This research we employed TaqMan real-time PCR to direct target quantification A.actinomycetemcomitans and cdtB gene in different severity of periodontitis sites of AgP or CP patients.and it is effective for detecting pathogen species that are extremely difficult to culture. Therefore, we chose different periodontal sites as targets to explore the occurrence and quantity of A.actinomycetemcomitans and its CDT encoding gene in Chinese subjects and different periodontal status. A.actinomycetemcomitans might not be present in all oral sites in an untreated periodontitis patient
. Taking subgingival samples from all teeth would be the most reliable way to detect A.actinomycetemcomitans. However, this method is money- consuming and time-consuming which is not suitable to be used in daily practice. Sampling from the deepest pocket of each quadrant has been demonstrated to be quite reliable for detecting the subgingival presence of periodontal pathogens in untreated patients
. This technique was based on individual level. While one suffered from periodontitis, aggressive or chronic mode, severity of periodontal disease might differ from site to site. So we evaluated relevance of A.actinomycetemcomitans and its CDT encoding gene with periodontitis stuates base on “site”.
The primers chosen for detection of A.actinomycetemcomitans and cdt gene were based on A.actinomycetemcomitans 16 s rDNA gene and cdtB gene respectively. The 16 s rDNA gene has been reported to be highly conserved and this pattern for detection of A.actinomycetemcomitans has been well preserved
[19, 20]. Moreover, recent data have shown that CdtB is the main component and indispensable for the expression of CDT holotoxin activity
[6, 8], and the sequence of cdtB was also highly conserved among A.actinomycetemcomitans species
. Thus, the prevalence of CDT encoding genes was evaluated using cdtB specific primers and probes.
Standard curves were used in this study to evaluate the absolute quantification of A.actinomycetemcomitans and cdt gene in samples. Plasmids containing cloned target sequences were used as standard substances in quantitative PCR, which enabled the measurements more precise and steady compared to using PCR amplicon as standard substances directly
Some studies have examined the prevalence of A.actinomycetemcomitans in some different populations
[4, 13, 18]. Our work is the first report on the analysis of both positive rate and absolute quantity of A.actinomycetemcomitans and its cdtB gene in Chinese periodontitis patients, and the association of the distribution of A.actinomycetemcomitans and cdtB with various periodontal status.
We found that A.actinomycetemcomitans and its cdtB gene were significantly more prevalent and with higher quantity in samples from patients suffering from AgP or CP than periodontal healthy subjects, and A.actinomycetemcomitans and its cdt gene were also more prevalent and with higher quantity in deep periodontal pockets than in moderate and shallow periodontal pockets.
Although A.actinomycetemcomitans is linked to the etiology of AgP, this bacterium is also found in subjects who are healthy or have other forms of periodontal disease
. Our results showed that only 7.0% periodontal healthy sites were A.actinomycetemcomitans positive; 92.4% CP samples and 87.6% AgP samples exhibited A.actinomycetemcomitans positive, demonstrating that the presence of A.actinomycetemcomitans was correlated with periodontitis.
Quantitative data showed that the amount of A.actinomycetemcomitans in CP and AgP samples were observably higher than in healthy samples, while no difference between CP and AgP. But the amount of cdtB genotype strain of A.actinomycetemcomitans in AgP samples were remarkablly higher than in CP and healthy samples (Table
3). These data may indicate quantitative results were more suitable to analyse the distribution of A.actinomycetemcomitans and its cdtB genotype strains. The cdtB genotype strain of A. actinomycetemcomitans may be more relevant with aggressive periodontitis.
Tan and his coworkers found A.actinomycetemcomitans with the cdt genotype were at a higher frequency from sites obtained from patients diagnosed with aggressive periodontitis
. Our study showed the cdtB genotype A.actinomycetemcomitans were at a higher quantity from sites obtained from deep pockets. The higher occurrence and amount of this bacterium in samples with severe periodontitis status was not a surprising observation. A.actinomycetemcomitans CDT toxin may be similar to H.ducreyi CDT toxin, which may contribute to the pathogenicity of bacteria at a higher concentration
. In vivo studies are still needed to explore the exact pathogenic role of A.actinomycetemcomitans CDT in the future. Our data showed A.actinomycetemcomitans and its cdt genotype strain were prevalent in deep and moderate periodontal pockets. To move forward a single step, quantitative analysis showed cdtB genotype strain of A.actinomycetemcomitans were more prevalent in deep periodontal pockets. The quantities of cdt genotype strain of A.actinomycetemcomitans were correlated with severe forms of periodontitis (CP or AgP). Numerous studies have considered A.actinomycetemcomitans as an important etiological microorganism involved in AgP
[24–26], however, from a new perspective, our data showed that cdtB genotype strain of A.actinomycetemcomitans was mainly found among sites with severe forms of periodontitis. A.actinomycetemcomitans with cdtB genotype may be more virulent to human periodontium.
As expected, none of the A.actinomycetemcomitans 16 s rDNA negative samples were positive for cdtB. This result confirmed that A.actinomycetemcomitans was the exclusive member in the oral microbial flora identified to carry and express the cytolethal distending toxin locus. Besides, there were 5 of healthy samples in our study which were positive for A.actinomycetemcomitans while negative for cdt. This may be results of minute quantity of cdt genotype strain of A. actinomycetemcomitans in this 5 subgingival plaque samples, while may be a new supporting proof for the exist of cdt-negative genotype strain of A.actinomycetemcomitans in oral cavity.
The cdt gene as well as lktA (leukotoxin A) of A.actinomycetemcomitans is a single copy gene
. However, 16 s rDNA gene may generally have 4 to 6 copies per cell (e.g., 6 copies for E. coli)
. Our results showed that within the same sample the absolute quantity of A.actinomycetemcomitans 16 s rDNA were 1–10 times over that of cdtB gene, which indicated that one periodontal site might be infected with two or even more genotypes of A.actinomycetemcomitans simultaneously. Some genotypes of A.actinomycetemcomitans possess cdt gene, which could express CDT activity; while other genotypes of A.actinomycetemcomitans are cdt-negative, which would be less virulent than cdt-positive strains. Yamano
 reported that 89% of A.actinomycetemcomitans strains possessed the cdt gene. Another study
 discovered that 86% of A.actinomycetemcomitans isolates presented complete operon of cdt gene and its characteristic cytotoxic activity. Tan and his coworkers showed a close association between AgP and cdt-positive genotype A.actinomycetemcomitans strains
. These findings suggested that not all the strains of A.actinomycetemcomitans possessed cdt gene, in other words, not every A.actinomycetemcomitans strains presented cytotoxic CDT activity. Similar to A.actinomycetemcomitans strains, Campylobacter spp., C.jejuni, H.ducreyi and other CDT-producing bacteria don’t express CDT activity or contain all of the cdtABC genes in all strains