title: Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species.
abstract: To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5' end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of > or =98.7% similarity for a "distinct species," a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMerieux, Marcy I'Etoile, France) and VITEK 2 (bioMerieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants.
authors.affiliation: Institute of Medical Microbiology, University of Münster, D-48149 Münster, Germany. kbecker@uni-muenster.de
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.forename: Dag
authors.initials: D
authors.lastname: Harmsen
authors.forename: Alexander
authors.initials: A
authors.lastname: Mellmann
authors.forename: Christian
authors.initials: C
authors.lastname: Meier
authors.forename: Peter
authors.initials: P
authors.lastname: Schumann
authors.forename: Georg
authors.initials: G
authors.lastname: Peters
authors.forename: Christof
authors.initials: C
authors.lastname: von Eiff
mesh_terms: D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D030561:Databases, Nucleic Acid; D020459:Genes, rRNA; D006801:Humans; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013203:Staphylococcal Infections / Q000382:microbiology*; D013210:Staphylococcus / Q000145:classification* / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S