Necrotrophic growth of periodontopathogens is a novel virulence factor in oral biofilms

The oral use of antimicrobial agents embedded in toothpastes and mouth rinses results in an oral microbial massacre with high amounts of dead bacteria in close proximity to few surviving bacteria. It was hypothesized that this provides the surviving pathogenic bacteria a large amount of dead microbial biomass as a nutritional source for growth (necrotrophy). This study demonstrated the necrotrophic growth of periodontal pathogens in the presence of different dead oral species. In addition, the presence of dead bacteria resulted in an outgrowth of several periodontal pathogens in complex multispecies biofilms. Additionally, upon contact with dead oral bacteria, virulence genes of P. intermedia and P. gingivalis were up-regulated (necrovirulence). This resulted in a more pronounced epithelial cytotoxicity (necrotoxicity). These findings indicate that presence of dead bacteria induce necrotrophy, necrovirulence and necrotoxicity in several oral pathogens

Microbial protein out of thin air : fixation of nitrogen gas by an autotrophic hydrogen-oxidizing bacterial enrichment

For the production of edible microbial protein (MP), ammonia generated by the Haber-Bosch process or reclaimed ammonia from waste streams is typically considered as the nitrogen source. These processes for ammonia production are highly energy intensive. In this study, the potential for using nitrogen gas (N-2) as a direct nitrogen source for MP production by hydrogen-oxidizing bacteria (HOB) was evaluated. The use of N-2 versus ammonium as nitrogen source during the enrichment process resulted in differentiation of the bacterial community composition of the enrichments. A few previously unknown potential N-2-fixing HOB taxa (i.e., representatives of the genus Azonexus and the family Comamonadaceae) dominated the enrichments. The biomass yield of a N-2-fixing HOB enrichment was 30-50% lower than that of the ammonium-based HOB enrichment from the same inoculum source. The dried biomass of N-2-fixing HOB had a high protein content (62.0 +/- 6.3%) and an essential amino acid profile comparable to MP from ammonium-based HOB. MP from N-2-fixing HOB could potentially be produced in situ without entailing the emissions caused by ammonia production and transportation by conventional means. It could be a promising substitute for N-2-fixing protein-rich soybean because it has 70% higher protein content and double energy conversion efficiency from solar energy to biomass

Dysbiosis by neutralizing commensal mediated inhibition of pathobionts

Dysbiosis in the periodontal microbiota is associated with the development of periodontal diseases. Little is known about the initiation of dysbiosis. It was hypothesized that some commensal bacteria suppress the outgrowth of pathobionts by H2O2 production. However, serum and blood components released due to inflammation can neutralize this suppressive effect, leading to the initiation of dysbiosis. Agar plate, dual-species and multi-species ecology experiments showed that H2O2 production by commensal bacteria decreases pathobiont growth and colonization. Peroxidase and blood components neutralize this inhibitory effect primarily by an exogenous peroxidase activity without stimulating growth and biofilm formation of pathobionts directly. In multi-species environments, neutralization of H2O2 resulted in 2 to 3 log increases in pathobionts, a hallmark for dysbiosis. Our data show that in oral biofilms, commensal species suppress the amounts of pathobionts by H2O2 production. Inflammation can neutralize this effect and thereby initiates dysbiosis by allowing the outgrowth of pathobionts

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

An Alternative Approach to Non-Log-Linear Thermal Microbial Inactivation: Modelling the Number of Log Cycles Reduction with Respect to Temperature

A mathematical approach incorporating the shoulder effect during the quantification of microbial heat inactivation is being developed based on »the number of log cycles of reduction « concept. Hereto, the heat resistance of Escherichia coli K12 in BHI broth has been quantitatively determined in a generic and accurate way by defining the time t for x log reductions in the microbial population, i.e. txD, as a function of the treatment temperature T. Survival data of the examined microorganism are collected in a range of temperatures between 52–60.6 °C. Shoulder length Sl and specific inactivation rate kmax are derived from a mathematical expression that describes a non-log-linear behaviour. The temperature dependencies of Sl and kmax are used for structuring the txD(T) function. Estimation of the txD(T) parameters through a global identification procedure permits reliable predictions of the time to achieve a pre-decided microbial reduction. One of the parameters of the txD(T) function is proposed as »the reference minimum temperature for inactivation«. For the case study considered, a value of 51.80 °C (with a standard error, SE, of 3.47) was identified. Finally, the time to achieve commercial sterilization and pasteurization for the product at hand, i.e. BHI broth, was found to be 11.70 s (SE=5.22), and 5.10 min (SE=1.22), respectively. Accounting for the uncertainty (based on the 90 % confidence intervals, CI) a fail-safe treatment of these two processes takes 20.36 s and 7.12 min, respectively

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