Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of IktA–specific sequences

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligo-nucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubac-terial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans , yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans , failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.     

Detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction

Background: In recent years, increasing evidence regarding the potential association between periodontal diseases and cardiovascular diseases has been identified. The available evidence underlines the importance of detecting periodontal pathogens on atheromatous plaque as the first step in demonstrating the causal relationship between the two conditions. The main aim of this investigation is to detect periodontitis‐associated bacteria from carotid artery atheromatous plaque from patients who received an endarterectomy using strict sample procurement and laboratory procedures. Methods: Atheromatous plaque from endarterectomies from carotid arteries were scraped and homogenized, and bacterial DNA was extracted. To obtain a representative concentration of amplicons, two amplifications of the bacterial 16S ribosomal‐RNA gene were carried out for each sample with universal eubacteria primers by a polymerase chain reaction (PCR). A nested PCR with specific primers for the target bacteria was performed next. Statistical tests included the χ² test. Results: Forty‐two atheromatous plaque were analyzed. All of them were positive for ≥1 target bacterial species. The bacterial species most commonly found was Porphyromonas gingivalis (78.57%; 33 of 42), followed by Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (66.67%; 28 of 42), Tannerella forsythia (previously T. forsythensis) (61.90%; 26 of 42), Eikenella corrodens (54.76%; 23 of 42), Fusobacterium nucleatum (50.00%; 21 of 42), and Campylobacter rectus (9.52%; four of 42). The simultaneous presence of various bacterial species within the same specimen was a common observation. Conclusion: Within the limitations of this study, the presence of DNA from periodontitis‐associated bacteria in carotid artery atheromatous plaque retrieved by endarterectomy is confirmed.     

Rapid detection of Actinobacillus actinomycetemcomitans using a loop‐mediated isothermal amplification method

Introduction: Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we applied a novel nucleic acid amplification method, called loop‐mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, allowing the rapid detection of A. actinomycetemcomitans. Methods: We designed the primers for detecting A. actinomycetemcomitans and evaluated the specificity and sensitivity of the assay. Results: The LAMP primers used in this study successfully amplified serotypes a–e of A. actinomycetemcomitans, while other oral bacteria were not amplified. By measuring the precipitation of magnesium pyrophosphate, we could quantify the chromosomal DNA of A. actinomycetemcomitans. The detection limits using the real‐time turbidimetry analysis were 5.8 × 10²–5.8 × 10⁷ copies of A. actinomycetemcomitans template DNA per reaction tube. In addition, the LAMP assay was used for the rapid detection of A. actinomycetemcomitans in clinical specimens from eight individuals. The results with the LAMP method were similar to those using conventional polymerase chain reaction. Conclusion: Our results suggest that the LAMP‐based assay is very useful for the rapid detection of A. actinomycetemcomitans.     

Quantification of Periodontal Pathogens in Vascular,Blood, and Subgingival Samples From Patients With Peripheral Arterial Disease or Abdominal Aortic Aneurysms

Background: The aim of this investigation is to quantify periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, and Tannerella forsythia) in vascular, blood, and subgingival samples. As a secondary objective, two molecular bacterial identification methods (nested polymerase chain reaction [PCR] and quantitative PCR [qPCR]) are compared. Methods: Seventy consecutive patients provided a vascular lesion, a blood sample, and 36 subgingival samples. Bacterial DNA was extracted, and qPCR was used to determine the prevalence and amounts of the target pathogens in each sample. Nested PCR was performed only in the samples from vascular lesions. Periodontal examination was performed in 42 patients. Mann‐Whitney U or χ² tests were used to compare microbiologic results according to periodontal diagnosis. Results: All targeted periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, T. forsythia, or C. rectus) were detected in subgingival samples, with a prevalence rate of 72.2%, 47.2%, 74.3%, and 82.9%, respectively. In 7.1% and 11.4% of vascular and blood samples, bacterial DNA was detected. One patient was positive for A. actinomycetemcomitans in the three types of samples. No differences were found in the levels of targeted bacteria when comparing patients with and without periodontitis. Prevalence rates obtained with nested PCR were significantly higher than those obtained with qPCR. Conclusions: The presence of A. actinomycetemcomitans was demonstrated in vascular, blood, and subgingival samples in one of 36 patients. These results, although with a very low frequency, may support the hypothesis of a translocation of periodontal pathogens from subgingival microbiota to the bloodstream and then to atheromatous plaques in carotid or other peripheral arteries. Nested PCR is not an adequate method for identifying DNA of periodontal pathogens in low quantities because of the high number of false‐negative results.     

Detection of Eikenella corrodens and Actinobacillus actinomycetemcomitans by use of the polymerase chain reaction (PCR) in vitro and in subgingival plaque

Abstract The purpose of the present investigation was to identify 2 putative penodontal pathogens: Eikenella corrodens and Actinobacillus actinoinycetemcoiniiuns by polymerase chain reaction (PCR) in vilro and in subgingival plaque. On the basis of published sequences coding for 16S rRNA two primer pairs were designed which amplify a 410 bp sequence from E. corrodens DNA and a 547 bp fragment from A. actinomycetemcomitans DNA. respectively. As few as 50 cells could be detected from pure bacterial cultures. Each of the two primer pairs was found to be specific in that it did not give any amplification product neither with cell lysates from the respective alternative bacterium nor with lysates obtained from other putative periodontal pathogens and other bacteria. The PCR method developed turned out to be a simple, rapid and reliable diagnostic tool for the detection of the target microorganisms in clinical samples.     

Discrepancy between culture and DNA probe analysis for the detection of periodontal bacteria

Abstract The purpose of this study was to compare a commercially available DNA probe technique with conventional cultural techniques for the detection of Actinobacillus actinomvcelemconntans. Porplivrotnonas gingivalis and Prevotella intermedia in subgingival plaque samples. Samples from 20 patients with moderate to severe periodontitis were evaluated at baseline and during a 15 months period of periodonlal treatment. Paperpoints from 4 periodontal pockets per patient were forwarded to Omnigene for DNA probe analysis, and simultaneously inserted paperpoints from the same pockets were analyzed by standard culture techniques. In addition, mixed bacterial samples were constructed harbouring known proportions of 25 strains of A. actinomycetemcomitans, P. gingivalis and P. intermedia each. A relatively low concordance was found between both methods. At baseline a higher detection frequency was found for A. actinomycetemcomitans and P. gingivalis for the DNA probe technique; for P. intermedia the detection frequency by culture was higher. For A. actinomycetemcomitans, 21% of the culture positive samples was positive with the DNA probe. Testing the constructed bacterial samples with the DNA probe method resulted in about 16% false positive results for the 3 species tested. Furthermore. 40% of P. gingi-valis strains were not detected by the DNA probe. The present data suggest that at least part of the discrepancies found between the DNA probe technique used and cultural methods are caused by false positive and false negative DNA probe results. Therefore, the value of this DNA probe method for the detection of periodontal pathogens is questionable.     

Specific primer for AP-PCR identification of Actinobacillus actinomycetemcomitans

We used arbitrarily-primed polymerase chain reaction (AP-PCR) to design and construct a specific primer pair for the identification of Actinobacillus actinomycetemcomitans. We analyzed 25 DNA samples of A. actinomycetemcomitans isolated from patients with localized-juvenile periodontitis. From 90 AP-PCR primers screened, one amplification product was selected, cloned in pCR II vector, and sequenced. The sequence was used to design a single pair of specific primers. The sequence was compared with GenBank entries using BLAST and showed no significant matches. PCR amplification using the new primer pair AA1416 produced a characteristic 3.5-Kb band in all A. actinomycetemcomitans DNAs tested. Primer pair AA16S produced no or different amplicon profiles using DNA samples from bacterial species other than A. actinomycetemcomitans. Our results show that this single primer pair AA1416 can be used in PCR to identify A. actinomycetemcomitans isolates and differentiate them from other periodontal bacteria. These approaches appear promising in facilitating laboratory identification and taxonomy of putative periodontopathogens.     

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans

Ganeshnarayan K, Velliyagounder K, Furgang D, Fine DH. Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans. J Periodont Res 2012; 47: 661–673. © 2012 John Wiley & Sons A/S Background and Objective: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. Material and Methods: Saliva from 10 healthy volunteers was tested individually for its anti‐A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti‐A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI‐TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans–BEC binding assay. Results: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. Conclusion: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.     

Suppression of the periodontopathic microflora in localized juvenile periodontitis by systemic tetracycline

Abstract. Since recent studies have implicated Actinobacillus actinomycetemcomitans in the etiology of localized juvenile periodontitis, this investigation determined the effectiveness of subgingival debridement, topical Betadine Solution®, and systemic tetiacycline in suppressing subgingival A. actinomycetemcomitans and other microorganisms. A total of 20 deep periodontal pockets and 10 normal periodontal sites of 6 localized juvenile periodontitis patients was included in the study. Each patient was treated in 3 stages over a period of 22 weeks, and the result of treatment was monitored for an additional 38 weeks. The first stage of treatment included plaque control, as well as thorough scaling and root planing, composed of at least 6 h of debridement. No concomitant periodontal surgery was performed. In the second stage, Betadine saturated cotton gauze was inserted into the periodontal pockets for 10 min. Stage 3 involved systemic tetracycline therapy (1 g/day) for J4 days. The subgingival microflora was determined at frequent intervals by selective culturing of A. actinomycetemcomitans and Capnocytophaga and by direct microscopic examination. The clinical effect was assessed by measuring changes in probing periodontal attachment level, probing periodontal pocket depth, radiographic alveolar bone mass, and other relevant clinical parameters. Scaling and root planing reduced the total subgingival bacterial counts and the proportions of certain Gram-negative bacteria, but no periodontal pocket became free of A actinomycetemcomitans. Betadine application had little or no effect on the subgingival microflora. In contrast, tetracycline administered via the systemic route suppressed. A actinomycetemcomitans, Capnocytophaga, and spirochetes to low or undetectable levels in all test periodontal pockets. A, actinomycetemcomitans reappeared in 9 of the deep periodontal pockets after the administration of tetracycline. Most of these 9 pockets became free of detectable A. actinomycetemcomitans during the second week of tetracycline administration, whereas pockets which yielded no A. actinomycetemcomitans after tetracycline therapy became free of the organisms during the first week of tetracycline treatment. This data suggests that systemic tetracycline therapy of localized juvenile periodontitis should, as a practical rate, be continued for 3 weeks. Periodontal destruction continued in 4 deep pockets which all showed high posttetracycline A, actinomycetemcomitans counts. All 6 pockets which demonstrated a marked gain in periodontal attachment yielded no cultivable A. actinomycetemcomitans. No association was found between periodontal disease status and subgingival Capnocytophaga, spirochetes or motile rods. The present study indicates that A. actinomycetemcomitans is an important etiologic agent in localized juvenile periodontitis. Also, this study demonstrates that the effectiveness of therapy can be monitored by subgingival A. actinomycetemcomitans counts, and that periodontal A, actinomycetemcomitans infections cannot be resolved by root surface debridement alone but can be cured by systemic tetracycline therapy.     

Molecular survey of atheromatous plaques for the presence of DNA from periodontal bacterial pathogens, archaea and fungi

Aquino ARL, Lima KC, Paiva MS, Rôças IN, Siqueira JF Jr. Molecular survey of atheromatous plaques for the presence of DNA from periodontal bacterial pathogens, archaea and fungi. J Periodont Res 2011; 46: 303–309. © 2011 John Wiley & Sons A/S Background and Objective: Chronic infections, such as periodontitis, have been associated with the development and progression of atherosclerosis. The mechanisms through which this occurs have yet to be elucidated. This study was carried out to detect periodontopathic bacteria as well as archaea and fungi in atheromatous plaques and search for factors associated with their occurrence in atheromas. Material and Methods: A cross‐sectional study was carried out including 30 patients diagnosed with atherosclerosis in the carotid, coronary or femoral arteries. Plaques were collected during surgery and analysed using PCR to detect Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and members of the Synergistetes group. Samples were also surveyed with universal primers for bacterial, archaeal and fungal DNA. Patients responded to a questionnaire to determine factors associated with PCR results. Results: All dentate individuals (66.7%) had periodontal disease, 95% of which was severe and 65% extensive. None of the targeted periodontopathic bacteria was found in the atheromas. No sample yielded positive results for fungal and archaeal DNA. Four samples (13%) were positive for the presence of bacterial DNA. Of these, three participants were dentate (two with severely chronic generalized periodontitis and one with severely chronic localized periodontitis). Conclusion: This study did not confirm previous findings of periodontal pathogens in atheromas, making it impossible to establish factors associated with their presence in plaques. Presence of bacterial DNA in some samples indicates that periodontal or nonoral bacterial species other than the ones targeted in this study may be involved with some cases of atherosclerosis.     

Occurrence and serotype distribution of Aggregatibacter actinomycetemcomitans in subjects without periodontitis in Turkey

ObjectiveTo determine the occurrence and serotype distribution of Aggregatibacter actinomycetemcomitans in subjects without periodontitis.DesignSystemically healthy dental students without periodontitis (n = 94), who had not used antibiotics within the last 3 months or received any form of periodontal therapy within the last 6 months, were included in the study. Pooled subgingival microbiological samples were collected from 4 first molars and 4 central incisors in each subject using sterile paper points. All samples were tested for the presence and the serotype of A. actinomycetemcomitans through PCR analysis of the 16S rRNA genes and the serotype-specific gene clusters in the DNA extracted from the samples.ResultsOf the 94 samples that were tested, 43 (46%) were positive for A. actinomycetemcomitans. No statistically significant differences in clinical parameters were found between subgingival sites with or without detectable A. actinomycetemcomitans (t-test, P > 0.01). Among the 43 A. actinomycetemcomitans-positive samples, the serotype was identified in 21 samples. Fifteen were positive for A. actinomycetemcomitans serotype a, 1 for serotype b, 1 for serotype c, and 4 for serotype f, while serotypes d and e were not detected.ConclusionA. actinomycetemcomitans serotype a is the most commonly found serotype among Turkish dental students without periodontitis.     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3′ ends with respect to the corresponding regions of the 16S rRNA gene of P. nigrescens, its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease Rsal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque

OBJECTIVES: Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. MATERIALS AND METHODS: The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. RESULTS: Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. CONCLUSION: Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.     

PCR primers for the amplification of the 16S rRNA gene of oral bacteria and for the specific identification of Actinobacillus actinomycetemcomitans

The aim of this study was to develop a PCR reaction specific to Actinobacillus actinomycetemcomitans , which targets a widely conserved gene of this bacterium. Two sets of primers were designed based on published sequences of the 16S rRNA of several microorganisms. The first set amplifies a major part of the 16S small subunit rRNA gene of several strains of bacteria commonly found in the periodontal pocket. This reaction produced a 1306 bp-long product and served as a positive control. The second set was specific to A. actinomycetemcomitans and produced a 449 bp-long product. H. tiphniphilus and E. coli yielded positive results with the control primers and negative results with the A. actinomycetemcomitans -specific primers. DNA-DNA hybridization was used to validate the identity of the amplified sequences. B. cereus. which is a common contaminator in the laboratory, and human DNA did not generate PCR products in either reaction. The developed primers seem useful for the identification of A. actinomycetemcomitans strains.     

Quantitative aspects of the subgingival distribution of Actinobacillus actinomycetemcomitans in a patient with localized juvenile periodontitis

Abstract The subgingival microflora in a patient with localized juvenile periodontitis was studied. Of the 97 sites investigated, 28 (29%) showed attachment loss. A correlation was found between the number of Actinobacillus actinomycetemcomitans cells and the clinical attachment level and probing pocket depth. Of the 97 test sites, 70 (73%) were positive for A. actinomycetemcomitans. Of the total number of A. actinomycetemcomitans cells isolated from this patient, more than 99% were found at sites with attachment loss, <1 % being present at sites without attachment loss. The mean percentage of A. actinomycetemcomitans was 21.2% at sites with attachment loss and 0.45% at sites without attachment loss. The distribution of Porphyromonas gingilis showed a symmetrical pattern, being present at the 1st molar and 2nd premolar sites in all quadrants and at the lower incisor sites. This species was absent at multiple sites showing overt attachment loss.     

The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans in localized and generalized forms of aggressive periodontitis

Abstract

Objective: To investigate the presence of A. actinomycetemcomitans, including the highly virulent JP2 clone, in young adult patients with aggressive periodontitis, and associate the findings with the two forms of the disease. Materials and methods: Seventy Moroccan subjects with aggressive periodontitis, aged less than 35 years, were recruited. Among these, 41 had LAgP and 29 had GAgP. Plaque samples were collected from periodontal pockets and examined using a PCR that detects the presence of A. actinomycetemcomitans and which differentiates between JP2 and non-JP2 genotypes of the bacterium. Results : total of 58 (83%) from the 70 AgP patients were positive for A. actinomycetemcomitans, among whom 77% were positives for the JP2 clone. The JP2 clone was detected in 34 (83%) of the LAgP patients compared to 20 (69%) of the GAgP patients (p = 0.17). Fourteen (20%) of the patients harbored non-JP2 genotypes of A. actinomycetemcomitans, although most of these patients (10/14) also harbored the JP2 clone. Conclusions: The presence of the JP2 clone of A. actinomycetemcomitans is strongly associated with both LAgP and GAgP in young adults in Morocco. This implies that treatment of AgP in this population should include microbiological screening and aim at eradication of the bacterium when present.     

Host–microbial co-evolution in periodontitis associated with Aggregatibacter actinomycetemcomitans infection

Periodontitis is caused by bacterial biofilms composed of more than 800 bacterial species. Not all biofilm bacteria are equally pathogenic; Aggregatibacter actinomycetemcomitans is recognized as one of the most virulent periodontopathic bacteria. A. actinomycetemcomitans is also associated with systemic diseases, including cardiovascular disease and brain abscesses. Molecular mimicry between A. actinomycetemcomitans and human β2-glycoprotein I may be associated with systemic diseases such as threatened preterm labor (TPL), preterm birth (PB), and Buerger's disease. Patients with certain genetic neutrophil dysfunction disorders (e.g., Kostmann and Papillon-Lefevre syndromes) also suffer from severe periodontitis. These patients are frequently infected with A. actinomycetemcomitans, suggesting that an impaired immune system might be relevant in the pathogenesis of A. actinomycetemcomitans-associated periodontitis. Virulence of A. actinomycetemcomitans differs amongst clones. JP2 is a highly pathogenic A. actinomycetemcomitans clone that has a deletion in the leukotoxin promoter, resulting in the production of large amounts of leukotoxin. Dissemination of the JP2 clone is skewed to certain regions and ethnic groups. JP2 has not been reported in East Asia, e.g., Japan, Korea, and China. However, an A. actinomycetemcomitans clone with an insertion sequence in the leukotoxin promoter was reported as a highly pathogenic clone in Japanese periodontitis patients. This evidence suggests that host–microbial co-evolution might exist in A. actinomycetemcomitans-associated periodontitis, and elucidation of the co-evolution of A. actinomycetemcomitans and its host might enable molecular diagnosis and treatment of A. actinomycetemcomitans-associated periodontitis.     

Prevalence of Aggregatibacter actinomycetemcomitans in Sudanese patients with aggressive periodontitis: a case-control study

Elamin A, Albandar JM, Poulsen K, Ali RW, Bakken V. Prevalence ofAggregatibacter actinomycetemcomitansin Sudanese patients with aggressive periodontitis: a case–control study. J Periodont Res 2011; 46: 285–291.©2011 John Wiley & Sons A/S Background and Objective: Aggregatibacter actinomycetemcomitans is considered a possible etiological agent for aggressive periodontitis. The aim of this study was to determine the prevalence of the JP2 clone and non‐JP2 genotypes of A. actinomycetemcomitans in the subgingival plaque of patients with aggressive periodontitis and controls among Sudanese high‐school students. Material and Methods: In a previous study we examined a large representative sample of students attending high schools in Khartoum, Sudan. In this population, 17 patients with aggressive periodontitis and 17 controls (14–19 years of age) consented to participate in the present study. The subjects underwent a clinical periodontal examination, and subgingival dental plaque samples were collected using paper points. The presence of the A. actinomycetemcomitans JP2 clone and non‐JP2 genotypes were assessed using loop‐mediated isothermal amplification (LAMP) and the PCR. Results: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of either the cases or the controls. Non‐JP2 types of A. actinomycetemcomitans were detected in the subgingival plaque of 12 (70.6%) patients with aggressive periodontitis and from only one (5.9%) control subject, showing a significantly higher frequency of detection in cases than in controls (p = 0.0001). The odds ratio for the detection of A. actinomycetemcomitans in the subgingival plaque of the patients with aggressive periodontitis was 38.4 (95% confidence interval: 4.0–373.0; p = 0.002). The PCR and LAMP methods showed identical results pertaining to the identification of non‐JP2 types of A. actinomycetemcomitans. Conclusions: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of high school subjects in Sudan. The detection of non‐JP2 types of A. actinomycetemcomitans may be a useful marker of increased risk for development of aggressive periodontitis in young subjects.     

Detection of a single bacterial cell using a 16S ribosomal RNA‐specific oligonucleotide probe designed to investigate periodontal pathogens

Introduction: The current detection methods for periodontopathogens mainly use polymerase chain reactions. However, there are few methods available for visualizing the bacteria that impact on patients with periodontal disease for use in health education. The purpose of this study was to develop a specific detection method to visualize periodontopathogenic bacteria. Methods: Fluorescently‐labeled oligonucleotide probes directed to specific 16S ribosomal RNA (rRNA) sequences of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were synthesized. Cultured individual bacterial species were fixed with 4% paraformaldehyde and smeared on glass slides. Fluorescein isothiocyanate‐labeled oligonucleotide probes were hybridized under stringent conditions with smeared whole cells, and then probe specificity was investigated by epifluorescence microscopy. Results: Comparatively long (50‐mer) oligonucleotide probes for P. gingivalis and A. actinomycetemcomitans were designed. These probes clearly hybridized with 16S rRNA of the target species in situ and single bacterial cells were detectable visually. The probes exhibited no cross‐hybridization against the additional organisms that were closely related to the target species. Conclusions: The fluorescence in situ hybridization technique is a specific and reliable method by which to visually identify the target organisms. The oligonucleotide probes designed in this study will be useful for detecting P. gingivalis and A. actinomycetemcomitans populations.     

Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in juvenile periodontitis

Abstract The occurrence of Actinobacillus actinomycetemcomitans, Porphyromanas gingivalis and Prevotella intermedia in subgingival plaque in 24 juvenile periodontitis patients was determined using DNA probe. 36 samples of subgingival plaque from 36 pockets having ≥6 mm depth, ≥3 mm of loss of attachment, and Weeding on probing anchor suppuration were taken from 18 patients with localized juvenile periodontitis (LJP, age range 12-24 years); and 12 samples from-6 patients with generalized juvenile periodontitis (GJP, age range 23–26 years). As control, an equal numbers of samples from health sites in the same patients were studied. P. gingivalis was found in 17 of 18 LJP patients, and in 31 of 36 diseased sites in those patients. P. intermedia was found in 15 out of the 18 LJP patients and in 28 of the 36 diseased sites. A, actinomycetemcomitans was present in 7 of the 18 LJP patients, and in 9 of the 36 diseased sites, and was not found in any GJP patients. All GJP patients had P. gingivalis 1 out of 12 diseased sites) and P. intermedia (all of the diseased sites). None of the three bacterial species was detected in healthy sites of GJP patients, and were found in healthy sites in only 2 of 18 LJP patients. The high prevalence and high levels of P. gingivalis and P. intermedia found in the LJP and GJP patients studied, suggest that there are populations affected by juvenile periodontitis in which this type of periodontitis is more associated with these species than with A. actinomycetemcomitans.