in one clinical isolate, respectively. Among the enterococci with vanC genotype, acid production from α-D-cyclodextrin and hippurate hydrolysis were positive only in VanC-1 genotype (E. gallinarum), acid production from glycerol and methyl- α-D-mannopyranoside were positive only in VanC-2 genotype (E. casseliflavus), and acid production from rhamnose and pigment production were negative only in VanC-2 genotype. Acid production from α-D-cyclodextrin was negative only in VanC-2 genotype. The positive rate of ribose fermentation of VanC-1, VanC-2, and VanC-2/3 (E. flavescens) genotype were 100%, 82%, and 0%, respectively.

Conclusion: Acid production from rhamnose, α-D-cyclodextrin, β-D-cyclodextrin, glycerol and methyl- α-D-mannopyranoside, pigment production, and hippurate hydrolysis test were useful biochemical tests for differentiating E. gallinarum from E. casseliflavus. The production of acid from α-D-cyclodextrin, glycerol, methyl-α-D-mannopyranoside and ribose were suitable biochemical tests for differentiating E. casseliflavus from E. flavescens. (Korean J Clin Microbiol 2000;3(1):30-35)