Evaluation of 16S rRNA Gene Restriction Analysis for the Identification of Cultured Organisms of Clinically ImportantClostridiumSpecies

Abstract In order to evaluate the usefulness of restriction analysis of the amplified 16S rRNA gene for the genotypic identification of cultured Clostridium organisms, a collection of a total of 52 isolates belonging to the following 19 species was studied: Clostridium bifermentans (n = 4), C. butyricum (3), C. cadaveris (3), C. clostridioforme (2), C. difficile (3), C. ghonii (2), C. glycolicum (3), C, histolyticum (3), C. innocuum (3), C. novyi A (1), C. paraputrificum (3), C. perfringens (3), C. ramosum (3), C. septicum (3), C. tetani (3), C. sordelli (3), C. sporogenes (3), C. subterminale (1), and C. tertium (3). Restriction analysis was carried out for all species with the enzymes Alu I, Cfo I and Rsa I, Bfa I and Mbo I. Restriction digestion with Alu I alone enabled differentiation between all of the species, except for C. bifermentans, C. ghonii and C. sordelli , which could not be differentiated from each other by any of these enzymes or by restriction with Bst UI, Hae III or Hin fI. Finally, one C. histolyticum isolate could not be identified with this method since it yielded a 1000 bp fragment after amplification (instead of the expected 1500 bp fragment), most probably due to a reproducible mispriming event.