Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients

Abstract

Fimbriae (FimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues. We previously demonstrated that fimA can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the fimA gene. In the present study, we attempted to detect the four different fimA genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method. Four sets of fimA type-specific primers were designed for the PCR assay. These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the fimA gene. Positive PCR results were observed with reference strains of P. gingivalis in a type-specific manner. All other laboratory strains of oral and nonoral bacteria gave negative results. The sensitivity of the PCR assay for fimA type-specific detection was between 5 and 50 cells of P. gingivalis. Clinical samples were obtained from saliva and subgingival plaque from deep pockets (>/=4 mm) of 93 patients with periodontitis. Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR. The presence of P. gingivalis was demonstrated in 73 patients (78.5%), and a single fimA gene was detected in most patients. The distribution of the four fimA types among the P. gingivalis-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6. 8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8%. P. gingivalis with type II fimA was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (>/=8 mm) pockets. These results suggest that P. gingivalis strains that possess type II fimA are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.

FIG. 1

Sensitivity of PCR assay for detection of our fimA types of P. gingivalis. The sensitivity of the PCR assay was studied with titrated cultures of P. gingivalis of four fimA types (types I to IV; 10⁹ cells of strains 381, HW24D-1, OMZ314, and HG564, respectively, per ml). The detection limit was determined for the simultaneous PCR by the use of known numbers of bacterial cells diluted in sterile distilled water. The following numbers of cells were added: 5 × 10⁵ (lane 1), 5 × 10⁴ (lane 2), 5 × 10³ (lane 3), 5 × 10² (lane 4), 5 × 10 (lane 5), and 5 (lane 6). Lane 7, a negative control from the PCR without any bacterial cells; lane 8, a PCR product obtained with P. gingivalis species-specific 16S rRNA primers (5 × 10³ cells); lane M, molecular size marker (a 100-bp DNA ladder).