Antimicrobial potential of known and novel probiotics on in vitro periodontitis biofilms

Abstract

Several oral diseases are characterized by a shift within the oral microbiome towards a pathogenic, dysbiotic composition. Broad-spectrum antimicrobials are often part of patient care. However, because of the rising antibiotic resistance, alternatives are increasingly desirable. Alternatively, supplying beneficial species through probiotics is increasingly showing favorable results. Unfortunately, these probiotics are rarely evaluated comparatively. In this study, the in vitro effects of three known and three novel Lactobacillus strains, together with four novel Streptococcus salivarius strains were comparatively evaluated for antagonistic effects on proximal agar growth, antimicrobial properties of probiotic supernatant and the probiotic's effects on in vitro periodontal biofilms. Strain-specific effects were observed as differences in efficacy between genera and differences within genera. While some of the Lactobacillus candidates were able to reduce the periodontal pathobiont A. actinomycetemcomitans, the S. salivarius strains were not. However, the S. salivarius strains were more effective against periodontal pathobionts P. intermedia, P. gingivalis, and F. nucleatum. Vexingly, most of the Lactobacillus strains also negatively affected the prevalence of commensal species within the biofilms, while this was lower for S. salivarius strains. Both within lactobacilli and streptococci, some strains showed significantly more inhibition of the pathobionts, indicating the importance of proper strain selection. Additionally, some species showed reductions in non-target species, which can result in unexpected and unexplored effects on the whole microbiome.

Fig. 1. Antimicrobial properties of probiotic’s cell-free supernatant.

Flow cytometric quantification of antimicrobial properties of the cell-free supernatant (CFS) of the probiotic candidates (postbiotic) on the membrane permeability, as a proxy for viability, of periodontal pathobionts after 6 h of incubation in the postbiotic. Data presented as mean and standard deviation of log₁₀ of total events per mL (Log(Events/mL)) and percentage composition of the total divided into classes according to their permeability/viability: green = intact/live, red = permeable/dead and yellow = intermediate permeability/partially damaged membrane. Gates were defined according to the degree of red fluorescence through the entry of propidium iodide into healthy- and killed cultures (Supplementary Figs. 2 & 3). Statistical differences in remaining live cells compared to the control (n = 3, ANOVA with Tukey HSD) are depicted with an asterisk.

Fig. 2. Probiotic prevalence does not affect total biofilm counts.

Probiotic prevalence in biofilms (a), total cells in the biofilms (b) and both plotted against each other (c). Probiotic abundance and total biofilm are presented as a boxplot of the log₁₀ cells per milliliter (log(cells/mL)) recovered from the discs of each probiotic per experiment (Tukey style boxplot with median centre line, IQR at 1.5, upper and lower hinges at 25th and 75th percentiles and whiskers at 1.5 IQR from the hinges). No correlation between the prevalence of the probiotic and the total biofilm was observed. Statistical differences in probiotic prevalence between conditions can be found in Supplementary Table 2. For the total biofilms, statistical differences from the control are depicted by an asterisk and otherwise as n.s. (n = 9; nonparametric Kruskal-Wallis with Dunn’s test).

Fig. 3. Probiotic effects on oral bacteria in biofilms.

Changes in prevalence of (a) pathobionts and (b) commensals in multispecies biofilms caused by the addition of the probiotic candidates during biofilm formation. Relative changes in abundance are presented as a boxplot of the change in log₁₀ cells per milliliter (log(cells/mL)) from the control (Tukey style boxplot with median centre line, IQR at 1.5, upper and lower hinges at 25th and 75th percentiles and whiskers at 1.5 IQR from the hinges). n.s.: nonsignificant from control biofilm. Statistically significant differences from the control biofilm are depicted as an asterisk (n = 9, ANOVA with Tukey HSD).

Fig. 4. Probiotic effects on the proportional composition of the biofilms.

Combined data of Figs. 1 & 2 presented as pie charts of the percentage composition of these biofilms divided into probiotics, periodontal pathobionts, cariogenic pathobionts, anaerobic commensals and streptococcal commensals.