DNA probes for identification of clinically important Bacteroides species

Abstract

Conventional procedures for identifying gram-negative anaerobes such as Bacteroides spp. are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. Since many different species of gram-negative anaerobes are isolated from clinical specimens, the most useful DNA probes would be probes that identify a few clinically significant groups of anaerobes. To obtain such probes, we cloned HindIII-digested chromosomal DNA from various Bacteroides species and then screened these probes, labeled with 32P by nick translation, for hybridization to DNA from various species of Bacteroides, Fusobacterium, and other gram-negative bacteria. We identified three DNA probes (pBFII-4, pBFII-5, and pBFII-6) that hybridized specifically to DNA from Bacteroides fragilis, the species that accounts for about half of all isolates from clinical specimens containing anaerobes. We identified one DNA probe (pBE-3) that hybridized to all members of the B. fragilis group, a group of species that resemble B. fragilis with respect to fermentation pattern and antibiotic resistances. We also identified one probe (pBO-21) that hybridized to DNA from all Bacteroides sp. strains as well as to DNA from strains of Fusobacterium necrophorum and Fusobacterium nucleatum. pBO-21, but not pBE-3, cross-hybridized with a cloned Bacteroides sp. 16S rRNA gene. The limit of detection for these probes was 10(6) bacteria. The probes could detect B. fragilis in blood culture medium and in mixed cultures with other gram-negative bacteria. Attempts to use biotin-labeled DNA probes instead of 32P-labeled probes were not successful because the Bacteroides sp. extracts contained material that bound the streptoavidin-peroxidase detection reagent.