Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

Abstract

BackgroundAggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone.MethodsBased on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97–100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer–probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis.ResultsThe JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 108 (mean 1.28 × 107) for JP2 clones and from 0 to 1.6 × 106 for non-JP2 clones (mean 1.84 × 105). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p < 0.01). Our new qPCR-based JP2- and non-JP2-specific quantitative detection assay is applicable to the diagnosis of aggressive periodontitis with A. actinomycetemcomitans.ConclusionsWe successfully developed a quantitative and discriminative PCR-based method for the detection of A. actinomycetemcomitans JP2 and non-JP2 clones. This technique will contribute to future analyses of the quantitative relationship between this organism and aggressive periodontitis.

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

“…P. gingivalis ATCC 33277, P. intermedia ATCC 25611, and F. nucleatum ATCC 25586 were grown in modified Gifu anaerobic medium broth (Nissui, Tokyo, Japan) for 48 hr. A. actinomycetemcomitans HK1651 was grown in trypticase soy broth supplemented with 0.6% yeast extract and 0.04% sodium bicarbonate for 12 hr . The overnight cultures were then inoculated into the relevant culture medium and allow to grow until they reached exponential growth phase (OD 620 0.1–0.5).…”

“…A. actinomycetemcomitans HK1651 was grown in trypticase soy broth supplemented with 0.6% yeast extract and 0.04% sodium bicarbonate for 12 hr. 31 The overnight cultures were then inoculated into the relevant culture medium and allow to grow until they reached exponential growth phase (OD 620 0.1-0.5). Bacteria were subsequently used for antimicrobial and bactericidal activity assays.…”

LL-37 Opsonizes and Inhibits Biofilm Formation of Aggregatibacter actinomycetemcomitans at Subbactericidal Concentrations

“…The JP2 genotype produces large amounts of leukotoxin due to a 530-bp mutational deletion in the promoter region of the lkt/ltx gene, which encodes leukotoxin (34). Strains of this genotype were associated with aggressive periodontitis in subjects of African origin (35,36).…”

High salivary levels of JP2 genotype of Aggregatibacter actinomycetemcomitans is associated with clinical attachment loss in Moroccan adolescents

“…Briefly, PCR mixture (10 μl) contained 5-μl Kapa Syber Green (KK 4702; Kapa Biosystems, Boston USA), 3-μl template, and 1 μl of a primer mix targeting JP2 or non-JP2 genotypes specific sequences within the leukotoxin promoter region of the leukotoxin operon (0.5 μM each; Table 1). The PCR mixtures also contained 1 μl of JP2 TABLE 1 Aggregatibacter actinomycetemcomitans-specific primers according to Kirakodu et al (2008) and Yoshida et al (2012) or non-JP2 genotypes related probes (0.2 μM; Table 3). The PCR program is shown in…”

“…Cycle settings for quantification of Aggregatibacter actinomycetemcomitans according toKirakodu et al (2008) andYoshida et al (2012) …”