Novel Options to Counteract Oral Biofilm Formation: In Vitro Evidence

Biofilm production on biotic and abiotic surfaces is crucial in the pathogenesis of most infections, particularly those occurring in the oral cavity. Its prevention and/or control may greatly facilitate the management of patients with oral diseases. Here, the antibiofilm activity of a biomimetic hydroxyapatite and a natural compound, MicroRepair (MicroR) and pomegranate (PomeGr), respectively, was assessed. By luminescence/fluorescence-based assays, Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were tested for biofilm production in the presence of MicroR and/or PomeGr. We found that both MicroR and PomeGr could affected biofilm production; however, the efficacy of the two, given alone or in combination, varied according to the microbial agent considered. These data open to clinical studies aimed at defining the most efficacious protocols to counteract oral biofilm-associated infections.


Introduction
The oral cavity is described as a highly complex habitat, involving resident microbial communities that play a key role in local homeostasis. Various parameters, such as poor oral hygiene, dental appliances, diet, drugs, systemic diseases, etc., are also known to influence a health condition [1]. Alterations in oral homeostasis may allow opportunistic pathogens to locally predominate and then organize themselves into microbial biofilms. This homeostatic imbalance leads to frequent oral diseases, such as caries, periodontitis, mucositis, etc. [1].
MicroRepair (MicroR) is a recently described biomimetic compound made of carbonatehydroxyapatite-zinc crystals; thanks to its biomimetic properties, it is able to interact with tooth hydroxyapatite, favoring enamel remineralization [2]. Moreover, due to its presence in Zn 2+ ions, MicroR is expected to mediate direct antimicrobial effects. Accordingly, an in vitro study has recently demonstrated the efficacy of MicroR to counteract the adhesion and persistence of oral bacteria onto orthodontic elastics [3].
The dramatic increase in drug-resistance, mostly due to improper use of antibiotics [4,5], has encouraged research into alternative drugs, such as natural compounds, including in the field of dentistry. In this respect, antimicrobial agents such as herbs, spices, and probiotics demonstrate effective properties against dental diseases [6]. In particular, pomegranate, the fruit of Punica granatum L. (PomeGr), has received significant attention due to its high content of phenolic compounds, likely mediating antimicrobial effects against oral pathogens [7,8].
Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) are opportunistic pathogens responsible of different clinical conditions involving the oral cavity [9]. Due to their strong ability to adhere and persist on abiotic and biotic surfaces, they often cause biofilm-associated infections, which are clinically relevant and difficult-to-treat [10]. In particular, P. aeruginosa is responsible for apical periodontitis, pulp necrosis, pulpitis or mandibular/maxillary alveolitis [11] and its organization as a biofilm causes a 4-fold enhancement in drug resistance [12]. Additionally, the high prevalence of S. aureus has been described in plaque and tongue samples from systemically healthy subjects with periodontal health, gingivitis or chronic periodontitis [13]. In addition to being a systemic opportunistic pathogen, C. albicans also influence mucosal bacterial microbiome at the oral and intestinal mucosa level [14].
Here, we investigated the effects of MicroR and PomeGr, either alone or in combination, on biofilm production by P. aeruginosa, S. aureus and C. albicans, as widely used prototypes of Gram-negative, Gram-positive and fungal pathogens. Using in vitro models employing ductile and sensitive luminescence and fluorescence-based assays [15,16], the antibiofilm activity of MicroR and PomeGr has been established.

Compounds
The biomimetic hydroxyapatite (HA) MicroR and the PomeGr peel extract were supplied by Coswell S.p.A. (Bologna, Italy); the former was a commercially available product, while the latter had been supplied as a suspension containing PomeGr peel extract, Saccharomyces ferment lysate filtrate, citric acid, sodium benzoate and potassium sorbate. The MicroR was sterilized by filtration and the PomeGr was treated by autoclave prior to being used in the assays described below.

Microbial Strains and Growth Condition
The bioluminescent bacterial strains, P. aeruginosa (strain P1242) and S. aureus (Xen29) and the fluorescent fungal strain (GFP-tagged strain derived from C. albicans SC5314) were used. According to previously detailed protocols [11], the viable bacterial or fungal cells constitutively emitted bioluminescent or fluorescent signals that could be recorded and quantified by a Fluoroskan reader (Thermo Fischer Scientific, Waltham, MA, USA). Such values, expressed as Relative Luminescence Units (RLU for bacterial cells) or Relative Fluorescence Units (RFU for fungal cells), allowed us to directly measure the amounts of viable microorganisms present in the control and experimental groups.
Operationally, in line with other studies [16], bacterial and fungal cells from −80 • C glycerol stocks were initially seeded onto Tryptic Soy Agar (TSA) or Sabouraud Dextrose Agar (SAB) (OXOID, Milan, Italy) plates, respectively, and incubated overnight at 37 • C. Then, isolated colonies were collected, added to 10 mL of Tryptic Soy Broth (TSB) or Sabouraud broth (OXOID, Milan, Italy) and allowed to grow overnight at 37 • C under gentle shaking, prior to being used for biofilm production.

Assessment of MicroR and PomeGr Effects on Microbial Biofilm Formation
According to previously described protocols [15,16], overnight bacterial or fungal cultures were diluted by the appropriate medium and seeded in 96 black well-plates (10 6 /mL; 100 µL/well); 100 µL of medium (untreated), MicroR or PomeGr were added to each well (the compounds were tested either alone or in combination, at different doses). Then, the plates were incubated at 37 • C for 24 h. Thereafter, the samples were washed twice with phosphate-buffered saline (PBS) to remove the planktonic cells and the bioluminescence or fluorescent signal/well was measured and expressed as RLU (bacterial biofilm) or RFU (fungal biofilm); these values represented the amounts of biofilm produced, under the different experimental conditions.

Statistical Analysis
The Shapiro-Wilk test was used to analyze the distribution of the data within each experimental group. Differences between groups were analyzed by the Kruskal-Wallis's test, followed by uncorrected Dunn's multiple comparisons test. Statistical analysis was performed by using GraphPad Prism 8 software. Values of p ≤ 0.05 were considered statistically significant (Family-wise significance and Confidence level, 0.05).

Antibiofilm Efficacy of MicroR and/or PomeGr
In the present study, MicroR and PomeGr, either alone or in combination, have been investigated for their effects against biofilm production by three major prototypes Gramnegative and Gram-positive bacterial cell and fungal cell pathogens, namely P. aeruginosa, S. aureus and C. albicans. Preliminarily, the antimicrobial activity of MicroR and PomeGr was evaluated against planktonic cells, employing the RLU/RFU assay to assess the Mi-croR and the standard CLSI/NCCLS micro-broth dilution assay to assess the PomeGr. The microbial cells, P. aeruginosa, S. aureus or C. albicans, were exposed to serial dilutions of each compound for 24 h and their Minimal Inhibitory Concentration (MIC) was established. The MIC values were 2.8% (PomeGr) and 2.35 mg/mL (MicroR), irrespective of the microorganism assessed (data not shown). Based on these results, the 2xMIC conditions, namely MicroR at 4.7 mg/mL and PomeGr at 5.6%, were tested hereafter for their effects against biofilm. In particular, P. aeruginosa, S. aureus and C. albicans (10 6 /mL) were exposed to MicroR and/or PomeGr for 24 h at 37 • C in order to allow biofilm formation [14,15]. After washing, the luminescence or fluoresce signals were detected as a measure of biofilm was produced. Figure 1 shows that microbial exposure to PomeGr resulted in a drastic decrease in P. aeruginosa biofilm, as assessed by RLU measurement; a partial but non-significant reduction was observed in S. aureus, while a significant impairment occurred in C. albicans exposed to PomeGr. The MicroR treatment reduced biofilm formation in all the strains, especially in C. albicans, where this reduction was highly significant (p < 0.0001). The microorganisms (10 6 /mL) were exposed to MicroR (4.7 mg/mL) or PomeGr (5.6%) or both and incubated at 37 • C for 24 h; then, biofilm was quantified by luminescence or fluorescence analysis, as detailed elsewhere. The data are the mean ± standard deviation of 6 to 9 determinations from 3 to 4 experiments. Statistical analysis was performed by using GraphPad Prism 8 software. The asterisks indicate statistically significant differences of medium vs. treated samples (* p = 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001).
The combination of MicroR and PomeGr significantly reduced biofilm both in S. aureus and C. albicans, while in P. aeruginosa, the RLUs were unexpectedly higher than those obtained with PomeGr alone (Figure 1).
When these data were expressed as a percent of biofilm decrease (Table 1), we found that, upon exposure to PomeGr, P. aeruginosa was inhibited down to 98%, while S. aureus and C. albicans biofilm were reduced to 37.5% and 41%, respectively. When exposed to MicroR alone, P. aeruginosa, S. aureus, and C. albicans showed a biofilm reduction ranging from 43% to 91%; yet, only in C. albicans was a decrease found to be significant. Finally, when the strains were exposed to PomeGr plus MicroR, the biofilm was reduced between 68% to 93%, with a partial additive effect observed only in C. albicans. The percent of biofilm decrease (treated and untreated samples) was calculated using the values depicted in Figure 1.

Discussion
Notoriously, the composition and structure of biomimetic HA are similar to biological systems such as human hard tissues, therefore it is able to integrate into host structures without altering or dissolving its own or neighboring elements. Several studies have shown the possibility of replacing some ions within HA to strengthen its properties; for example, the replacement of Ca 2+ with Zn 2+ significantly increases the antimicrobial properties of such biomimetic material [2]. Nowadays, HA is used in dental practice as it has been added to the new formulations of oral hygiene products (mouthwashes, toothpastes, etc.) [2,3]. Here, we provide the first evidence that MicroR, a biomimetic HA, acts as a anti-biofilm agent. In particular, MicroR was capable to reduce P. aeruginosa and S. aureus biofilm, as measured by the luminescence assay; however, such a decrease did not reach statistical significance. On the other hand, the reduction observed in MicroR-treated C. albicans returned a p < 0.001; when these variations were expressed as a percentage of biofilm decrease, we obtained 43% for P. aeruginosa, 91% for S. aureus and 72% for C. albicans. Using other doses of MicroR or PomeGr, we failed to detect better results (data not shown).
Furthermore, the combination of the two, MicroR and PomeGr, provided heterogeneous and somehow unexpected results. Indeed, intermediate levels of biofilm inhibition were observed with P. aeruginosa, suggesting that MicroR might partially mask the elevated anti-P. aeruginosa biofilm efficacy observed with PomeGr alone. Differently, the combination MicroR and PomeGr exerted anti-S. aureus effects that reached statistically significant values. Finally, when used together against C. albicans, MicroR and PomeGr clearly showed additive effects as inhibitors of fungal biofilm formation. The heterogeneous trends observed with the three microbial agents remain unexplained. Undoubtedly, major differences in the composition and structure of the three microbial agents assessed partially explain the results obtained.
Additionally, we may envisage that MicroR and PomeGr might have differently affected the secretory profile of P. aeruginosa, S. aureus and C. albicans, which are known to release profoundly different sets of crucial molecules, including auto-inducers [15], to produce and establish their biofilm. In any case, by novel in vitro assays, we provide the first evidence that microR and PomeGr exert antibiofilm activity and that their action may be additive. Thus, although there are limitations to any in vitro model, this work opens the door to further studies on the antibiofilm activity of MicroR and PomeGr against clinical isolates and, even more, multispecies microbial biofilms, including cariogenic bacterial species [17], that commonly afflict the oral cavity.

Conclusions
By means of an in vitro model, the antibiofilm activity of a biomimetic hydroxyapatite, MicroRepair, and a natural compound, pomegranate, has been established. Given the crucial role of biofilm formation in the pathogenesis of many oral diseases, these data offer a basic rationale for the design of trials aimed at maintaining health conditions or recovering homeostatic imbalance in the oral cavity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.