ENDODONTOLOGY. Introduction. Original Research ABSTRACT

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1 Original Research Antibacterial efficacy of five different indian varieties of grape extracts against two antimicrobial resistant strains in endodontics - An in vitro study Sureshchandra B. Amrita Mujumdar Roma M. ABSTRACT Aim :- To evaluate the antibacterial efficacy of five different Indian varieties of grape extracts against two antimicrobial resistant strains of Enterococcus Faecalis and Candida Albicans in Endodontics and the effects of the extracts on the growth and survival of the same organisms during storage upto 48 hours. Materials and Method :- Five different Indian varieties of grape extract (Sharad, Dilkhush, Kali-Sahebi, Bangalore Blue, Anab-e-shahi) were obtained using Soxhlet s extractor and water bath (water extracts) in standardized concentrations. Surface plating and Test Tube inoculation method was used to test the antibacterial activity of the extracts on the pathogenic bacteria such as E.Faecalis and Candida Albicans respectively. Results :- Amongst the bacteria tested, Enterococcus Faecalis exhibited high sensitivity to Anab-e-shahi and Dilkhush grape extracts compared to the rest of the group extracts. Candida Albicans showed resistance to all the extract varieties. Conclusion :- Grape extract exhibits antibacterial efficacy against Enterococcus Faecalis found in the pulp space. Clinical Relevance :- Biologically active compounds, in the form of Grape extracts are effective in combating antimicrobial resistant strain of E.faecalis which can survive in the pulp space, post adequate cleaning and shaping and calcium hydroxide treatment leading to endodontic failures. Toxicity concerns of the commonly used pulp space irrigants are eliminated in the case of grape extracts. Keywords :- Grape extract, Enterococcus faecalis, Candida Albicans, Antimicrobial resistant strains, Introduction Debridement of the pulp space system by instrumentation and irrigation in collaboration with prevention of re-infection is considered the most important factor in prevention and treatment of endodontic diseases. Elimination of endodontic infection is quite different from most other sites in the human body. Host measures that are sufficient to eliminate infectious microorganisms in other sites do not suffice for complete elimination of endodontic infection mainly because of complex anatomy of the pulp space system. Microorganisms are able to survive in periods of starvation and predominate even post thorough cleaning and shaping of the pulp space. The most frequently isolated antimicrobial resistant species, by far in previously root filled teeth with apical periodontitis are Enterococcus Faecalis and Candida Albicans. E.Faecalis forms biofilms in medicated pulp spaces, endures prolonged periods of starvation and acquires gene coding antibiotic resistance and natural resistance to antimicrobial agents. Candida Albicans also demonstrates Quorum Sensing with Dentinophilic properties and affinity to smear layer. Intracanal medicaments and irrigants add to the overall predictability of pulp space therapy but due to their relevant toxic nature, their use is controlled and restricted. Hence, the need for an antimicrobial # Department of Conservative Dentistry & Endodontics, A.J. Institute of Dental Sciences, Mangalore, Karnataka, India 6

ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT STRAINS IN ENDODONTICS - AN IN VITRO STUDY intracanal medicament to eliminate the

2 ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT STRAINS IN ENDODONTICS - AN IN VITRO STUDY intracanal medicament to eliminate the resistant microorganisms. Extensive research worldwide over grape extracts, such as skin and seeds have exhibited them each to be rich source of bioactive polyphenolic compounds such as Anthocyanins, Flavonols, Flavanols and Resveratrol. These bioactive compounds demonstrate a variety of biological activities such as being:- 1. Anti-oxidant 2. Antimicrobial 3. Cardio-protective 4. Anti-carcinogenic 5. Anti-inflammatory 6. Anti-aging India is ranked 12 th in the world in terms of grape production. Total polyphenols in Indian grapes ranged between 3-58%. Hence, this evidence suggests the ample availability of grape extracts and the utilization of their antibacterial efficacy against resistant strains of E. Faecalis and Candida Albicans in endodontics. The effect of the grape extract on the growth and survival of the same organisms is tested during storage upto 48 hours. Materials and Method :- A) Selection Of Grape Varieties: Following Five Different Indian Grape Varieties Were Selected:- Group No Variety Province Color Presence/ Absence of Seeds 1. Sharad Sangli Purplish Black Seedless 2. Dilkhush Bangalore Green Seeded 3. Bangalore Blue Bangalore Purplish Blue Seeded 4. Kali- Sahebi Bijapur Purplish Black Seeded 5. Anab-E-Shahi Hyderabad Light Green Seeded 6. Control Group Distilled Water B) Standardization by Weight :- 150 gram of each grape variety was premeasured and prepared for extraction procedure Each group of extract was squashed using a mortar and pestle. 7

SURESHCHANDRA B., AMRITA MUJUMDAR, ROMA M.

D) Transport :- The extract concentrates are carried to the microbiology

3 SURESHCHANDRA B., AMRITA MUJUMDAR, ROMA M. Post squashing, each extract group was diluted with water as a liquid extract medium in an equal ratio. C) Extraction:The diluted solution was poured into Soxhlet s apparatus to yield the concentrated compound. Extract concentration is reduced to maximum dryness in a water bath. D) Transport :- The extract concentrates are carried to the microbiology department in sealed labeled bottles. E) Microbiological Tests Conducted To Evaluate Antimicrobiology Efficacy :a) Disc Diffusion Strains tested :1. E.Faecalis ATCC Candida Albicans ATCC b) Test tube inoculation 8

ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT

Blood Agar for Brain Heart Infusion (BHI) broth for Enterococcus Enterococcus Faecalis Faecalis Sabouraud s

First the inoculum was prepared by inoculating E faecalis into BHI broth and C.

faecalis and 10-12 hours for Candida albicans.

4 ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT STRAINS IN ENDODONTICS - AN IN VITRO STUDY F) Armamentarium: Culture Medias used- Muller Hinton (MH) Agar and Blood Agar for Brain Heart Infusion (BHI) broth for Enterococcus Enterococcus Faecalis Faecalis Sabouraud s Dextrose Agar (SDA) for Candida Sabouraud s Dextrose broth for Candida albicans albicans Inoculum Prepeartion: First the inoculum was prepared by inoculating E faecalis into BHI broth and C. albicans into Sabouraud s Dextrose broth. The broths were incubated at 37oc for 4-6 hours for E. faecalis and hours for Candida albicans. Enterococcus Faecalis Candida Albicans Muller Hinton (MH) agar and Blood Agar Sabouraud s Dextrose Agar (SDA) Surface Plating Done : The inoculum was streaked onto the corresponding solid media as a lawn or carpet culture. 9

5 SURESHCHANDRA B., AMRITA MUJUMDAR, ROMA M. Disc Diffusion: 6 mm filter paper discs dipped in different grape extracts were placed on the surface of the media marked as 1,2,3,4,5 corresponding to the type of grape extract. The media was incubated at 370c for 24 hours All extracts in one media Individual extract media 10

ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE

- AN IN VITRO STUDY Test Tube Inoculation: lated.

hours and repeated subcultures were done Muller Hinton (MH) Agar

rococcus faecalis Sabouraud s Dextrose Agar (SDA) for Candida

6 ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT STRAINS IN ENDODONTICS - AN IN VITRO STUDY Test Tube Inoculation: lated. The tubes were than incubated at 37oc for Culture media : hours and repeated subcultures were done Muller Hinton (MH) Agar and Blood Agar for Ente- onto corresponding solid media. rococcus faecalis Sabouraud s Dextrose Agar (SDA) for Candida albicans One ml of each extract was taken in a set of sterile test tubes in duplicates. To one set E. faecalis was inoculated with a 4 mm loop which holds about 0.01 ml of broth and to other set same amount of C. albicans was inocu- Incubation: 11

7 SURESHCHANDRA B., AMRITA MUJUMDAR, ROMA M. Results: Maximum Zone of Inhibition- Zone II And V No Zone of Inhibition Exhibited in Any Group Candida Albicans CONTROL GROUP- no growth observed 12

8 Discussion: The present study for in-vitro Antimicrobial activity exhibited the presence of Phytochemicals with biological activity in selected variety of grape extracts. Enterococcus faecalis demonstrated sensitivity to all grape varieties particularly maximum with varieties such as Dilkhush (13 mm) and Anab-e-shahi (15 mm), according to the Disc Diffusion and Test tube inoculation method. Whereas Candida Albicans proved resistant to all extracts. The high sensitivity of E.Faecalis towards extracts of Dilkhush and Anab-e-shahi variety may suggest high amount of polyphenols being present in the seeded varieties compared to the seedless variety. Disc diffusion and Test tube inoculation methods are generally accepted procedures but have some limitations such as ph of the substrate, incubation period and diffusion capacity of the drug having effect on activity of tested materials. However evidence also suggests that agar diffusion tests show good correlation with other antimicrobial susceptibility tests. Conclusion: Biologically active compounds, in the form of Grape extracts are effective in combating antimicrobial resistant strain of E.faecalis which can survive in the pulp space, post adequate cleaning and shaping and calcium hydroxide treatment leading to endodontic failures. Toxicity concerns of the commonly used pulp space irrigants are eliminated in the case of grape extracts. ANTIBACTERIAL EFFICACY OF FIVE DIFFERENT INDIAN VARIETIES OF GRAPE EXTRACTS AGAINST TWO ANTIMICROBIAL RESISTANT STRAINS IN ENDODONTICS - AN IN VITRO STUDY References : 1. Shiny Benjamin, Roshni, Sabeena Susan Thomas, et al. Grape Seed Extract as a Potential Remineralizing agent: A comparative in-vitro study. Journal of Contemporary Dental Practice, 2012;19(4): Mahkameh Mirkarimi, S.Mahmoud Amin-Marashi, Majid Bargrizan, Amir Abtahi, Abbas Ali Imani Fooladi. The Antimicrobial Activity of Grape Seed Extract against Two Important Oral Pathogens. Zahedan J Res Med Sci 2013; 15(1): Manalika Ringshia. The Effect of Grape Juice on the Adherence of Streptococcus mutans on Tooth Surfaces. John F. Kennedy Memorial High School, Woodbridge, NJ 4. Christian Hanniga, Julia Sorga, Bettina Spitzmüllera, Matthias Hannigb, Ali Al-Ahmada. Polyphenolic beverages reduce initial bacterial adherence to enamel in situ. published online 06 April Xie Q, Bedran-Russo AK, Wu CD. In vitro remineralization effects of grape seed extract on artificial root caries. J Dent. 2008;36(11): Joseph C. Brown, Guohui Huang, Vivian Haley- Zitlin, and Xiuping Jiang. Antibacterial Effects of Grape Extracts on Helicobacter pylori. Appl Environ Microbiol. 2009; 75(3): Binit Shrestha, M.L. Srithavaj Theerathavaj, Sroisiri Thaweboon, Boonyanit Thaweboon. In-vitro antimicrobial effects of grape seed extract on peri-implantitis microflora in craniofacial implants. Asian Pacific Journal of Tropical Biomedicine 2012; 2(10): Veronica Sanda Chedea, Cornelia Braicu, Flore ChirilÎ, Ciprian Ober and Carmen Socaciu. Antibacterial action of an aqueous grape seed polyphenolic extract. African Journal of Biotechnology.2011;10(33): Ajay Kumar Sharma, P. G. Adsule and G. S. Karibsappa. Grape juice..take a sip for health. Krishisewa. Com 10. Wallace. Red Wine Can Help Prevent The Tooth Aging. 11. Jennifer Viegas. Drinking Red Wine May Prevent Cavities, When you can t brush, have a drink. Wed Nov 25, Stacy Kish. Grapes: A New Way to Fight Tooth Decay? CSREES Staff (July 23, 2008). 13

The Effect of Fragaria x ananassa Infusum on Salivary Mutans Streptococci

The Effect of Fragaria x ananassa Infusum on Salivary Mutans Streptococci Harun.A Gunawan.,AR. Putri.,H.Widodo., S Mangundjaja Department of Oral Biology Faculty of Dentistry Universitas Indonesia Jakarta