Abstract
The JP2 clone of Actinobacillus actinomycetemcomitans is associated with early-onset periodontitis in certain ethnic populations of African origin. Here, we describe and evaluate a set of primers for PCR to assay for the presence of A. actinomycetemcomitans and to discriminate between JP2-like strains and other genotypes in subgingival plaque samples.
Actinobacillus actinomycetemcomitans has long been implicated in the etiology of early-onset periodontitis (EOP) (13, 14). Production of leukotoxin, which kills host immune cells, is considered to be a key virulence factor in A. actinomycetemcomitans. The leukotoxin belongs to the RTX family of toxins and is encoded by the ltx gene operon (9, 10, 11). A particular clonal type of A. actinomycetemcomitans serotype b, called the highly leukotoxic JP2 clone, has a 530-bp deletion called Δ530 in the ltx promoter region, resulting in enhanced production of leukotoxin (1). The JP2 clone shows pronounced racial tropism, as members of this clone have been isolated almost exclusively from adolescents of African descent (2, 4, 5, 6, 7). In this population it is strongly associated with EOP.
In a previous study of children in Morocco, cultivation of plaque samples was used to show a strong association between the presence of the JP2 clone and EOP (6). However, cultivation and identification are time-consuming. Also, it was found that many samples had to be excluded because the primary cultures were either overgrown by other microorganisms or showed no growth after shipment to Denmark. In such studies, PCR is an attractive alternative to cultivation.
Microbiological sampling.
Subgingival samples on paper points were obtained from adolescents in Rabat, Morocco; placed in selective medium and sent by mail to Aarhus, Denmark; and analyzed by cultivation immediately after receipt as described previously (6). In parallel, subgingival samples on paper points transferred to a tube containing 1 ml of 0.9% (wt/vol) NaCl were sent to Aarhus for PCR analysis. Prior to PCR, the paper points were removed after thorough mixing, the suspension was centrifuged at 20,000 × g at 4°C for 30 min, the supernatant was discarded, and the pellet was resuspended in 100 μl of H2O and heated for 5 min in a boiling water bath.
The primers ltx1 (5′-CAGATCAAAACCTGATAACAGTATT-3′) and ltx2 (5′-TTTCTCCATATTAAATCTCCTTGT-3′) were previously used in a PCR assay performed on isolated colonies to identify members of the JP2 clone among A. actinomycetemcomitans strains (5, 6, 7) (Fig. 1). However, we found that in our assay, PCR with these primers performed directly on subgingival plaque samples often resulted in a smear of bands when analyzed by 1% agarose gel electrophoresis, indicating that the primers were not specific for the A. actinomycetemcomitans ltx promoter region (results not shown). The same primers were used by Contreras and coworkers in direct PCR on subgingival samples, but they did not comment on problems with nonspecific products (2).
FIG. 1.
DNA fragments of the ltx region amplified in PCR with the four sets of primers used. (A) Strains of the JP2 clone with the Δ530 deletion. The deletion is indicated. (B) Strains without the Δ530 deletion. (C) Strains with the 886-bp insertion sequence (IS).
Specificity and sensitivity of primers ltx3 and ltx4 in PCR on isolates.
We designed another set of primers, ltx3 (5′-GCCGACACCAAAGACAAAGTCT-3′) and ltx4 (5′-GCCCATAACCAAGCCACATAC-3′), that span the Δ530 deletion present in the JP2 clone (Fig. 1). In PCR on genomic DNAs from strains of the JP2 clone, the primers are expected to amplify a product of 686 bp, whereas in strains with the insertion sequence described by He et al. (8), the amplicon is 2,117 bp, and in other strains it is 1,216 bp.
For PCR in a volume of 25 μl containing 23 μl of sample and 10 pmol each of two primers, we used Ready-To-Go PCR Beads (Amersham Biosciences, Uppsala, Sweden). The thermocycling program was as follows: denaturation for 5 min at 94°C and 30 cycles of 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min, followed by a final extension at 72°C for 8 min.
The primers were tested in PCR on genomic DNAs from 65 strains of A. actinomycetemcomitans without Δ530 selected from the collection described previously (12) and chosen to represent the different evolutionary lineages within the population of A. actinomycetemcomitans. All of the strains gave rise to a single PCR product of 1.2 kb. In addition, PCR on genomic DNAs from all 38 JP2 clone isolates from different subjects (5) resulted in a single amplicon of 0.7 kb. Thus, in A. actinomycetemcomitans, PCR with ltx3 and ltx4 is specific, and the sizes of the amplified fragments identify the various structures of the ltx promoter region.
The detection limit of PCR with ltx3 and ltx4 was ∼103 CFU of A. actinomycetemcomitans. The presence of genomic DNA from a strain without the Δ530 deletion in 100-fold excess did not interfere with amplification of a 0.7-kb fragment from a highly leukotoxic strain with Δ530 (results not shown).
Direct amplification with primers ltx3 and ltx4 from plaque samples compared to cultivation.
The primers ltx3 and ltx4 were tested in direct PCR on 89 fresh subgingival plaque samples from subjects who were also tested for the presence of A. actinomycetemcomitans in subgingival plaque using a cultivation technique (6). In addition to those of 1.2 and 0.7 kb, no bands were observed for any of the samples, demonstrating that the primers were highly specific for the ltx promoter region in A. actinomycetemcomitans (Fig. 2A). In general, the results of PCR correlated with the results of cultivation (Table 1). Out of 89 samples, there was agreement between the two methods for 69 (78%). PCR failed to detect A. actinomycetemcomitans in three samples that were positive by culture. Conversely, among samples positive in PCR, cultivation failed to detect A. actinomycetemcomitans in 16 samples. In addition, in a single sample, A. actinomycetemcomitans with the Δ530 deletion was detected by culture, whereas PCR detected both a strain with and a strain without Δ530. Thus, PCR was superior to cultivation in demonstrating the presence of A. actinomycetemcomitans, including the JP2 clone, in these samples. However, the method missed the bacterium in three samples, indicating that it is moderately sensitive.
FIG. 2.
Agarose gel analysis of DNA fragments amplified from isolates and directly from plaque samples. The combinations of primers used are indicated below the gels. (A) Lanes 1 and 11, sample 506F; lanes 2 and 12, sample 401F; lanes 3 and 13, sample 336F; lanes 4 and 14, sample 364; lanes 5 and 15, sample 310; lanes 6 and 16, sample 305; lanes 7 and 17, sample 304; lanes 8 and 18, isolate JP2 with Δ530; lane 9, empty; lane 10, isolate HK1605 without Δ530. The estimated sizes of the fragments are shown on the right. Weak bands corresponding to a size of 1.2 kb and hardly visible in the photographic reproduction are present in lanes 15 and 17. (B) Lanes 1 and 11, isolate HK1605 without Δ530; lanes 2 and 12, sample 586F; lanes 3 and 13, sample 585F; lanes 4 and 14, sample 377F; lanes 5 and 15, sample 369F; lanes 6 and 16, sample 332F; lanes 7 and 17, sample 582; lanes 8 and 18, sample 490; lanes 9 and 19, isolate JP2 with Δ530; lane 10, empty; lane 20, molecular weight marker II from Roche Molecular Biochemicals, Hvidovre, Denmark (lambda DNA digested with HindIII). (C) Lanes 1 to 13, different plaque samples; lane 14, isolate HK1605 without Δ530; lane 15, isolate JP2 with Δ530; lane 16, molecular weight marker II.
TABLE 1.
Detection of A. actinomycetemcomitans with and without Δ530 by cultivation compared to detection by PCR with primers ltx3 and ltx4 on 89 fresh plaque samples
| Culture results (A. actinomycetemcomitans) | Results for PCR with primers ltx3 and ltx4a
|
Total | |||
|---|---|---|---|---|---|
| No product | 1.2-kb product | 0.7-kb product | 1.2- and 0.7-kb products | ||
| None | 56 | 13 | 3 | 0 | 72 |
| Without Δ530 | 3 | 11 | 0 | 0 | 14 |
| With Δ530 | 0 | 0 | 2 | 1 | 3 |
| Total | 59 | 24 | 5 | 1 | 89 |
No. of samples. PCR was performed within 1 week after receipt of the sample.
To test whether PCR with ltx3 and ltx4 could be used on samples stored for prolonged periods, we applied the method to another 157 subgingival plaque samples stored for up to 2 years at −20°C and for which results of culture were available. The PCR and previous culture results agreed on the presence versus the absence of A. actinomycetemcomitans with the different ltx promoter structures for 115 (73%) of the samples (Table 2). Notably, 27 samples were falsely negative in PCR, and among these, 24 were also negative in PCR with the primers TT-15 and TT-16 (see below). Thus, prolonged storage of the plaque samples under the conditions used here significantly lowered the sensitivity of the PCR method.
TABLE 2.
Detection of A. actinomycetemcomitans with and without Δ530 by cultivation compared to detection by PCR with primers ltx3 and ltx4 on 157 old plaque samples
| Culture results (A. actinomycetemcomitans) | Results for PCR with primers ltx3 and ltx4a
|
Total | |||
|---|---|---|---|---|---|
| No product | 1.2-kb product | 0.7-kb product | 1.2- and 0.7-kb products | ||
| None | 51 | 8 | 0 | 0 | 59 |
| Without Δ530 | 25 | 57 | 1 | 2 | 85 |
| With Δ530 | 2 | 0 | 7 | 1 | 10 |
| With and without Δ530 | 0 | 1 | 2 | 0 | 3 |
| Total | 78 | 66 | 10 | 3 | 157 |
No. of samples. PCR was performed on samples that were stored for up to 2 years at −20°C.
One advantage of the PCR method compared to cultivation is that it does not rely on the viability of the bacteria, and furthermore, the competing influence of large numbers of other microorganisms on the analysis is expected to be less. We used PCR with ltx3 and ltx4 to analyze an additional 99 plaque samples for which cultivation results were excluded either because of no growth at all (23 samples) or because of overgrowth with contaminating microorganisms (76 samples). Among these 99 samples, PCR showed the presence of A. actinomycetemcomitans in 34, of which 7 were positive for the JP2 clone. Lack of a “gold standard” to detect the presence of A. actinomycetemcomitans prevented calculation of the specificity and sensitivity of the PCR method.
Direct amplification from plaque samples with ltx3 and ltx4 compared to primers TT-15 and TT-16.
PCR using the primers TT-15 (5′-TCGCGAATCAGCTCGCCG-3′) and TT-16 (5′-GCTTTGCAAGCTCCTCACC-3′) amplified a 285-bp fragment of the structural ltxA gene in all strains of A. actinomycetemcomitans (3) (Fig. 1). These primers were used in PCR on the 345 plaque samples discussed above in order to compare their performance with PCR using ltx3 and ltx4. Using primers TT-15 and TT-16 as described by Flemmig et al. (3), we occasionally encountered problems with artificial products (Fig. 2B). Consequently, in this PCR assay, a sample was considered positive when a band corresponding to 285 bp was present in the agarose gel analysis. This resulted in false positives. Sequence analysis of the amplified fragment in three of six samples that were positive in this PCR and negative in both PCR with ltx3 and ltx4 and cultivation revealed that none of these amplicons of the expected size represented the ltxA gene. However, there was a high degree of concordance between the results of PCR with ltx3 and ltx4 and those of PCR with TT-15 and TT-16, indicating that false positives with the TT primers were infrequent (Table 3). Notably, the three fresh samples that were falsely negative in PCR with primers ltx3 and ltx4 were all negative in PCR with TT-15 and TT-16, indicating either that the limits of detection for the two PCR methods are very similar or that some substances inhibitory to PCR were present in these samples. Twenty-eight samples were positive in PCR with ltx3 and ltx4 and negative with TT-15 and TT-16, and among 22 of these samples that were also analyzed by cultivation, the presence of A. actinomycetemcomitans was demonstrated in 13.
TABLE 3.
Results for PCR with primers ltx3 and ltx4 compared to PCR with primers TT-15 and TT-16 on plaque samples
| Results with primers TT-15 and TT-16 | Results for PCR with primers ltx3 and ltx4b
|
Total | |||
|---|---|---|---|---|---|
| No product | 1.2-kb product | 0.7-kb product | 1.2- and 0.7-kb products | ||
| Negativea | 189 | 26 | 1 | 1 | 217 |
| Positivea | 13 | 91 | 19 | 5 | 128 |
| Total | 202 | 117 | 20 | 6 | 345 |
See text for definitions.
No. of samples.
In conclusion, primers ltx3 and ltx4 were superior to primers TT-15 and TT-16 in PCR for the detection of A. actinomycetemcomitans, being both slightly more sensitive and, importantly, more specific. Furthermore, PCR with ltx3 and ltx4 allowed distinction between the JP2 clone and other genotypes of A. actinomycetemcomitans.
Direct amplification from plaque samples with ltx3 and ltx4 compared to primers A and B.
Zambon et al. (15) have also described a set of primers, A (5′-TCCATATTAAATCTCCTTGT-3′) and B (5′-AACCTGATAACAGTATT-3′), for PCR that amplifies a fragment covering the Δ530 deletion (Fig. 1). However, PCR with primers A and B was not very sensitive, as we found that these primers did not amplify a product from 1 ng of genomic DNA using an annealing temperature of 55°C and 26 cycles in the PCR as recommended (15). Using a temperature gradient revealed that the optimal annealing temperature was ∼50°C. Applying a different temperature-cycling program, including annealing at 50°C and 30 cycles in the PCR, resulted in amplification of the expected DNA fragments from 1 ng of genomic DNA from A. actinomycetemcomitans. However, for strains without Δ530, the primers amplified a fragment in addition to the expected one of 1.0 kb (Fig. 2C). PCR with primers A and B was performed directly on 46 subgingival samples using the modified temperature-cycling program. Two of the samples showed a product of 1.0 kb, indicating the presence of A. actinomycetemcomitans without the Δ530 deletion; one sample showed a product of 0.5 kb, indicating the presence of the JP2 clone; and some of the samples showed a smear in the agarose gel electrophoresis analysis (Fig. 2C). In addition to the three samples shown to be positive by this method, PCR with primers ltx3 and ltx4 showed the presence of A. actinomycetemcomitans in 27 of the 46 samples. Insufficient material in other samples prevented further evaluation of primers A and B. However, despite the limited number of samples analyzed, we conclude that PCR with primers A and B was less sensitive and less specific than PCR using ltx3 combined with ltx4.
In summary, PCR with ltx3 and ltx4 directly on plaque samples is an easy and reliable assay for detecting the presence of A. actinomycetemcomitans in fresh plaque samples and for differentiation of the highly leukotoxic JP2 clone and other clones without the Δ530 deletion. The assay facilitates comprehensive studies of the presence of the JP2 clone and A. actinomycetemcomitans without the Δ530 deletion in periodontal pockets. Such additional studies may provide more information on the epidemiology of this clone and its relation to periodontal disease.
Acknowledgments
We thank Mogens Kilian for helpful discussions and Lise Hald for excellent technical help.
Financial support was obtained from the Aarhus University Research Foundation and the Danish Dental Association.
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