Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that HO production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.

There is evidence that pathogenic bacteria can adapt to antiseptics upon repeated exposure. More alarming is the concomitant increase in antibiotic resistance that has been described for some pathogens. Unfortunately, effects of adaptation and cross-adaptation are hardly known for oral pathogens, which are very frequently exposed to antiseptics. Therefore, this study aimed to determine the in vitro increase in minimum inhibitory concentrations (MICs) in oral pathogens after repeated exposure to chlorhexidine or cetylpyridinium chloride, to examine if (cross-)adaptation to antiseptics/antibiotics occurs, if (cross-)adaptation is reversible and what the potential underlying mechanisms are. When the pathogens were exposed to antiseptics, their MICs significantly increased. This increase was in general at least partially conserved after regrowth without antiseptics. Some of the adapted species also showed cross-adaptation, as shown by increased MICs of antibiotics and the other antiseptic. In most antiseptic-adapted bacteria, cell-surface hydrophobicity was increased and mass-spectrometry analysis revealed changes in expression of proteins involved in a wide range of functional domains. These in vitro data shows the adaptation and cross-adaptation of oral pathogens to antiseptics and antibiotics. This was related to changes in cell surface hydrophobicity and in expression of proteins involved in membrane transport, virulence, oxidative stress protection and metabolism.

Peri-implantitis (PI) is an inflammatory disease of peri-implant tissues, it represents the most frequent complication of dental implants. Evidence revealed that microorganisms play the chief role in causing PI. The purpose of our study is to evaluate the cleaning of contaminated dental implant surfaces by means of the Q-switch Nd:YAG (Neodymium-doped Yttrium Aluminum Garnet) laser and an increase in temperature at lased implant surfaces during the cleaning process. Seventy-eight implants (titanium grade 4) were used (Euroteknika, Sallanches, France). Thirty-six sterile implants and forty-two contaminated implants were collected from failed clinical implants for different reasons, independent from the study. Thirty-six contaminated implants were partially irradiated by Q-switch Nd:YAG laser (1064 nm). Six other contaminated implants were used for temperature rise evaluation. All laser irradiations were calibrated by means of a powermetter in order to evaluate the effective delivered energy. The irradiation conditions delivered per pulse on the target were effectively: energy density per pulse of 0.597 J/cm2, pick powers density of 56 mW/cm2, 270 mW per pulse with a spot diameter of 2.4 mm, and with repetition rate of 10 Hz for pulse duration of 6 ns. Irradiation was performed during a total time of 2 s in a non-contact mode at a distance of 0.5 mm from implant surfaces. The parameters were chosen according to the results of a theoretical modeling calculation of the Nd:YAG laser fluency on implant surface. Evaluation of contaminants removal showed that the cleaning of the irradiated implant surfaces was statistically similar to those of sterile implants (p-value ≤ 0.05). SEM analysis confirmed that our parameters did not alter the lased surfaces. The increase in temperature generated at lased implant surfaces during cleaning was below 1 °C. According to our findings, Q-switch Nd:YAG laser with short pulse duration in nanoseconds is able to significantly clean contaminated implant surfaces. Irradiation parameters used in our study can be considered safe for periodontal tissue.

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.

Commensal species suppress pathobionts by producing H O . Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to dysbiosis by allowing the outgrowth of pathobionts.

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.

(1) Background: The relatively high prevalence of peri-implantitis (PI) and the lack of a standard method for decontamination of the dental implant surface have pushed us to conduct further research in the field. Bacterial biofilms were found to play a primordial role in the etiology of PI. Therefore, the aim is to evaluate the efficacy of a laser-assisted elimination of biofilm protocol in the removal of a multi-species biofilm on titanium surfaces. (2) Methods: In total, 52 titanium discs (grade 4) were used. The study group consisted of 13 titanium disks contaminated with multi-species biofilms and subsequently irradiated with the laser (T + BF + L). The control groups consisted of the following types of titanium disks: 13 contaminated with multi-species biofilms (T + BF), 13 sterile and irradiated (T + L), 13 sterile and untreated (T). Q-Switch Nd:YAG laser Irradiation parameters were the following: energy density equal to 0.597 J/cm2 per pulse, power equal to 270 milliwatt per pulse, 2.4 mm of spot diameter, and 10 Hz repetition rate for pulse duration of six nanoseconds (ns). The laser irradiation was made during 2 s of total time in non-contact and at 0.5 mm away from the titanium disc surface. After treatment, presence of biofilms on the disks was evaluated by staining with crystal violet (CV), which was measured as optical density at six hundred thirty nm, and statistical analyses were done. (3) Results: the optical density values were 0.004 ± 0.004 for the study group T + BF + L, 0.120 ± 0.039 for group T + BF, 0.006 ± 0.003 for group T + L, and 0.007 ± 0.007 for group T. For the study group, laser treatment resulted in a total elimination of the biofilm, with mean values statistically significantly lower than those of contaminated titanium surfaces and similar to those of sterile titanium surfaces. (4) Conclusions: Our irradiation protocol provided a significant elimination of the multi-species biofilm on titanium surfaces. Laser treated titanium surfaces were biofilm-free, similar to the sterile ones.

Peri-implantitis (PI) is a relatively frequent pathology that compromises the overall survival of the dental implant. Adjunctive approaches for the conventional mechanical debridement are being suggested to optimize the treatment of PI. The goal of the study was the assessment of the disinfection potential of the Q-Switch Nd: YAG laser on contaminated titanium implant surfaces. A total of 72 sterile titanium discs were used and divided into three groups: 24 contaminated titanium discs treated with the laser (study Group L), 24 contaminated titanium discs with no treatment (control 1—Group C), and 24 sterile titanium discs with no treatment (control 2—Group S). Multi-species biofilm was used: Porphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus sobrinus, and Prevotella intermedia. Commensal bacteria were included also: Actinomyces naeslundii, Actinomyces viscosus, Streptococcus cristatus, Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, and Veillonella parvula. Parameters delivered per pulse on the targeted surfaces of the titanium discs were an energy density of 0.597 J/cm2 each pulse, a pulse power of 270 mW, a laser beam spot of 2.4 mm in diameter, and a rate of repetition of 10 Hertz (Hz) for a pulse duration of 6 nanoseconds (ns). The mode was no contact, and a distance of 500 micrometers was used with a total time of irradiation equal to 2 s (s). The collection of microbiological samples was made for all groups; colony-forming units (CFU) were identified by two different practitioners, and the average of their examinations was considered for each sample. The average of the TBC (CFU/mL) was calculated for each group. Values were 0.000 CFU/mL, 4767 CFU/mL, and 0.000 CFU/mL for Group L, Group C, and Group S, respectively. Therefore, the suggested treatment protocol was able to provoke a total disinfection of the contaminated titanium surfaces. A statistical difference was only found between Group L vs. Group C and between Group S vs. Group C. The difference was not significant between Group S and Group L. In conclusion, the present study confirmed that the Q-Switch Nd: YAG laser under our specific conditions can provide a total disinfection of the contaminated titanium surfaces.