fimA genotypes and multilocus sequence types of Porphyromonas gingivalis from patients with periodontitis

Abstract

Fimbriae are important virulence factors of pathogenic bacteria, facilitating their attachment to host and bacterial cells. In the periodontal pathogen Porphyromonas gingivalis, the fimA gene is classified into six types (genotypes I, Ib, II, III, IV, and V) on the basis of different nucleotide sequences, with fimA genotypes II and IV being prevalent in isolates from patients with periodontitis. The aims of this study were to examine the distribution of fimA genotypes in a collection of 82 P. gingivalis isolates from adult periodontitis patients of worldwide origin and to investigate the relationship between the fimA genotypes and the sequence types (STs), as determined by multilocus sequence typing (MLST), of the isolates. The fimA gene was amplified by PCR with primer sets specific for each genotype. The STs of all strains were assigned according to the MLST database for P. gingivalis (www.pubmlst.org/pgingivalis). The 82 strains showed extensive genetic diversity and were assigned to 69 STs. Only isolates with closely related STs harbored the same fimA genotype. Twenty-eight (34.1%) strains harbored fimA genotype II, while only the reference strain for fimA genotype V reacted with the primers specific for this genotype. Twenty-one isolates (25.6%) were positive by more than one of the fimA PCR assays; the most frequent combinations were genotypes I, Ib, and II (eight isolates) and genotypes I and II (four isolates). Sequencing of the fimA gene from selected isolates did not support the observed specific fimA genotype combinations, suggesting that the genotyping method used for the major fimbriae in P. gingivalis should be reevaluated.

FIG. 1.

Sequences of forward and reverse primers specific for fimA types I to V superimposed on the multiple-sequence alignment of the reference sequences obtained with the ClustalW program. (A) Forward primer specific for fimA type V; (B) forward primer specific for fimA type Ib; (C) forward primer specific for fimA type I; (D) forward primers specific for fimA types II, III, and IV; (E) reverse primer specific for fimA type V; (F) reverse primer specific for fimA type Ib; (G) reverse primer identical to the sequences of fimA types I, II, III, and IV.

FIG. 2.

Distance matrix of selected sequenced isolates obtained by UPGMA after multiple-sequence alignment with the ClustalW program (http://www.ebi.ac.uk./clustalw/) of different fimA genotypes of P. gingivalis. Clusters at a genetic distance of 0.02 were designated A to E. The corresponding fimA genotypes are shown in the right column. The fimA nucleotide sequences from GenBank are marked with an asterisk.

FIG. 3.

Genetic relationships among 82 P. gingivalis isolates. A distance matrix was calculated by UPGMA on the basis of the differences in the allelic profiles. Clusters at a genetic distance of 0.5 were designated A to G. Columns to the right of the tree give the ST, the strain designation, the fimA genotype (see below), and the continents where the strains originated (EU, Europe; NA, North America; AF, Africa; AS, Asia).

FIG. 4.

SplitsTree graph illustrating the degree of recombination between 82 isolates of P. gingivalis for the seven ubiquitous chromosomal gene fragments used for MLST.