Comparison of several salt water concentrations on salivary flora

doi:10.21203/rs.3.rs-4388413/v1

Objective :Mouth washing involves soaking the mouth's mucosa, gums, and teeth in a solution. Its objectives include treating certain oral diseases and enhancing oral hygiene. Examining the impact of salt water mouthwashes on oral flora in vitro is the goal of this study.

Methodology : in the Bacteriology Division of the Central Laboratory of Tishreen university Hospital, we carried out a cross-over clinical trial. From January 2021to July 2022, this study has a 6-month duration. We performed convenience sampling throughout this work.

In our article, the same mouthwash was given to every participant at the same time, and the results were tracked throughout time (H1–H5). In order to prevent carry-over effects, a wash-out time of 3 days was observed between the interventions (mouthwashes with various saline solutions: 0.9%; 2%; 5.8% and 23%). To identify the bacterial species impacted by the mouthwashes, gram staining was done before and seven hours after the mouthwashes . We selected 10 subjects, from whom we collected 240 samples of saliva. There was a 1:1 sex ratio.

Results :Our findings demonstrated that all solutions reduced the oral flora 2 minutes after rinsing (e.g., 5.8% saline reduced the original flora by half). The handmade saline (2%) exhibited an antibacterial impact on the oral flora that lasted for 3 hours, but the action of the 0.9% saline was only present for this period. However, only the 5.8% and 23% concentrations significantly reduced the oral flora as the sampling process went on. 5.8% salt content exhibited a 5-hour bactericidal effect, while 23% salt concentration had a 7-hour effect. We also observed a more pronounced action on gram positive bacteria.

Conclusion: the 5.8% saline is more well-tolerated by the subjects and has long-lasting efficacy. Since the prepared saline in home has an antibacterial effect that lasts for three hours, it can be recommended for short-term purposes. Since the 23% saline has a strong, unpleasant taste, it should be avoided.

In Vitro Study

Mouthwash

Oral flora

Saline Solution

The act of soaking the mouth's mucous membranes, gums, and teeth in a solution is known as mouth washing. This procedure's goals are to treat some oral diseases and enhance oral hygiene(1) . Oral hygiene has traditionally been practiced with salt water, various herbal infusions, and an alkaline sodium bicarbonate solution. However, the antiseptic qualities of more modern solutions like chlorhexidine, povidone iodine, etc. have reduced the use of these natural solutions (1). Natural alternatives like saline mouthwash, which have less negative effects, would appear to be preferable. In fact (2) Kim et al. showed that using mouthwash made of salt water (at certain concentrations) could enhance dental cleanliness. He also demonstrated that using a salt water mouthwash will reduce halitosis, and the amount of bacteria in the mouth . In order to determine the greatest alternative for dental care with the least amount of danger, we conducted this study to compare the in vitro effects of various concentrations of saline solution to that of 0.9% saline.

Between January 2021to July 2022, we carried out a cross-over clinical trial. Central Laboratory of Tishreen university Hospital is where this study was conducted. From among the dental medicine students at the University of Tishreen Faculty of dental Medicine, we chose 10 participants. Participants had to meet six requirements: be between the ages of 20 and 25; appear to be in good health; have good oral health; not be wearing dentures; not perform any oral hygiene act 12 hours prior to the start of the clinical trial; and have had a scaling within the previous six months.

Any participant with a condition that might affect the oral flora, who neglected oral hygiene, or who was unavailable during the study period was also excluded. Participants who took medications that might affect the flora, who ate within one hour of the sampling, and who ate during the sampling period were also excluded. Following that, we received approval for our research from the Yaoundé General Hospital and institutional ethics clearance from the Faculty of Medicine and Biomedical Sciences at the University of Yaoundé 1. Participants received an explanation of the information leaflet and an informed consent form. Participants received free mouthwash and were given and given an explanation of the clinical trial's findings.

Each participant in our study washed their mouth between 9:00 and 9:30am using 10ml of the solution for 30 seconds. At regular intervals throughout the day, effects were noticed, including 2 minutes, 1 hour, 3 hours, 5 hours, and 7 hours after mouthwash. The flora was allowed to reassemble ad integrum by waiting three days between applications of mouthwash. Gram staining was carried out on the saliva samples before and 7 hours after mouthwash in order to identify any changes in the bacterial flora. By using the viable plate count method, saliva samples were examined for bacterial colony count(3) . One milliliter of unstimulated saliva was collected in a dry, sterile tube to make up the saliva samples. For our work, we used 4 distinct saline concentrations (selected in accordance with the bacterial halotolerance categorization)(4) . These included 5.8% hypertonic saline (1M), 23% hypertonic saline (4M), 0.9% saline (0.16M), and 2% saline (5mg of salt in 250ml of water) (5). Traditional table salt and sterile distilled water were used to prepare our solutions. The concentration of the salt used was as follows: Calcium: 25 mg/kg max; Magnesium: 150 mg/kg max; Sulphate: 400 mg/kg max; Sodium chloride: 97% min; Iodine: 100 ppm; Anti-caking agent E-536: 10 ppm; Insoluble: 0.01% max; Moisture: 0.1% maximum. Using the following formulas, we calculated the molar concentration of each solution prior to use: molar concentration = number of moles (mol)/volume (l), and mole = mass (g)/molar mass (mol/g).

Analysis of data

Data were entered into Microsoft Excel for recording. The repeated measures analysis of variance allowed us to compare the means obtained by the paired samples, and Student's paired-sample t-test was used to evaluate differences between groups. P values less than 0.05 were regarded as statistically significant. However, because there were numerous statistics run, the p-value was adjusted using the Bonferoni correction. These various statistical tests were run using SPSS 26.0 software.

A total of 240 saliva samples (24 samples per patient) were collected from the study's 10 subjects. The patients ranged in age from 22 to 25 years, with girls being on average 23.6 years old and boys being on average 22.6 years old. As shown in Table 1, the bacterial load of our patients before mouthwash (H0) varied from patient to patient. Additionally, this table displays the impact of several mouthwashes on the oral flora at every possible interval, from H1 to H5. Although the solutions' actions vary, these variations are not statistically significant. Gram-stained smear data (Table 2) reveal that at H0, the flora is composed of 60–70% gram–positive bacteria and 30–40% gram–negative bacteria. Following mouthwash with the following solutions: saline 5.8% and saline 23%, we saw a substantially larger reduction in gram-positive bacteria on analysis of the smears at H5. We gathered the participants' subjective opinions on the various mouthwashes during our clinical trial. This demonstrates:

Table 1: Effects of different saline mouthwashes on oral flora with time

Mouthwash
Time	0,9% saline	Homemade saline 2%	5,8% saline	23% saline
H0	56,5 ± 62,8	36,1 ± 12,7	36,2 ± 10,8	66,9 ± 105,5
H1	25,9 ± 15,4	26,9 ± 9,2	23,2 ± 15,6	33,4 ± 26,7
H2	86,1 ± 116	58,2 ± 92,8	32,1 ± 14,3	26,8 ± 11,7
H3	118,6 ± 129,1	35,6 ± 16,7	34,7 ± 10,7	34,2 ± 15,1
H4	42,7 ± 22,9	36,5 ± 9,7	27,7 ± 11,9	31,1 ± 9,1
H5	63,03 ± 70,6	35,7 ± 13,4	37,8 ± 13,9	40,2 ± 9,8
P value	0,097	0,438	0,003	0,317

The aforementioned numbers are all multiplied by 107 and given in CFU/ml.

H0: using a sample before mouthwash; H1: sampling two minutes after using mouthwash; H2: sampling an hour later; H3: sample three hours following mouthwash; sample five hours after using mouthwash; H5: 7 hrs following mouthwash

Table 2: Gram-stained swabs Results at H0 and H5.

Time	Stains	0.9% saline	Homemade saline 2%	5,8% saline	23% saline
H0	G+	69 ± 3,2	63 ± 4,8	64 ± 5,2	68 ± 9,2
H0	G-	31±6,8	37±5,2	36±4,8	32±0,8
H5	G+	66 ± 5,2	64 ± 5,2	53 ± 4,8	49 ± 8,8
H5	G-	34±4,8	36±4,8	47±5,2	51±1,2
P		0.081	0.726	<0,001	<0,001

Gram negative bacteria appear in blue; gram positive bacteria appear in black. (Show as%)

H0: sampling before to using mouthwash; H5: seven hours later

a) Following the 0.9% saline and homemade saline mouthwashes, all subjects complained of a salty taste. They characterized this flavor as typical and common.

b) According to 50% of the participants, the 5.8% saline was extremely salty but nevertheless bearable. While the other participants said its flavor was comparable to the above-mentioned solutions.

c) During our clinical experiment, none of the subjects were able to tolerate the 23% saline. Everyone noticed the flavor, which was harsh, abrasive, and lingered for a while.

Dental professionals commonly recommend mouthwash. Some 'comfort mouthwashes' are even sold in supermarkets. All of these industrial or medical solutions have gradually taken the place of a previous one, saline mouthwash, which had great results (2) (6) (7). Since a solution with a high salt concentration can have antibacterial effects, the usage of salt water mouthwash is, in fact, primarily supported by empirical arguments. It is sometimes referred to as a "grandmother's solution," is easy to prepare, and contains inexpensive ingredients like table salt and drinking water. A kilogram of table salt costs far less than a bottle of hydrogen peroxide, povidone iodine, or chlorhexidine. It would be prudent to utilize this solution once more because it has a low level of toxicity and has previously been successful(1) (6) .We conducted this study to examine the antibacterial impact of various salt water concentrations on the oral flora due to the dearth of scholarly research on this topic. We employed a variety of saline solutions for this investigation, including 0.9%, homemade saline (2%), 5.8%, and 23%.These remedies were selected in accordance with Vreeland's description of the halo tolerance categorization of bacteria(8) . According to the halo tolerance categorization, each solution represented a certain group of bacteria. The effects of these various solutions were contrasted with those of 0.9% saline.

Population Characteristics in General

Due to their accessibility, simplicity of comprehending the study process, and similar oral flora, our population was made up primarily of young oral health students with little age difference(9, 10).The results of Addy and Smith's study(9, 11) on students and administrative employees at the University of Cambridge and Shapiro et al.'s study (9, 11)on administrative personnel at the University of Bergen are analogous to the differences in flora across individuals.

Mouthwash's Impact on Oral Flora

The findings of our investigation demonstrated that all solutions decreased oral flora 2 minutes after mouthwash (e.g., 5.8% saline reduced the initial bacteria by half). This reduction may be due to two factors. The first is the physiological hyper-salivation that occurs after using mouthwash. The oral flora would be diluted by this serous saliva, primarily from the parotid region, which would lower the counted bacteria. The second explanation is that mouthwash has an instant antiseptic impact on bacteria because saline solutions can kill cells by causing them to dry up and lose their structure and function. Due to their limited ability to adapt to a saline environment, light halophiles exhibit this bacterial cell depletion most prominently.

Due to the fact that it is isotonic for bacteria and mucous membranes, the 0.9% saline solution only has a limited effect in this range. This outcome is comparable to Shapiro and Smith's work (10, 11) .However, as sampling went on, only two chemicals (saline 5.8% and saline 23%) significantly decreased oral flora. First, a 5.8% saline solution lowered oral flora for 5 hours, but a 23% saline solution had a 7-hour effect .The 2% saline solution had a 3-hour impact. These findings imply that these mouthwashes affect the oral flora in a sustained manner. This is likely because it took the salivary hyper secretion a long time to dilute these high salt concentrations. As a result, the high NaCl level's strong osmotic pressure is maintained. The growth and survival of the bacteria will be adversely affected by this in the long run.

5.8% and 23% saline's long-lasting effects may be a result of their adherence to dental plaque. Indeed, 12 hours before to the start of the mouthwashes, none of the trial participants had undertaken any oral care tasks. Plaque can form in this amount of time (around 30 minutes) (12). The glycoprotein matrix (exogenously acquired film) is where the 5.8% and 23% salts are most likely to bind and kill the plaque bacteria. It's also possible that the salt's effect on plaque is prolonged by the failure to remove salt-rich bacterial cell debris. It is believed that this concentration of Na+ in combination with that obtained after using salt water mouthwash enhances the alkalinity of the medium and hence inhibits bacterial development (13). This is because the concentration of Na+ in saliva rises with salivary flow to the point of approaching plasma levels.

When comparing the actions of these saline solutions with those of other mouthwashes described in the literature, it can be seen that these mouthwashes have a greater or equal effect (for example: Triclosan and Hexeditin have an antibacterial effect that lasts 3 hours according to Jenkins et al, Shapiro et al, and Tartaglia et al (11, 14, 15) , Acidified sodium chlorite and sodium lauryl sulphate .

After using mouthwash, bacteria change The solution that had the greatest impact on the oral flora, according to the results of the investigation, was the 23% saline solution. During the course of the entire experiment (7 hours), this lowered the oral flora by 50%. However, compared to homemade saline (2%), and saltwater (5.8%), the subjects found its taste to be offensive, grating, and unpleasant. The hyper stimulation of the taste cells' ionophores is responsible for this reaction. Since there are numerous sodium ion receptors on the tongue, mouthwash containing 23% saline will result in an excessive stimulus discharge because the Na+ is, in fact, what gives food its salty flavor (16).

After using mouthwash, bacteria change

The solution that had the greatest impact on the oral flora, according to the results of the investigation, was the 23% saline solution. During the course of the entire experiment (7 hours), this lowered the oral flora by 50%. However, compared to homemade saline (2%), and saltwater (5.8%), the subjects found its taste to be offensive, grating, and unpleasant. The hyper stimulation of the taste cells' ionophores is responsible for this reaction. Since there are numerous sodium ion receptors on the tongue, mouthwash containing 23% saline will result in an excessive stimulus discharge because the Na+ is, in fact, what gives food its salty flavor (16). Even though they are opinions, these evaluations suggest that 5.8% saline would be preferable since it is more tolerable and has a 5-hour antibacterial impact. However, homemade saline can be used or prescribed for short-term reasons (up to 3 hours). Gram-stained smear analysis revealed that the flora at H0 was composed of 60–70% gram positive bacteria and 30–40% gram negative bacteria. Following mouthwash with the following solutions: saline 5.8% and saline 23%, we saw a substantially larger reduction in gram-positive bacteria on analysis of the smears at H7. This finding would suggest that the saline solutions of 5.8% and 23% would be substantially more effective against gram positive bacteria.

The 5.8% saline is more well-tolerated by the subjects and has long-lasting efficacy. Since the prepared saline has an antibacterial effect that lasts for three hours, it can be recommended for short-term purposes. Since the 23% saline has a strong, unpleasant taste, it should be avoided.

Author Contributions Statement

Abdurrahman Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Validation, Visualization, Writing - Original Draft Preparation, Writing - Review & Editing Chaza Kouchaji Roles: Conceptualization, Formal Analysis, Funding Acquisition, Project Administration, Supervision, Validation, Visualization, Writing - Original Draft Preparation, Writing - Review & Editing

Funding

non

Data Availability

The datasets used and/or analyzed during the current study are available. from the corresponding author on reasonable request

Ethics approval and consent to participate

Ethical Approval was obtained from the Ethics Committee and the Board of Scientific Research at the Faculty of Dentistry at Tishreen university (No. 1203) dated 19-12-2020, to confirm all the methods were carried out in accordance with relevant guidelines and regulations. Official written permissions were also obtained from the authority bodies (Ministry of Health, Ministry of Higher Education and Research). Also, informed consent was obtained from children’s parents/careers to recruit their children to this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

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No competing interests reported.

Comparison of several salt water concentrations on salivary flora

Status:

Version 1

Abstract

INTRODUCTION

METHODOLOGY

RESULTS

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Status:

Version 1