Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction.

BACKGROUND: Saliva has been studied for the presence of subgingival pathogens in periodontitis patients. With the anaerobic culture technique, the discrepancy between salivary recovery and subgingival presence has been significant, which makes this approach not suitable for practical use in the microbial diagnosis of periodontitis patients. The real-time polymerase chain reaction (PCR) technique represents a very sensitive technique to detect and quantify bacterial pathogens. The aim of the study was to compare the presence and numbers of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, and Micromonas micros in subgingival plaque and mouthwash samples by the anaerobic culture and real-time PCR techniques. METHODS: Pooled subgingival plaque samples and 10-ml mouthwash samples were collected from 21 adult patients with periodontitis and analyzed by quantitative anaerobic culture and real-time PCR for A. actinomycetemcomitans, P. gingivalis, T. forsythensis, P. intermedia, and M. micros. RESULTS: The detection frequency of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis in subgingival plaque was identical by culture and real-time PCR and was higher for P. intermedia and M. micros by real-time PCR. The highest detection frequencies for the target bacteria were found in mouthwash samples by real-time PCR. The additional value of the real-time PCR to detect target bacteria was 38% for P. gingivalis, 73% for T. forsythensis, 77% for P. intermedia, and 71% for M. micros. The sensitivity to detect target species in mouthwash by real-time PCR was 100% for all test species except for P. intermedia (93.8%). CONCLUSIONS: Rapid detection and quantification of periodontal pathogens in mouthwash samples are possible by real-time PCR. The procedure is significantly less time-consuming than subgingival sampling with paper points. This approach to detect major periodontal pathogens in mouthwash samples may simplify microbial diagnosis in periodontitis patients and may be used to monitor periodontal treatment.

Periodontal pathogens: a quantitative comparison of anaerobic culture and real-time PCR.

Periodontitis is a multi-factorial chronic inflammatory and destructive disease of the tooth-supporting tissues. Quantitative anaerobic culture techniques have been used for microbial diagnosis of the different forms of the disease. The aim of this study was to compare real-time PCR with quantitative anaerobic culture for detection and quantification of 5 prominent periodontal pathogens. Real-time PCR assays with the 16s rRNA genes of Actinobacillus actinomycetemcomitans, Prevotella intermedia, Tannerella forsythensis, Peptostreptococcus micros and Fusobacterium spp. were developed. The PCR was validated on pure cultures of various bacterial strains. Subsequently, subgingival plaque samples from 259 adult patients with periodontitis were analyzed with quantitative anaerobic culture and real-time PCR. A standard curve for DNA quantification was created for each primer-probe set based on colony-forming units equivalents. All bacterial species were correctly identified. The lower limits of detection by PCR varied between 1-50 colony-forming units equivalents depending on the species. No cross-reactivities with heterologous DNA of other bacterial species were observed. Real-time PCR results showed a high degree of agreement with anaerobic culture results. Real-time PCR is a reliable alternative for diagnostic quantitative anaerobic culture of subgingival plaque samples.

The additional value of real-time PCR in the quantitative detection of periodontal pathogens.

BACKGROUND AND AIM: For the analysis of subgingival plaque, anaerobic bacterial culture has been the gold standard for many years. Currently, molecular microbial techniques have become available to identify and quantify target organisms with high specificity and sensitivity. The technique of real-time polymerase chain reaction(RT-PCR) provides a new tool to detect oral pathogens both in oral and non-oral human infections. The aim of this study was to compare the RT-PCR and anaerobic culture for detection and quantification of six periodontal pathogens in periodontal health and disease. MATERIAL AND METHODS: Subgingival plaque samples from 259 adult patients with periodontitis and 111 healthy controls were analysed with quantitative anaerobic culture and quantitative RT-PCR for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Micromonas micros and Fusobacterium spp. RESULTS: All species were more frequently isolated from patients than controls with both culture and RT-PCR. P. gingivalis, T. forsythia and M. micros appeared significant markers for disease with both techniques. P. intermedia was significantly associated with periodontitis by RT-PCR only (OR 9.7), whereas A. actinomycetemcomitans showed a significant relationship by culture only. The critical differences between culture and RT-PCR were culture-negative/PCR-positive samples which amounted to 7% for A. actinomycetemcomitans, 3% for P. gingivalis, 7% for T. forsythia, 20% for P. intermedia, 6% for M. micros, and 0.8% for Fusobacterium spp. in periodontitis patients and 12%, 3%, 2%, 35%, 14% and 0%, respectively, in the periodontally healthy group. Furthermore, periodontitis individuals had significantly higher amount of all of the test species in the subgingival plaque samples compared with healthy subjects. CONCLUSION: RT-PCR provides a new rapid diagnostic tool and opens the opportunity to detect small numbers of oral pathogens in clinical specimens, which are under the detection limit by culture technique.

Identification and characterization of the capsular polysaccharide (K-antigen) locus of Porphyromonas gingivalis.

Capsular polysaccharides of gram-negative bacteria play an important role in maintaining the structural integrity of the cell in hostile environments and, because of their diversity within a given species, can act as useful taxonomic aids. In order to characterize the genetic locus for capsule biosynthesis in the oral gram-negative bacterium Porphyromonas gingivalis, we analyzed the genome of P. gingivalis W83 which revealed two candidate loci at PG0106-PG0120 and PG1135-PG1142 with sufficient coding capacity and appropriate gene functions based on comparisons with capsule-coding loci in other bacteria. Insertion and deletion mutants were prepared at PG0106-PG0120 in P. gingivalis W50-a K1 serotype. Deletion of PG0109-PG0118 and PG0116-PG0120 both yielded mutants which no longer reacted with antisera to K1 serotypes. Restriction fragment length polymorphism analysis of the locus in strains representing all six K-antigen serotypes and K(-) strains demonstrated significant variation between serotypes and limited conservation within serotypes. In contrast, PG1135-PG1142 was highly conserved in this collection of strains. Sequence analysis of the capsule locus in strain 381 (K(-) strain) demonstrated synteny with the W83 locus but also significant differences including replacement of PG0109-PG0110 with three unique open reading frames, deletion of PG0112-PG0114, and an internal termination codon within PG0106, each of which could contribute to the absence of capsule expression in this strain. Analysis of the Arg-gingipains in the capsule mutants of strain W50 revealed no significant changes to the glycan modifications of these enzymes, which indicates that the glycosylation apparatus in P. gingivalis is independent of the capsule biosynthetic machinery.

Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples.

Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of P. gingivalis in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the P. gingivalis 16S rRNA gene. By the real-time PCR assay, as few as 1 CFU of P. gingivalis could be detected. Subgingival plaque samples from 259 adult patients with severe periodontitis were analyzed. P. gingivalis was detected in 111 (43%) of the 259 subgingival plaque samples by culture and in 138 (53%) samples by PCR. The sensitivity, specificity, and positive and negative predictive values of the real-time PCR were 100, 94, 94, and 100%, respectively. We conclude that real-time PCR confirms the results of quantitative culture of P. gingivalis and offers significant advantages with respect to the rapidity and sensitivity of detection of P. gingivalis in subgingival plaque samples.

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle.

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.