Association of Filifactor alocis and its RTX toxin gene ftxA with periodontal attachment loss, and in synergy with Aggregatibacter actinomycetemcomitans.

The Gram-positive bacterium, Filifactor alocis is an oral pathogen, and approximately 50% of known strains encode a recently identified repeat-in-toxin (RTX) protein, FtxA. By assessing a longitudinal Ghanaian study population of adolescents (10-19 years of age; mean age 13.2 years), we recently discovered a possible correlation between deep periodontal pockets measured at the two-year follow-up, presence of the ftxA gene, and a high quantity of F. alocis. To further understand the contribution of F. alocis and FtxA in periodontal disease, we used qPCR in the present study to assess the carriage loads of F. alocis and the prevalence of its ftxA gene in subgingival plaque specimens, sampled at baseline from the Ghanaian cohort (n=500). Comparing these results with the recorded clinical attachment loss (CAL) longitudinal progression data from the two-year follow up, we concluded that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and concomitant presence of the ftxA gene were two factors that were both associated with an enhanced prevalence of CAL progression. Interestingly, CAL progression appeared to be further promoted upon the simultaneous presence of F. alocis and the non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promotes CAL during periodontal disease.

Aggregatibacter actinomycetemcomitans and Filifactor alocis as Associated with Periodontal Attachment Loss in a Cohort of Ghanaian Adolescents.

The aims of the present study were to document the presence of Aggregatibacter actinomyctemcomitans and the emerging oral pathogen Filifactor alocis, as well as to identify genotypes of these bacterial species with enhanced virulence. In addition, these data were analyzed in relation to periodontal pocket depth (PPD) and the progression of PPD from the sampled periodontal sites during a two-year period. Subgingival plaque samples were collected from 172 periodontal pockets of 68 Ghanaian adolescents. PPD at sampling varied from 3-14 mm and the progression from baseline, i.e., two years earlier up to 8 mm. The levels of A. actinomycetemcomitans and F. alocis were determined with quantitative PCR. The highly leukotoxic JP2-genotype of A. actinomycetemcomitans and the ftxA a gene of F. alocis, encoding a putative Repeats-in-Toxin (RTX) protein, were detected with conventional PCR. The prevalence of A. actinomycetemcomitans was 57%, and 14% of the samples contained the JP2 genotype. F. alocis was detected in 92% of the samples and the ftxA gene in 52%. The levels of these bacterial species were significantly associated with enhanced PPD and progression, with a more pronounced impact in sites positive for the JP2 genotype or the ftxA gene. Taken together, the results indicate that the presence of both A. actinomycetemcomitans and F. alocis with their RTX proteins are linked to increased PPD and progression of disease.

Multilocus Sequence Typing of Non-JP2 Serotype b Aggregatibacter actinomycetemcomitans Strains of Ghanaian and Swedish Origin.

Objective and Methods: The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans is associated with periodontitis affecting young individuals. The geographic dissemination of the highly leukotoxic JP2 genotype of serotype b of this species was previously studied by multilocus sequence typing (MLST). Here, we have used MLST to genetically characterize non-JP2 genotype strains of serotype b, isolated from individuals living in Ghana (n=41), and in Sweden (n=13), respectively. Results: The MLST analysis revealed a total of nine sequence types (ST). Both Ghanaian and Swedish isolates were distributed in ST 1-3. ST 5 and 6 were only identified among the Ghanaian strains, whereas ST 4, 7, 8 and 9 were uniquely represented among the Swedish strains. Previously, we characterized these non-JP2 genotype strains of A. actinomycetemcomitans serotype b by arbitrarily-primed (AP)-PCR, which distributed them into three groups, AP-PCR type 1, 2, and 3, respectively. AP-PCR type 1 strains are generally highly leukotoxic, and are associated with progression of periodontal attachment loss. As AP-PCR type 1 includes both JP2 genotype strains and a proportion of non-JP2 genotype strains of serotype b, a straightforward diagnostic procedure has been sought. This has revealed a gene, cagE, which appears to be conserved only in this AP-PCR type. According to our results, MLST was not a highly discriminatory method to identify AP-PCR type 1, as strains of this AP-PCR type could be found within three different ST: ST 2, ST 3 and ST 8. Conclusion: According to MLST, a geographic dissemination of non-JP2 genotype A. actinomycetemcomitans serotype b appears to exist. However, aiming to identify carriers of AP-PCR type 1, non-JP2 genotype serotype b, PCR with cagE-specific primers is likely the most efficient diagnostic procedure known today.

Characterization of Aggregatibacter actinomycetemcomitans Serotype b Strains with Five Different, Including Two Novel, Leukotoxin Promoter Structures.

The JP2 genotype of A. actinomycetemcomitans, serotype b has attracted much interest during the past three decades due to its close association with periodontitis in young individuals and the enhanced expression of a leukotoxin (LtxA). A typical feature of this genotype is a 530-base pair (bp) deletion in the ltxCABD promoter region controlling leukotoxin expression. In the present work, we have characterized serotype b strains with four additional promoter types. Two novel types have been recognized, that is, one with a 230-bp deletion and one with a 172-bp duplication. Moreover, a strain with a 640-bp deletion and three strains with a full-length promoter, including the type strain Y4, were included in the present study. The seven strains were characterized by multi locus sequence typing (MLST) and arbitrarily primed polymerase chain reaction (PCR) and assessed for LtxA production. MLST showed that the strains with the non-JP2-like deletions represented distinct monophyletic groups, whereas the JP2 strain, HK1651, represented a separate branch. LtxA production was high in all three strains with a promoter deletion, whereas the other four strains showed significantly lower levels. It can be concluded that the genetic characterization and determination of LtxA production of A. actinomycetemcomitans isolates from individuals with periodontitis can contribute to the identification of novel virulent genotypes of this bacterium.

Genetic Profiling of Aggregatibacter actinomycetemcomitans Serotype B Isolated from Periodontitis Patients Living in Sweden.

The bacterium Aggregatibacter actinomycetemcomitans is associated with aggressive forms of periodontitis and with systemic diseases, such as endocarditis. By assessing a Ghanaian longitudinal adolescent cohort, we earlier recognized the cagE gene as a possible diagnostic marker for a subgroup of JP2 and non-JP2 genotype serotype b A. actinomycetemcomitans strains, associated with high leukotoxicity as determined in a semi-quantitative cell assay. This group of A. actinomycetemcomitans is associated with the progression of attachment loss. In the present work, we used conventional polymerase chain reaction (PCR) and quantitative PCR to perform the cagE genotyping of our collection of 116 selected serotype b A. actinomycetemcomitans strains, collected over a period of 15 years from periodontitis patients living in Sweden. The A. actinomycetemcomitans strains carrying cagE (referred to as cagE+; n = 49) were compared to the cagE-negative strains (n = 67), present at larger proportions in the subgingival plaque samples, and were also much more prevalent in the young (≤35 years) compared to in the old (>35 years) group of patients. Our present results underline the potential use of cagE genotyping in the risk assessment of the development of periodontal attachment loss in Swedish adolescents.

Tools of Aggregatibacter actinomycetemcomitans to Evade the Host Response.

Periodontitis is an infection-induced inflammatory disease that affects the tooth supporting tissues, i.e., bone and connective tissues. The initiation and progression of this disease depend on dysbiotic ecological changes in the oral microbiome, thereby affecting the severity of disease through multiple immune-inflammatory responses. Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium associated with such cellular and molecular mechanisms associated with the pathogenesis of periodontitis. In the present review, we outline virulence mechanisms that help the bacterium to escape the host response. These properties include invasiveness, secretion of exotoxins, serum resistance, and release of outer membrane vesicles. Virulence properties of A. actinomycetemcomitans that can contribute to treatment resistance in the infected individuals and upon translocation to the circulation, also induce pathogenic mechanisms associated with several systemic diseases.

Age-related prevalence and characteristics of Aggregatibacter actinomycetemcomitans in periodontitis patients living in Sweden.

Background: The presence of Aggregatibacter actinomycetemcomitans in patients with periodontitis has been extensively studied for decades. Objective: To study the prevalence of A. actinomycetemcomitans in younger and older periodontitis patients and to genetically characterize isolates of this bacterium. Design: Data from microbiological analyses of 3459 subgingival plaque samples collected from 1445 patients, 337 'younger' patients (≤35 yrs) and 1108 'older' patients (>35 yrs) during 15 years (2000-2014), has been summerized. Isolates of A. actinomycetemcomitans were serotyped, leukotoxin promoter typed (JP2 and non JP2) and arbitrarily primed PCR (AP-PCR) genotyped. The origin of the JP2 genotype detected in the study population was determined. Results: The prevalence of A. actinomycetemcomitans was higher among younger than older patients and samples from the younger patients contained higher proportions of the bacterium. Serotype b was more prevalent among younger patients and the majorty of these isolates was from the same AP-PCR genotype. The JP2 genotype was detected in 1.2% of the patients, and the majority of these carriers were of non-African origin. Conslusions: For presence and charcteristics of A. actinomycetemcomitans in clinical samples the age of the carriers were a discriminating factor. Additional, apparently non-African carriers of the JP2 genotype of A. actinomycetemcomitans were identified.

Leukotoxic activity of Aggregatibacter actinomycetemcomitans and periodontal attachment loss.

Aggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. This leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a well-characterized specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been isolated. In particular, the presence of the JP2 genotype significantly increases the risk for the progression of periodontal attachment loss (AL). Based on these findings we hypothesized that variations in the leukotoxicity are linked to disease progression in infected individuals. In the present study, the leukotoxicity of 239 clinical isolates of A. actinomycetemcomitans was analysed with different bioassays, and the genetic peculiarities of the isolates were related to their leukotoxicity based on examination with molecular techniques. The periodontal status of the individuals sampled for the presence of A. actinomycetemcomitans was examined longitudinally, and the importance of the observed variations in leukotoxicity was evaluated in relation to disease progression. Our data show that high leukotoxicity correlates with an enhanced risk for the progression of AL. The JP2 genotype isolates were all highly leukotoxic, while the isolates with an intact leukotoxin promoter (non-JP2 genotypes) showed substantial variation in leukotoxicity. Genetic characterization of the non-JP2 genotype isolates indicated the presence of highly leukotoxic genotypes of serotype b with similarities to the JP2 genotype. Based on these results, we conclude that A. actinomycetemcomitans harbours other highly virulent genotypes besides the previously described JP2 genotype. In addition, the results from the present study further highlight the importance of the leukotoxin as a key virulence factor in aggressive forms of periodontitis.

Progression of attachment loss is strongly associated with presence of the JP2 genotype of Aggregatibacter actinomycetemcomitans: a prospective cohort study of a young adolescent population.

AIM: To assess the progression of attachment loss (AL) during a 2-year period according to the presence of JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans in a Ghanaian adolescent population. METHODS: A total of 500 adolescents (mean age 13.2 years, SD ± 1.5) were enrolled in the study. After 2 years, 397 (79.4%) returned for a periodontal re-examination, including the measurement of AL. The carrier status of the JP2 and non-JP2 genotypes of A. actinomycetemcomitans was evaluated in a baseline examination 2 years earlier. RESULTS: Individuals who carried the JP2 genotype of A. actinomycetemcomitans had a significantly increased risk [relative risk (RR) = 7.3] of developing AL ≥ 3 mm. The mean AL at the follow-up and the mean 2-year progression of AL were significantly higher in the JP2 genotype-positive group (n = 38) compared with the group positive for the non-JP2 genotypes of A. actinomycetemcomitans (n = 169), and the group of A. actinomycetemcomitans-negative individuals (n = 190). The JP2 genotype was strongly associated with the progression of AL ≥ 3 mm (OR = 14.3). The non-JP2 genotypes of A. actinomycetemcomitans were also, however, less pronounced, associated with the progression of AL ≥ 3 mm (OR = 3.4). CONCLUSION: The JP2 genotype of A. actinomycetemcomitans is strongly associated with the progression of AL.

Cytolethal distending toxin in isolates of Aggregatibacter actinomycetemcomitans from Ghanaian adolescents and association with serotype and disease progression.

BACKGROUND AND OBJECTIVES: The cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL). MATERIALS AND METHODS: A total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up. RESULTS: Presence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994-8.779], p<0.001). CONCLUSION: A. actinomycetemcomitans isolated from the Ghanaian adolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.

Detection of the highly leucotoxic JP2 clone of Aggregatibacter actinomycetemcomitans in members of a Caucasian family living in Sweden.

BACKGROUND: carriers of the JP2 clone of Aggregatibacter actinomycetemcomitans exhibit an enhanced risk for developing aggressive periodontitis compared with individuals carrying non-JP2 clones. While the JP2 clone is almost exclusively detected among adolescents of African descent, reports on Caucasians colonized with the JP2 clone are remarkably few. OBJECTIVE: the aim of this paper is to report on the history of periodontal disease and microbiological findings in a Caucasian family. MATERIAL AND METHODS: a. actinomycetemcomitans and other periodontitis-associated bacterial species in subgingival plaque samples were quantified by conventional culture technique. Leucotoxin promoter typing, serotyping and further characterizations of A. actinomycetemcomitans isolates were performed by PCR. DNA sequencing of the pseudogene, hbpA was performed to determine the origin of the detected JP2 clones. Further, genetically ancestry testing of family members was carried out. RESULTS: the JP2 clone was detected in samples from two of the family members, a 33-year-old daughter and her 62-year-old mother. Relationship of their JP2 clones with JP2 clone strains from the Mediterranean area of Africa was indicated. Genotyping confirmed the Caucasian origin of all family members. CONCLUSIONS: Caucasian JP2 carriers exist and older subjects can carry the JP2 clone of A. actinomycetemcomitans.