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The Gram-positive organism Filifactor alocis is implicated in multiple oral diseases including periodontitis, and approximately 50% of known strains encode and produce a recently identified repeat-in-toxin (RTX) protein, FtxA, partly homologous to the Aggregatibacter actinomycetemcomitans leukotoxin. By assessing a longitudinal Ghanaian study population of adolescents, we recently identified a possible correlation between F. alocis levels, ftxA gene carriage, and progression of clinical attachment loss (CAL). To extend knowledge on the possible significance of F. alocis and its FtxA in periodontal disease, we have in the present work analyzed saliva samples in an independent cohort of periodontitis (n=156), collected at two private periodontal specialist practices in Perth, Western Australia. The present results corroborate that high loads of F. alocis and the presence of its ftxA gene together are associated with parameters of periodontal tissue destruction and severity. Moreover, among the individuals carrying A. actinomycetemcomitans, a majority also exhibited an ftxA-positive F. alocis, supporting the notion of the synergistic behavior of these two species. This emphasizes that F. alocis and its ftxA are involved in the pathogenesis of periodontitis and may have ecological roles, with diagnostic and prognostic implications for the disease.

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.

Microbial ecosystems experience spatial and nutrient restrictions, leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here we demonstrate first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring newer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing presence of an active T6SS in the oral cavity.

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

This paper aims to investigate the presence of Aggregatibacter actinomycetemcomitans and to assess potential indicators of the risk of severe form(s) of periodontitis. A descriptive cross-sectional study of 156 consecutive patients with periodontitis was conducted. Subgingival plaque samples were collected from the participants. The identification of A. actinomycetemcomitans was performed using quantitative polymerase chain reaction. A descriptive analysis, a chi-square test, and a binary logistic regression statistical evaluation were performed. The prevalence of A. actinomycetemcomitans in this population of 156 participants was 17.30% (27 patients). The prevalence of stage-III periodontitis was 75.6% and greater in older men, while the prevalence of stage-IV periodontitis was 22.4% and greater in younger women. We observed a significant relation between the risk of severe periodontitis (stage-IV) and poor oral hygiene (p = 0.006), attendance at dental appointments (p ≤ 0.001), and familial history of periodontitis (p = 0.032). In conclusion, twenty-seven individuals were positive for A. actinomycetemcomitans. Poor oral hygiene, family history of periodontitis, and irregular attendance at dental appointments were identified as potential risk factors for severe periodontitis in this cohort.

Here, we present a protocol for evaluating type VI secretion system (T6SS)dependent fitness of the oral symbiont A. aphrophilus using biofilm competition assays and metaproteomics. We describe steps for designing T6SS-specific mutants. We then detail procedures for using them in competition assays with the pathobiont A.actinomycetemcomitans and in biofilm models, analyzing metaproteomes to assess the impact of the T6SS on multiple pathobionts. The biofilm modelis designed to mimic the oral plaque ecosystem and includes seven species. For complete details on the use and execution of this protocol, please refer to Oscarsson et al.

The objective was to evaluate using unstimulated saliva in detecting Aggregatibacter actinomycetemcomitans and to compare the saliva and subgingival and mucosa membrane occurrence of this periodontal pathogen in patients diagnosed with advanced periodontitis. Patients with advanced forms of periodontitis (n = 220; mean age: 54.03 ± 03 years) at stage III/IV were sampled. Unstimulated saliva, buccal cheek mucosa, and pooled subgingival plaque samples were collected. The identification of A. actinomycetemcomitans was performed using qPCR. A descriptive analysis and Wilcoxon test and analysis of variance were performed. A. actinomycetemcomitans was isolated from 28.18% of the subjects. A total of 660 samples were obtained, 220 from unstimulated saliva, 220 from buccal cheek mucosa surfaces, and 220 from pooled subgingival plaque samples. A. actinomycetemcomitans was isolated from 21.80% of unstimulated saliva, 19.50% of buccal cheek swabs, and17.70% of subgingival samples. There was a statistically significant difference between the presenceof A. actinomycetemcomitans in the unstimulated saliva samples and in the buccal cheek mucosa swab samples and pooled subgingival plaque samples (p < 0.001). These results suggest that in advanced periodontitis, unstimulated saliva is representative of pooled subgingival plaque/buccal cheek mucosa samples and its use is adequate in the oral detection of A. actinomycetemcomitans in a cohort of patients with stage III and IV periodontitis.

Oral bacteria naturally secrete extracellular vesicles (EVs), which have attracted attention for their promising biomedical applications including cancer therapeutics. However, our understanding of EV impact on tumor progression is hampered by limited in vivo models. In this study, we propose a facile in vivo platform for assessing the effect of EVs isolated from different bacterial strains on oral cancer growth and dissemination using the larval zebrafish model. EVs were isolated from: wild-type Aggregatibacter actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; and wild-type Porphyromonas gingivalis. Cancer cells pretreated with EVs were xenotransplanted into zebrafish larvae, wherein tumor growth and metastasis were screened. We further assessed the preferential sites for the metastatic foci development. Interestingly, EVs from the CDT-lacking A. actinomycetemcomitans resulted in an increased tumor growth, whereas EVs lacking the lipopolysaccharide O-antigen reduced the metastasis rate. P. gingivalis-derived EVs showed no significant effects. Cancer cells pretreated with EVs from the mutant A. actinomycetemcomitans strains tended to metastasize less often to the head and tail compared to the controls. In sum, the proposed approach provided cost- and labor-effective yet efficient model for studying bacterial EVs in oral carcinogenesis, which can be easily extended for other cancer types. Furthermore, our results support the notion that these nanosized particles may represent promising targets in cancer therapeutics.

Background: Periodontitis in adolescents has historically been rare in the Nordic countries but could be expected to increase due to changes in demographics. The primary aim was to cross-sectionally examine the presence of radiographic bone loss in adolescents in Västerbotten County, Sweden. The secondary aim was to compare periodontal and microbial parameters, as well as demographic patterns, in controls without bone loss with cases with bone loss.

Methods: Adolescents born 2001 and who had a dental examination 2016 (n = 1656) were screened for proximal bone loss using bitewing radiographs taken during dental examinations (2014-2016). Individuals that exhibited proximal bone loss (> 2 mm) were invited to participate in a complete periodontal examination. Subgingival plaque and saliva were also sampled. For each adolescent with bone loss, two healthy individuals as controls were examined. Selected bacterial species in saliva and subgingival plaque were quantitatively examined by quantitative PCR. The subgingival plaque samples were also analysed with cultivation technique.

Results: Proximal bone loss was identified in 24 individuals (1.45%) based on all the radiographs. Thirteen of these cases were periodontally examined and matched with 26 controls. Most cases were diagnosed with periodontitis (12/13 [92%]), whereas none of the controls had periodontitis. Higher concentrations and higher prevalence of the bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Filifactor alocis were generally found in the cases.

Conclusion:The results suggest that periodontitis among adolescents, because of demographic differences (an increasingly heterogenous population), in Sweden is increasing and emphasizes the importance of radiographs for early detection of this disease.

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.