RAPID DISTINCTION OF BREVIBACTERIUM SPECIES BY RESTRICTION ANALYSIS OF RDNA GENERATED BY POLYMERASE CHAIN-REACTION

Twelve reference strains, including the type strains, of species either belonging or related to the genus Brevibacterium, and nineteen well-characterized clinical isolates assigned to this genus were studied. Polymerase chain reaction was used for partial amplification of the gene coding for their 16S rRNA (rDNA genes) which were digested with nine different restriction endonucleases. The amplified rDNAs were approximately 1300 bp long. The type strains of B. casei and B. epidermidis exhibited different BstNI rDNA restriction patterns. Furthermore, these patterns differed from those observed for B. linens and B. iodinum. Of the clinical isolates fifteen had the same BstNI restriction pattern as the B. casei type strain, two the same as the B. epidermidis type strain, one the same as B. linens, and one strain had a particular pattern. Results obtained by rDNA restriction analysis were confirmed by DNA-DNA hybridization studies, which revealed that representatives of the fifteen isolates resembling B. casei shared more than 75% homology with B. casei CIP102111(T). Combined with a rapid DNA extraction procedure, PCR-amplified-rDNA restriction fragment analysis allowed the distinction of the different Brevibacterium species in less than seven hours. The results suggested that most brevibacteria from clinical samples belongs to B. casei rather than B. epidermidis, as commonly thought. This approach may also be of value for differentiation within other genera of Gram-positive bacteria.