IDENTIFICATION OF CLOSTRIDIUM-TYROBUTYRICUM AND RELATED SPECIES USINGSUGAR FERMENTATION, ORGANIC-ACID FORMATION AND DNA PROBES BASED ON SPECIFIC 16S-RIBOSOMAL-RNA SEQUENCES

Spores of Clostridium tyrobutyricum are considered as the causative ag ents for butyric acid fermentation (late blowing) in brine-salted, sem i-hard and hard cheeses. In order to enable the early and specific det ection of these bacteria, clostridia commonly present in raw milk and farm environments were classified using phenotypic and genotypic prope rties. In addition specific DNA probes were designed for the identific ation of clostridia commonly present in these environments. Phenotypic characterization based on the API 20A system, the formation and conve rsion of organic acids and the litmus milk reaction, only allowed the differentiation of 71 clostridial strains into three groups, comprisin g the species C. tyrobutyricum (group A), C. acetobutylicum, C. beijer inckii and C. butyricum (group B) and C. sporogenes and C. bifermentan s (group C) respectively. Strains belonging to the C. tyrobutyricum gr oup formed a well-defined and homogenous collection. Using DNA probes based on specific 16S rRNA sequences and DNA-DNA hybridizations a perf ect correlation was obtained with the phenotypic classification. The 1 6S rRNA probes allowed the specific identification of this species. Us ing specific 16S rRNA probes, the species in phenotypic group B could be readily differentiated from each other and from other clostridia. A 16S rRNA probe was also developed for C. sporogenes belonging to grou p C. The probe allowed the differentiation of this species from all ot her clostridia. The identification based on the probes was fully compa tible with the results from DNA-DNA hybridization studies.