Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.

Rapid molecular identification and subtyping of Helicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions.

We describe here the use of real-time DNA sequence analysis of Helicobacter pylori 16S rRNA gene fragments by pyrosequencing for rapid molecular identification and subtyping of clinical isolates based on DNA sequence heterogeneity within the variable V1 and V3 regions. Six individual 16S rDNA V1 alleles (position 75-100) were identified in 23 clinical isolates obtained from gastric biopsy specimens. Eleven of these revealed sequence identities with H. pylori 26695 and one was identical with the rrn genes in strain J99. The other V1 alleles showing single or double nucleotide mutations or single nucleotide insertions could be divided into four groups with 5, 4, 1, and 1 isolates each. Two out of 25 isolates demonstrated single C to T transitions in the V3 region (position 990-1020). The present findings show that subtle DNA sequence variation occurs sufficiently often in the 16S rDNA variable V1 and V3 regions of H. pylori to provide a consistent system for subtyping.