Development of antiseptic adaptation and cross-adapatation in selected oral pathogens in vitro

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

Evaluating the intrinsic capacity of oral bacteria to produce hydrogen peroxide (H2O2) in liquid cultures: Interference by bacterial growth media

This work highlights the issue of interference by growth media when measuring bacterial H2O2 production. H2O2 was shown to be stable in phosphate buffered saline (PBS) but not in growth media. The protocol used for evaluating the intrinsic capacity of oral streptococci to produce H2O2 was shown to be reliable

In vitro dysbiotic oral biofilms deregulate the host immune response

EUROBIOFILMS 2017 - 5th European Congress on Microbial BiofilmsAn imbalance in the periodontal microbiota and dysbiosis deregulate the host immune response, leading to chronic inflammation. Since little is k nown about the initiation of dysbiosis, it can be hypothesized that some commensal bacteria can suppress the outgrowth of pathobionts by H 2 O 2 production. However, serum and blood components released during inflammation can neutralize this suppressive effec t, leading to the initiation of dysbiosis. The aim of this study is to determine if the neutralizing effect of serum, hemoglobin and hemin on the inhibitory effect of the commensal bacteria on pathobiont growth is translated in a more pronounced immune res ponse. Bacterial quantitative PCR, expression analysis of bacterial virulence and cellular inflammatory genes and cytokine quantification by ELISA were performed to quantify the pathobiont outgrowth and to detect possible differences in inflammatory respon se after exposure of cell cultures to homeostatic or dysbiotic biofilms. Peroxidases, serum and blood components neutralized the inhibitory effect of H 2 O 2 by exogenous peroxidase activity, increasing the pathobiont outgrowth. Moreover, the addition of seru m, peroxidase and blood compounds upregulated the main virulence genes of P. gingivalis and P. intermedia in multi - species biofilms. Exposure of epithelial and fibroblast cultures to these dysbiotic biofilms increased the expression of IL6, IL1β, TNFα and MMP8, but especially of IL8. Moreover, higher amounts of IL8 were produced after the challenge with dysbiotic biofilms. Conversely, homeostatic and commensal biofilms had a minor inflammatory response at expression and protein level. Overall, serum, peroxi dases or blood compounds allowed the outgrowth of pathobionts and increased their virulence. Dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilmsinfo:eu-repo/semantics/publishedVersio

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, alpha-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

Q-Switch Nd:YAG Laser-Assisted Elimination of Multi-Species Biofilm on Titanium Surfaces.

peer reviewed(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

Controlling oral biofilms through modulation of the commensal oral microbiota : an alternative for antiseptics?

Modulation of the commensal oral microbiota is a promising preventive or therapeutic strategy to battle the initiation and progression of biofilm-related oral pathologies such as periodontal diseases. Although established classical therapies, based on mechanical removal of dental plaque and sometimes supplemented with the use of antimicrobials, have proven clinical and microbiological benefits, their limitations justify the exploitation of alternative approaches. For instance, there is an increasing awareness that the widespread use of antimicrobials in the oral cavity could lead to phenomena like antiseptic adaptation or resistance development. Furthermore, due to the often aspecific removal and killing of oral biofilms, it can be hypothesized that these interventions might disrupt the oral health-associated homeostatic relationships that exist within the oral microbiota and between the oral microbiota and host. Therefore, the focus shifted to pro-microbial strategies like the use of pro- and prebiotics to modulate the commensal oral microbiota and the host response. Especially the use of probiotics for oral health is nowadays well-established, and the associated microbiological (e.g. decreased abundances of periodontal pathogens) and clinical (e.g. reduced periodontal pocket depth and clinical attachment loss) benefits have been well-documented. Since the use of probiotics is based on the administration of live, exogenous microorganisms, this approach also comes with certain risks and limitations. To circumvent these, the nutritional and/or metabolic stimulation of the endogenous commensal oral microbiota by prebiotic substrates has been recently introduced in the field and forms an important research topic. Finally, exploitation of important features of the commensal microbiota, such as H2O2 production that plays an important role in shaping oral biofilm ecology, represents another modulatory approach increasingly gaining interest. In this PhD project, it is hypothesized that the pathogenicity of oral biofilms can be reduced by modulation of the commensal oral microbiota, whereas repeated exposure of pathogens to antiseptics increases their virulence and hence pathogenicity. This modulation could be achieved through (in)direct stimulation of commensal/beneficial bacteria with potential prebiotics and/or stimulation of endogenous H2O2 production. The main goal of this project is to increase the ‘host-compatibility’ of oral biofilms (reduced amounts of pathogens, less virulence and lower inflammatory potential) through emerging strategies as an alternative for the use of antimicrobials. Therefore, this PhD project aims to (1) determine the effects of repeated in vitro exposure to antiseptics on oral pathogens, (2) investigate the effects of previously identified and novel potential prebiotic substrates and substrates that might affect endogenous H2O2 production on several aspects of in vitro multi-species oral biofilms and (3) evaluate the effects of these substrates on planktonic and biofilm H2O2 production

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 (approximate to 10(07) 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 (>= 10(10) 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

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 by resulted in increasing quantification cycle (C-q) differences between live and killed cells while maintaining a good qPCR efficiency. Longer amplicon lengths, up to approximately 400 bp, further increased the C-q difference but at the cost of qPCR efficiency. Above 400 bp, no valuable increase in C-q 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

Development of antiseptic adaptation and cross-adapatation in selected oral pathogens in vitro

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.status: publishe