Comparative evaluation of salt water rinse with... : Journal of Indian Society of Pedodontics and Preventive Dentistry (2024)

Introduction

Worldwide, 60%–90% of schoolchildren have dental cavities, often leading to pain and discomfort.[¹] Dental caries is the most prevalent oral disease in Member States of the South-East Asia Region, affecting 70%–95% of school-aged children and the vast majority of adults with mean decayed, missing, and filled teeth 1.87 in 2011.[²] The overall prevalence of dental caries among 12–15-year-old children range from 40% to 80% across various regions in India; the overall impression is that prevalence of dental caries has increased in India.[³]

Globally, severe periodontal disease, which may result in tooth loss, is found in 15%–20% of middle-aged (35–44 years) adults.[¹] According to National Oral Health Survey and Fluoride Mapping 2002–2003, the prevalence of children aged 12 years was 55.4% and it peaked at 89.2% in the 35–44-year age group. The prevalence was lower in 65–74-year age group (79.4%), possibly due to the presence of a high number of fully and partially edentulous patients in that age group.[⁴]

Combating dental caries and periodontal disease is the need of the hour for oral health professionals. Plaque is the common etiological factor for dental caries and periodontal disease.[⁵] Thus, plaque control remains one of the primary preventive modalities since ages. Mechanical plaque control at individual and professional level is the most effective way of maintaining good oral hygiene. The removal of plaque by normal brushing method alone is less effective. Chemical plaque control methods such as mouth rinses act as an adjunct to the primary mechanical methods of cleaning. The use of antimicrobial agents with significant antiplaque activity would be a meaningful, cost-effective measure in addition to mechanical oral hygiene methods.[⁶]

Mouth rinses are used in dentistry for prevention as well as curative purpose. Efficacy of various commercially available mouth rinses has been tested and proven. Majority of the commercially available mouth rinses are alcohol based possessing potent side effects. However, the affordability for daily usage of mouth rinses when it comes to a country like India is low.[⁷] Mouth rinse that is natural, safe, cost effective, readily available, and culturally acceptable is essential for oral health promotion in India. Thus, the present study is planned to verify if salt water (laboratory graded) rinse is effective in reducing oral disease causing microbial count. Chlorhexidine mouth rinse is the most commonly used anti plaque agent and hence used as a bench mark control for comparison. The aim of the present study is to assess and compare the effectiveness of salt water rinse with chlorhexidine mouth rinse in reducing oral disease causing microbial count in saliva.

Methods

An in vitro study was conducted to determine the minimum inhibitory concentration (MIC) of salt water against Streptococcus mutans, Lactobacillus acidophilus, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Various concentrations of salt solution were prepared by adding the required amount of solid sodium chloride (laboratory-graded NaCl) to 100 ml of distilled water in a beaker.

Amount of NaCl = Molecular weight of NaCl/1000 × required molarity × required volume

Molecular weight of NaCl = 58.44

For 0.2 M = 58.44/1000 × 0.2 × 1000 = 1.2 g

S. mutans (MTCC no. 497) and L. acidophilus (MTCC no. 10307) strains were obtained from Microbial Type Culture Collection (MTCC) and Gene Bank, Chandigarh, India. The S. mutans culture broth from MTCC was streaked on brain-heart infusion plates and incubated at 37°C for 48 h under 5%–10% CO₂. L. acidophilus culture broth from MTCC was streaked on Rogosa agar plate and incubated anaerobically using gas pack for 96 h at 37°C as per MTCC instructions.

Isolation of A. actinomycetemcomitans and P. gingivalis was done by collecting gingival crevicular fluid of patients with aggressive periodontitis and chronic periodontitis, respectively. Sterile paper points were inserted into the bottom of periodontal pocket in various areas around the tooth and removed after 10 s.[⁸] These paper points were transferred to sterile microcentrifuge tubes containing 1.5 ml of prereduced broth and stored in an ice pack containing thermocol box which was transported to Madras Veterinary College and Hospital, Vepery, within 4 h of sample collection and later streaked on the respective media.[⁹] The samples were placed on tryptic soy-serum bacitracin vancomycin (TSBV) medium and incubated anaerobically for 72 h at 37°C for A. actinomycetemcomitans.[¹⁰] The sample was placed on Wilkins Chalgren (WC) plate and incubated anaerobically for 7 days at 37°C for P. gingivalis.[¹¹] Gram-staining was done and visualized through microscopy. Biochemical test was carried out for all the bacteria for confirmation.

According to A. A. Miles and S. S. Misra method, tenfold dilutions were made from the overnight broth. One hundred microliters of culture broth was added to 900 μl of saline. Twenty microliters of ten dilutions was placed on the respective media using a sterile pipette, kept dried for 15–20 min, and then incubated in aerobic condition for S. mutans and L. acidophilus at 37°C for 48 h. Remaining three bacteria were incubated anaerobically at 37°C for 7 days. The drop areas from the higher concentrations of culture yield circular patches of confluent growth. Counts were made in drop areas containing the largest number of colonies without signs of confluence or of gross diminution in colony size due to overcrowding. The similar process was repeated again for all the bacteria. The number of colonies was estimated from the mean of each dilution as colony-forming units (CFU/ml).

The MIC of the salt water was determined by macrobroth dilution method [Table 1]. Five hundred microliters of 0.2 M to 1.0 M salt water was added in separate test tube and labeled as 0.1 M to 1.0 M. Five hundred microliters of the test material (respective broth culture contains 10⁶ CFU/ml) which includes S. mutans, L. acidophilus, A. actinomycetemcomitans, and P. gingivalis was taken in each test tube.[¹²] The test tubes were incubated at 37°C overnight.

After incubation, the lowest concentration level of salt which inhibits the visible growth of bacteria is considered as MIC.[¹³] The first concentration of incubated test tube showing blurriness is considered as MIC. The blurriness was visualized by drawing lines behind the test tube.[¹⁴] Table 2 summarizes the MIC for S. mutans, L. acidophilus, A. actinomycetemcomitans, and P. gingivalis.

This double-blind, randomized controlled trial was approved from the Institutional Ethics Committee of Tamil Nadu Dental College and Hospital, Chennai. Out of the total schools in Chennai city, one school was randomly selected. Official permission was obtained from the concerned school authorities. Thirty schoolchildren satisfying the eligibility criteria were recruited from the selected school by simple random sampling employing lottery method.

Inclusion criteria

Participants belonging to 12–15 years of age group were included in the study.

Exclusion criteria

• Participants with a history of systemic diseases
• Participants who have undergone oral prophylaxis or any kind of periodontal treatment within 1 week
• Participants with a history of antibiotics usage within 1 week
• Participants using any chemical antiplaque agents within 1 week
• Participants wearing fixed or removable orthodontic appliances or partial dentures
• Participants with a known history of allergy to chlorhexidine.

Written informed consent was obtained from the participants' parents/legal guardians. The study participants were randomized into two groups, 15 participants in each group. The two groups were referred as study group (0.8 M salt water) and control group (0.2% chlorhexidine mouth rinse).

Demographic data, plaque index (Silness and Loe, 1964), and dental caries by DMFS Index (Klein, Palmer, and Knutson, 1938) and defs Index (A. O. Gruebbel in 1944) were recorded by trained and calibrated chief investigator.

To minimize the influence of circadian cycles on the salivary secretion and composition, salivary collection was done at a fixed time throughout the study from 9:00 am to 9:30 am. The baseline unstimulated saliva samples were obtained from all the participants by spitting method into a sterile test tube and immediately transported to the Department of Microbiology, Madras Veterinary College and Hospital, Chennai through thermacol box containing ice packs and assessed for salivary S. mutans, L. acidophilus, A. actinomycetemcomitans, and P. gingivalis.

After baseline saliva collection, oral prophylaxis was done by the chief investigator to all the participants. A standard toothpaste and toothbrush were provided to the participants. All the participants were instructed to brush twice daily once in the morning and once before bedtime by Modified Bass technique. The selected participants were instructed not to eat or drink anything 2 h before saliva sample collection to circumvent alteration of the microbial flora.[¹⁵]

One day after oral prophylaxis, plaque index was recorded. Later, pretest unstimulated saliva samples were obtained from all the participants by spitting method and immediately transported to the Department of Microbiology, Madras Veterinary College and Hospital, Chennai, through thermacol box containing icepacks and assessed for microbial counts.

Triple blinding was done. The randomization was done by the supervision of coinvestigator. The participants were randomly and evenly allocated into study group and control group using Sequentially Numbered Opaque Sealed Envelopes (SNOSE). The study and control group participants were instructed to rinse with 0.8 M salt water and 0.2% chlorhexidine for 5 days, respectively.

Ten milliliters of mouth rinses were dispensed in similar color. The study participants were instructed to use 10 ml of assigned mouth rinse (study and control group) under co-investigator's supervision for 30 s daily morning between 9:00 and 9:30 am for a period of 5 days. The posttest unstimulated saliva samples were collected on the 5^th day and assessed for salivary microbial count.

Microbial analysis

One hundred microliters of vortexed salivary samples was pipetted using standard 100 μl pipette. The salivary sample was serially diluted (/10¹–/10⁶) by adding isotonic saline in a sterile tube. Twenty microliters of each dilution was pipetted and placed over the respective media for each bacterium. MSB (Mitis Salivarius Bacitracin) plates were incubated at 37°C for 48 h under 5%–10% CO₂ for the enumeration of S. mutans count.[¹⁵] Rogosa agar plates were incubated anaerobically using gas pack for 96 h at 37°C for L. acidophilus count.[¹⁶] The dilution was placed on TSBV plate and incubated anaerobically at 37°C for 72 h and on WC plate at 37°C for 7 days for A. actinomycetemcomitans[¹⁰] and P. gingivalis,[¹¹] respectively. Colony counting was done. The number of CFU was multiplied by the number of times the original milliliter of sample was diluted (the dilution factor of the plate counted) and expressed as the number of colony-forming units per milliliter (CFU/mL) of saliva.

The collected data were analyzed using Statistical Package for the Social Sciences software version 16 (SPSS - Inc, Released 2007, Chicago, IL, USA). Normality was analyzed using Shapiro–Wilks test. Dental caries experience was assessed by Mann–Whitney U-test. Independent Student's t-test was used for intergroup comparison of plaque scores and salivary microbial count. Mean rank difference in the baseline, pre-, and post-rinse values was assessed by Mann–Whitney U-test. Intragroup comparison was done using repeated measures ANOVA. Post hoc comparisons were done by Bonferroni analysis.

Results

The present study comprised two sections; the first part being the in vitro estimation of MIC of salt water against S. mutans, L. acidophilus, P. gingivalis, and A. actinomycetemcomitans and the second part was a comparison of plaque scores and salivary microbial count between salt water and chlorhexidine. Table 2 denotes the MIC of salt water against S. mutans, L. acidophilus, P. gingivalis, and A. actinomycetemcomitans.

Demographic characteristics of study and control group are presented in Table 3. Dental caries experience at baseline was statistically insignificant between the study and control group [Table 4]. Baseline plaque score, salivary S. mutans, L. acidophilus, and P. gingivalis count [Table 5] between study and control group were statistically not significant except A. actinomycetemcomitans (P = 0.01). However, after oral prophylaxis, salivary A. actinomycetemcomitans count was statistically insignificant (P = 0.34). The postrinse plaque scores and salivary microbial count were statistically not significant. Mean rank difference in the baseline, pre-, and post-rinse values between study and control group was statistically significant for S. mutans, L. acidophilus, and P. gingivalis [Table 6].

Intragroup comparison by repeated measures ANOVA revealed a statistically significant reduction in plaque scores (P<0.05) and salivary microbial count (P = 0.00) at baseline, pre- and post-rinse [Table 7]. Pairwise comparison reflects statistically significant results between the various tested timelines [Table 8].

Discussion

Commercially available chemical plaque control agents are limited for routine usage owing to its potent side effects. Herbal mouth rinses such as neem,[⁷] shallot extract,[¹⁷] garlic,[¹⁸] white pepper extract,[¹⁴] and aloe vera [^19,20] have been tested and proven to be effective against oral microbes. However, preparation of majority of herbal mouth rinses demands intense laboratory preparation. Thus, the present study evaluated the effectiveness of readily available salt water against oral microbes. 0.2% chlorhexidine was used as the benchmark control in the present study.

Salt solution has a different mechanism in inhibiting the growth of oral bacteria. In low concentration of salt solution, the surrounding environment is hypotonic. Oral bacteria have the ability to pump in ions with the energy from adenosine triphosphate by respiratory enzyme found in mesosomes. Water moves into the cell by osmosis and this gives an aqueous environment which is favorable for growth and reproduction of oral bacteria. At high concentration of salt solution, the solute concentration in the surrounding solution is greater than the cytoplasm of oral bacteria. Water moves out from cell by osmosis. Oral bacteria become dehydrated and eventually die within a minute.[²¹]

Salt water though proven to be effective, the MIC against oral microbes has not been scientifically established. Among the oral microflora, S. mutans and L. acidophilus are implicated in the initiation and progression of dental caries. P. gingivalis and A. actinomycetemcomitans are involved in chronic and aggressive periodontitis, respectively. Therefore, an in vitro study was conducted to determine the MIC of salt water against these bacteria. It was found that MIC of salt water was 0.7 M for S. mutans, A. actinomycetemcomitans, and P. gingivalis and 0.8 M for L. acidophilus. Based on the in vitro study results, 0.8 M of salt water was chosen as the intervention for the study group.

Although plaque is the representative sample for evaluation of oral microbes, saliva collection is more preferable than plaque owing to its practical feasibility. Saliva was used as one of the sites for sample collection since it is a fluid wherein all the other intraoral sites are immersed and could thus contain dislodged oral microorganisms from various intraoral sites.[²²] The salivary microbial species reflect the oral microbial community composition and could serve as a biomarker of the health and disease status of the oral cavity. Saliva allows dental plaque to flourish and also detaches layers of plaque.[²³] Saliva could act as an oral circulating fluid for bacterial transmission and act as a reservoir for bacterial colonization. Bacteria can survive in saliva and utilize salivary constituents for growth.[²⁴] The levels of cariogenic species in saliva have been investigated as a potential tool for caries risk assessment.[²⁴] Whole saliva can be easily collected with stimulating agents (using paraffin for mastication or using citric acid or sour candy drops on the tongue) or without stimulation. The unstimulated whole saliva is often used in diagnostics as stimulated whole saliva contains a diluted concentration of biomarkers that may be difficult to detect.[²⁵]

In the present study, 0.2% chlorhexidine was used as the intervention for the control group. The amount of chlorhexidine for rinsing depends on its concentration. Fifteen milliliters for 0.12% solution and 10 ml for the 0.2% solution of chlorhexidine were effective against microbes causing oral diseases.[²⁶] Thirty seconds is an acceptable length of time for mouth rinsing to achieve good compliance and efficacy.[²⁷] Therefore, it was planned to use 10 ml of salt water and 0.2% chlorhexidine as the interventions for duration of 30 s.

The present study revealed statistically significant reduction in the plaque scores (P = 0.00), salivary S. mutans (P = 0.00), L. acidophilus (P = 0.00), A. actinomycetemcomitans (P = 0.00), and P. gingivalis (P = 0.00) count with salt water rinsing. There was statistically insignificant difference between the study and control group for all the tested baseline values except A. actinomycetemcomitans. However, after oral prophylaxis (prerinse value), A. actinomycetemcomitans count became statistically insignificant between the study and control group (P = 0.34). Such a result means the tested outcomes can be attributed only to the intervention induced.

There was a statistically insignificant difference between the study and control group (prerinse vs. postrinse) in plaque scores (P = 0.19). On evaluation of the difference in the bacterial count between study group and control group, it revealed that the mean rank (prerinse vs. postrinse) is higher in the control group with the difference being statistically significant for all the microbes (P = 0.00) except A. actinomycetemcomitans (P = 0.35). This demonstrates that salt water was as effective as chlorhexidine in reducing dental plaque and A. actinomycetemcomitans count whereas chlorhexidine was superior against S. mutans, L. acidophilus, and P. gingivalis. This may be because chlorhexidine is a well-known bactericidal agent whereas salt is bacteriostatic which means it can only inhibit the growth of bacteria.

The present study results are in agreement with a study conducted by Rupesh et al., 2010, which showed statistically significant reduction in salivary S. mutans count after 21 days of saturated saline rinsing.[¹⁵] A study conducted by Gupta et al. which evaluated the effect of aloe vera mouthwash with chlorhexidine and saline as the placebo on dental plaque, concluded that saline rinse was not as effective as aloe vera and chlorhexidine.[²⁰] The concentration of saline in molarity has not been quantitatively mentioned in both the studies discussed. There are limited studies assessing the effect of salt water against L. acidophilus, A. actinomycetemcomitans, and P. gingivalis.

Dental plaque formation occurs de novo in the oral cavity. Complete elimination of dental plaque is practically not feasible. Thus, the intent of plaque control is to prevent the growth and maturation of dental plaque microorganisms. In this regard, salt though being bacteriostatic may be considered as effective as chlorhexidine in plaque control which is also reflected in the outcome of the present study.

Strengths

• The study participants were recruited with strict selection criteria which minimize the known confounders. Schoolchildren were recruited in the present study to enable easy supervision and compliance. Schoolchildren of age group 12–15 years were selected as it is the global index age for dental caries for international comparisons and monitoring of disease trends [²⁸]
• The mouth rinses were not given to the participants for rinsing outside school hours and weekends to eliminate contamination of interventions. As the interventions were carried out only at the school premises under the supervision of the co-investigator, compliance was assessed
• Concealed randomization was done by a different investigator through SNOSE to minimize allocation bias. The chief investigator was blinded about the randomization procedure. The microbiologist who performed the salivary analysis was also blinded about the details of the intervention which minimizes investigator bias [²⁹]
• After baseline salivary sample collection, oral prophylaxis was done for all the participants to make study and control group comparable before the intervention. Similar toothbrushes and dentifrices were provided to them. The participants were instructed to brush twice daily with the provided toothbrush and dentifrice by modified bass brushing technique throughout the study period. This was done to make all the parameters comparable during the study period. Thus, the final outcome can be attributed only to the intervention.

Limitations

• The study participants would have experienced some improvement not specifically associated with the therapeutic properties of the test agent, but rather related to behavioral change known as Hawthorne effect
• The study outcome could have been influenced by novelty effect, which is the motivation to improve oral hygiene practices induced by the use of a new substance
• Microbial analysis was done using salivary samples from the participants rather than plaque as a collection of saliva was practically feasible.

The present study concluded that 0.8 M salt water is effective against reducing dental plaque and the salivary oral microbial count. Despite the fact that salt water is less effective than chlorhexidine, considering the potential adverse effects of chlorhexidine, the presently tested 0.8 M salt water rinse can be an adjunct to mechanical plaque control for prevention of dental diseases as it is safe, readily available, cost effective and most importantly culturally acceptable. However, the effects of salt water rinsing among hypertensive patients have to be further evaluated.

Briefly, salt water can be recommended as a prophylactic antiplaque agent for routine usage while chlorhexidine may be used for short-term therapeutic purpose. 0.8 M salt water can be prepared by dissolving 4.7 g of sodium chloride (approximately equivalent to a three-fourth teaspoon of table salt) in 100 ml of distilled water. Thus, oral health-care professionals can recommend this formula at individual, community, and population level for the prevention of dental caries and periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgment

We acknowledge Dr. B. Samuel Masilamoni Ronald, MVSc., PhD., Professor and Head, Vaccine Research Centre – Bacterial Vaccine, TANUVAS, Dr. John Kirubakaran, MVSc., PhD., Professor and Head, Mr. V. Dilhibabu, M. Sc., Microbiologist, Department of Veterinary Microbiology, Madras Veterinary College, Chennai, for their unrelenting support and guidance in microbiological analysis.

REFERENCES