A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNAglycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-offlight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yetuncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria. E merging antibiotic-resistant pathogens, the actual threat of bioterrorist attacks, and bioremediation or bioprospecting efforts all require fast and accurate identification of the bacteria involved. Conventional diagnoses rely on the characterization of phenotypic traits of pure cultures obtained from specimens after cultivation and isolation of bacteria on appropriate laboratory media. These culture-based methods are time-consuming especially for slow-growing pathogens such as mycobacteria and generally are restricted to yet-culturable bacteria, which constitute only a small fraction (1-10%) of the global bacterial diversity (1). In contrast, genotypic analyses such as comparative sequencing of PCR-amplified 16S rRNA genes (rDNAs) allow for the identification of both cultured and as-yet-uncultured bacteria (1). Currently, 16S rDNA sequences constitute the largest gene-specific data set, and the number of entries in generally accessible databases is increasing continually (currently Ϸ30,000), making 16S rDNA-based identification of unknown bacteria isolates more and more likely. Conventional 16S rDNA sequencing is expensive and time-consuming because of the enzymatic reactions and electrophoresis or chromatography steps involved. Hence, it still is not suited for massive parallel testing as required in clinical, pharmaceutical, or environmental microbiology. Compared with standard 16S rDNA sequencing, alternative nonelectrophoretic techniques such as sequencing by MS (2) or pyrosequencing (3) are faster but generate only short read lengths, limiting their utility in 16S rDNA-based bacterial identification.Here we introduce the concept of base-specific fragmentation of PCR-amplified DNA follow...

Molecular analysis of the gastric microflora in mice revealed that Helicobacter pylori infection causes an increase in microbial diversity. The stomachs of H. pylori-infected animals were colonized by bacteria which are naturally restricted to the lower intestinal tract. Clostridia, Bacteroides/Prevotella spp., Eubacterium spp., Ruminococcus spp., streptococci and Escherichia coli were detected exclusively in the stomachs of infected animals, whereas lactobacilli dominated the gastric flora in noninfected mice. The H. pylori-induced shifts in the gastric microbiota were independent from histological pathology and from changes in the gastric pH but were prevented by immunization of mice with live Salmonella expressing H. pylori urease. Immunized mice displayed reduced H. pylori levels in the gastric epithelium and developed a normal gastric microflora, indicating that vaccination may be protective against H. pylori-induced changes in the gastric flora.