Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species

Abstract

Abstract:The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by peR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B.fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp, Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for I1e-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-Iabeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B.fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.Key words: Bacteroides, 16S-23S rDNA spacer, Molecular diagnosisThe genus Bacteroides, which consists of Gram-negative, saccharolytic nonpigmenting obligate anaerobes, is frequently isolated from human infected tissues. One species, B. fragilis, is considered to be the most medically important because this species accounts for over half of the anaerobes from clinical specimens (7) and sometimes causes severe septicemia with a high mortality rate in compromised hosts (9). Various biochemical tests, including analysis of electrophoretic patterns of dehydrogenase (33), cellular fatty acid and sugar composition (4, 5, 25), lipid analysis (26, 29), serology (13,19,36) and bacteriophage typing (3), have been used to discriminate Bacteroides species. However, these analytical procedures require many troublesome steps, and in some cases it is difficult to differentiate each Bacteroides species due to their similar behavior in many bio-*Addresscorrespondenceto Dr. YoshinariOhnishi, Department of Bacteriology,School of Medicine, The University of Tokushima, Kuramoto-cho, Tokushima,Tokushima770-8503, Japan. Fax:+81-88-633-7069. E-mail: ohnishi@basic.med.tokushima-u.ac.jp 191 chemical tests (12,32). Despite the similarities in biochemical characteristics, none of the Bacteroides species has been found to share more than 30% DNA homology with any other species in DNA-DNA hybridization studies (30). These findings indicate that molecular geneticsbased techniques are more suitable for the differentiation of Bacteroides species.In the past decade, molecular genetics-based techniques, including hybridization assays...

Succession in the intestinal microbiota of preadolescent turkeys

“…Real‐time PCR was performed to confirm the B. uniformis bloom detected at week 11. Primers 16F2 and 23R4 (Table 1) were used to amplify the rrn ITS from week 11 DNAs and amplicons were cloned into pGEM‐T (Kuwahara et al , 2001). Bacteroides uniformis ITS‐containing plasmid pBu25 was identified by sequence analysis and subsequently used as the positive control for these experiments.…”

The Medically Important Bacteroides spp. in Health and Disease

“…The application of 16S rDNA PCR-RFLP to species identification offers a rapid and accurate approach for identification of Bacteroides to the species level. A similar strategy was used by Kuwahara et al (2001) to examine the 16S and 23S rRNA internal transcribed spacer region among Bacteroides spp. Amplification products of the spacer region digested with MspI produced distinctive species-specific RFLP patterns, and it was possible to differentiate 90 strains to the species level, including B. fragilis, B.…”

Physical and genetic map of the Bacteroides fragilis YCH46 chromosome

“…A DNA^DNA hybridization study by Johnson and Ault led to the distinction of two DNA homology groups, I and II [3]. We also showed the heterogeneity of this anaerobe by PCR-ribotyping using 16S^23S rDNA internal-transcribed spacer sequences [4]. It has also been reported that the pathogenic potential and antimicrobial susceptibility of B. fragilis vary among strains [5,6].…”