Construction and Use of Species-Specific Dna Probes for the Detection, Identification, and Enumeration of Human Colonic Bacteroides Species (Anaerobes, Hybridization, Microecology, Feces)

105 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.Bacteroides is a genus of Gram negative, obligately anaerobic bacteria which are among the most numerous organisms in the human colon. Studies of the colonic microbial flora have been hampered by the cumbersome and time consuming techniques that are currently used to isolate and enumerate organisms from colon contents or feces. Consequently, it has been virtually impossible to address questions concerning the effects of host diet, age, or other factors on the species composition of the colonic flora. Studies that involve colonic Bacteroides species are particularly difficult because these organisms are not readily distinguished by conventional biochemical identification tests. These organisms do differ significantly, however, at the genetic level. Accordingly, an alternate method for identification and enumeration was devised which exploits differences in chromosomal DNA sequences among Bacteroides species. Random fragments of chromosomal DNA that are unique to each of 8 major colonic Bacteroides species were obtained and used to construct species-specific DNA probes. A procedure was devised whereby Bacteroides species could be detected and enumerated directly in pure and mixed cultures by DNA-DNA hybridization with the species-specific DNA probes. Slight modifications in this procedure allowed for the detection and enumeration of Bacteroides species in a crude extract from feces. The concentrations of these organisms in feces that were estimated by the DNA probe method are in agreement with estimates that have been previously obtained by conventional bacteriological methods. Moreover, with the DNA probe method it was possible to obtain concentrations for several Bacteroides species for which estimates have not been previously reported. Because Bacteroides species are important human pathogens, the DNA probe method was also applied to the rapid detection of these organisms in simulated clinical specimens.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD