Evaluation of partial rpoB and 16S rDNA sequencing for identification of Enterococcal species and the comparison to phenotypic methods

Abstract number: 1733_77

Haase G., Lütticken R., Weber-Heynemann J., Ramminger I., Mellmann A., Harmsen D.

Objectives: Enterococci can cause a variety of serious infections in humans and animals. As of spring 2005 this genus compromise 32 species whereas many of the newer species have not yet been included in the database of commercially available identification systems. Therefore, in this study a reference databases for partial RNA polymerase B (rpoB) and the 16S rDNA gene sequences comprising all type strains of the genus Enterococcus was established. Subsequently, the performance of sequence-based identification in comparison to two commercially available, phenotypic based identification systems was evaluated using 44 clinical isolates.

Methods: In addition to the 32 type strains, a panel of 44 strains (mainly human isolates compromising 9 different species) was analysed in order to reveal the relative performance of the different identification methods tested. Direct sequencing was performed using ABI Prism 3100 Analyzer (Applied Biosystems). For quality assured sequence analysis (rpoB: 551 bp, position 2491 to 3041, AF535187; 16S rDNA: 446 bp, position 54 to 510 of E. coli) the Ridom TraceEditPro version 1.0 software (Ridom GmbH, Würzburg, Germany) was used. For phenotypic identification the rapid ID 32 Strep (bioMérieux, Nürtingen, Germany) and the Gram positive panel of the PhoenixTM system (BD, Heidelberg, Germany) were used according to the instructions of the manufactures. ID 32 panels were examined using the MiniAPI reader.

Results: Of the 32 type species, 28 exhibit a unique sequence in the respective 16S rDNA/rpoB reference database whereas only the two pairs E. porcinus/E. villorum and E. italicus/E. saccharominimus had identical sequences. Only 7 type strains were correctly identified by ID 32 Strep or the Phoenix system. Analyzing the 44 clinical isolates, 36 (81.8%) could be identified univocally by sequencing (≥99% similarity) using the 16S rDNA/rpoB reference database. Using the rpoB based identification as reference, only 25 (56.8%) and 32 (72.7%) of these strains were correctly identified by the ID 32 Strep or Phoenix test system, respectively.

Conclusion: Sequence based identification, in particular rpoB sequencing turned out as the most discriminatory identification procedure for achieving reliable species identification of Enterococci.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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