Porphyromonas gingivalis Strain Diversity

Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains; however, the extent of variability remains unclear and regions of variability remain largely unidentified. To assess P. gingivalis strain diversity, we previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type strains in clinical samples and identified 22 heteroduplex types. Additionally, we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of the strains. In the current study, heteroduplex analysis of the ISR was used to determine the worldwide genetic variability and distribution of P. gingivalis, and microarray-based comparative genomic hybridization (CGH) analysis was used to more comprehensively examine the variability of major heteroduplex type strains by using the entire genome. Heteroduplex analysis of clinical samples from geographically diverse populations identified 6 predominant geographically widespread heteroduplex types (prevalence, ≥5%) and 14 rare heterodpulex types (prevalence, <2%) which are found in one or a few locations. CGH analysis of the genomes of seven clinically prevalent heteroduplex type strains identified 133 genes from strain W83 that were divergent in at least one of the other strains. The relatedness of the strains to one another determined on the basis of genome content (microarray) analysis was highly similar to their relatedness determined on the basis of ISR sequence analysis, and a striking correlation between the genome contents and disease-associated phenotypes of the strains was observed.Porphyromonas gingivalis is a gram-negative anaerobe that has been strongly implicated as a pathogen in adult (chronic) periodontitis (3, 15, 20, 32, 39, 40), a destructive disease that affects the gingiva and supporting structures of the teeth. The bacterium is found under conditions of both health and disease, with prevalences that range from 10% to 25% in healthy individuals and 79% to 90% in individuals with periodontitis being found (20, 23, 24). Previous epidemiologic studies have demonstrated that P. gingivalis strains vary with respect to their levels of human disease association (4, 5, 21). Studies have also demonstrated that P. gingivalis strains vary in their virulence (soft tissue destruction and death) in animal models, with some strains being classified as virulent, e.g., strains W83, W50, ATCC 49417, and A7A1, and others being classified as avirulent, e.g., strains 381, 33277, and 23A4 (19, 25, 37).Many studies have assessed the genetic diversity that exists among P. gingivalis strains, resulting in the finding of a high degree of diversity in some cases (17, 29, 31, 34) and a considerably lower degree of diversity in others (2, 28). The amount of variability found may be due to the different techniques used in the studies. In a previous clinical study, heteroduplex analysis of the intergenic spacer region (ISR) between the 16S and 23S rRNA genes was used to identify the clonal types of P. gingivalis that were present in 661 subgingival plaque samples (28). The rRNA ISR was used because, unlike the 16S gene, it possesses enough variability to distinguish between strains within the same species. Heteroduplex analysis allows similar but nonidentical DNA fragments, in this case, ISR sequences, to be distinguished by polyacrylamide gel electrophoresis (41). By that assay, 22 distinct heteroduplex types of P. gingivalis were identified in the samples, with many of them matching previously characterized laboratory strains. Multiple heteroduplex types were found in 34% of the samples (28). Additional rare heteroduplex types not found in this study have been identified in other study populations.Various methods have been used to examine the evolutionary relationships among P. gingivalis strains and to try to correlate specific clonal types with disease status (17, 30, 34, 43). In a previous clinical study conducted to determine the distribution of P. gingivalis heteroduplex types in chronic periodontitis and health, 130 adults with clear indicators of periodontitis and 181 healthy, age-matched controls were sampled, and the heteroduplex types present in each sample that was positive for P. gingivalis were identified (21). Six heteroduplex types were present at levels high enough for statistical analysis. By using a multivariate model for the relationship of heteroduplex type to disease status, heteroduplex type hW83 was by far the most strongly associated with periodontitis (P = 0.0000), and two additional types, h49417 and hHG1691, were also statistically significantly associated with disease. The prevalence of the remaining types, h23A4, h381, and hA7A1, in health or disease was not statistically different, suggesting that they are equally likely to be detected in both groups.Phylogenetic reconstruction of the P. gingivalis heteroduplex type strains on the basis of the sequence of the ribosomal operon ISR correlated with these findings, placing the two type strains most associated with disease (strains W83 and ATCC 49417) in a clade that was well separated from the others (42). The proximity of the two type strains most strongly associated with disease and their separation from type strains less associated with disease suggest that there is a link between ISR sequence phylogeny and the disease-associated phenotype of P. gingivalis heteroduplex types.All these studies demonstrate that there is genetic variability among P. gingivalis strains; however, the degree of variability that exists remains unclear and the regions of variability remain largely unidentified. The availability of P. gingivalis W83 whole-genome microarrays allows a more comprehensive assessment of the genetic heterogeneity that exists within the species and can identify regions of variability by using the entire genome. Comparative genomics by microarray analysis has been used to examine the genetic diversity among strains of numerous bacterial species (10, 11, 14, 18, 26, 36, 44, 45, 47, 49), and in some cases, this has led to the identification of genes that are associated with pathogenic strains of the species (10, 14, 47).The objectives of this study were to examine the worldwide genetic variability of P. gingivalis using heteroduplex analysis, to examine the relatedness of clinically prevalent P. gingivalis heteroduplex type strains on the basis of the entire genome (i.e., comparative genomic hybridization [CGH] analysis), to compare the relatedness of the strains on the basis of their genome content to phylogeny on the basis of the sequence of a single locus (ISR), and to identify the genes that are associated with strongly disease-associated, virulent strain W83 that could be important for virulence. Heteroduplex analysis showed that six P. gingivalis heteroduplex types (types hW83, h49417, h381, h23A4, hA7A1, and hHG1691) that matched previously characterized laboratory strains were predominant (prevalence, ≥5%) in a diverse sample population and were geographically widely distributed. Comparative genomic analysis of seven clinically prevalent P. gingivalis heteroduplex type strains with W83 whole-genome microarrays identified 133 W83 genes that were divergent in at least one of the test strains. The results of hierarchical cluster analysis of the P. gingivalis strains on the basis of their genome content (microarray data) correlated well with phylogenetic predictions from ISR sequence analysis, and a striking correlation between the genome content of the strains and their disease-associated phenotype was observed.