Whole cell protein and partial 16S rRNA gene sequence analysis suggest the existence of a second Rothia species

OBJECTIVE: To subject ten clinical isolates grouped together based on their biochemical and microbiological profile to further investigations aimed at correct species identification. METHODS: The 16S rRNA gene was partially sequenced using nested amplification. Whole cell protein analysis (SDS-PAGE) and cluster analysis were performed on the 10 strains and also for comparison on 31 reference strains. The API Coryne biochemical kit as well as API 20 Strep were used for analysis of the phenotypic diversity of the strains by use of computerized numerical identification procedures. Antibiotic susceptibility testing was performed using a standardized disk diffusion test. RESULTS: The 265--556-bp-long 16S rRNA gene sequences of all 10 strains showed highest similarity to Rothia dentocariosa. Three strains showed complete identity between the sequences obtained and the sequence of the type strain of Rothia dentocariosa 16S rRNA gene (M59055), and the other seven ranged between 99.7% and 98.3% similarity. Detailed analysis of the sequences revealed a clustering of the strains into two groups. One group consisted of four isolates with the highest degrees of similarity with the reference strain (type I), while the members of another group (type II) showed differences in their nucleotide sequence at four distinct positions in the variable V7 region. T was replaced by C at position 597, C by T at position 608, T by C at position 610, and G by A at position 684 (position numbers according to reference sequence M59055, EMBL/GenBank). Whole cell protein analysis (SDS-PAGE) and cluster analysis also segregated the 10 Rothia dentocariosa strains into two different clusters, with one cluster containing all four strains belonging to 16S rRNA gene type I, and a second cluster containing all six strains belonging to 16S rRNA gene type II. CONCLUSIONS: Partial sequence data of the 16S rRNA gene as well as whole cell protein analysis showed a subdivision of the Rothia species into two groups, genomovar I (Rothia dentocariosa sensu stricto) and genomovar II, a possible new Rothia species.