Comparing the efficacy of Neem extract, Aloe vera juice, and 0.2% Chlorhexidine against dental unit waterline pathogens. an In-vitro study

 

Dr. Shivashankar Kengadaran¹, Dr. Srisakthi. D.², Dr. Divvi Anusha³, Dr. Gopinath Prakasam⁴, Dr. Kavitha. M⁵, Dr. Vigneshwari. S.K⁶

^1,3Department of Public Health Dentistry, Indira Gandhi Institute of Dental Sciences,

Sri Balaji Vidyapeeth, Pondicherry.

²Reader, Department of Public Health Dentistry, Saveetha Dental College, Chennai.

⁴Associate Professor, Department of Microbiology, Royal Dental College, Palakkad, Kerala.

⁵Department of Pediatric and Preventive Dentistry, Indira Gandhi Institute of Dental Sciences,

Sri Balaji Vidyapeeth, Pondicherry.

⁶Department of Conservative and Endodontics, Indira Gandhi Institute of Dental Sciences,

Sri Balaji Vidyapeeth, Pondicherry.

 

ABSTRACT:

Introduction: Nosocomial infections are caused by the retrograde spread of viral, bacterial and fungal pathogens from the hospital environment to the patients. Despite the efforts to avoid cross-infection in the dental office using sterilized instruments, individual protection equipment, and disinfection procedures, there is an increased risk of cross-infection through dental units, since one dental chair is used to treat many patients. Materials and methods: Seven strains of micro-organisms- Streptococcus, Lactobacillus, Pseudomonas aeruginosa, Klebsiella, Enterococcus, Staphylococcus aureus, Candida albicans, E. coli, and Legionella were taken. Neem extract, Aloe Vera Juice, and 0.2% chlorhexidine were tested against all these organisms. Sterile discs were incorporated with an equal amount of prepared formulations using a micropipette. Then these discs were placed equidistant to each other following which these plates were incubated for 24 hours. Results: Zone of inhibition was higher in chlorhexidine 12mm, 22mm, 11mm, 8mm, 18mm, 12mm, 11mm, 14mm and 15 mm followed by neem extract 7mm, no inhibition, 10mm, 7mm, 11mm, 7mm, 11mm and 9 mm and no inhibition by Aloe Vera juice against Streptococcus, Lactobacillus, Pseudomonas aeruginosa, Klebsiella, Enterococcus, Staphylococcus Aureus, Candida albicans, E.coli and Legionella respectively. Conclusion: Results from this study have shown that chlorhexidine was most effective against dental unit waterline pathogens followed by neem extract and least by Aloe Vera juice.

 

 

 

 

INTRODUCTION:

Nosocomial infections are caused by the retrograde spread of viral, bacterial and fungal pathogens from the hospital environment to the patients. Despite the efforts to avoid cross-infection in the dental office using sterilized instruments, individual protection equipment, and disinfection procedures, there is an increased risk of cross-infection through dental units, since one dental chair is used to treat many patients. Specific parts of the dental unit are responsible for the spread of microbial infection, of which Dental Unit Waterlines have been shown to the harbour, in significant numbers, a wide variety of microorganisms including bacteria, fungi, and protozoans.¹

 

Dental unit waterlines (DUW) deliver water to the high-speed handpieces, scalar unit, and three-way syringe. The water may circulate in a closed system, where it is taken from a container belonging to a unit. The quality of dental water is of considerable importance since patients and dental staff are regularly exposed to water and aerosols generated from the dental equipment². The microorganisms in Dental unit waterline colonize and replicate on the interior surfaces of the waterline tubing, inevitably resulting in adherent heterogeneous microbial accumulations termed “biofilms”.³

 

The goal of infection control is to minimize the risk from exposure to potential pathogens and create a safe working environment to treat patients.⁴ With the increasing stress on infection control, because of contaminated water in dental units, the dental profession is understandably interested in preventing any problems that might arise in this area. Recently, interest in this biofilms has reawakened. This can be attributed to increased awareness of potential occupational hazards in the dental office and concern about increasing numbers of dental patients considered to have diminished resistance to overt and opportunistic microbial pathogens. ⁴

 

Dental unit waterlines must be maintained regularly to deliver water of optimal microbiologic quality. Although infection associated with microbial contamination of waterlines appears to be rare, it has been shown that the level of microorganisms in untreated dental unit waterlines is greater than 500 CFU/mL, which exceeds the drinking water standard.⁵ Colonization of microorganisms within the waterlines may not be a concern to healthy individuals however, it might place elderly or immunocompromised patients at unnecessary risk. ⁴

 

Glutaraldehyde, chlorine, Sodium Hypochlorite and Hydrogen peroxide are some of the agents used in dental unit waterline disinfection. Chlorhexidine is one of the most widely used biocides in antiseptic products, in both handwashing and oral products and as a disinfectant and preservative. ^{5, 6} Thus, it seems that it is important to study the effects of disinfectants such as chlorhexidine (used as a mouthwash by most patients). Studies show that 0.2% chlorhexidine has a significant antiseptic effect on dental unit waterline pathogens.⁷ Aloe vera is a succulent from the Aloe family with its origin in the African continent. Several studies have proven that Aloe vera extract has antimicrobial properties. ⁸

 

Azadirachta indica, commonly known as neem, has attracted worldwide prominence in recent years, owing to its wide range of medicinal properties. Neem has been broadly used in Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern medicine.⁹ Neem elaborates on a vast array of biologically active compounds that are chemically diverse and structurally multifaceted. ^10,11

 

The responsibility for ensuring that dental chair units provide good quality water output has, by and large, been considered to rest on the shoulders of dental practitioners and/or dental clinic management.¹² Hence an attempt has been made to find the efficiency of herbal products against dental unit waterline pathogens. This study aims to compare the efficacy of Neem extract, Aloe Vera Juice, and 0.2% chlorhexidine against the dental unit waterline pathogens.

 

MATERIALS AND METHODS:

An In-vitro study was conducted to find the efficacy of anti-microbial agents against dental unit waterline pathogens. More than 25 different bacterial species as well as several species of fungi, and protozoa have been isolated from DUWL¹³ of which 7 most predominant organisms were included in this study. Before the start of the study ethical clearance was obtained from the institutional ethics committee.

 

Study groups:

 

Figure 1: Steps in neem extract preparation.

 

Neem extracts preparation:

Fresh leaves of neem were collected neem trees on the campus of the Saveetha Dental College, Chennai and used to prepare alcoholic extract: neem leaves were washed with distilled water and shadowed dried at room temperature for 48 hours to maintain the antimicrobial activity of the leaves. A powder was prepared by grinding the dried leaves to form a fine mixture. 5 mg of neem powder was then mixed with 1 ml of absolute alcohol to form alcoholic neem extract. (Figure. 1)

 

 

Anti-microbial assay:

Seven strains of micro-organisms-Streptococcus mutans, Escherichia coli, and Candida albicans were taken. The media used were Mutans media, Mueller Hilton agar, and Sabouraud Dextrose agar media respectively for each microorganism. Nutrient agar media was used to inoculate Pseudomonas aeruginosa, Klebsiella and Staphylococcus aureus. Legionella was inoculated in legionella media. These bacteria were inoculated in their respective medium by swab method.

 

Sterile discs were incorporated with an equal amount of different dilutions of prepared extracts using a micropipette. Then these discs were placed equidistant to each other following which these plates were incubated for 24 hours (Figure 2)

Inoculation of the microorganisms by lawn culture method

Incubated for 24 hours

Sterile Disc placed

Disc diffusion method

Laminar air flow chamber

Completed plates

. The test plates were held in front of a desk lamp, and the zones were measured with a ruler held against the back of the Petri plate. The diameters of the zones of inhibited growth were measured to the nearest whole millimetre.

 

Table 1 depicts the Zone of Inhibition of various disinfectants against cariogenic organisms.

 

This in-vitro-study shows that the zone of inhibition was higher in chlorhexidine 12mm and 22mm and 20mm followed by neem extract 7mm and the no inhibition by Aloe Vera juice against Streptococcus and Lactobacillus respectively. (Table. 1).

 

 

Figure 2: Steps in Antimicrobial assay.

 

RESULTS:

Table 1: Zone of Inhibition of various disinfectants against cariogenic organisms

 

 

Table 2: Zone of Inhibition of various disinfectants against Opportunistic pathogens

Product	Zones of inhibition (mm)
	Pseudomonas aeruginosa	Klebsiella	Enterococcus	Staphylococcus aureus	Candida albicans	E.coli	Legionella
Neem extract	10	7	11	7	11	9	9
Aloe Vera juice	0	0	0	0	0	6	0
0.2% Chlorhexidine	11	8	18	12	11	14	15

Table 2 depicts the Zone of Inhibition of various disinfectants against Opportunistic pathogens.

 

Data were entered in Microsoft Excel spreadsheet and analyzed using SPSS software (version 20). Descriptive statistics were used. For significance level, a p-value of <0.05 was considered statistically significant.

 

The Zone of inhibition is found to be higher in Chlorhexidine 11mm, 8mm, 18mm, 12mm, 11mm, 14mm and 15 mm followed by Neem extract 10mm, 7mm, 11mm, 7mm, 11mm and 9 mm and no inhibition by Aloe Vera juice against Pseudomonas aeruginosa, Klebsiella, Enterococcus, Staphylococcus aureus, Candida albicans, E. coli and Legionella respectively. (Table.2)

 

DISCUSSION:

Dental unit water systems (DUWS) are used to irrigate the oral cavity during dental treatment and provide cooling to certain items of equipment such as air rotors and mechanical scalars. Water delivered from these devices is not sterile and has been shown to contain relatively high numbers of bacteria.

 

Several agents were used as Dental unit waterline Disinfectants which included Glutaraldehyde, chlorine, Sodium Hypochlorite, Hydrogen peroxide, etc.¹³ Integral automated flush system employing glutaraldehyde is commercially available but its sensitization of the human lung and skin has severely inhibited the use of this compound in dentistry.¹³ The disadvantage of long term exposure to chlorine include bacteria developing resistance, corrosion damage even at 1ppm, the formation of trihalomethanes (potential carcinogens) and that high chlorine levels are unpalatable.¹³ Hydrogen peroxide may lead to internal corrosion to the dental tubing.

 

0.2% chlorhexidine and 3% sodium hypochlorite disinfectants were almost equally effective with no significant difference among them. Hence, chlorhexidine was selected as the control group. ⁷

 

As there is a paucity of literature regarding the use of herbal products such as Neem extract and Aloe Vera juice in disinfection of dental unit waterlines, this study was conducted to evaluate the efficacy of Neem extract, Aloe Vera juice, and 0.2% chlorhexidine against the dental unit waterline pathogens.

 

The efficacy of these biocides was tested against 8 micro-organisms which include cariogenic organisms such as Streptococcus mutants and Lactobacillus; opportunistic pathogens such as Klebsiella, Candida albicans, Staphylococcus aureus, Enterococcus, and E. coli; waterborne pathogens such as Pseudomonas aeruginosa and Legionella. These organisms were found predominantly in the dental unit waterlines.¹³ The organisms were inoculated in the respective media by the lawn culture method. The antimicrobial efficacy of the disinfectants is then tested using the disc diffusion method.

 

In our study, the antimicrobial efficacy of the disinfectants was measured as a zone of inhibition. The zone of inhibition is found to be higher in chlorhexidine followed by neem extract and the no inhibition by Aloe Vera juice against cariogenic microorganisms. A similar range of zone of inhibition was obtained by using ethanolic extract of neem against S. mutans.⁹

 

In the current study, the zone of inhibition is found to be higher in Chlorhexidine followed by Neem extract and no inhibition by Aloe Vera juice against opportunistic pathogens. A study conducted by Olaleye et al., also elicited the poor antimicrobial activity of Aloe Vera. ⁸

 

Chlorhexidine exhibited the highest antimicrobial activity followed by Neem extract and least or no effect by Aloe Vera juice. Studies have reported that high-speed handpieces and air/water syringes bacterial and fungal contamination were almost non-existent after adding 0.2% chlorhexidine which was in constant with our study.^7,14

 

After, chlorhexidine, Neem exhibited antimicrobial activity against all the tested micro-organisms. With the increasing rise in multi-resistant strains of micro-organisms, herbal products are more preferred since they contain phytochemicals such as alkaloids, tannins, essential oils and flavonoids which have pronounced antimicrobial activity and developing resistance against all these compounds is not possible. Hence neem can be considered as an alternative to chlorhexidine.

 

CONCLUSION:

The present study was based on in-vitro experiments. Results from this study have shown that chlorhexidine was most effective against dental unit waterline pathogens followed by neem extract and least by Aloe Vera juice. Thus turning to nature can be as good as the latest advances. These traditional methods have numerous other claims also which are of great benefit for health. Exploring and harvesting these herbal products and blending the same with the advances may make oral and health care better. Thus the present article also provides insight into the dentists that the herbal products are equally effective against oral microorganisms.

 

CONFLICT OF INTEREST:

None declared.

 

REFERENCES:

1.        Szymańska J. Control methods of the microbial water quality in dental unit waterlines. Ann Agric Environ Med. 2003;10:1-4.

2.        Kengadaran S, Srisakthi D, Arumugham IM, Pradeepkumar R. Knowledge, attitude, and practice regarding dental unit waterline disinfection among dental practitioners of India. Journal of Advanced Pharmacy Education and Research| Jul-Sep. 2017;7(3).

3.        Padma RM. Control & prevention of biofilm in dental office. Indian Journal of Dental Advancements. 2011 Apr 1;3(2):512-21.

4.        American Dental Association. Oral health topic. Dental unit waterline. Available from: http://www.ada.org/en/member-center/oral-health-topics/dental-unit-waterlines. last accessed on 14-7-2017

5.        McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews. 1999 Jan 1;12(1):147-79.

6.        Kathariya MD, Kashinath PS, Kulkarni S, Singh D, Akkareddy B, Kathariya R. Evaluation of bacterial contamination of dental unit waterlines and the efficacy of commercially available disinfectants. Int. J. Pub. Health Dentistry. 2013;4(1):23-8.

7.        Olaleye MT, Bello-Michael CO. Comparative antimicrobial activities of Aloe vera gel and leaf. African Journal of Biotechnology. 2005;4(12).

8.        Roberson MC, Heggers JP, Hagstrom Jr WJ. Myth, magic, witchcraft, or fact? Aloe vera revisited. Journal of Burn Care and Research. 1982. May 1;3(3):154-63.

9.        Lekshmi P, Sowmia N, Viveka S, Brindha JR, Jeeva S. The inhibiting effect of Azadirachta indica against dental pathogens. Asian J Plant Sci Res. 2012;2(1):6-10.

10.     Achio S, Ameko E, Kutsanedzie F, Alhassan S. Insecticidal effects of various neem preparations against some insects of agricultural and public health concern. International Journal of Research in BioSciences. 2012;1(2):11-9.

11.     Coleman DC, O’Donnell MJ, Shore AC, Swan J, Russell RJ. The role of manufacturers in reducing biofilms in dental chair waterlines. J Dent. 2007;35:701-11

12.     Kotaka CR, Garcia LB, Ito FA, Fuganti MR, Carnio J, Pelayo JS. Evaluation of the level of microbial contamination and prevalence of gram-negative non-fermentative rods in dental unit waterlines. RSBO (Online). 2012 Sep;9(3):245-53.

13.     Kamma JJ, Bradshaw DJ, Fulford MR, Marsh PD, Frandsen E, Østergaard E, Schel AJ, Cate JM, Moorer WR, Mavridou A, Mandilara G. Attitudes of general dental practitioners in Europe to the microbial risk associated with dental unit water systems. International Dental Journal. 2006 Aug 1;56(4):187-95.

14.     Tilak AH, Geetha Priya S, Raja K. Microbial Contamination in Dental unit water line. Int. J. Pharma Bio. Sci. 2012;3(4):1032-44.

 

 

 

Received on 15.04.2020            Modified on 23.05.2020

Accepted on 29.06.2020         © RJPT All right reserved