In Vitro Anti-Biofilm and Antibacterial Properties of Streptococcus downii sp. nov.

The aim of this study was to evaluate the potential anti-biofilm and antibacterial activities of Streptococcus downii sp. nov. To test anti-biofilm properties, Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were grown in a biofilm model in the presence or not of S. downii sp. nov. for up to 120 h. For the potential antibacterial activity, 24 h-biofilms were exposed to S. downii sp. nov for 24 and 48 h. Biofilms structures and bacterial viability were studied by microscopy, and the effect in bacterial load by quantitative polymerase chain reaction. A generalized linear model was constructed, and results were considered as statistically significant at p < 0.05. The presence of S. downii sp. nov. during biofilm development did not affect the structure of the community, but an anti-biofilm effect against S. mutans was observed (p < 0.001, after 96 and 120 h). For antibacterial activity, after 24 h of exposure to S. downii sp. nov., counts of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020) were significantly reduced in well-structured biofilms. Although moderate, anti-biofilm and antibacterial activities of S. downii sp. nov. against oral bacteria, including some periodontal pathogens, were demonstrated in an in vitro biofilm model.


Introduction
The oral cavity has one of the richest microbial communities within the human microbiome, with around 200 cultivable bacterial species and approximately 1000 phylotypes detected by 16S rRNA gene sequencing [1,2]. In healthy subjects, the majority of these species are commensal and maintain a symbiotic relation with the host, being the predominant phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria [3]. Among Firmicutes the most abundant species belongs to Streptococcus, Veillonella, and Lactobacillus genera [4]. However, under specific environmental conditions, existing pathobionts may overgrow and become pathogenic, mainly by disturbing the homeostasis between the bacterial challenge and the host immune responses (dysbiosis) [5], which can also be associated with changes in microbial metabolism and/or shifts in bacterial diversity. Moreover, within the oral cavity, overgrow of microorganisms usually occurs forming highly structured poly-microbial communities, as biofilms, what may hinder the efficacy of host defenses and natural antimicrobial strategies. Figure 1 shows the experimental design followed for the study of the anti-biofilm properties of S. downii sp. nov. strain CECT 9732T against bacteria in an oral biofilm model. Microorganisms 2021, 9, x FOR PEER REVIEW 3 of 16 Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid, Basingstoke, UK), 5.0 mg mL −1 hemin (Sigma-Aldrich, St. Louis, MO, USA) and 1.0 mg mL -1 menadione (Merck, Darmstadt, Germany) for 72 h at 37 °C. Figure 1 shows the experimental design followed for the study of the anti-biofilm properties of S. downii sp. nov. strain CECT 9732T against bacteria in an oral biofilm model.
Sterile HA discs [7-mm diameter and 1.8 (standard deviation-SD = 0.2) mm thickness (Clarkson Chromatography Products, Williamsport, PA, USA)] were placed in a multi-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany). Each well was inoculated with 1.5 mL of the mixed bacterial suspension, and to assess the anti-biofilm
Sterile HA discs [7-mm diameter and 1.8 (standard deviation-SD = 0.2) mm thickness (Clarkson Chromatography Products, Williamsport, PA, USA)] were placed in a multiwell tissue culture plate (Greiner Bio-one, Frickenhausen, Germany). Each well was inoculated with 1.5 mL of the mixed bacterial suspension, and to assess the anti-biofilm activity, treated wells were inoculated with 10 3 CFU mL −1 of S. downii sp. nov. final concentration (recovered at late-exponential phase by centrifugation of an overnight culture and resuspended in fresh modified BHI medium). Control biofilms, not exposed to S. downii sp. nov., and treated ones were then incubated in anaerobiosis at 37 • C, for 12, 24, 48, 72, 96, and 120 h. Plates only containing culture media were also incubated to check for sterility. Three independent trials (on three different occasions) were carried out. Before the DNA isolation, discs were sequentially rinsed in 2 mL of sterile buffer saline (PBS) (immersion time per rinse, 10 s), three times, in order to remove non-adherent bacteria. Biofilms were then disrupted by vortex for 2 min in 1 mL of PBS. The DNA from the biofilm was extracted using an ATP Genomic DNA Mini Kit ® (ATP biotech. Taipei, Taiwan) according to the manufacturer's recommendations. The bacterial 16S rRNA gene sequence was amplified by qPCR, according to the hydrolysis probes 5 nuclease method ( Table 1). The qPCR amplification was performed in a total reaction mixture volume of 10 µL. The reaction mixtures contained 5 µL of 2× master mixture (LC 480 Probes Master; Roche, Mannheim, Germany), optimal concentrations of primers and probes (300, 300 and 300 nM, respectively, for S. mutans, Streptococcus spp., A. naeslundii and P. gingivalis; 750, 750, and 400 nM for V. parvula; 300, 300, and 200 nM for A. actinomycetemcomitans; and, finally, 600, 600, and 300 nM for F. nucleatum) and 2 µL of DNA from samples. The negative control was 2 µL of sterile water (Water PCR grade, Roche Mannheim, Germany). The samples were subjected to an initial amplification cycle of 95 • C for 10 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 1 min. Analyses was performed with a LightCycler ® 480 II thermocycler (Roche Mannheim, Germany). LightCycler ® 480 Multiwell Plates 384 and sealing foils were used (Roche Mannheim, Germany). Each DNA sample was analyzed in duplicate. Table 1. Primers and probes used for quantification of genomic DNA from the target bacteria. Primers and probes were targeted against 16S rRNA gene (Obtained from Life Technologies Invitrogen (Carlsbad, CA, USA) and Roche (Roche Diagnostic GmbH; Mannheim, Germany)).

Analysis of Biofilms by Confocal Laser Scanning Microscopy (CLSM)
Biofilms grown on HA discs were stained with LIVE/DEAD ® BacLight TM Bacterial Viability Kit solution (Molecular Probes B. V., Leiden, The Netherlands) at room temperature, with 1:1 fluorocromes ratio, in the dark for 10 min (SD = 1). The obtained fully hydrated biofilms were studied with a confocal laser scanning microscope, using a fixed-stage Ix83 Olympus inverted microscope, coupled to an Olympus FV1200 confocal system (Olympus; Shinjuku, Tokyo, Japan) with a ×63 water-immersion lenses (Olympus, Shinjuku, Tokyo, Japan). At least three separate and representative locations were selected from the HA discs covered with biofilm. With the use of a dedicated software (Olympus ® software (Olympus, Shinjuku, Tokyo, Japan) and Image analysis FIJI ® software (Image J v. 2.0.0-rc-65 /1.52b) a z-series of scans (xyz) of 0.5 µm thickness (8 bits, 1024 × 1024 pixels) were analyzed.

Analysis of Biofilms by Scanning Electron Microscope (SEM)
Specimens were fixed in a solution at 4% paraformaldehyde and 2.5% glutaraldehyde for 4 h, at 4 • C. The discs were washed once in phosphate buffer saline (PBS) and another time in sterile water (immersion time per washed 10 min) and then dehydrated through a series of graded ethanol solutions (30,50,70, 80, 90, and 100%; immersion time per series 10 min). After that, specimens were critical point dried, sputter-coated with gold and analysed by electron microscopy JSM 6400 (JSM6400; JEOL, Tokyo, Japan) with a back-scattered electron detector and an image resolution of 25 kV. Figure 1 presents the experimental design of the study on the antibacterial activity of S. downii sp. nov. strain CECT 9732T against bacteria, in an in vitro oral biofilm model.

Biofilm Development
Biofilms were developed as described previously (Section 2.2.1), and a mixed bacterial suspensions in supplemented BHI medium were prepared in order to develop biofilms, containing 10 3 CFU mL −1 of S. mutans, 10 5 CFU mL −1 of V. parvula and A. naeslundii, and 10 6 CFU mL -1 of F. nucleatum, A. actinomycetemcomitans, and P. gingivalis. HA discs (Clarkson Chromatography Products, Williamsport, PA, USA) and 1.5 mL of the bacterial suspension were placed in the multi-well tissue culture plate (Greiner Bio-one, Frickenhausen, Germany) and incubated at 37 • C in anaerobiosis for 24 h. After that, the 24 h developed biofilms were: (i) exposed to 1 mL of S. downii sp. nov. at 10 8 CFU mL −1 in supplemented BHI medium or (ii) in the case of controls, exposed to 1 mL of fresh supplemented BHI medium. Biofilms were then incubated for additional 24 and 48 h at 37 • C in anaerobiosis. Three independent trials (on three different occasions) were carried out.

Biofilm Analysis
The 24 h biofilms and the biofilms re-incubated another 24-48 h in the presence or not (controls) of S. downii sp. nov., were analysed. Confocal laser scanning microscopy and scanning electron microscopy (Sections 2.2.3 and 2.2.4) displayed the biofilms structures and bacterial viability. Quantitative polymerase chain reaction was used to assess the effect of S. downii sp. nov. in bacterial load (amounts of each bacterium expressed as CFU mL −1 ; Section 2.2.2).

Statistical Analysis
The primary outcome variable was the count of viable bacteria present in the in vitro developed biofilms, expressed as viable CFU mL −1 of S. downii sp. nov., S. mutans, V. parvula, A. naeslundii, A. actinomycetemcomitans, P. gingivalis, and F. nucleatum. An Microorganisms 2021, 9, 450 6 of 16 experiment-level analysis was performed for each parameter of the study (n = 3). Shapiro-Wilk goodness-of-fit tests and distribution of data were used to assess normality. Data were expressed as means and standard deviations (SD).
In order to evaluate the impact of S. downii sp. nov. and the time of biofilmdevelopment (up to 120 h) and their interaction with the primary outcome variable (counts expressed in CFU mL −1 ), a general linear model was constructed for each bacterial species, using the method of maximum likelihood and Bonferroni corrections for multiple comparisons. A p-value < 0.05 was considered statistically significant. A software package (IBM SPSS Statistics 21.0; IBM Corporation, Armonk, NY, USA) was used for all data analysis.

Anti-Biofilm Activity of S. downii sp. nov. in an in Vitro Biofilm Model
The impact of S. downii sp. nov. on the obtained multispecies biofilms was assessed from 12 to 120 h. Figure 2 depicts the kinetic profiles of the six bacterial species used in this in vitro biofilm model (strains S. mutans, A. naeslundii, V. parvula, F. nucleatum, P. gingivalis, and A. actinomycetemcomitans), quantified by qPCR, when forming biofilms with or without the presence of S. downii sp. nov. In biofilms exposed to S. downii sp. nov., it could be detected this bacterial species incorporated in the biofilm as early as 12 h of biofilm evolution and up to 120 h, with an average concentration of 2.7 × 10 7 CFU mL −1 (SD = 1.5 × 10 7 ).
In both obtained biofilms, with or without the presence of S. downii sp. nov, the six bacterial species were incorporated already after 12 h of incubation, and were present for up to 120 h. Only the growth of S. mutans was significantly different when S. downii sp. nov. was present in the biofilms (Figure 2). This impact was not observed in the early stages of biofilm formation, since at 12 h, both biofilms were similar (p > 0.05). From 24 to 72 h there was a clear trend, but without being statistically significant (p > 0.05 in all cases). However, differences were evident and statistically significant in the stationary phase (96 and 120 h) of the biofilm formation (p < 0.001, in both cases) ( Figure 2). The kinetics of the rest of initial colonizers (V. parvula and A. naeslundii), and that of the periodontal pathogens (F. nucleatum, A. actinomycetemcomitans, and P. gingivalis) was not affected by the presence of S. downii sp. nov. during the 120 h of incubation (p > 0.05 in all cases).
Biofilm structure and bacterial viability were studied by CLSM. During the initial growth phase, and after 24 h of incubation, biofilms containing S. downii sp. nov. depicted a well-structured bacterial community, similar to biofilms without S. downii sp. nov. (Figure 3a,b), with a live/dead ratio of 2.75 (SD = 0.2) for biofilms without S. downii sp. nov. and 2.4 (SD = 0.8) for biofilms containing S. downii sp. nov. This tendency was maintained during the exponential phase of biofilm development and, after 72 h, both modalities of biofilms showed a similar architecture, corresponding to a mature bacterial community (Figure 3c,d), with a live/dead ratio of 3.0 (SD = 0.1), for biofilms without S. downii sp. nov., and 2.7 (SD = 0.2), for biofilms containing S. downii sp. nov. However, and in agreement with the previously described qPCR data, in the stationary phase of biofilms (from 96 to 120 h, Figure 3e   The analysis by SEM showed similar findings (Figure 4). After 120 h or gro would seem that the proportion of S. mutans forming chains of cocci was reduced films containing S. downii sp. nov., compared to grown biofilms without S. downii s (Figure 4e,f). The analysis by SEM showed similar findings (Figure 4). After 120 h or growth, it would seem that the proportion of S. mutans forming chains of cocci was reduced in biofilms containing S. downii sp. nov., compared to grown biofilms without S. downii sp. nov. (Figure 4e

Antibacterial Effect of S. downii sp. nov. Against Oral Species in an Already Vitro Biofilm
After 24 h of incubation, well-structured biofilms were developed. Th the six inoculated bacterial species was confirmed by qPCR, and their li measured by CLSM, was 2.0 (SD = 0.1) ( Figure 5).

Antibacterial Effect of S. downii sp. nov. Against Oral Species in an Already Established in Vitro Biofilm
After 24 h of incubation, well-structured biofilms were developed. The presence of the six inoculated bacterial species was confirmed by qPCR, and their live/dead ratio, measured by CLSM, was 2.0 (SD = 0.1) ( Figure 5).
These biofilms were then exposed to 10 8 CFU mL −1 of S. downii sp. nov. for 24 and 48 h and examined by CLSM ( Figure 6). The obtained 24 h biofilms showed similar well-structured bacterial communities when compared those non-exposed versus exposed to S. downii sp. nov. (Figure 6a,b, respectively) and with live/dead ratio of 3.0 (SD = 0.3) and 4.2 (SD = 1.7), respectively. In those exposed to S. downii sp. nov., a thicker live biomass could be observed, possibly caused by the additional bacteria incorporated (Figure 6a,b). After 48 h of exposition, in spite of having a similar biofilm structure, the percentage of dead bacteria was significantly higher in the biofilms exposed to S. downii sp. nov. These biofilms were then exposed to 10 8 CFU mL −1 of S. downii sp. nov. for 24 and 48 h and examined by CLSM ( Figure 6). The obtained 24 h biofilms showed similar wellstructured bacterial communities when compared those non-exposed versus exposed to S. downii sp. nov. (Figure 6a,b, respectively) and with live/dead ratio of 3.0 (SD = 0.3) and 4.2 (SD = 1.7), respectively. In those exposed to S. downii sp. nov., a thicker live biomass could be observed, possibly caused by the additional bacteria incorporated (Figure 6a,b). After 48 h of exposition, in spite of having a similar biofilm structure, the percentage of dead bacteria was significantly higher in the biofilms exposed to S. downii sp. nov. when compared with those without S. downii sp. nov. (Figure 6c,d; live/dead ratio of 1.1 (SD = 0.2) and 1.8 (SD = 0.2), respectively.)  These biofilms were then exposed to 10 8 CFU mL −1 of S. downii sp. nov. for 24 and 48 h and examined by CLSM ( Figure 6). The obtained 24 h biofilms showed similar wellstructured bacterial communities when compared those non-exposed versus exposed to S. downii sp. nov. (Figure 6a,b, respectively) and with live/dead ratio of 3.0 (SD = 0.3) and 4.2 (SD = 1.7), respectively. In those exposed to S. downii sp. nov., a thicker live biomass could be observed, possibly caused by the additional bacteria incorporated (Figure 6a,b). After 48 h of exposition, in spite of having a similar biofilm structure, the percentage of dead bacteria was significantly higher in the biofilms exposed to S. downii sp. nov. when compared with those without S. downii sp. nov. (Figure 6c : (a,b) 24-h biofilms exposed for 24 h to 1 mL of brain heart infusion (BHI) medium (control biofilms) and to 1 mL of 10 8 colony forming units (CFU) mL −1 of S. downii sp. nov. in fresh BHI medium (exposed biofilms), respectively; (c,d) 24-h biofilms exposed for 48 h to 1 mL of BHI medium (control biofilms) and to 1 mL of 10 8 CFU mL −1 of S. downii sp. nov., in fresh BHI medium (exposed biofilms), respectively. Figure 7 depicts the concentration (expressed in CFU mL −1 ) of the six inoculated bacteria when comparing those biofilms exposed and non-exposed to S. downii sp. nov. for 24 and 48 h. After 24 h of contact with S. downii sp. nov., the concentration of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020) were significantly reduced, when compared to non-exposed biofilms (Figure 7a). After 48 h of contact, a similar tendency was observed, but with statistically significant higher concentrations for A. naeslundii, in exposed biofilms (p = 0.003) (Figure 7b). Figure 7 depicts the concentration (expressed in CFU mL −1 ) of the six inoculated bacteria when comparing those biofilms exposed and non-exposed to S. downii sp. nov. for 24 and 48 h. After 24 h of contact with S. downii sp. nov., the concentration of S. mutans (p = 0.019) and A. actinomycetemcomitans (p=0.020) were significantly reduced, when compared to non-exposed biofilms (Figure 7a). After 48 h of contact, a similar tendency was observed, but with statistically significant higher concentrations for A. naeslundii, in exposed biofilms (p = 0.003) (Figure 7b).
With longer time of incubation (from 24 to 48 h), the concentration of the selected bacteria increased in both types of biofilms, but for P. gingivalis, a significant increase (p = 0.018) was observed in non-exposed biofilms, while the increase in exposed biofilms was not statistically significant (p = 0.395). A similar trend was observed for V. parvula (p = 0.050 in non-exposed biofilms and p = 0.295 in exposed biofilms, respectively).

Discussion
The novel species S. downii sp. nov. has been recently isolated from a supragingival dental biofilm sample of an individual with Down syndrome [29]. The orofacial and skeletal developmental disturbances associated with Down syndrome contribute to frequent oral conditions and diseases in these patients, as periodontal diseases, malocclusion, mouth breathing, macroglossia, delayed teeth eruption, missing and malformed teeth, microdontia, diastema, and bruxism [31]. In spite of this, Down syndrome individuals have With longer time of incubation (from 24 to 48 h), the concentration of the selected bacteria increased in both types of biofilms, but for P. gingivalis, a significant increase (p = 0.018) was observed in non-exposed biofilms, while the increase in exposed biofilms was not statistically significant (p = 0.395). A similar trend was observed for V. parvula (p = 0.050 in non-exposed biofilms and p = 0.295 in exposed biofilms, respectively).

Discussion
The novel species S. downii sp. nov. has been recently isolated from a supragingival dental biofilm sample of an individual with Down syndrome [29]. The orofacial and skeletal developmental disturbances associated with Down syndrome contribute to frequent oral conditions and diseases in these patients, as periodontal diseases, malocclusion, mouth breathing, macroglossia, delayed teeth eruption, missing and malformed teeth, microdontia, diastema, and bruxism [31]. In spite of this, Down syndrome individuals have shown significantly lower prevalence of dental caries when compared with matched individuals without the syndrome [31]. In this regard, previous evidence has indicated antibacterial activity of S. downii sp. nov. against pathogens involved, not only in caries, but also in periodontal diseases, carried out by inhibition assays on solid culture medium [29].
Based on the previous findings, and in order to provide additional information, but with a model that better resembles what occurs in the oral cavity (bacteria organized in biofilms), the potential effect on oral health of S. downii sp. nov. was studied using a validated in vitro biofilm model. The present manuscript describes the in vitro antibacterial and anti-biofilm effects of S. downii sp. nov. when inoculated with oral bacteria included in a multispecies biofilm. Although there was no significant impact in the early stages of biofilm formation, during the stationary phase of biofilm growth, there was a significant reduction in the growth of S. mutans and A. actinomycetemcomitans that was evident with qPCR analysis.
This antibacterial activity on oral bacteria forming biofilms are in accordance with previous studies reported antibacterial activity of S. mitis group [23][24][25]. Similarly, other streptococci have demonstrated an impact on periodontal pathogens [22,28,[32][33][34][35][36]. However, most of these in vitro studies were done using planktonic in vitro cultures, unlike the present study, carried out with an in vitro multispecies biofilm model. This difference may be relevant since not only translates closer the real bacterial growth in the oral cavity, but also demonstrates the antibacterial effect in an environment where these bacteria are more tolerant against antimicrobial agents. Another reason for choosing this in vitro biofilm model was to test the potential of the tested bacteria to adhere to oral surfaces, which can only be mimicked in such a model. Finally, the role of dental biofilms in the etiology of caries and periodontal diseases justifies the use of the present validated in vitro biofilm model [30], which was modified by changing the early colonizer S. mutans instead of S. oralis, since S. mutans has demonstrated a stronger association with both the onset and progression of dental caries [37][38][39].
When assessing the anti-biofilm capacity of S. downii sp. nov., an indigenous species of the oral cavity in subjects with Down syndrome, it was observed that it was able to effectively colonize the in vitro developed biofilms, without interfering with its structure and bacterial composition, since it did not affect the kinetics of colonization of secondary colonizers (V. parvula and A. naeslundii) or periodontal pathogens (F. nucleatum, A. actinomycetemcomitans, and P. gingivalis). However, its presence significantly interfered with the colonization kinetics of S. mutans, suggesting that the presence S. downii sp. nov. may control S. mutans levels, thus promoting health compatible biofilms or modifying their pathogenic potential [40].
Our findings agree with previous publications, which describe that some bacterial species, closely related to S. downii sp. nov., and isolated from caries-free subjects, exhibited antibacterial capacity against S. mutans. Bao et al. and Tong et al. [32,33] reported the inhibition of S. mutans by Streptococcus oligofermentans, due to the production of hydrogen peroxide, while Ogawa et al. [34], observed that Streptococcus salivarius inhibited the development of S. mutans biofilms, via bacteriocin production. More recently, the antibacterial capacity of Streptococcus A12 [35] and S. dentisani [22] against S. mutans was also described. Other studies reported that exogenous bacteria from the oral cavity could affect S. mutans, such as Bifidobacterium spp. isolated from human intestine [41], Lactobacillus reuteri strains, which were able to inhibit the growth of S. mutans in different degrees [42] or different Lactobacillus spp., which exhibits a notable degree of antagonism against S. mutans [41,43].
In regard to the ability to inhibit the formation and/or biofilm growth, as observed in the present research, this has been documented for other oral exogenous bacteria, including Enterococcus faecium WB2000, Bifidobacterium adolescentis SPM1005, or heat-inactivated Bifidobacterium BB12 [44][45][46]. Söderling et al. [47] observed that the probiotic strains L. reuteri SD2112 and L. reuteri PTA5289, Lactobacillus rhamnosus GG, and Lactobacillus plantarum 229v inhibited S. mutans biofilm formation on glass surfaces. Marttinem et al. [48] confirmed that L. reuteri ATCC PTA5289 could interfere with the adhesion of S. mutans to HA discs and inhibited biofilm formation. Lin et al. [49] refereed to the ability to inhibit S. mutans formation and biofilm growth on glass surfaces of other Lactobacillus strains, including L. rhamnosus HN001, L. plantarum ST-III, Lactobacillus casei strain Shirota, L. casei LC01, and Lactobacillus paracasei Lpc-37.
When assessing the antibacterial capacity of S. downii sp. nov. against a well-structured biofilm, this bacterial species was able to induced a significant lower concentrations of S. mutans (p = 0.019) and A. actinomycetemcomitans (p = 0.020), significant higher concentrations of A. naeslundii (p = 0.003) and prevented a statistically significant increment for P. gingivalis and V. parvula (p = 0.395 and p=0.295, respectively), compared to non-exposed biofilms. These results may suggest a potential probiotic effect of S. downii sp. nov. in the maintenance of oral homeostasis, decreasing the amounts of potential pathogenic species, and stimulating the growth of health-associated, potentially beneficial bacteria, such as A. naeslundii.
In periodontitis, both indigenous and exogenous bacterial strains have been in vivo and in vitro assessed, testing the hypothesis that they could help in the suppression of periodontal pathogens by competitive exclusion mechanisms and/or via the production of antimicrobial substances [28,36,50]. In vitro studies have shown positive results for different endogenous bacterial strains. S. dentisani showing inhibition of growth of periodontal pathogens and other pathogens implicated in root canals infections by the production of antimicrobial substances, although the study was conducted in planktonic in vitro cultures [22]. S. mitis demonstrated a successful antagonism in the adhesion of P. gingivalis [23], and also reduced the growth of P. gingivalis in presence of salivary bifidobacteria by reducing growth factor(s) from the environment [26]. Among exogenous strains, in vitro viability of A. actinomycetemcomitans was affected by human intestinal Bifidobacterium spp. [41]. Bdellovibrio bacteriovorus has been shown to significantly reduce the number of viable A. actinomycetemcomitans, both in planktonic and mono-species in vitro biofilm cultures [51]. However, it was observed that the efficiency decreased as the complexity of the model increased, i.e., with a model including six bacterial species (P. intermedia, A. actinomycetemcomitans, P. gingivalis, F. nucleatum, S. mitis, and A. naeslundii) [52].
This study presents clear limitations: an in vitro biofilm model consisting of six bacterial species has been used, and the effects of S. downii sp. nov. could be less evident in more diverse biofilms, closer to in vivo conditions; the application of the present findings could be limited due if there were relevant differences in the oral microbiota among Down syndrome subjects and other systemically healthy individuals and/or periodontitis patients [53,54], in whom S. downii sp. nov. may exhibit less capacity to colonize oral biofilms in vivo. The selected biofilm model consists in a common consortium of bacteria, including commensal bacteria and common periodontal pathogens strongly associated with periodontitis and caries, and the present study has shown, for the first time, antibacterial and antibiofilm activities of the tested novel species; thus, more studies are granted to better understand the mechanisms of action behind these findings.

Conclusions
In summary, the results of the present study have shown that an indigenous bacterial strain, S. downii sp. nov., had a moderate impact in oral biofilm formation and composition, as tested in a validated multi-species in vitro biofilm model. Firstly, it was shown that it was able to colonize the formed biofilms; secondly, and although the structural development was not affected by the presence of S. downii sp. nov., significantly lower amounts of A. actinomycetemcomitans (a periodontal pathogen, strongly associated with periodontitis) and of S. mutans (strongly associated with caries) were incorporated in the biofilms, in the presence of S. downii sp. nov. The present results, together with the previous findings showing αand β-galactosidase activities of S. downii sp. nov. [29], which indicates that S. downii sp. nov. can use several saccharides as carbon source, including lactose, represents an advantage for its potential application as an oral probiotic [55].  Data Availability Statement: Data available on request due to restrictions. The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.