16S ribosomal DNA sequence analysis of a large collection of environmentaland clinical unidentifiable bacterial isolates

Some bacteria are difficult to identify with phenotypic identification sche mes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)- based identification of bacteria potentially offers a useful alternative wh en phenotypic characterization methods fail. However, as yet, the usefulnes s of 16S rDNA sequence analysis in the identification of conventionally uni dentifiable isolates has not been evaluated with a large collection of isol ates. In this study, we evaluated the utility of 16S rDNA sequencing as a m eans to identify a collection of 177 such isolates obtained from environmen tal, veterinary, and clinical sources. For 159 isolates (89.8%) there was a t least one sequence in GenBank that yielded a similarity score of greater than or equal to 97%, and for 139 isolates (78.5%) there was at least one s equence in GenBank that yielded a similarity score of greater than or equal to 99%. These similarity score values were used to defined identification at the genus and species levels, respectively, For isolates identified to t he species level, conventional identification failed to produce accurate re sults because of inappropriate biochemical profile determination in 76 isol ates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase ac tivity determination in 5 isolates (3.6%) and growth requirement determinat ion in 2 isolates (1.5%), Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0 .07), The 16S rDNA approach failed to identify Enterobacter and Pantoea iso lates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence int erval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with > 1% undetermined p ositions in the databases. Unlike phenotypic identification, which can be m odified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible i n and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of c ertain taxa should further improve the usefulness of molecular identificati on of bacteria.