Periodontitis is a bacterial-induced oral inflammatory disease, which over time degrades the periodontal tissues, ultimately causing tooth loss (Pihlstrom et al., 2005). Research is indicating that when the natural balance among the resident bacterial species is disturbed, causing a dysbiotic environment, some of the bacteria might exhibit potential pathogenic characteristics, leading to an inflammatory response in the periodontal tissues, which together with factors related to the host susceptibility promotes the degradation (Darveau, 2010). Periodontal diseases affecting adolescents and young individuals typically have been typically referred to as aggressive periodontitis and are at present defined as grade C indicating a rapid progress (Tonetti et al., 2018). There is a wide geographical spread (Bouziane et al., 2020). Among adolescents from the western and northern parts of Africa, the disease has been observed to have a high incidence (Kissa et al., 2022; Yoshida et al., 2021). A highly leukotoxin (LtxA)-producing genotype of the Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, JP2, is associated with the periodontal disease progress in these geographical regions (Haubek et al., 2008; Höglund Åberg et al., 2014). It carries a typical deletion of 530 base pairs (bp) in the ltxCABD promoter (Brogan et al., 1994). To the best of our knowledge, few studies executed in the northern and western geographical regions of Africa have been reported on regarding the relation between periodontitis and the progression of this disease in young individuals, and concomitantly focusing on bacterial species other than A. actinomycetemcomitans (Yoshida et al., 2021; Dahlén et al., 2014), thus including the Gram-positive, anaerobic bacterium Filifactor alocis. This species has recently been identified in the oral microbiome by the method called high throughput DNA sequencing. F. alocis is culturable and is regarded as an emerging oral pathogen with significant roles in the etiology of periodontal (Aruni et al., 2014; Greenwood et al., 2020), peri-implantitis (Sanz-Martin et al., 2017), and endodontic (Zehnder et al., 2017) infections. It is a potential biomarker for active disease in young children (Aruni et al., 2014). Interestingly, A. actinomycetemcomitans and F. alocis seem to have a synergistic relationship in active periodontal disease (Fine et al., 2013; Wang et al., 2013). However, A. actinomycetemcomitans appears thereafter to be outcompeted by F. alocis in the deeper pockets (Dahlén et al., 2014). This suggests that F. alocis has mechanisms to be able to adapt and drive the disease process forward in the absence of A. actinomycetemcomitans. Mechanisms for this are yet unknown.

In the present work, we have assessed longitudinal data from a study population of adolescents in Ghana. This study population was followed up after two years (Höglund Åberg et al., 2014; Höglund Åberg et al., 2012). In the previous studies, cultivation and PCR were used to analyse the pooled subgingival plaque samples, which revealed a 54.2% prevalence of A. actinomycetemcomitans at baseline (Höglund Åberg et al., 2012). Among the individuals, it was also found that 8.8% in the studied population carried the JP2 genotype of A. actinomycetemcomitans (Höglund Åberg et al., 2012). At baseline, an association between clinical attachment loss (CAL) and carriage of A. actinomycetemcomitans was observed, and in addition an increased extent regarding individuals carrying the JP2 genotype (Höglund Åberg et al., 2012).

F. alocis was recently found to encode and express a putative repeats-in-toxin (RTX) protein, FtxA (Bao et al., 2022; Oscarsson et al., 2020). Interestingly, only approximately 50% of the isolated F. alocis strains appeared to carry the ftxA gene (Oscarsson et al., 2020), suggesting the presence of a potentially more virulent genotype of this species. In a recent study (Razooqi et al., 2022), a possible correlation between deep periodontal pockets, presence of the ftxA gene, and a high quantity of F. alocis in the Ghanaian study population could be seen. Moreover, there seemed to be an association between the presence of the ftxA gene, the JP2 genotype of A. actinomycetemcomitans, high levels of F. alocis, and deep periodontal pockets (Razooqi et al., 2022).

To further understand the potential contribution of F. alocis and its ftxA gene in periodontal disease, we aimed at determining the loads of carried F. alocis at baseline among the individuals in the Ghanaian study cohort, and the prevalence of ftxA, and relate these data to the presence and/or absence, respectively of periodontal attachment loss progress observed at the two-year follow-up.

In the present work, we have used qPCR and PCR, respectively, to characterize the prevalence and carriage loads of the emerging oral pathogen F. alocis, and its RTX-toxin encoding ftxA gene, in subgingival plaque specimens, sampled at baseline from a longitudinal cohort of Ghanaian adolescents (n=500) (Höglund Åberg et al., 2014; Höglund Åberg et al., 2012). The obtained results were thereafter compared with the recorded CAL and CAL progression data, collected at baseline and at the two-year follow up of the Ghanaian cohort, respectively (Höglund Åberg et al., 2014; Höglund Åberg et al., 2012). The rationale for using this cohort in the present study was that this collection is unique due to being linked to clinical longitudinal data showing attachment loss progression over a two-year time, and it was therefore very suitable for analyzing associations between F. alocis carriage, presence of the ftxA gene, and periodontal disease progression. Our results revealed that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis, and presence of the ftxA gene were two factors both associated with an enhanced prevalence of CAL progression.

The high proportion of individuals with CAL, and CAL progression at the follow up, in the Ghanaian cohort can be partly explained by the presence of A. actinomyctemcomitans at baseline, especially the high proportion (8.8%) of individuals carrying the highly leukotoxic JP2 genotype (Höglund Åberg et al., 2014). However, A. actinomyctemcomitans cannot itself explain all cases of CAL, and CAL progression in this cohort. One reason for assessing F. alocis and its ftxA in relation to CAL in the present work was that there appears to be a synergism between F. alocis and A. actinomycetemcomitans in active periodontal disease, based on observations, such as that A. actinomycetemcomitans could stimulate F. alocis accumulation, albeit that this effect appeared to be dependent on the strain of F. alocis it interacted with (Fine et al., 2013; Wang et al., 2013). Evidently, isolates of A. actinomycetemcomitans from the oral cavity are also heterogeneous with respect to their expression levels of LtxA and production of fimbriae (Haubek, 2010; Rylev et al., 2011), and as suggested (Wang et al., 2013), different strains of A. actinomycetemcomitans might therefore also exhibit different patterns of interaction with F. alocis.

Indeed, the results from our present work would support a scenario where CAL progression is further promoted upon simultaneous presence of F. alocis and a non-JP2 genotype of A. actinomycetemcomitans. This would be consistent with the notion that there may occur genotype-specific synergy between F. alocis and A. actinomycetemcomitans. Mechanism(s) for such interbacterial interactions between these two species, and whether it could be a dependence on FtxA, are yet unknown, but could potentially include interplay based on reduction of the oxidative environment, which evidently was conducted via F. alocis in synergy between this organism and Porphyromonas gingivalis (Mishra et al., 2024). In contrast, CAL progression was not further promoted upon simultaneous presence of F. alocis and the highly leukotoxic JP2 genotype of A. actinomycetemcomitans, which most likely was a result of an apparent distinctive influence of this genotype to drive CAL progression itself, regardless F. alocis was present or not. This would be consistent with the strong leukotoxic activity of the JP2 genotype, as evidenced by the observation that only one bacterial cell was sufficient to lyse the majority of macrophage cells in in vitro experiments (Kelk et al., 2008).

Interestingly, A. actinomycetemcomitans seems to be outcompeted by F. alocis in the deeper periodontal pockets (Dahlén et al., 2014), suggesting that this bacterium can have the capability to adapt and drive the disease process forward in later stages, in the absence of A. actinomycetemcomitans. Concomitantly, we recently discovered a possible correlation between deep periodontal pockets at the two-year follow up of the Ghanaian cohort, presence of the ftxA gene, and a high quantity of F. alocis (Razooqi et al., 2022). As the relatively newly discovered FtxA toxin of F. alocis belongs to the same family of RTX proteins as the A. actinomycetemcomitans leukotoxin, LtxA (Oscarsson et al., 2020; Linhartova et al., 2010), we hypothesized that high F. alocis levels, and carriage of F. alocis encoding ftxA, might correlate to CAL and to CAL progression in the cohort. Indeed, our present results supported the idea that carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Whether this depends on mechanism(s) directly executed by FtxA is not known, but as presence of the ftxA gene as well as high carriage loads of F. alocis were two factors both associated with an enhanced prevalence of CAL progression, we suggest that FtxA represents a factor promoting the disease progression, at least in younger individuals in these ages.

Carriers of ftxA-positive F. alocis typically exhibited higher loads of the bacterium. Moreover, high carriage loads of F. alocis and the 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 simultaneous presence of F. alocis and non-JP2 genotype of Aggregatibacter actinomycetemcomitans. Taken together, our present findings are consistent with the notion that F. alocis and its ftxA gene promote CAL during periodontal disease. Hence, ftxA appears to have the potential to be used as a PCR-based gene marker for the identification of F. alocis that are more prone to promote disease progress. This finding might be of importance in the risk assessment in young individuals and is therefore a relevant discovery for development of new diagnostic tools and/or treatment strategies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Medical Research, University of Ghana (IRB 000 1276), and from the local Ethical committee of Umeå University, Sweden (Dnr 2010-188-31M). The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from primarily isolated as part of your previous study for which ethical approval was obtained. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

ZR: Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. IT: Formal analysis, Investigation, Validation, Writing – original draft, Writing – review & editing. CHÅ: Writing – review & editing, Resources, Supervision. FK: Resources, Writing – review & editing. RC: Conceptualization, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. DH: Resources, Writing – review & editing. AJ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JO: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.