The development of viability qPCR (v-qPCR) has allowed for a more accurate assessment of the viability of a microbial sample by limiting the amplification of DNA from dead cells. Although valuable, v-qPCR is not infallible. One of the most limiting factors for accurate live/dead distinction is the length of the qPCR amplicon used. However, no consensus or guidelines exist for selecting and designing amplicon lengths for optimal results. In this study, a wide range of incrementally increasing amplicon lengths (68-906 bp) was used on live and killed cells of nine bacterial species treated with viability dye (PMA). Increasing amplicon lengths up to approximately 200 bp resulted in increasing quantification cycle (Cq) differences between live and killed cells, while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased Cq difference, but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. Importance Viability qPCR (v-qPCR) has evolved to a valuable, mainstream technique for determining the number of viable micro-organisms in samples by qPCR. Amplicon length is known to be positively correlated with the ability to distinguish between live and dead bacteria but is negatively correlated with qPCR efficiency. This trade-off is often not taken into account and might have an impact on the accuracy of v-qPCR data. Currently there is no consensus on the optimal amplicon length. This paper provides methods to determine the optimal amplicon length and suggests an amplicon length range for optimal v-qPCR, taking into consideration the trade-off between qPCR efficiency and live-dead distinction.

Objective Chlorhexidine mouthrinses are marketed in different formulations. This study aimed at investigating qualitative and quantitative changes in in‐vitro multispecies oral biofilms, induced by different chlorhexidine‐containing mouthrinses. Background data Earlier studies comparing chlorhexidine mouthrinses are either clinical studies or in‐vitro studies assessing the antimicrobial efficacy of the mouthrinses. However, no clear investigations are available regarding ecological impact of different chlorhexidine formulations on in‐vitro multispecies oral biofilms after rinsing with different chlorhexidine formulations. Methods Nine commercially available chlorhexidine mouthrinses were selected. Multispecies oral communities (14 species) were grown for 48 h in a Biostat‐B Twin bioreactor. After that, they were used to develop biofilms on the surface of hydroxyapatite disks in 24‐well pates for 48 h. Biofilms were then rinsed once or multiple times with the corresponding mouthrinse. Biofilms were collected before starting the rinsing experiment and every 24 h for 3 days and vitality quantitative PCR was performed. The experiment was repeated 3 independent times on 3 different days and the results were analyzed using a linear mixed model. Results The mouthrinses provoked different effects in terms of change in total viable bacterial load (VBL), ecology, and community structure of the multispecies biofilms. There was no relation between chlorhexidine concentrations, presence, or absence of cetylpyridinium chloride and/or alcohol, and the observed effects. Some tested chlorhexidine mouthrinses (MC, HG, HH, and HI) strongly lowered the total VBL (≈1007 Geq/ml), but disrupted biofilm symbiosis (≥40% of the biofilms communities are pathobionts). On the other hand, other tested chlorhexidine mouthrinses (MD, ME, and HF) had limited impact on total VBL (≥1010 Geq/ml), but improved the biofilm ecology and community structure (≤10% of the biofilms communities are pathobionts). Conclusion Not all chlorhexidine mouthrinses have the same effect on oral biofilms. Their effect seems to be strongly product dependent and vary according to their compositions and formulations.

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.

Background : Modulation of the commensal oral microbiota constitutes a promising preventive/therapeutic approach in oral healthcare. The use of prebiotics for maintaining/restoring the health-associated homeostasis of the oral microbiota has become an important research topic. Aims : This study hypothesised that in vitro 14-species oral biofilms can be modulated by (in)direct stimulation of beneficial/commensal bacteria with new potential prebiotic substrates tested at 1 M and 1% (w/v) , resulting in more host-compatible biofilms with fewer pathogens, decreased virulence and less inflammatory potential. Methods : Established biofilms were repeatedly rinsed with N-acetyl-D-glucosamine, α-D-lactose, D-(+)-trehalose or D-(+)-raffinose at 1 M or 1% (w/v) . Biofilm composition, metabolic profile, virulence and inflammatory potential were eventually determined. Results : Repeated rinsing caused a shift towards a more health-associated microbiological composition, an altered metabolic profile, often downregulated virulence gene expression and decreased the inflammatory potential on oral keratinocytes. At 1 M, the substrates had pronounced effects on all biofilm aspects, whereas at 1% (w/v) they had a pronounced effect on virulence gene expression and a limited effect on inflammatory potential. Conclusion : Overall, this study identified four new potential prebiotic substrates that exhibit different modulatory effects at two different concentrations that cause in vitro multi-species oral biofilms to become more host-compatible.

Stem cell therapy might be a promising method to stimulate alveolar bone regeneration, which is currently a major clinical challenge. However, its therapeutic features largely depend on pretreatment and transplantation preparation. Herein, a novel biomimetic periodontal ligament transplantation composed of human periodontal ligament stem cells (hPDLSCs) pretreated with gold nanocomplexes (AuNCs) and embedded in a type-I collagen hydrogel scaffold is developed to protect alveolar bone from resorption. AuNCs are readily absorbed by primary hPDLSCs, with limited cytotoxicity, and promote osteogenic differentiation of hPDLSCs effectively in vitro. In addition, the AuNCs-induced hPDLSCs are encapsulated with type-I collagen hydrogel scaffold to mimic their native physiological niche, and then are transplanted into a rat model of alveolar bone resorption. Both micro-computed tomography (micro-CT) and immunohistochemical assays demonstrate that alveolar bone loss is significantly prevented. Furthermore, the underlying therapeutic mechanism is elucidated, in which transplantation-activated osteogenesis is associated with autophagy, which enables bone remodeling and regeneration. This study provides critical insight into the role of PDLSCs in bone homeostasis and proposes an innovative AuNCs-based strategy for stem cell therapy in bone regeneration.

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Oral cryotherapy is used in dentistry as a safe, simple, and low-cost treatment for a variety of oral lesions. It is well known for its ability to aid in the healing process. However, its effect on oral biofilms is unknown. As a result, the purpose of this study was to assess the effects of cryotherapy on in vitro oral biofilms. In vitro multispecies oral biofilms were grown on the surface of hydroxyapatite discs in symbiotic or dysbiotic states. CryoPen X+ was used to treat the biofilms, whereas untreated biofilms served as control. One set of biofilms was collected for study immediately after cryotherapy, whereas another group was reincubated for 24 h to permit biofilm recovery. Changes in biofilm structure were analyzed with a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM), while biofilm ecology and community compositional changes were analyzed with viability DNA extraction and quantitative polymerase chain reaction (v-qPCR) analysis. One cryo-cycle immediately reduced biofilm load by 0.2 to 0.4 log10 Geq/mL, which increased with additional treatment cycles. Although the bacterial load of the treated biofilms recovered to the same level as the control biofilms within 24 h, the CLSM detected structural alterations. Compositional alterations were also detected by SEM, corroborating the v-qPCR findings that showed ≈≤10% incidence of pathogenic species compared to nontreated biofilms that encompassed ≈45% and 13% pathogenic species in dysbiotic and symbiotic biofilms, respectively. Spray cryotherapy showed promising results in a novel conceptual approach to the control of oral biofilms. Acting selectively by targeting oral pathobionts and retaining commensals, spray cryotherapy could modify the ecology of in vitro oral biofilms to become more symbiotic and prevent the evolution of dysbiosis without the use of antiseptics/antimicrobials.

Both in vitro and in vivo studies have shown that the probiotic Limosilactobacillus reuteri can improve oral health. Limosilactobacillus reuteri species are known to produce the antimicrobial “reuterin” from glycerol. In order to further increase its antimicrobial activity, this study evaluated the effect of the combined use of glycerol and Limosilactobacillus reuteri (ATCC PTA 5289) in view of using a synergistic synbiotic over a probiotic. An antagonistic agar growth and a multispecies biofilm model showed that the antimicrobial potential of the probiotic was significantly enhanced against periodontal pathobionts and anaerobic commensals when supplemented with glycerol. Synbiotic biofilms also showed a significant reduction in inflammatory expression of human oral keratinocytes (HOK‐18A), but only when the keratinocytes were preincubated with the probiotic. Probiotic preincubation of keratinocytes or probiotic and synbiotic treatment of biofilms alone was insufficient to significantly reduce inflammatory expression. Overall, this study shows that combining glycerol with the probiotic L. reuteri into a synergistic synbiotic can greatly improve the effectiveness of the latter.