Bacterial 16s rRNA Gene Sequencing in the Routine Diagnostic Laboratory.
Jason D Pimentel, Linoj P Samuel, Robert J Tibbetts, Lisa Whitely, Sally M Dubedat, Evan LN Dodds. Henry Ford Hospital, Detroit, MI; Royal Prince Alfred Hospital, Sydney, NSW, Australia; Sydney University Prince Alfred Macromolecular Analysis Centre, Sydney, NSW, Australia
Background: Sequencing DNA for bacterial identification has become more practical due to automated genetic analyzers and alignment software. The literature suggests that identification of bacterial isolates by 16S rRNA gene sequence is more accurate than commercial biochemical kits and should be used to identify any clinically significant isolate with less-than-excellent commercial identification. However, debate has arisen regarding the selection of databases used for identification. Our aim was to evaluate the accuracy of the free databases available with the online BLAST program and evaluate the utility of sequence-based identification in a routine diagnostic laboratory.
Design: 44 isolates (aerobes, anaerobes, mycobacteria) with known identifications (18 ATCC, 26 patient samples) from two different institutions were evaluated. DNA was extracted from isolated colonies using commercial spin columns or by 10 minutes at 95oC. A monochrome (SYBR green I), real-time PCR was used to amplify and detect 500 base pairs of the 16S rRNA gene using either a Rotor-Gene RG-3000 (Corbett) or a SmartCycler 16 (Cepheid). BigDye Terminator 3.1 chemistry (Applied Biosystems,) and either a 3100 or 3730xl DNA analyzer (Applied Biosystems) was used to sequence the real-time PCR product. A BLAST match was then performed with the following parameters: Database nucleotide collection (nr/nt); Exclude models (XM/XP) and uncultured/environmental sample sequences; Optimize for highly similar sequences (megablast). Matches with the highest max scores were evaluated for clinical relevance (e.g. non-human pathogens excluded) and reputable source (reference strain or published biochemical characters). Close matches were further evaluated by comparison of biochemical characters.
Results: Sequence alone provided genus-level identification in 98% of isolates. Species/species complex/species group-level identification was achieved in 93%. There was no differentiation between the genera Leclercia and some Enterobacter spp; Enterococcus durans (ATCC 6056) and E. faecium; or Bordetella bronchiseptica, B. pertussis, B. parapertussis and B. holmesii.
Conclusions: The methods and results described were reproducible and practically applied in two different routine diagnostic laboratories using different types of equipment. Moreover, use of the free databases provided reliable identifications for pure colonies with a completed biochemical profile.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 223, Wednesday Afternoon