The emerging oral pathogen, Filifactor alocis, extends the functional lifespan of human neutrophils.

Periodontitis is a chronic inflammatory infectious disease that affects the integrity of tooth-supporting tissues and has adverse systemic consequences. Advances in sequencing technologies have uncovered organisms that are exclusively found in high numbers in periodontal lesions, such as the gram-positive anaerobic rod, Filifactor alocis. F. alocis can manipulate neutrophil effector functions, which allows the organism to survive within these granulocytes. Several neutrophil functions have been tested in the context of F. alocis challenge, but the effect of the organism on neutrophil apoptosis is still unknown. RNA sequencing of human neutrophils challenged with F. alocis showed that apoptosis pathways were differentially regulated. Compared to media-cultured controls, F. alocis-challenged neutrophils maintain their nuclear morphology, do not stain for Annexin V or 7-AAD, and have decreased DNA fragmentation. Inhibition of apoptosis by F. alocis involved reduced caspase-3, -8, and - 9 activation and upregulation of important anti-apoptotic proteins. Prolonged lifespan was dependent on contact through TLR2/6, and F. alocis-challenged neutrophils retained their functional capacity to induce inflammation for longer timepoints. This is the first in-depth characterization of neutrophil apoptotic programs in response to an oral pathogen and provides key information on how bacteria manipulate immune cell mechanisms to maintain a dysregulated inflammatory response.

Tobacco smoke exacerbates Filifactor alocis pathogenicity.

AIM: Filifactor alocis has recently emerged as a periodontal pathobiont that appears to thrive in the oral cavity of smokers. We hypothesized that identification of smoke-responsive F. alocis genes would provide insight into adaptive strategies and that cigarette smoke would enhance F. alocis pathogenesis in vivo. MATERIALS AND METHODS: F. alocis was grown in vitro and cigarette smoke extract-responsive genes determined by RNAseq. Mice were exposed, or not, to mainstream 1R6F research cigarette smoke and infected with F. alocis, or not, in an acute ligature model of periodontitis. Key clinical, infectious, and immune data were collected. RESULTS: In culture, F. alocis growth was unaffected by smoke conditioning and only a small number of genes were specifically regulated by smoke exposure. Reduced murine mass, differences in F. alocis-cognizant antibody production, and altered immune profiles as well as altered alveolar bone loss were all attributable to smoke exposure and/or F. alocis infection in vivo. CONCLUSIONS: F. alocis is well-adapted to tobacco-rich conditions and its pathogenesis is enhanced by tobacco smoke exposure. A smoke-exposed ligature model of periodontitis shows promise as a tool with which to further unravel mechanisms underlying tobacco-enhanced, bacteria-induced disease.

The first case of deep neck abscess due to Filifactor alocis co-infected with Eggerthia catenaformis, Parvimonas micra, and Streptococcusconstellatus.

Filifactor alocis, an anaerobic Gram-positive rod, has garnered interest from its association with periodontal disease. Extraoral infections by F. alocis are rare; only seven cases have been reported. We report the first case in which we identified F. alocis as one of the causative organisms of a deep neck abscess. A 71-year-old male on hemodialysis came to our hospital with a fever and left buccal pain. The patient's left neck was swollen, and contrast-enhanced computed tomography showed an abscess with gas extending from the left cheek to the deep neck. We diagnosed the patient with a deep neck abscess and performed an urgent neck drainage. We isolated F. alocis, Eggerthia catenaformis, Parvimonas micra, and Streptococcus constellatus in the abscess and identified them using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Blood cultures were negative. We initiated treatment with piperacillin-tazobactam and vancomycin. The patient improved but developed a hemorrhagic duodenal ulcer on the third day of admission. We attempted endoscopic hemostasis, but the patient's bleeding continued. Ultimately, he died of the duodenal ulcer hemorrhage on the sixth day of admission. This is the first case of F. alocis detected in a deep neck abscess.

Brain abscess mimicking acute stroke syndrome caused by dual Filifactor alocis and Porphyromonas gingivalis infections: A case report.

This case report is about a woman who had a brain abscess in the left parietal lobe that atypically presented as acute stroke-like syndrome. Pus samples from brain abscess aspiration revealed the periodontal pathogens Porphyromonas gingivalis and Filifactor alocis. After dental health care and 8 weeks of combined antimicrobial therapy, the patient recovered completely.

Filifactor alocis and Tumor Necrosis Factor-Alpha Stimulate Synthesis of Visfatin by Human Macrophages.

There is little known about the effect of the periodontopathogen Filifactor alocis on macrophages as key cells of the innate immune defense in the periodontium. Therefore, the aim of the present study was to investigate the effect of F. alocis and additionally of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) on visfatin and other pro-inflammatory and proteolytic molecules associated with periodontitis in human macrophages. The presence of macrophage markers CD14, CD86, CD68, and CD163 was examined in gingival biopsies from healthy individuals and periodontitis patients. Human macrophages were incubated with F. alocis and TNFα for up to 2 d. The effects of both stimulants on macrophages were determined by real-time PCR, ELISA, immunocytochemistry, and immunofluorescence. F. alocis was able to significantly stimulate the synthesis of visfatin by human macrophages using TLR2 and MAPK pathways. In addition to visfatin, F. alocis was also able to increase the synthesis of cyclooxygenase 2, TNFα, and matrix metalloproteinase 1. Like F. alocis, TNFα was also able to stimulate the production of these proinflammatory and proteolytic molecules. Our results highlight the pathogenetic role of F. alocis in periodontal diseases and also underline the involvement of visfatin in the aetiopathogenesis of periodontitis.

Filifactor alocis brain abscess identified by 16S ribosomal RNA gene sequencing: A case report.

We report a clinical case of Filifactor alocis brain abscess in an 85-year-old man who had decayed teeth 1 week prior. In this case, the abscess was surgically drained after empirical antibiotics had been initiated. Although the causative organism could not be identified by culture, F. alocis was detected via 16S ribosomal RNA (16S rRNA) gene sequencing of the pus isolated from the abscess. The patient recovered without serious sequelae after surgical drainage and prolonged antibiotic treatment, including metronidazole, ceftriaxone and meropenem for 8 weeks. The findings in this case emphasize that 16S rRNA gene sequencing allows bacterial diagnosis of brain abscess when phenotypic identification fails, such as in cases where patients are undergoing antimicrobial treatment at the time of sampling or where patients are infected with fastidious organisms.

Regulation of matrix metalloproteinase-1 by Filifactor alocis in human gingival and monocytic cells.

OBJECTIVES: Periodontitis is a highly prevalent chronic inflammatory disease caused by periodontopathogens, such as Filifactor alocis. This study sought to examine the matrix metalloproteinase (MMP)-1 synthesis by monocytic and fibroblastic cells in response to F. alocis and to unravel the underlying cellular mechanisms. MATERIAL AND METHODS: Gingival biopsies from periodontally healthy and periodontitis individuals were analyzed for the presence of F. alocis and MMP-1 by RT-PCR. Human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells were stimulated with F. alocis in the presence and absence of a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. MMP-1 expression and protein levels were studied by RT-PCR and ELISA, respectively. RESULTS: F. alocis was highly prevalent in biopsies from periodontitis patients but barely present in the healthy gingiva. Significantly higher MMP-1 expression levels were found in the inflamed gingiva as compared with healthy biopsies. F. alocis caused a significant and dose-dependent MMP-1 upregulation in both cells. The stimulatory effect of F. alocis on MMP-1 was TLR2- and MAPK-dependent and more pronounced on THP-1 cells as compared with HGF-1 cells. CONCLUSIONS: Our results demonstrate that F. alocis and MMP-1 are more prevalent at periodontitis sites. Additionally, our study provides original evidence that F. alocis can stimulate MMP-1 production by fibroblastic and monocytic cells, suggesting that F. alocis may contribute to periodontal breakdown through MMP-1. CLINICAL RELEVANCE: F. alocis and MMP-1 are linked to each other and key players in periodontitis, which may have significant implications for future diagnostic and treatment strategies.

[Filifactor alocis and its role in the etiology of chronic periodontitis]. / Filifactor alocis i ego rol' v etiologii khronicheskogo parodontita.

The review is devoted to the analysis of modern ideas about the role of bacteria Filifactor alocis in the etiology of chronic periodontitis. The study of these bacteria, discovered in 1985, is complicated by the difficulty of their detection with cultural methods. According to modern researches, the bacteria F.alocis with good reason can be included in the red complex of periodontal pathogens as the most important pathogens of chronic periodontitis. F.alocis is a synergist of such a key pathogen Porphyromonas gingivalis, as well as a frequent satellite of Fusobacterium nucleatum and, somewhat less frequently, Aggregatibacter actinomycetemcomitans. F.alocis is practically not found in healthy people (except for smokers), with a high frequency accompanies the aggressive course of periodontal disease, and also recorded in endodontitis. Due to the ability to participate in the metabolism of arginine, expressed protease activity, a wide range of virulence factors, F.alocis not only colonizes the periodontal tissues, but also significantly affects the formation of the community of periodontal microorganisms (including viruses), contributing to their invasion of epithelial tissues. F. alocis has a number of unique properties, including resistance to oxidative stress conditions in the home defeat, induction of apoptosis of epithelial cells, extracellular matrix degradation of periodontal tissues, activation of proinflammatory cytokines formulation in sites of its presence, suppression of protective reactions of neutrophilic granulocytes, inhibition of the process of complement activation.

Cigarette smoke reduces short chain fatty acid production by a Porphyromonas gingivalis clinical isolate.

OBJECTIVES: We hypothesized that short chain fatty acid (SCFA) production by oral pathogens is suppressed by exposure to cigarette smoke extract (CSE). BACKGROUND: Tobacco smoking is a major risk factor for plaque-induced periodontal diseases. Despite increased disease susceptibility, overt oral inflammation is suppressed in smokers, presenting a diagnostic conundrum. Bacterial-derived SCFAs can penetrate into oral tissues where they influence multiple components of immune and healing responses. Indeed, the SCFA burden has been correlated with the inflammatory condition of the gingiva. However, the influence of cigarette consumption on SCFA production is unknown. METHODS: GC/MS was employed to monitor the production of several SCFAs (propionic acid, isobutyric acid, butyric acid, and isovaleric acid) by representative anaerobic oral pathogens (Filifactor alocis 35896, Fusobacterium nucleatum 25586, Porphyromonas gingivalis 33277) that were exposed, or not, to a physiologically relevant dose of CSE (2000 ng/ml nicotine equivalents) generated from 3R4F reference cigarettes. RESULTS: The growth of all three bacterial species was unaffected by CSE. The capacity to produce SCFAs by these bacteria was highly varied. F alocis produced the highest concentration of a specific SCFA (butyrate); P gingivalis provided the most robust overall SCFA signal, while F alocis and F nucleatum did not release detectable levels of isobutyrate or isovalerate. As P gingivalis 33277 was the broadest SCFA producer, three low-passage clinical isolates (10208C, 5607, and 10512) were also examined. Compared to unconditioned microbes, reduced SCFA release was apparent in CSE-exposed low-passage clinical isolates of P gingivalis which reached significance for one of the three isolates (propionic, isobutyric, butyric, and isovaleric acids, all P < 0.05). CONCLUSIONS: There is high disparity in the SCFA profiles of variant chronic periodontitis-associated bacteria, while CSE exposure reduces SCFA production by a specific clinical strain of P gingivalis. If the latter phenomenon occurs in vivo, a reduced SCFA burden may help explain the reduced vascular response to dental plaque in tobacco smokers.

Salivary microbiota reflecting changes in subgingival microbiota.

The purpose of this study was to determine whether subgingival microbial changes according to the severity of periodontal disease and following the non-surgical periodontal treatment of periodontitis are reflected in the saliva microbiota. Subgingival and saliva samples were collected from 7 periodontally healthy controls, 14 patients with gingivitis, 12 with moderate periodontitis, and 18 with severe periodontitis. Among subjects who received treatment, seven moderate and seven severe periodontitis patients were selected for post-treatment microbial analysis, and their samples were analyzed at baseline and 6 months after treatment. The V3 and V4 regions of the 16S rRNA gene were sequenced, and correlations of the relative abundance of phyla and health- or periodontitis-dominant species between subgingival plaque and saliva samples were analyzed using Spearman signed-rank tests. Alpha diversity was higher in saliva than subgingival plaque samples, and beta diversity was significantly different between the two samples. However, levels of phyla and most health- or periodontitis-dominant species in salivary microbiota were significantly correlated with those in subgingival plaque. The improvement in clinical parameters following treatment was accompanied by a microbial shift not only in subgingival plaque but also in saliva. The abundance of 2 phyla including Bacteroidetes, 6 genera including Porphyromonas and Treponema, and 11 species including Porphyromonas gingivalis, Tannerella forsythia, and Filifactor alocis was significantly reduced in saliva following treatment. These results indicate that the salivary microbiota can reflect changes in the subgingival microbiota, suggesting that saliva can be used as a diagnostic tool to monitor the periodontal health status of individuals. IMPORTANCE: The salivary microbiota has attracted increasing attention as a promising method for monitoring periodontal disease. With regard to the pathogenesis of periodontal disease, however, subgingival plaque microbiota is the dominant etiological factor. Although it has been established that periodontopathogenic bacteria exist in saliva and their distribution differs, depending on the severity of the disease, it is necessary to analyze the extent to which the salivary microbiota reflects the subgingival microbiota. This study explored whether subgingival microbial changes according to the severity of periodontal disease and following the non-surgical periodontal treatment of periodontitis are reflected in the saliva microbiota and concluded that the salivary microbiota can reflect changes in the subgingival microbiota. Saliva can be used as a diagnostic tool to monitor the periodontal health status of individuals.

Filifactor alocis enhances survival of Porphyromonas gingivalis W83 in response to H2 O2 -induced stress.

A dysbiotic microbial community whose members have specific/synergistic functions that are modulated by environmental conditions, can disturb homeostasis in the subgingival space leading to destructive inflammation, plays a role in the progression of periodontitis. Filifactor alocis, a gram-positive, anaerobic bacterium, is a newly recognized microbe that shows a strong correlation with periodontal disease. Our previous observations suggested F. alocis to be more resistant to oxidative stress compared to Porphyromonas gingivalis. The objective of this study is to further determine if F. alocis, because of its increased resistance to oxidative stress, can affect the survival of other 'established' periodontal pathogens under environmental stress conditions typical of the periodontal pocket. Here, we have shown that via their interaction, F. alocis protects P. gingivalis W83 under H2 O2 -induced oxidative stress conditions. Transcriptional profiling of the interaction of F. alocis and P. gingivalis in the presence of H2 O2 -induced stress revealed the modulation of several genes, including those with ABC transporter and other cellular functions. The ABC transporter operon (PG0682-PG0685) of P. gingivalis was not significant to its enhanced survival when cocultured with F. alocis under H2 O2 -induced oxidative stress. In F. alocis, one of the most highly up-regulated operons (FA0894-FA0897) is predicted to encode a putative manganese ABC transporter, which in other bacteria can play an essential role in oxidative stress protection. Collectively, the results may indicate that F. alocis could likely stabilize the microbial community in the inflammatory microenvironment of the periodontal pocket by reducing the oxidative environment. This strategy could be vital to the survival of other pathogens, such as P. gingivalis, and its ability to adapt and persist in the periodontal pocket.

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.

Induction of TNF-α by Filifactor alocis in THP-1 macrophagic cells.

OBJECTIVES: Filifactor alocis is an emerging periodontal pathogen, and macrophage-produced tumor necrosis factor-α (TNF-α) plays important roles in periodontal pathogenesis. In this study, we investigated F. alocis-stimulated TNF-α production in THP-1 macrophagic cells. DESIGN: Phorbol 12-myristate 13-acetate-differentiated THP-1 macrophagic cells were challenged with F. alocis ATCC 35896 for various durations. TNF-α mRNA expression and protein secretion were determined using RT-PCR and ELISA, respectively. Activation of protein kinases and transcription factor proteins was evaluated by Western blot analysis. RESULTS: Live F. alocis stimulated THP-1 cells to produce TNF-α in a dose-dependent manner. However, glutaraldehyde-killed or heat-killed F. alocis showed no effectiveness for TNF-α induction. In contrast, both live and killed Porphyromonas gingivalis robustly increased TNF-α expression. Furthermore, F. alocis was unable to stimulate TNF-α expression in Toll-like receptor 2 (TLR2) knockout THP-1 cells. F. alocis activated all three mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Pharmacological inhibition of ERK and JNK, but not p38, significantly reduced F. alocis-induced TNF-α production. Finally, increased levels of phospho-c-Jun were detected in F. alocis-stimulated THP-1 cells. CONCLUSIONS: These results suggest that F. alocis induces TNF-α production in THP-1 macrophagic cells primarily by activating the TLR2, JNK, and c-Jun pathways.

Comparative analysis of the red-complex organisms and recently identified periodontal pathogens in the subgingival plaque of diabetic and nondiabetic patients with severe chronic periodontitis.

Aim: This analytical case-control study sought to evaluate the presence of the recently established putative periodontal pathogen organisms, Filifactor alocis and Fretibacterium fastidiosum, against the levels of the already established red-complex pathogens, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, in chronic periodontitis sites of patients with and without diabetes mellitus. Materials and Methods: Fifty-six subgingival plaque samples were obtained from the deepest sites of subjects diagnosed with severe chronic periodontitis with and without diabetes mellitus. These patients were categorized into two groups of 28 each. Clinical parameters were recorded and microbial analysis was done with quantitative polymerase chain reaction, and the bacterial counts of F. alocis and F. fastidiosum were determined and then compared with that of the red-complex organisms. Results: The bacterial counts were found to be higher in the diabetic group than that in the nondiabetic group, which was statistically significant for T. forsythia (P < 0.037) and T. denticola (P < 0.003). The study found very less number of F. alocis, which was slightly higher in the diabetic group. When correlating the bacterial levels within the nondiabetic groups, the red complex species had a strong positive correlation both individually with F. alocis (P < 0.0001) and F. fastidiosum (P < 0.001) and also when the newer species was clubbed together as a cohort (P < 0.0001). Whereas, in the diabetic group, although there was a positive correlation, there was no statistical significance. Conclusion: The results of this study highlighted the presence of a definite difference in the subgingival microbiota of both the patient groups evaluated. They also indicate that of the newly identified microorganisms, both the cohorts had higher levels of F. fastidiosum, suggesting a pathobiont-like role of this bacteria among both these periodontitis groups. F. alocis was comparatively lesser in number among the cohorts evaluated, and the cause for this decreased level of F. alocis needs to be further evaluated. The results of the present study depict a higher bacterial load in the diabetic group when compared to the nondiabetic group. Further, the study demonstrates a strong correlation between the red-complex species and the newer organisms in the nondiabetic group.

Characteristics of the Salivary Microbiota in Periodontal Diseases and Potential Roles of Individual Bacterial Species To Predict the Severity of Periodontal Disease.

The purposes of this study were to examine the compositional changes in the salivary microbiota according to the severity of periodontal disease and to verify whether the distribution of specific bacterial species in saliva can distinguish the severity of disease. Saliva samples were collected from 8 periodontally healthy controls, 16 patients with gingivitis, 19 patients with moderate periodontitis, and 29 patients with severe periodontitis. The V3 and V4 regions of the 16S rRNA gene in the samples were sequenced, and the levels of 9 bacterial species showing significant differences among the groups by sequencing analysis were identified using quantitative real-time PCR (qPCR). The predictive performance of each bacterial species in distinguishing the severity of disease was evaluated using a receiver operating characteristic curve. Twenty-nine species, including Porphyromonas gingivalis, increased as the severity of disease increased, whereas 6 species, including Rothia denticola, decreased. The relative abundances of P. gingivalis, Tannerella forsythia, Filifactor alocis, and Prevotella intermedia determined by qPCR were significantly different among the groups. The three bacterial species P. gingivalis, T. forsythia, and F. alocis were positively correlated with the sum of the full-mouth probing depth and were moderately accurate at distinguishing the severity of periodontal disease. In conclusion, the salivary microbiota showed gradual compositional changes according to the severity of periodontitis, and the levels of P. gingivalis, T. forsythia, and F. alocis in mouth rinse saliva had the ability to distinguish the severity of periodontal disease. IMPORTANCE Periodontal disease is one of the most widespread medical conditions and the leading cause of tooth loss, imposing high economic costs and an increasing burden worldwide as life expectancy increases. Changes in the subgingival bacterial community during the progression of periodontal disease can affect the entire oral ecosystem, and bacteria in saliva can reflect the degree of bacterial imbalance in the oral cavity. This study explored whether the specific bacterial species in saliva can distinguish the severity of periodontal disease by analyzing the salivary microbiota and suggested P. gingivalis, T. forsythia, and F. alocis as biomarkers for distinguishing the severity of periodontal disease in saliva.

Characterization of FA1654: A putative DPS protein in Filifactor alocis.

The survival/adaptation of Filifactor alocis, a fastidious Gram-positive asaccharolytic anaerobe, to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Moreover, its pathogenic characteristics are highlighted by its capacity to survive in the oxidative-stress microenvironment of the periodontal pocket and a likely ability to modulate the microbial community dynamics. There is still a significant gap in our understanding of its mechanism of oxidative stress resistance and its impact on the virulence and pathogenicity of the microbial biofilm. Coinfection of epithelial cells with F. alocis and Porphyromonas gingivalis resulted in the upregulation of several genes, including HMPREF0389_01654 (FA1654). Bioinformatics analysis indicates that FA1654 has a "di-iron binding domain" and could function as a DNA starvation and stationary phase protection (DPS) protein. We have further characterized the FA1654 protein to determine its role in oxidative stress resistance in F. alocis. In the presence of hydrogen peroxide-induced oxidative stress, there was an ∼1.3 fold upregulation of the FA1654 gene in F. alocis. Incubation of the purified FA1654 protein with DNA in the presence of hydrogen peroxide and iron resulted in the protection of the DNA from Fenton-mediated degradation. Circular dichroism and differential scanning fluorimetry studies have documented the intrinsic ability of rFA1654 protein to bind iron; however, the rFA1654 protein is missing the intrinsic ability to reduce hydrogen peroxide. Collectively, the data may suggest that FA1654 in F. alocis is involved in oxidative stress resistance via an ability to protect against Fenton-mediated oxidative stress-induced damage.

Whole Genome Sequencing and Phenotypic Analysis of Antibiotic Resistance in Filifactor alocis Isolates.

There is scarce knowledge regarding the antimicrobial resistance profile of F. alocis. Therefore, the objective of this research was to assess antimicrobial resistance in recently obtained F. alocis clinical isolates and to identify the presence of antimicrobial resistance genes. Isolates were obtained from patients with periodontal or peri-implant diseases and confirmed by sequencing their 16S rRNA gene. Confirmed isolates had their genome sequenced by whole genome sequencing and their phenotypical resistance to nine antibiotics (amoxicillin clavulanate, amoxicillin, azithromycin, clindamycin, ciprofloxacin, doxycycline, minocycline, metronidazole, and tetracycline) tested by E-test strips. Antimicrobial resistance genes were detected in six of the eight isolates analyzed, of which five carried tet(32) and one erm(B). Overall, susceptibility to the nine antibiotics tested was high except for azithromycin in the isolate that carried erm(B). Moreover, susceptibility to tetracycline, doxycycline, and minocycline was lower in those isolates that carried tet(32). The genetic surroundings of the detected genes suggested their inclusion in mobile genetic elements that might be transferrable to other bacteria. These findings suggest that, despite showing high susceptibility to several antibiotics, F. alocis might obtain new antimicrobial resistance traits due to its acceptance of mobile genetic elements with antibiotic resistance genes in their genome.

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.

Virulence of Filifactor alocis lipoteichoic acid on human gingival fibroblast.

OBJECTIVES: The purpose of this study was to investigate virulence of lipoteichoic acid extracted from Filifactor alocis (F. alocis) through comparison of previously known bacterial virulence factors. DESIGN: F. alocis was cultured in columbia media including L-arginine and L-cysteine, and lipoteichoic acid (LTA) from F. alocis was purified using organic solvent and bead extraction. Human gingival fibroblasts (HGFs) were treated with the extracted LTA and other Gram-positive LTA or lipopolysaccharide of other periodontopathogens. The induction of cytokine expression was examined by real-time RT-PCR and ELISA, and the stimulated signaling pathway by the LTA was investigated by immunoblotting and various inhibitors. RESULTS: LTA induced expression of pro-inflammatory cytokines and Matrix-metalloprotein 2. Also, F. alocis LTA induced expression pro-inflammatory cytokines similar to Porphyromonas gingivalis lipopolysaccharide. The LTA activated NF-κB and MAP kinase pathway. Furthermore, the induction of TNF-α, IL-6, IL-8, and MMP-2 expression by F. alocis LTA was reduced by the inhibitor of NF-κB, ERK, JNK, and p38 pathway. CONCLUSIONS: LTA of F. alocis, a bacterium recently detected in periodontal patients, may play an important role in inducing periodontitis by induction of expression of pro-inflammatory cytokines.

Aggregatibacter actinomycetemcomitans and Filifactor alocis: Two exotoxin-producing oral pathogens.

Periodontitis is a dysbiotic disease caused by the interplay between the microbial ecosystem present in the disease with the dysregulated host immune response. The disease-associated microbial community is formed by the presence of established oral pathogens like Aggregatibacter actinomycetemcomitans as well as by newly dominant species like Filifactor alocis. These two oral pathogens prevail and grow within the periodontal pocket which highlights their ability to evade the host immune response. This review focuses on the virulence factors and potential pathogenicity of both oral pathogens in periodontitis, accentuating the recent description of F. alocis virulence factors, including the presence of an exotoxin, and comparing them with the defined factors associated with A. actinomycetemcomitans. In the disease setting, possible synergistic and/or mutualistic interactions among both oral pathogens might contribute to disease progression.