Automated fluorescent in situ hybridization for the specific detection andquantification of oral streptococci in dental plaque

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) ass ay for the identification of different oral groups of streptococci in denta l plaque and to combine it with digital image analysis for the automated en umeration of target cells. Cy3-labeled oligonucleotide plobes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius gro ups of streptococci were hybridized under stringent conditions with bacteri al cultures or supragingival plaque samples that had been permeabilized wit h lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity fur the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR40 5, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. pr obes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque s amples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaq ues. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque. (C) 2001 Elsevier Science B.V. All rights reserved.