Metabolism-independent phenomenon in ethanolic propolis inhibitory capacity towards enterococcus spp proteolytic activity

Background : Root canal bacteria produce many virulence factors which are responsible for endodontic pathological states. Bacteria are assumed to utilize energy from bacterial cell metabolism activity for producing these virulence factors. Propolis extracts are commonly reported to have antibacterial abilities against dental pathogens. The purpose of this study is to investigate the possible correlation between bacterial proteolytic and metabolism activities under the treatment of ethanolic extract of propolis (EEP). Method : The 0.00125%; 0.05%; 0.1%; 0.2%; 0.4%; and 0.8% ethanolic propolis were prepared for recovery rate confirmative procedure, proteolytic, and metabolism activity assay, with 2% of chlorhexidine gluconate (CHX) was used as a positive control. The bacteria were cultured in brain heart infusion (BHI) media after EEP treatment. Bacterial suspension was initially prepared in broth culture dilution with BHI media, followed by the gelatin liquefaction measurement for proteolytic assay. Phenol-red and arginine dehydrogenase enriched media for observing both carbohydrate and arginine metabolism activities, respectively, in the clinical Enterococcus spp. and E. faecalis ATCC 29212. Result : The recovery rate of the bacteria was not terminated after several EEP treatments. Proteolytic activity of the bacteria was likely decreased in several EEP treatments. EEP tended to affect the carbohydrate and arginine metabolism of the bacteria in certain fashions. Conclusion : This study suggested that the EEP treatment affected both proteolytic and metabolism activity in negative regulation tendencies.


INTRODUCTION
Endodontic treatment is a challenging procedure due to the possibility for reinfection and developing periapical pathologic condition after the treatment process. This pathologic state could be affected by several factors such as chemical, iatrogenic error, filling problem, incomplete sterilizing procedure and persistent microorganisms (microbiome dysbiosis concern). 1,2 One of bacteria which is presumably found to be resistant against root canal medicaments is Enterococcus faecalis. 3,4 E. faecalis is a bacterium that is involved in persistent endodontic infections with a prevalence of 24% to 77%. 5 E. faecalis is found in dentinal tubules radicular area in both colony or solely form. 6 E. faecalis is a gram-positive biofilm producing bacterium, 7 and commonly to be found in colony with consist of cocci or chain forms of the bacteria. 8 These bacteria in a biofilm matrix are able to confront stresses derived from antibiotic application and highly alkaline environment. 7 On the other hand, E.
faecalis produce virulence factors in order to confer damage on endodontic tissue. 8 One of the factor is gelatinase, a metalloprotease which may cleavages peptides component in endodontic surroundings, 9 and is associated with microbial adhesion to the dentin surface. 10 The gelatinase expression by E. faecalis is known to be important in the collagen hydrolysis mechanism which plays an critical role in initiating periapical pathogenesis, attract nonspecific For gelatinase assay, the cultures were added to the prefilled gelatine tubes. The tubes were then incubated in anaerobic jar at 35°C.
The tubes were placed in the refrigerator for 1h prior measurement, and the gelatin liquefaction height was measured using the sliding caliper.

Metabolic Activity
To analyze the metabolic activities of carbohydrate and arginine, the cultures of bacteria were incubated in phenol red and arginine dehydrolase based-broth culture media for 24h and 48h, respectively, at 37°C.
Color changes were observed qualitatively and then followed by quantification using ImageJ software 1.53o (NIH, Bethesda, MD, USA).

Statistical Analysis
Data were analyzed using GraphPad Prism 9.3.1 (GraphPad Software, USA). The normality test was conducted using Shapiro-Wilk, followed by ANOVA with Dunnet's as the post hoc analysis.

RESULTS
Recovery rate refers to a capacity of bacteria to resolve its population after the given treatment. Based on Figure 1, the data showed no significant reduction in resolving capacity after 1h treatment with either 0.00125%, 0.4% and 10% EEP on clinical bacteria recovery rate compared to the untreated group (p>0.9999).
Based on Figure  1, several concentrations of EEP did not affect the bacteria recovery capacity, since they were not significantly different to the untreated group.
The results confirm that the EEP-treated bacteria could re-grow after culture media replacement.

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Metabolism-independent phenomenon in ethanolic propolis inhibitory capacity towards enterococcus spp proteolytic activity Odonto : Dental Journal. Volume 9. Number 2. December 2022 Figure 1. Bacterial recovery rate. Enterococci was treated using 0.00125%, 0.4%, 10% EEP solution, and positive control using 2% of CHX digluconate for 1h. The recovery rate was obtained from the OD difference between 24h post incubation BHI broth resuspension and the post 1h of each treatment (ΔOD600). The ΔOD600 was divided by the negative control/untreated (BHI only) to obtain the percentage of recovery rate. Data were stated as mean ± standard error of the mean (SEM), with the significance level for each annotation: ns=nonsignificant, *p<0.05, **p<0.01, ***p<0.001, *p<0.0001.
Based on Figure 2, it showed that the bacterial proteolytic activity decreased gradually along with increasing EEP concentration. Interestingly, the proteolytic activity increased in 0.8% EEP treatment. It seems that EEP has inhibitory capacity in bacterial proteolytic activity, which was gradually decreasing until a certain concentration. However, it can be inferred that the lowest bacterial proteolytic activity was in 0.4% of EEP compared to other concentrations.    Figure 3a

Metabolism Activity
Based on bacterial metabolic activity on In our previous initial hypothesis, EEP was thought to affect the bacterial metabolism than it will block bacterial growth and proteolytic activity.
Interestingly, in our findings, EEP seems not to affect the bacterial growth, but tends to inhibit proteolytic activity by reducing the synthesis of gelatinase for several concentrations. However, this did not seem to correlate with the effect of propolis on metabolic activities of the bacteria.
Significant effect on proteolytic activity was only shown in 0.1% and higher EEP treatments, whereas EEP lower than 0.1% had significant effect on metabolic activities of bacteria. It seems that the inhibitory capacity of EEP is likely affecting the cell metabolic activity rather than inhibiting the population growth.

CONCLUSION
According to our findings, it could be concluded that EEPs may affect E faecalis proteolytic activity and their metabolism in an independent manner.