Determination and comparison of antimicrobial activity of aqueous and ethanolic extracts of Amorphophallus paeoniifolius on periodontal pathogens: An in vitro study.

Background: People globally are turning to herbal products to reconnect with nature. Cost efficacy and minimal side effects are the reasons for this changeover. This study assessed the effect of Amorphophallus paeoniifolius as an antimicrobial agent against Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Aim: To determine and compare the antimicrobial activity of aqueous and ethanolic extracts of A. paeoniifolius on periodontal pathogens. Materials and Methods: Aqueous and ethanolic extracts of A. paeoniifolius were tested against the standard strains of the selected bacteria. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were used. These tests assessed the lowest concentrations of test agent, either by showing a lack of turbidity or by no or few bacterial growth colonies, respectively. In this study, tetracycline hydrochloride was used as the control group. Results: Aqueous and ethanolic extracts of A. paeoniifolius showed antibacterial activity at various concentrations against the selected organisms. While assessing the MBC, the aqueous and ethanolic extracts of A. paeoniifolius and tetracycline hydrochloride exhibited bactericidal activity against F. nucleatum at all concentrations. The ethanolic extract of Amorphophallus paeoniifolius and tetracycline hydrochloride showed bactericidal action, whereas the aqueous extract exhibited bacteriostatic action against P. gingivalis. The aqueous and ethanolic extracts of A. paeoniifolius showed bacteriostatic action, whereas tetracycline hydrochloride showed bactericidal action against P. intermedia. Conclusion: Both aqueous and ethanolic extracts of A. paeoniifolius showed antibacterial activity against standard strains of P. gingivalis, P. intermedia, and F. nucleatum. The ethanolic extract showed a significant antibacterial effect against the selected microorganisms when compared to the aqueous extract of A. paeoniifolius.

Detection and comparison of prevalence of Porphyromonas gingivalis through culture and Real Time-polymerase chain reaction in subgingival plaque samples of chronic periodontitis and healthy individuals.

Introduction: The micro-flora of oral cavity is a myriad of micro-organism. Any infection of oral cavity leads to diseased condition which is a transitional transformation of the micro-organism in a specific paradigm depending upon the diseased condition. Periodontitis is one of the predominant chronic diseases which is a multifactorial infection. Porphyromonas gingivalis is a key etiological agent in causing periodontitis. To study the predominance of these bacteria in the diseased condition is important to detect, quantify and to find its efficacy by comparing different methods for identification. Aim and Objectives: The aim of the study is to determine the prevalence of P. gingivalis by anerobic culture and by real-time polymerase chain reaction (PCR) from subgingival plaque samples of chronic periodontitis and healthy individual and to compare efficacy of two methods. Materials and Methods: A total of 400 subjects were considered, and subgingival plaque was collected using paper points. Individual were equally divided into two groups: chronic periodontitis (200) and healthy individuals (200). Each plaque sample collected was divided into two aliquots of which the first aliquot was subjected for anerobic culture to isolate P. gingivalis. Phenotypical identification was done morphologically and biochemically further quantification of P. gingivalis was done by colony-forming unit. The second aliquot was subjected for DNA extraction and real-time PCR was conducted to detect and quantify P. gingivalis using specific primer. Results: Out of 400 samples, 73% showed detection of P. gingivalis by culture method and through reverse transcription-PCR (RT-PCR), the detection was 75%. Individual detection of P. gingivalis by culture in chronic periodontitis was 89.5% and 54.4% in healthy individuals, while detection by RT-PCR was found to be 91.5% in chronic periodontitis and 58% in healthy individuals. However, comparison between two techniques in detection of P. gingivalis was statistically insignificant. Conclusion: When we compared RT-PCR with culture RT-PCR showed higher positivity. RT-PCR is more sensitive and requires less time to detect. However, in the present study, culture also showed good positivity, suggesting proper dilution and with extended incubation, the specificity of culture can be improved to a great extent.

Coculture method for in vitro cultivation of uncultured oral bacteria.

PURPOSE: The purpose of the study is to culture uncultured oral bacteria with helper strains using the coculture method from the subgingival plaque samples of chronic periodontitis patients. MATERIALS AND METHODS: The samples were processed and inoculated on a blood agar medium enriched with hemin and Vitamin K. A helper strain Propionibacterium acnes (ATCC 6919) was cross-streaked across the inoculums to facilitate coculture. The plates were then incubated for 7 days with subsequent subculturing and further incubation. RESULTS: Satellite colonies around helper strain showed one colony type of Porphyromonas gingivalis, one was of nonpigmented Prevotella, three were of Fusobacterium nucleatum and five isolates remained unidentified. CONCLUSIONS: Coculture could be used effectively as one of the methods in the isolation and in vitro cultivation of oral bacteria. Incubation using the anaerobic jar technique was found to be economical and efficient for the growth of anaerobic oral bacteria.

Antimicrobial susceptibility pattern of oral gram negative anaerobes from Indian subjects.

OBJECTIVES: There is paucity of information on the antimicrobial susceptibility pattern of oral anaerobic bacteria. In this study, an attempt has been made to evaluate the antimicrobial susceptibility/resistance trend of oral Gram negative bacteria from Indian subjects. METHODS: Minimum inhibitory concentrations (MIC) of 304 isolates against twelve different antibiotics were determined using gradient diffusion MIC strips. The organisms were isolated and identified based on phenotypic characteristics and included Porphyromonas gingivalis, Prevotella species, Tannerella forsythia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcoitans, Eickenella corrodens and Capnocytophaga species. For each antimicrobial agent, MIC50 and MIC90 were calculated and expressed. RESULTS: Resistance to azithromycin, clindamycin, and amoxicillin was observed in most of the anaerobic bacterial species studied. High degree of susceptibility was observed to amoxillin-clavulanic acid, doxycycline and moxifloxacin. A single strain of P. melaninogenica was resistant to moxifloxacin. The susceptibility pattern varied with cephalosporins among species. Ceftriaxone showed highest and cefazolin least efficacy among cephalosporins. All anaerobic bacteria tested were susceptible to metronidazole. Strains of T. forsythia were more resistant to several antibiotics than other anaerobic bacteria. All three species of capnophilic bacteria displayed high degree of resistance to metronidazole and significant resistance to amoxicillin, azithromycin, clindamycin, cefazolin and cefuroxime. CONCLUSIONS: Amoxicillin-clavulanic acid, doxycycline, moxifloxacin and metronidazole appeared to be the most effective drugs against gram negative anaerobic bacteria. However, the MIC50 and MIC90 values against metronidazole were on the higher side of the normal indicating a potential for developing resistance.

Comparison of cluster analysis of Porphyromonas gingivalis by arbitrarily primed-polymerase chain reaction between healthy and chronic periodontitis subjects.

INTRODUCTION: Periodontitis is a chronic destructive inflammatory disease of the oral cavity. The main causative agent is presence of biofilm formed due to different micro-organisms. Among different micro- organisms "red complex" bacteria is known to be the main causative agent in progression of periodontitis. Porphyromonas gingivalis out of the red the complex organism plays a major role in progression of periodontitis. P. gingivalisis present in both in healthy and diseased individuals. The difference in the strains will determine the virulence factor of the organism and also progression of disease. Only few studies have been done showing variation in strains present between healthy and diseased. AIMS: To check the difference in heterogeneity of P. gingivalis in chronic periodontitis and healthy individuals through Arbitrarily Primed-PCR (AP-PCR). MATERIALS AND METHODS: A total of 400 subjects (200 each of chronic periodontitisandhealthy individuals) were included. Sub-gingival plaque was collected in the Reduced transport fluid (RTF) medium and processed at the institutional central research laboratory. Presence of P. gingivalis was, confirmed by culture andphenotypical analysis. Further confirmed cases were processed for PCR after DNA extraction using 16S rRNA. Positive cases of P. gingivalis were subjected for AP-PCR for clonal analysis using the specific 272 primer. RESULTS: In 152(76%) and 98(49%) were confirmed for P. gingivalis in chronic periodontitis and healthy individual respectively by PCR. AP-PCR analysis showed 6 clusters with similarity index in CP and 3 clusters with similarity index in Healthy individuals. CONCLUSION: The present study showed difference in clusters between chronic periodontitis and healthy individual'sthussuggestive variantin genetic heterogeneity of P. gingivalis strain between healthy and chronic periodontitis. AP- PCR appears to be a promising tool for clonal analysis of P. gingivalis.

Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections.

Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion . The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections.