Isolation and identification of fluoride sensitive and fluoride resistant Streptococcus mutans strains from dental carries of diabetic patients using conventional methods in Sudan.

Introduction

Bacteria have the ability to form biofilms on the surface of teeth and produce high levels of lactic acid, leading to the emergence of dental caries, which is the most prevalent dental disease today.¹ Fluoride (F⁻) is an inorganic, monatomic anion, which was discovered in 1931. Fluoride-based salts and minerals are important industrial chemicals as fluoride has been widely used in dental products to prevent caries by inhibiting demineralization and increasing remineralization. Furthermore, it affects bacterial growth by directly inhibiting metabolic activity through inhibition of enolase and ATPase enzymes, leading to a reduction in sugar uptake and glycolysis.² When the PH is 7, fluoride is in an ionic state, it cannot enter the bacterial cell membrane. Cariogenic bacteria ferments carbohydrates, leading to the formation of an acidic PH. Fluoride then binds to a hydrogen atom to form a HF (hydrogen fluoride) compound which can enter the bacterial cell membrane and interfere with the ability of the bacterium to tolerate acid. Consequently, the antimicrobial action of fluoride is known to be pH-dependent.³

One of the risks of prolonged use of fluoride is the occurrence of bacterial resistance. Fluoride resistant strains have been discovered in many studies, for example Streptococcus mutans can gain fluoride resistance either by acquiring resistance genes or through developing phenotypically, altering their physical appearance on the culture media containing flouride.⁴ When comparing the resistant strains with the wild type, S. mutans UA130 and S. mutans U159, they were found to be less cariogenic. It has been discovered in many clinical studies that fluoride resistant strains are created in the laboratory by subculturing colonies many times the presence of 400–600 mg/L fluoride.⁴ Microbes are excellent at adapting to stressful situations which is why antibiotic resistance is such a significant problem today.

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia and disturbances in the metabolism of carbohydrates, fats, and proteins.⁵ Poorly controlled DM has been associated with an increased susceptibility to oral infections including periodontal disease.⁶ The hypothesis of this research is that S. mutans develop fluoride resistance among Sudanese diabetic patients with dental caries.

The objective of this study is to isolate and identify S. mutans and characterize its antimicrobial properties against different concentrations of sodium fluoride and to estimate the prevalence of S. mutans among diabetic patients with dental caries in Sudan.

Methods

This was a descriptive, cross-sectional study involving 80 diabetic patients aged from 19 to 80 years old which was conducted in Khartoum-Sudan from patients attending diabetics center in Bahari and Army dental clinic, between September 2019–June 2022. The patients were 46% male and 54% female.

The samples collected from dental caries by using sterile cotton swap which was well rotated in the teeth cavity after mouth wash, from patients in the dental clinic and the diabetic’s center. The sample was inoculated onto mitis salivarius bacitracin agar containing 0.2 units of bacitracin and 20% of sucrose, as a selective media for S. mutans and onto chocolate agar. To detect alpha (partial) hemolysis of streptococcus mutans, they were incubated in a candle jar (10% CO₂) at 37°C from 24–48 hours. The purified isolates were then sub-cultured on appropriate slopes prepared from brain heart infusion agar medium for 24 hours at 37°C and stored in a refrigerator at 4°C for further identification. All isolates were Gram stained according to gram stain protocol⁷ and examined by microscopy using an 100× magnification oil immersion lens to study their staining properties and their shape and arrangements were recorded.

The isolates were identified based on their biochemical production of catalase enzyme by using 3% hydrogen peroxide (H₂O₂).⁸ A sterile wooden stick was used to transfer a small amount of a colony to the surface of a clean and dry glass slide. A drop of 3% H₂O₂ was placed onto the colony. Evolution bubbles were not observed. The acidogenic sugar fermentation (glucose, sucrose, mannitol, and lactose)⁹ solutions were prepared by dissolving 2 g peptone powder in one liter of distilled water, and then sterilized by autoclaving at 121°C for 15 minutes. Twenty-five mL of sterile 0.2% bromocresol purple solution was added to 950 mL of peptone water. Sugar solutions were prepared by dissolving 10 g of each sugar in 100 mL of distilled water and sterilized by autoclaving at 100^oC for 10 minutes. Twenty-five mL of sugar solution was then added to peptone water indicator solution and poured into a sterile test tube. A few colonies from a pure culture of the test organism were transferred to the test tube containing the sugar solution and peptone water using a sterile transfer lobe, the sets of inoculated test tubes were incubated in an aerobic incubator for 24 hours at 37^oC. The antimicrobial properties against optochin, using a disc impregnated with 5 μg of optochin were applied to 5% sheep blood ager plate, streaked with the organism being tested. After overnight incubation at 37°C in a 10% CO₂ atmosphere, isolates either displayed a zone of inhibition or did not. The method used to detect optochin sensitivity was the Kirby-Bauer disc diffusion method.¹⁰

Data was collected using a structured questionnaire for socio-demographic data, while the samples were collected using sterile cotton swabs. Data was analyzed using Statistical Package for Social Sciences software, version 23.0 (IBM SPSSInc., Chicago, IL, SPSS (RRID:SCR_002865)). Initially, all information was gathered via questionnaires then coded into variables. Both descriptive and inferential statistics involving Fishers’ exact test and binary logistic regression were used to present the results. A p-value of less than 0.05 was considered statistically significant.

Results and discussion

From the 80 participants caries samples were collected from, 37 were male (46%) and 43 were female (54%), the majority were aged over 50, and 51 participants (64%) had type II DM while 29 (36%) had type I DM. Thirty-four patients (43%) had good control of their diabetes according to their HbA1C levels. Seventy-one (89%) patients do not regularly follow up with the doctor for 6 months or more and 34 (43%) reported cleaning their teeth once per day and 75 (94%) use toothpaste. Sixty-nine (86%) of the patients do not have a history of heart disease, 64 (80%) show the invasion of tooth caries, and 60 (75%) have not used antibiotics for 6 months before the sample was collected. The sample was cultured and 68 (85%) had a positive growth while only 12 (15%) had a negative growth. S. mutans grew on MSA medium containing bacitracin inoculated with samples from 32 (40%) patients and 48 (60%) patients had samples which showed growth for organisms other than S. mutans.

To assess the sensitivity of the isolates to sodium fluoride, the technique stated by Ying et al. (2018)⁴ was used to prepare four different concentration of sodium fluoride (80, 125, 250, and 500 mg/L). Half of the isolates were found to be resistant to 80 mg/L NaF, as displayed in Figure 1.

Figure 1. The antimicrobial effect of fluoride against Streptococcus mutans.

Patients who had suffered with DM for longer time, were more likely to have a S. mutans infection AOR (adjustment odds ratio): 2.233 (CI 95%; 1.272–3.920) times statistically significant with a p value of 0.005<0.05.

Patients with previous antibiotic use were more likely to have S. mutans infections, with AOR: 3.413 (CI95%; 1.048–11.117), which is statistically significant with a p value of 0.042 which is <0.05. All other predictors contributed to the prediction of an S. mutans infection but had statistically insignificant p values of >0.05.

Fluoride concentrations in oral hygiene products vary from 250 to 1500 ppm the fluoride concentration remaining in dental biofilms after the application of these products was reported to be only between 1.14 and 5.7 ppm.¹¹ The biofilm fluoride level may induce resistance in oral bacteria when the pH of the environment remains low for an extended period of time.¹² In the present study, we used fluoride salt to investigate its antimicrobial activities against isolated S. mutans.

Conclusion

It can be concluded that 40% of the 80 samples collected showed the isolation of Streptococcus mutans. There was a significant correlation between having an S. mutans infection and patients suffering from diabetes mellitus disease for more than 20 years and with previous antibiotic use. Fifty percent of isolated S. mutans were found to be resistant to 80 ppm sodium fluoride, while 28% were resistant to 125 ppm NaF, 16% to 250 ppm and 3% to 500 ppm NaF.

It’s necessary to create a protocol for a superior examination of study results, which can improve the identification of fluoride-resistant strains. Further studying fluoride resistant S. mutans may highlight novel molecular methods to study the prevalence of fluoride resistance in the oral bacterial community. The role and importance of these mutations on gene regulation in the presence or absence of fluoride will be the subjects of follow-up studies.

Ethical approval

Ethical approval was obtained from ministry of health – Khartoum state general administration of strategic and information innovation, developments and scientific research. Serial number: KMOH-REC-018.2-2022

Consent

Written informed consent for publication of the patients’ data was obtained from the participants.

Data availability