Testing the sensitivity of periodontal pathogens requires the selection of an easier and more reliable method to be used with such anaerobic bacteria that need a long period of time for growth. Natural materials are a new era of antibacterial agents to control periodontal infections. The aims of the current study were to test the antibacterial activity of two natural agents, namely olibanum and alum, against three types of red complex periodontal pathogens and compare the application of agar diffusion and microdilution methods for testing the susceptibility. Gingival crevicular fluid from pockets with chronic infections was sampled as a source for the three types of bacteria, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola (the red complex pathogens). The samples (n= 30) were cultured on three types of media, namely Schaedler Anaerobe Agar, Tannerella forsythia (TF) agar, and Trypton Yeast extract Gelatin Volatile fatty acids and Serum (TYGVS) agar for the three types of red complex pathogens. After anaerobic growth, the isolates of red complex pathogens were identified by cultural and cellular morphological characteristics and confirmed by molecular diagnosis. The antibacterial activity of the two natural materials was tested by agar (disk and well) diffusion and microdilution method (spectrophotometer- and resazurin- based). Ciprofloxacin (CIP) and chlorhexidine (CHX) were used as controls. Minimal Inhibitory Concentrations (MICs) of the four reagents (olibanum, alum, CIP, and CHX) were determined on the three types of bacteria. MIC values by each susceptibility method were compared and analyzed statistically at p- value ≤ 0.05. The results showed that resazurin- based microdilution method was the easiest and simplest approach which gave reliable reads. MIC values of the four regents differed from a method to another and from one bacterium to another. Statistically, there were no significant values among these differences, except for olibanum which was statistically more significant than the other reagents.

Background and Purpose: For enhancing the efficiency of diagnostic tools and overcome drawbacks faced in diagnosis attempt, molecular methods have been developed aid accurate and rapid identification of bacterial species particularly those which are difficult to study with. The current study searched to find a more specific and rapid method serves researchers for the identification of periodontal pathogens and overcome disturbances encountered in study such anaerobic fastidious organisms. Methods and Results: Three periodontal pathogens, Porphyromonas gingivalis , Tannerella forsythia and Treponema denticola were characterized and identified by phenotypic features and Loop- Mediated Isothermal Amplification (LAMP) techniques which involved 4 sets of primers targeted to 16SrRNA genes and loop primers and the Colorimetric Master Mix containing Bst DNA polymerase and Phenol Red for the detection of amplicon formation. There was a variance in phenotypic characters of the isolates of the same species. LAMP, as a novel molecular technique was of usefulness value in identifying the target weather as extracted DNA or whole cells in a high specific and very rapid manner within 30 min. by visual reading of the results. Conclusion: It is strongly recommended the use of the novel LAMP method in researches work with periodontal pathogens as its advantages make it superior to other molecular techniques in respects of a higher specificity, rapidity, sensitivity and overcome DNA extraction step and special equipment in that it can be used at chair- side identification.

Maintaining the level of periodontal bacteria under control represents the basis for reducing periodontal infections. Therapeutic therapy along with scaling aids to prevent the causative agent from recolonizing the treated surface. As natural substitutes, this study aimed to verify the validity of two natural products, olibanum and alum as inhibitors of periodontal pathogens and also as supporting agents to elevate the anti-action of the pre-validated antimicrobials, ciprofloxacin and chlorhexidine. The study chose three periodontal bacteria, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola as representative taxa because they are considered the more virulent with high proteolytic activity. The antimicrobial activity was studied to find out the minimal inhibitory concentration (MIC) values using resazurin-based microdilution assay. The cooperative interaction between reagents was studied by calculating the fractional inhibitory concentration (FIC) values, analyzing the statistical difference between the single and combinational use and comparing the inhibition zone by agar diffusion. The results proved the inhibitory activity of olibanum and alum against the three pathogens and their high efficacy in improving the inhibitory action of the two standard drugs which was evidenced by the lowered MIC values, calculated FIC values, enlarged inhibition zone and statistical significance of the combinational use. The study concluded the successful use of olibanum and alum in reducing the red complex pathogens either in a single use or in combination as a pure natural preparation and also raising the anti-action of lower concentrations of ciprofloxacin and chlorhexidin.

This study is concerned with the isolation of red complex pathogens, identifying them by a new molecular method as the first locally used and described procedure and characterizing these pathogens by their phenotypic features and biofilms. Gingival fluids were sampled from chronic periodontitis and inoculated into three types of culture media, Schaedler Anaerobe Agar, Tannerella forsythia (TF) agar, and Trypton Yeast extracts Gelatin Volatile fatty acids and Serum (TYGVS) agar. The different appearing colonies were purified and identified by Loop-Mediated Isothermal Amplification protocol (LAMP) for the detection of red complex species. Bacterial biofilm was estimated in term of mono-and polytypic biofilm by measuring the absorbance of a crystal violet-stained biofilm formed in a microtiter plate. Different forms of colonies appeared at the primary isolation. LAMP method was of a significant value for perfect rapid identification of the target species of extracted DNA or intact cells within half an hour. The three types of red complex pathogens were simultaneously detected in the same gingival fluid sample. They formed mono-and polymicrobial biofilms in a synergistic manner particularly the two intimates P. gingivalis and T. denticola. In conclusion, the updated LAMP molecular protocol was attractive method for the diagnosis of red complex pathogens which showed great morphological variations. P. gingivalis fortified the growth of the other two to establish polymicrobial biofilms in a synergistic manner.