Oral Biofilm Composition, Dissemination to Keratinocytes, and Inflammatory Attenuation Depend on Probiotic and Synbiotic Strain Specificity.

Several inflammatory diseases are characterized by a disruption in the equilibrium between the host and its microbiome. Due to the increase in resistance, the use of antibiotics for the widespread, nonspecific killing of microorganisms is at risk. Pro-microbial approaches focused on stimulating or introducing beneficial species antagonistic toward pathobionts may be a viable alternative for restoring the host-microbiome equilibrium. Unfortunately, not all potential probiotic or synbiotic species and even subspecies (to strain level) are equally effective for the designated pathology, leading to conflicting accounts of their efficacy. To assess the extent of these species- and strain-specific effects, 13 probiotic candidates were evaluated for their probiotic and synbiotic potential with glycerol on in vitro oral biofilms, dissemination from biofilms to keratinocytes, and anti-inflammatory activity. Species- and strain-specific effects and efficacies were observed in how they functioned as probiotics or synbiotics by influencing oral pathobionts and commensals within biofilms and affected the dissemination of pathobionts to keratinocytes, ranging from ineffective strains to strains that reduced pathobionts by 3 + log. In addition, a minority of the candidates exhibited the ability to mitigate the inflammatory response of LPS-stimulated monocytes. For a comprehensive assessment of probiotic therapy for oral health, a judicious selection of fully characterized probiotic strains that are specifically tailored to the designated pathology is required. This approach aims to challenge the prevailing perception of probiotics, shifting the focus away from "form over function." Rather than using unproven, hypothetical probiotic strains from known genera or species, one should choose strains that are actually functional in resolving the desired pathology before labelling them probiotics.

pH-Triggered Controlled Release of Chlorhexidine Using Chitosan-Coated Titanium Silica Composite for Dental Infection Prevention.

Peri-implantitis is a growing pathological concern for dental implants which aggravates the occurrence of revision surgeries. This increases the burden on both hospitals and the patients themselves. Research is now focused on the development of materials and accompanying implants designed to resist biofilm formation. To enhance this endeavor, a smart method of biofilm inhibition coupled with limiting toxicity to the host cells is crucial. Therefore, this research aims to establish a proof-of-concept for the pH-triggered release of chlorhexidine (CHX), an antiseptic commonly used in mouth rinses, from a titanium (Ti) substrate to inhibit biofilm formation on its surface. To this end, a macroporous Ti matrix is filled with mesoporous silica (together referred to as Ti/SiO2), which acts as a diffusion barrier for CHX from the CHX feed side to the release side. To limit release to acidic conditions, the release side of Ti/SiO2 is coated with crosslinked chitosan (CS), a pH-responsive and antimicrobial natural polymer. Scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM/EDX) and Fourier transform infrared (FTIR) spectroscopy confirmed successful CS film formation and crosslinking on the Ti/SiO2 disks. The presence of the CS coating reduced CHX release by 33% as compared to non-coated Ti/SiO2 disks, thus reducing the antiseptic exposure to the environment in normal conditions. Simultaneous differential scanning calorimetry and thermogravimetric analyzer (SDT) results highlighted the thermal stability of the crosslinked CS films. Quartz crystal microbalance with dissipation monitoring (QCM-D) indicated a clear pH response for crosslinked CS coatings in an acidic medium. This pH response also influenced CHX release through a Ti/SiO2/CS disk where the CHX release was higher than the average trend in the neutral medium. Finally, the antimicrobial study revealed a significant reduction in biofilm formation for the CS-coated samples compared to the control sample using viability quantitative polymerase chain reaction (v-qPCR) measurements, which were also corroborated using SEM imaging. Overall, this study investigates the smart triggered release of pharmaceutical agents aimed at inhibiting biofilm formation, with potential applicability to implant-like structures.

Probiotics for oral health: do they deliver what they promise?

Probiotics have demonstrated oral health benefits by influencing the microbiome and the host. Although promising, their current use is potentially constrained by several restrictions. One such limiting factor lies in the prevailing preparation of a probiotic product. To commercialize the probiotic, a shelf stable product is achieved by temporarily inactivating the live probiotic through drying or freeze drying. Even though a lyophilized probiotic can be kept dormant for an extended period of time, their viability can be severely compromised, making their designation as probiotics questionable. Additionally, does the application of an inactive probiotic directly into the oral cavity make sense? While the dormancy may allow for survival on its way towards the gut, does it affect their capacity for oral colonisation? To evaluate this, 21 probiotic product for oral health were analysed for the number of viable (probiotic), culturable (CFU) and dead (postbiotic) cells, to verify whether the commercial products indeed contain what they proclaim. After isolating and uniformly lyophilizing three common probiotic species in a simple yet effective lyoprotective medium, the adhesion to saliva covered hydroxyapatite discs of lyophilized probiotics was compared to fresh or reactivated lyophilized probiotics. Unfortunately, many of the examined products failed to contain the claimed amounts of viable cells, but also the strains used were inadequately characterized and lacked clinical evidence for that unknown strain, questioning their label of a 'probiotic'. Additionally, lyophilized probiotics demonstrated low adhesive capacity compared to their counterparts, prompting the question of why fresh or reactivated probiotics are not currently used.

Mode of killing determines the necrotrophic response of oral bacteria.

Background: Bacteria respond to changes in their environment, such as nutrient depletion and antimicrobials exposure. Antimicrobials result not only in bacterial death, but also have a hand in determining species abundances and ecology of the oral biofilms. Proximity of dead bacterial cells to living ones is an important environmental change or stress factor. Dead bacteria represent high concentrations of nutrients, such as proteins, lipids, sugars, and nucleic acids. Living bacteria can use these biomasses as a nutrients source, which is termed necrotrophy. Aim: This study investigates the effect of exposing living oral bacteria (planktonic and biofilms) to their dead siblings after being killed by heat or hydrogen peroxide. Results: Tested bacterial species showed different responses towards the dead cells, depending on the mode of killing, the nutritional value of the culture media, and the the dead cells density. The multispecies oral biofilms showed different responses towards the supplementation of dead cells during biofilm development, while matured biofilms were more resilient. Conclusion: This study indicates that dead bacteria resulting from antiseptics use may imbalance the nutrient availability in the oral cavity, resulting in overgrowth of opportunistic species, and hence ecological changes in oral communities, or introducing new bacterial phenotypes.

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

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.

Glycerol strengthens probiotic effect of Limosilactobacillus reuteri in oral biofilms: A synergistic synbiotic approach.

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.

Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro oral biofilms.

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.

Comparison of the modulatory effects of three structurally similar potential prebiotic substrates on an in vitro multi-species oral biofilm.

Previous research identified potential prebiotic substrates for oral health like the structural analogues N-acetyl-D-mannosamine (NADM) and N-acetyl-D-glucosamine (NADG). The main hypothesis of the current study was twofold. Firstly, it was hypothesized that the modulatory effects of NADM are not limited to changes in multi-species oral biofilm composition, but also include effects on metabolism, virulence, and inflammatory potential. Secondly, the presence and orientation of their N-acetyl group could play a role. Therefore, a comparison was made between the effects of NADM, NADG and D-(+)-mannose on multi-species oral biofilms. Besides a beneficial compositional shift, NADM-treated biofilms also showed an altered metabolism, a reduced virulence and a decreased inflammatory potential. At a substrate concentration of 1 M, these effects were pronounced for all biofilm aspects, whereas at ~ 0.05 M (1%(w/v)) only the effects on virulence were pronounced. When comparing between substrates, both the presence and orientation of the N-acetyl group played a role. However, this was generally only at 1 M and dependent on the biofilm aspect. Overall, NADM was found to have different effects at two concentrations that beneficially modulate in vitro multi-species oral biofilm composition, metabolism, virulence and inflammatory potential. The presence and orientation of the N-acetyl group influenced these effects.

Potential prebiotic substrates modulate composition, metabolism, virulence and inflammatory potential of an in vitro multi-species oral biofilm.

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.

A Viability Quantitative PCR Dilemma: Are Longer Amplicons Better?

The development of viability quantitative PCR (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 to 906 base pairs [bp]) was used on live and killed cells of nine bacterial species treated with a viability dye (propidium monoazide [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 the Cq difference but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in Cq differences was observed. IMPORTANCE Viability quantitative PCR (v-qPCR) has evolved into a valuable, mainstream technique for determining the number of viable microorganisms 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.