Comparative Evaluation of Disinfection of Dental Water Unit Line Using Neem Extract and Sodium Hypochlorite: An In vitro Study

Introduction

The term “biofilm” refers to a highly organized complex arrangement of microbial cells well surrounded in a polysaccharide matrix that is irreversibly associated with a surface, possessing properties of high resistance and virulence.¹ Small-bore water tubing, such as that found in dental unit water lines (DUWLs) is among the equipment in a dental set-up which is highly susceptible to bacterial growth and biofilm contamination.²

DUWLs are narrow long flexible tubes made of plastic material which enhances biofilm formation along its internal surface. Various factors influence initiation and growth of biofilm within the waterlines such as the tube length, speed of water flow, any water stagnation as well as usage frequency of the dental chair.^3-5 When a new DUWL system is connected to mains water supply, biofilm forms within eight hours.⁶ Due to inadequate valves, patient saliva may back suck into the waterline in dental units, adding to the microbial load that results in biofilm formation. Bottled water systems may also be contaminated.⁷

Use of waterline flushing, independent water reservoir systems, distilled or pasteurised water, ultrasonics, ultraviolet light, inline micropore filtration, periodic or continuous chemical disinfection are the ways to keep the biofilm and contamination of the DUWLs under control or rather to reduce the risk of infection for both patients and the medical staff.⁸

Sodium hypochlorite is a potent disinfecting agent, being bactericidal and proteolytic in action. The cytoplasmic membrane integrity is inhibited by sodium hypochlorite's high pH of 6, an irreversible enzymatic inhibition, a biosynthetic shift in cellular metabolism, and phospholipid degradation seen in lipidic peroxidation.⁹ There are certain drawbacks, such as dental equipment corrosion, DUWL obstruction, and inflammatory lesions in the oral mucosa, despite the fact that utilising sodium hypochlorite (5,000 or 1,500 ppm) to disinfect dental treatment water is particularly effective in reducing biofilms and improving water quality.¹⁰

This has created the need to explore other natural and potentially less harmful disinfecting agents for the DUWL system. Neem leaves have historically been used to cleanse water.¹¹ Neem, also known as Azadirachta indica, has demonstrated antibacterial, antioxidant, antiviral, and cytotoxic properties. It has antimicrobial properties due to the presence of active compounds like Nimbidin, Azadirachtin and Nimbin which actively disrupts the integrity of bacterial cell membrane.¹²

Thus, the present study aimed to determine the microbial quantity of water in DWU and also the effectiveness of Neem extract and Sodium hypochlorite in reducing the microbial count in the DWU system.

Materials and Methods

Twenty dental units in the Department of Conservative Dentistry and Endodontics were allocated for water sample collection. The selected units had not undergone prior treatment for removal or depletion of planktonic bacteria. Water samples of 50 mL were collected from the outlet of air/water syringe and high-speed hand piece from each of the 20 dental units. At the start of the study, baseline samples were obtained. Before the beginning of the working day, samples were collected on the first day of the week. Before sample collection, 70% alcohol was used to disinfect the end of each hand piece and the three-way syringe to avoid other sources of contamination. Water splashing was minimised and any contact between the hand piece and the container was avoided when filling the sample container. Within two hours, samples were processed at the lab after being kept in a refrigerator. The total viable count was estimated to determine the level of microbial contamination in the dental unit water line.

Following this, the dental chairs were randomly allocated to two groups of 10 chairs each, for treatment with a particular disinfectant, either commercially available Neem extract (BSD organics) or 5% Sodium hypochlorite. The disinfectants were diluted with distilled water to obtain a 1:1 dilution. Cross disinfection was prevented by using a single dental chair and a same disinfectant each time.

The dental unit's reservoir bottle was filled with 200 to 250 mL of disinfectant as part of the cleaning procedure, and the solution was then pumped through the apparatus for two minutes. The unit was switched off and the disinfectant was left in place after the disinfectants were added over the weekend, right before the start of that day's work. Water samples of 10 to 50 mL from each treated unit's air/water syringe were taken after 48 hours under aseptic circumstances, marked, and collected in separate sterile containers before treating the first patient of the day and measuring total viable counts.

The collected water was subjected to serial dilution with sterile water up to dilution factor of 100. One mL of each sample was inoculated on to R2A agar plates and incubated for a week at 22-28˚C. After a week, the colony forming units were counted manually and the results (CFU/mL) were recorded for the samples at baseline (before intervention) and after intervention.

Statistical analysis

Prior to conducting the analysis, the assumptions of normal distributed scores were examined. The assumption was considered satisfied. The data was tabulated using SPSS version 23. Paired ‘t' test was used to analyse data before and after intervention. Unpaired ‘t' test was used to analyse data between the two groups. A P value of less than 0.05 was considered statistically significant.

Results

The study included collection of water samples from 20 dental chair units beginning with baseline collection and after DUWL exposure to disinfectant. In Group 1 (Neem group), the mean CFU/mL at baseline was 128.70 and after intervention, it reduced to 51.90, which was statistically significant (P<0.05). In Group 2 (Sodium hypochlorite group), the mean CFU/mL at baseline was 125.90 (28.661) and after intervention, it reduced to 0, which was statistically significant (P<0.05) (Table 1). There was no statistically significant difference between the two groups at baseline (P>0.05) (Figure 1). Post-intervention, the mean reduction in Neem group was 76.8 CFU/mL and the mean reduction in Sodium hypochlorite group was 125.90 CFU/mL. A statistically significant difference was observed between the two groups (P<0.05) (Graph 2).

Discussion

The inner surface of dental unit tubing acts as a conducive environment for biofilm formation and is a constant reservoir of germs. Patients with impaired immune systems and medical conditions are at a high risk of infection from contaminated dental unit output water.¹³ Opportunistic pathogens like Pseudomonas and Legionella could easily be transmitted via aerosol production in a dental set-up and pose serious risk to patients as well as dental professionals.¹⁴

The Centre for Disease Control and Prevention (CDC) recommends that the dental unit's water output include no more than 500 CFU/mL of aerobic bacteria for infection control in oral health care facilities.¹⁵ This has created the need to develop efficient ways to disinfect the dental unit water line system.

The optimal features for an agent needed to treat DUWL 16 include a broad spectrum of antimicrobial action, little toxicity, low cost, ease of treatment, and similarity with a variety of materials.¹⁶ In consideration of the ideal properties of dental waterline disinfectant, newer natural disinfectants have been explored which possess the desired antimicrobial properties, in addition to being non-toxic and biodegradable. Sodium hypochlorite has several undesirable characteristics such as potential corrosive damage induced in dental equipment due to prolonged contact, along with disagreeable smell and taste.¹⁰ Thus in the present study, commercially available neem extract was used as an experimental DUWL disinfectant in comparison with the gold standard, 5% sodium hypochlorite. Prior to disinfection, the mean CFU/mL was approximately 127.3. This was well within the minimum permissible level of bacteria for dental unit waterlines.

Post intervention, both the disinfectant groups showed significant reduction in the mean CFU count with sodium hypochlorite showing 100% disinfection, which is statistically significant as compared to neem extract. This is in agreement with studies conducted by Sims et al. who evaluated effectiveness of varying concentrations of sodium hypochlorite against biofilm in dental tubing samples.¹⁷ Sodium hypochlorite has been established as the biocide used in hospital water treatment systems, particularly effective in controlling Legionella proliferation.¹⁸ This could be attributed to the wide range of antibacterial activity of sodium hypochlorite against Gram positive and Gram negative bacteria, with the ability to completely destroy microbial biofilm.¹⁹

Azadirachta indica or Neem has shown strong biosorbent potential in waste water treatment.²⁰ Neem has shown consistent effectiveness against bacterial growth, specifically targeting biofilm-grown cells.²¹ It has also displayed significant antiviral properties, owing to the inherent phytochemicals blocking critical processes of the viral life-cycle.²² These properties definitely provide an edge to Neem as a potential disinfectant, particularly during the era of the global COVID-19 pandemic.²³ Various studies have demonstrated effectiveness of Neem extract against E. faecalis, Candida albicans and mixed flora.^24,25

In the present study, Sodium hypochlorite showed significantly greater disinfection capacity compared to Neem extract. Increasing the concentration and contact period of the novel neem disinfectant along with usage of improved delivery systems in the form of nano-particles or concentrated phytochemicals could enhance its disinfectant action to comparable levels with Sodium hypochlorite. Greater research is desired in this regard.

Conclusion

Disinfecting the dental water unit line is of prime importance to avoid nosocomial spread of infections. Herbal disinfectants with greater bio acceptability and minimal toxicity can be used in place of chemical disinfectants. Within the limitations of the present study, Sodium hypochlorite was found to be significantly effective compared to Neem extract in disinfecting DUWL system. It is suggested that more research be undertaken to improve the effectiveness of Neem extract in disinfecting dental water systems.

Conflict of interest

The authors have no conflicts of interest regarding this investigation.

Acknowledgments

The authors would like to thank Valencia Health care and Diagnostic Centre, Mangalore for their kind support during microbiological lab studies.