Effects of Biosurfactant-Producing Bacteria Bacillus pumilus MMLN01 on Major Virulence Factors of Streptococcus mutans

Abstract

Streptococcus mutans, primarily responsible for dental caries, results in tooth demineralization due to plaque buildup and high sugar consumption. Glucosyltransferases produced by S. mutans synthesize virulent extracellular polysaccharides, enhancing cariogenic biofilms. Sucrose activates glucosyltransferase, leading to glucan synthesis, a key factor in caries formation. This study analyzed the effect of biosurfactant-producing bacteria extracted from the oral microbiota on Streptococcus mutans MTCC 497 targeting glucosyltransferase and ATPase. Oral swabs from healthy individuals from different states in India were screened for biosurfactant production using various assays such as oil drop collapse, CTAB methylene blue agar, and emulsification activity. The inhibitory effect of biosurfactants on S. mutans MTCC 497 were analyzed with minimum inhibitory broth concentration, sucrose dependent and sucrose independent adhesion and biofilm formation assays. Of 100 oral swabs, 40 bacterial isolates demonstrated antagonistic activity against S. mutans MTCC 497. From that, 17 biosurfactant-producing bacteria were identified. Ten bacteria, including DMTMM46, were assessed through various assays. DMTMM46 exhibited significant activity against Streptococcus mutans MTCC 497. Biosurfactants demonstrated efficacy in glucan synthesis, adhesion, biofilm formation, and ATPase activity at 75 µg/mL. Identification through 16SrRNA sequencing revealed DMTMM46 as Bacillus pumilus. The results indicate that biosurfactant produced by the bacteria Bacillus pumilus MMLN01 have therapeutic potential for dental caries.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01393-5.

Introduction

Oral diseases, especially dental caries and periodontal diseases, are common public health challenges worldwide. Dental caries afflict almost all adults and up to 60–90% of school-aged children, representing a chronic bacterial infectious disease that affects tooth surfaces [1]. Traditional treatments for dental caries encompass mechanical plaque removal, extensive clinical interventions (e.g., root canal therapy, dental restorations, and surgery), and the use of broad-spectrum antibiotics. However, obstacles such as drug resistance and inadequate oral hygiene can compromise the effectiveness of preventive measures [2]. Moreover, dental treatment is expensive, and it averages 5% of total health expenditures and 20% of out-of-pocket health expenditures in most high-income countries, and is beyond the capacity of healthcare systems in many low- and middle-income countries. Consequently, there is an urgent need for more cost-effective and preventive therapeutic strategies [3].

Furthermore, dental caries are closely associated with biofilms formation, with cariogenic biofilms representing a primary contributor to bacterial infection. Streptococcus mutans, the predominant etiological agent of dental caries, plays a pivotal role in cariogenic biofilms formation [4]. This bacterium secretes three types of glucosyltransferases (GTFases)—GTF-I (also known as GtfB), GTF-SI (GtfC), and GTF-S (GtfD)–which utilize dietary sucrose to synthesize extracellular polysaccharides (EPSs), serving as virulence determinants in cariogenic biofilms. GTFases catalyze the synthesis of water-insoluble glucans, promoting bacterial adhesion and biofilms aggregation on tooth surfaces, while water-soluble glucans act as a source of metabolizable carbohydrates for plaque bacteria, inducing the formation of water-insoluble glucans [5]. Targeting GTFases offers a promising approach for preventing and treating dental caries and related diseases, such as infective endocarditis [6].

Dental caries affect a substantial proportion of the global population, with an estimated 2 billion experiencing caries in permanent teeth and 514 million children affected by caries in primary teeth. The burden of oral diseases is particularly significant in middle-income countries, where urbanization and changes in living conditions contribute to increased prevalence rates [7]. In 2022, the World Health Organization (WHO) launched a global strategy on oral health to achieve universal health coverage for oral health by 2030 [8]. Furthermore, dental caries prevalence is highest in middle-income countries with high sugar consumption rates, predominantly affecting adults. Socioeconomic status influences disease prevalence, with higher rates observed among disadvantaged populations. In India, dental caries prevalence is notably high, with variations across age groups and regions [8].

Likewise, inhibitors of S. mutans GTFases have demonstrated efficacy in reducing bacterial adhesion to tooth surfaces and inhibiting caries development. However, significant levels of penicillin resistance among oral streptococcal isolates emphasize the importance of judicious antibiotic use, while biosurfactants produced by microbial cells, with their antimicrobial properties, offer potential alternatives to synthetic antibiotics [9]. Microorganisms such as Pseudomonas, Bacillus, Candida, Rhodococcus, and Corynebacterium are major producers of biosurfactants [10].

In conclusion, this study aims to evaluate the efficacy of biosurfactant produced by the bacteria Bacillus pumilus MMLN01 isolated from individuals with good oral hygiene and immune resistance, targeting the enzymes GTFases secreted by S. mutans MTCC 497, along with ATPase by cell permeabilization and ATPase assay.

Materials and Methods

Sample Collection and Eligibility Criteria

Oral swabs from healthy individuals were vigorously rubbed against the cheek, roof of mouth, and sides of molar teeth for 3 min, then transferred into saline and inoculated into tryptic soy broth medium. After 24 to 48 h of incubation, single bacterial colonies were isolated using spread plate technique on De Man–Rogosa–Sharpe (MRS) agar with dilutions of 10^–3, 10^–4, and 10^–5 [11]. MRS agar was chosen over BHI agar to favor growth of beneficial gram-positive bacteria. Participants with specific criteria (no dental caries, non-smokers, teetotalers, antibiotic-free for a year) were selected from diverse Indian states such as Tamilnadu, Kerala, Karnataka, Andhra Pradesh, Maharashtra, Madhya Pradesh, Odissa, Assam, Uttar Pradesh, Punjab, and Haryana to capture oral microbiota variations influenced by regional practices. Swabs were collected prior to teeth brushing with participant consent.

Pathogen

The lyophilized culture of caries-forming S. mutans MTCC 497 isolates was purchased from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The lyophilized culture were revived by inoculating into Brain Heart Infusion (BHI) broth and incubated for 48 h under anaerobic condition in 5% CO₂. Then, the grown cultures were streaked on BHI agar plate and incubated again in the same condition, and the purity of the bacteria was confirmed by using bright-field light microscope under 100X oil immersion. The pure culture strain obtained was maintained in BHI agar medium. A glycerol stock can be used for revival in case of contamination of pure culture, which is prepared by adding equal volume of exponential phase bacterial broth culture and glycerol. The stock prepared was stored at -20 ℃ for later use.

Screening of Biosurfactant-Producing Bacteria

Oil Drop Collapse Assay

To screen biosurfactant production, a drop of supernatant of oral bacterial isolates which was collected by centrifugation at 4293 g for 15 min, was added on oil-coated solid surface like glass or tiles. The stability of the drop was observed by using a bright-field light microscope under 40X immersion. The stability is dependent on surfactant concentration and correlates with surface and interfacial tension [12].

Cetyltrimethylammonium Bromide (CTAB) Methylene Blue Agar Assay

Medium plates were prepared by incorporating mineral salt medium (composition in grams per L of distilled water: Na₂HPO₄–3.6; (NH)₄SO₄–1.0; KH₂PO₄–1.0; MgSO₄.7H₂O–1.0; Fe(NH₄) citrate–0.01; yeast extract–25.0; CTAB–0.2; trace element solution–10 mL) with 0.005 g methylene blue. Wells were cut in the prepared medium by sterile gel puncture method and 100 µL of oral bacterial culture supernatant collected by centrifugation at 1493 g for 15 min was added to each wells cut. Then the plates were incubated for 24 to 48 h at 37 ℃. The production of biosurfactant was confirmed by halo zones produced by the bacterial supernatant were observed by naked eye [13].

Emulsification Activity

Emulsification index (EI) was detected by mixing hydrophobic compounds such as castor oil, coconut oil, gingelly oil, olive oil, kerosene, and petroleum with cell-free supernatant of oral bacterial culture in a ratio of 2:3 (V/V). The mixture was vortexed for 2 min and allowed to stand for 24–48 h at room temperature. The emulsification activity, i.e., the ability of the oral bacteria to form emulsion layer, was calculated by the following formula:

Emulsification index (EI) = Height of the emulsion layer (H_E)/height of the total mixture (H_T)_X100 [14]

Lipase Activity

The lipase activity of the bacteria was detected by using trybutyrin agar (HiMedia Laboratories Pvt.Ltd., Maharashtra, India) with 1% trybutyrin. A simple streak was done on the center of the trybutyrin agar plate and incubated at 37 ℃ for 24–48 h until clear zones were formed, which can be seen observed by naked eyes [15]

Hemolytic Activity

The pathogenecity of the isolated oral bacteria can be identified by detecting their hemolytic activity. The bacteria were streaked on the sterilized blood agar plate supplemented with 5% sheep blood. The plates were then incubated at 37 ℃ for 24–48 h and the zones formed were observed [16]. Complete lysis of blood (β-hemolysis) by the bacteria results in a clear zone, and partial lysis (α-hemolysis) results in a partial or greenish zone. No zone will be observed in the absence of lysis (γ-hemolysis). Based on this, bacteria that do not cause lysis are considered non-pathogenic, whereas those that cause lysis are considered pathogenic.

Surface Tension Determination

A surface tensiometer (Kyowa Tensiometer Model DY-300) was used to measure the surface tension of biosurfactant at 25 ℃ according to du Nouy method [17]. The platinum ring present in the instrument was sterilized with flame until red hot and was hanged parallel to the sample to aid the sinking of ring into the sample after cooling. Surface tension of the isolated oral bacterial supernatant was measured by placing the glass lid with the supernatant on the stage of the instrument. Click start measurement button and values were recorded by using the DY-300 software installed and connected to the instrument with water as control.

Formula: γ₂ = γ₁ n₁ ρ₁ / n₂ ρ₂.

Where,

γ₁–surface tension of water.

γ₂–surface tension of biosurfactant.

n₁–number of water drops.

n₂–number of biosurfactant drops.

ρ₁–density of water.

ρ₂–density of biosurfactant.

Production of Biosurfactants

The biosurfactant-producing bacteria identified were incorporated into BHI broth and shaken at 180 rpm while being incubated at 37 ℃ for 72 h. Afterward, the broth culture was centrifuged at 4293 g for 30 min, and the cell-free supernatant was collected. It was then acidified with 6N hydrochloric acid and incubated for 24 h at room temperature. The precipitate was removed following incubation by centrifugation at 4293 g for 30 min, and it was then dissolved in sterile MilliQ water. Then, an equal amount of methanol and chloroform in a 2:1 ratio was added, mixed vigorously, and kept aside for a while. The organic phase was then collected. The steps were repeated twice or thrice until the solvent was clear. A rotary vacuum evaporator was used to concentrate the collected organic phase, which was then saved for further examination [18].

Minimum Inhibitory Concentration

The minimum inhibitory concentration of biosurfactants was assessed using the MIC broth dilution method [19]. The overnight exponential phase BHI broth culture of S. mutans MTCC 497 were diluted in freshly sterilized BHI broth to 0.1 OD ( ~ 1 × 10⁷ CFU/mL). Then, a stock solution of the extracted crude biosurfactants (3 mg/mL) was prepared. To a 96-well microtitre plate, 200 µL of the pathogen and 5–125 µg/mL (concentration increased at a rate of 5 µg/mL, i.e., 5, 10, 15,…125 µg/mL) of the crude biosurfactants taken from the stock solution were added. The mixture was incubated for 24 h at 37 ℃ in 5% CO₂ and the turbidity formed was observed at 600 nm with UV visible spectrophotometer and sub-minimum inhibitory concentration (sub-MIC) of the biosurfactants were identified [20].

Sucrose-Dependent and Sucrose-Independent Assays

The biofilm inhibitory action of biosurfactants was analyzed both in the presence and in the absence of sucrose. Overnight-grown broth culture of S. mutans MTCC 497 (0.1 OD) was inoculated in BHI broth with and without 5% sucrose, respectively. A total of 100 µL of the diluted broth culture and crude biosurfactants at sub-MIC level were added to a microtiter plate and incubated at 37 ℃ for 24 h in 5% CO₂. After incubation, the planktonic cells of the incubated broth culture (total cells) were removed and their absorbance was measured at 600 nm. Similarly, the cells attached to the microtiter plate were washed with 0.5 M NaOH and suspended in 0.85% sterile saline [21], and their absorbance was measured at 600 nm. The percentage of adherence based on the activity of the crude biosurfactants was calculated by the following formula:

Percentage of adherence = OD of adhered cells/OD of total cells × 100 [22]

Time-Killing Dynamics of Planktonic cells

A total of 100 µL of diluted 0.1 OD of overnight-grown culture of S. mutans MTCC 497 and crude biosurfactants at sub-MIC level were added into each well of the microtiter plate and incubated at 37 ℃ in 5% CO₂. The optical density values were recorded at 0, 2, 4, 8, 12, and 24 h at 600 nm [23] under UV Visible spectrophotometer.

Cell Permeabilization and F₁F₀ATPase Activity

The ATPase activity of the crude biosurfactants of oral bacterial isolates was determined by permeabilizing the cells of S. mutans MTCC 497. S. mutans MTCC 497 cells were permeabilized with 10% toluene by continuous freezing and thawing. To the permeabilized cells, 75 mMol Tris–maleate buffer (pH 7, containing 5 mM adenosine 5 triphosphate) and 10 mM MgCl₂ were added. The reaction mixture was incubated for 1 min and the readings were recorded at 340 nm. The F₁F₀ATPase activity of the biosurfactants was measured by determining the inorganic phosphate levels released by the cells [24]. All experiments were done in triplicates.

Biofilm Inhibition Assay

Crystal violet assay was used to measure the inhibition of biofilm [25]. The stationary phase culture of S. mutans MTCC 497 was adjusted to 0.1 OD with 5% sucrose containing BHI broth. 200 µL of adjusted culture was added to the wells of microtiter plate and incubated for 24 h at 37 ℃ with 5% CO₂. Planktonic cells were taken out after incubation, and the plates were rinsed with sterile phosphate buffer saline (PBS). Crude biosurfactants at sub-MIC levels were added to the well along with new sterile broth and reincubated for 24 h at the same condition. After incubation, the adhered biofilm in the plate was rinsed with sterile PBS and fixed with 95% methanol after the total cells were removed. Finally, the cells were stained with 0.1% crystal violet solution for 15 min at room temperature. After that, the stain was tapped off and dissolved with 100 µL of 100% ethanol. From that, 75 µL was transferred to a new plate and the absorbance was measured at 600 nm.

Formula: Percentage of biofilm Inhibition = absorbance of treated group/absorbance of control group × 100.

Inhibition of Synthesized Glucan

Glucosyltransferase (GTFs) was initially isolated from Streptococcus mutans MTCC 497. S. mutans MTCC 497 were inoculated into BHI broth and incubated under shaking condition at 180 rpm for 96 h, and the culture was centrifuged at 4293 g in 4ºC for 30 min. The supernatant obtained was collected, to which 60% saturated solid ammonium sulfate was added and incubated for 24 h. After incubation, the mixture was centrifuged for 30 min at 4293 g in 4⁰C. The crude protein was then recovered, suspended in saline, and kept at -80 ℃ [26].

The reaction mixture contains 0.25 ml of extracted enzyme crude, 0.25 ml of 0.4 mM sucrose, and sub-MIC level crude biosurfactants isolates in 20 mM phosphate buffer at pH 6.8. The reaction mixture was incubated at 37 ℃ for 18 h. Phenol sulfuric acid method was used to estimate the amount of glucan synthesized [27]. All experiments were performed in triplicates and average values were noted.

Microscopic Observation

The inhibition of biofilm formation by S. mutans MTCC 497 was assessed using sub–MIC level of effective oral bacterial biosurfactant. The biofilm formation over glass surface was visualized using two microscopy techniques. First, fluorescent microscopy with acridine orange staining was employed to observe live and dead cells within the biofilm structure. Second, bright-field microscopy with methylene blue staining under oil immersion was used to visualize the overall biofilm and cell distribution [28].

Identification of Bacteria

The DNA of the selected biosurfactant-producing bacteria was extracted by using QIAamp DNA Mini Kit (50 DNA preps) [29], and 16S rRNA gene was amplified with PCR by using Universal Primers 27F–5’AGAGTTTGATCMTGGCTC-AG3’ and 1492R–5’CTACGGCTACCTTGTTAC-GA3’ with the conditions of initial denaturation—95 ℃ for 3 min, denaturation—94 ℃ for 30 secs, annealing—53 ℃ for 1 min, extension—72 ℃ for 2 min and final extension—72 ℃ for 10 min. Based on the amplification data obtained, molecular identification was done [30].

Characterization of Biosurfactant

Analysis by Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra were recorded to identify the functional groups present in the biosurfactant produced by the bacteria exhibiting effective activity, acquired in the range of 4000 to 400 cm⁻¹[31].

Results

Isolation of Bacteria

Of the 100 oral swabs collected from healthy individuals, 40 oral bacteria that showed activity against S. mutans MTCC 497 were isolated by antagonistic test done by agar well diffusion method based on the formation of zone.

Screening Tests for Biosurfactant-Producing Bacteria

The 40 oral bacterial isolates that showed activity against S. mutans MTCC 497 were screened for biosurfactant-producing activity by various screening assays. Of the total isolates collected, 17 oral bacterial isolates (see Table 1) were found to have biosurfactant-producing activity. Of the 17 listed oral bacteria, 15 had the ability to collapse the oil drops streaked on the glass plate, which shows that the isolates were capable of producing biosurfactants. Biosurfactant-producing ability of the isolates was also confirmed by CTAB methylene blue agar assay, in which the isolates that produced biosurfactants formed zones. All 17 oral bacterial supernatants when mixed with hydrophobic compounds exhibited emulsifying property. Eight oral bacteria streaked on tributyrin agar with 1% tributyrin formed zones, which show their ability to break the lipids present in the agar medium by producing lipase enzyme. However, all the oral bacteria listed in Table 1 did not lyse the blood in the blood agar plates except DMTMMBA20. DMTMMBA20 completely lysed the blood (β-hemolysis) and therefore was considered pathogenic. The remaining 16 oral isolates were considered non-pathogenic as they did not lyse the blood (γ-hemolysis). Moreover, all the oral bacterial supernatants were found to have less surface tension when compared with water (70 dynes/cm).

Table 1.

Screening test results for biosurfactant producing oral bacterial isolates

S. no	Oral bacterial isolates	Drop collapse assay	CTAB assay	Emulsification index (EI)%	Lipase activity	Hemolytic activity	Surface tension (dynes/cm)
1	DMTMMAO02	+	+	53.125	_	No lysis	48.64
2	DMTMMPA09	+	+	33.333	_	No lysis	51.51
3	DMTMMEC12	+	+	46.875	+	No lysis	46.73
4	DMTMMLA16	+	+	53.125	+	No lysis	40.93
5	DMTMMBA20	_	+	53.125	_	β-hemolysis	53.2
6	DMTMMAB24	+	+	53.125	+	No lysis	45
7	DMTMMAS25	+	+	50.000	+	No lysis	48
8	DMTMMBC37	+	+	33.333	+	No lysis	33.3
9	DMTMMEH38	_	+	53.125	+	No lysis	46.2
10	DMTMMET39	+	+	53.125	_	No lysis	46.5
11	DMTMMEL42	+	+	50.000	+	No lysis	51.8
12	DMTMMBP46	+	+	20.000	+	No lysis	32
13	DMTMMEC51	+	+	33.33	_	No lysis	48
14	DMTMMCB56	+	+	36.67	_	No lysis	58.4
15	DMTMMAB67	+	+	55.88	_	No lysis	55
16	DMTMMEB75	+	+	46.67	_	No lysis	55
17	DMTMMPR80	+	+	10.00	_	No lysis	60.1

Production of Biosurfactants

The crude biosurfactant was extracted, dried, and powdered from the 17 biosurfactant producing oral isolates, was then stored, dissolved in DMSO, and used for subsequent inhibitory assays.

Minimum Inhibitory Concentration

The minimum inhibitory concentration of biosurfactants was assessed using the MIC broth dilution method. To a 96-well microtitre plate, 200 µL of the pathogen culture and crude biosurfactant at a concentration range 5–125 µg/mL were added. The mixture was incubated for 24 h at 37 ℃ in 5% CO₂ and the turbidity formed was observed at 600 nm. An initiation of turbidity reduction was observed at a concentration of 75 µg/mL and very less turbidity at concentrations starting from 90 µg/mL; therefore, 90 µg/mL was considered sub-MIC level. No change was observed for the negative control DMSO. This shows that the biosurfactants were effective at a concentration of 90 µg/mL and were capable of inhibiting S. mutans MTCC 497. Of the 17 oral bacterial biosurfactants, 10 biosurfactant crudes such as DMTMMAO02, DMTMMEC51, DMTMMLA16, DMTMMBA20, DMTMMAB24, DMTMMBC37, DMTMMEH38, DMTMMET39, DMTMMBP46, and DMTMMEB75 showed effective inhibitiory action against the pathogen (Fig. 1). Therefore, the 10 effective oral bacterial biosurfactants were used for inhibitory assays.

Fig. 1.

Minimum Inhibitory Broth Concentration of the biosurfactants produced by oral bacterial isolates against S. mutans MTCC 497

Sucrose-Dependent and Sucrose-Independent Adhesion Assays

The anti-adherence activity of the crude biosurfactants of oral bacteria was tested against S. mutans MTCC 497 by sucrose-dependent and sucrose-independent adhesion assays. Ten oral bacteria exhibited effective anti-adherence activity against the pathogen at sub-MIC level (Fig. 2). Of the 10 oral bacterial biosurfactants, DMTMMBP46 showed 19% and 51% inhibitory activity against S. mutans MTCC 497 both in the presence and absence of sucrose, respectively. However, the sucrose-dependent adherence activity of the biosurfactants was marginally more significant.

Fig. 2.

Anti-adherence activity of oral bacterial biosurfactants against S. mutans MTCC 497 using both Sucrose-dependent (BHI + 5% sucrose) and sucrose-independent (BHI) conditions

Time—Killing Dynamics of Planktonic cells

Time-kill experiments were performed to confirm the efficacy of oral bacterial biosurfactants in reducing pathogen viability (Fig. 3). Initial inoculation was done at 10⁷ CFU/mL. Over time, the biosurfactants derived from the selected oral isolates at Sub-MIC levels showed a decline in bacterial growth. However, the rate of pathogen decline varied among the different biosurfactants tested. After 24 h of incubation, the biosurfactants derived from oral isolates demonstrated significant reduction in pathogen viability.

Fig. 3.

Graph showing the effect of sub-MIC levels of biosurfactants produced by oral bacterial isolates on killing of S.mutans MTCC 497 over different time intervals

Cell Permeabilization and F₁F₀ATPase Activity

The efficiency of the crude biosurfactants was analyzed by examining the inorganic phosphate released when the biosurfactants react with permeabilized cells of S. mutans MTCC 497, and the absorbance was observed by UV spectrophotometer at 340 nm. Biosurfactants with increased efficiency decreases the ATPase activity of the permeabilized cells, which in turn decreases the amount of inorganic phosphate released. This shows that the biosurfactants studied were effective against S. mutans MTCC 497 and hence can be utilized in the treatment of dental caries. Of the 17 biosurfactant studied, DMTMMBP46 showed high efficiency in suppressing the ATPase enzyme present in S. mutans MTCC 497 compared to negative control and other oral bacterial biosurfactants (Fig. 4).

Fig. 4.

The efficacy of biosurfactants at sub-MIC level on the ATPase activity of S. mutans MTCC 497 at 340 nm

Biofilm Inhibition Assay

All the biosurfactants showed biofilm inhibitory action. However, DMTMMBP46 was found to be significantly more effective at a rate of 93% compared to other biosurfactants (Fig. 5).

Fig. 5.

Inhibition of biofilm formation by Crystal Violet Assay using sub-MIC levels of biosurfactants from the selected oral bacterial isolates against S.mutans MTCC 497

Inhibition of Synthesized Glucan

The ability of the biosurfactants to reduce the FTPase-mediated glucan synthesis in S. mutans MTCC 497 was analyzed by glucan synthesis assay. GTFase converts sucrose into sticky glucans, which helps S. mutans to adhere to the tooth's surface, which means that adherence of S. mutans to smooth surfaces has been attributed to the production of sucrose-derived glucans. Water-insoluble glucans help bacteria to adhere and aggregate on the tooth surface to form biofilms, while water-soluble glucan may supply a source of metabolizable carbohydrates for plaque bacteria and induce water-insoluble glucan formation. This study showed that the crude biosurfactants significantly reduced the production of GTFase. Of the 10 biosurfactants examined, DMTMMEH38, DMTMMET39, and DMTMMBP46 effectively prevented the activity of glucans. As shown in Fig. 6, the biosurfactants decreased the production of glucans by 55%. However, DMTMMBP46 was found to be more effective against S. mutans MTCC 497 compared to other biosurfactant-producing bacteria.

Fig. 6.

Inhibition of synthesized glucan by sub-MIC levels of oral bacterial biosurfactants against S.mutans MTCC 497

Microscopic Observation

The biosurfactant DMTMMBP46 demonstrated significant effectiveness in inhibiting the formation of biofilms by reducing cell to cell adherence was observed under the microscope. Figure 7 clearly shows the significant reduction in biofilm formation after the incubation of S. mutans MTCC 497 with the biosurfactant DMTMMBP46. This reduction is evident fron the decreased fluorescent signal in the acridine orange staining, indicating fewer viable cells, and less dense biofilm was observed with methylene blue staining.

Fig. 7.

A, B, and C Fluorescent imaging; D, E, and F bright field light microscope imaging of Streptococcus mutans alone, treated with positive control chlorhexidine, and treated with biosurfactant of DMTMMBP46

Identification of Biosurfactant-Producing Bacteria

After DNA extraction, the 16S rRNA gene of the chosen strain was sequenced, and the species was then tentatively identified. The BLAST programme of NCBI was used to determine the taxonomic affiliation of the isolates, and the representatives of the most homologous (96–98%) sequences of the isolates were collected. Phylogenetic affiliation was created by utilizing MEGA 6.06.

Based on the 16 s rRNA forward and reverse sequence data available in National Centre for Biotechnology Information (NCBI) BLAST nucleotide, the bacteria DMTMMBP46 was identified as Bacillus pumilus. Following submission to the NCBI, the sequence was assigned the accession number (OP090341) in the Bacillus pumilus MMLN01 nomenclature.

Characterization of Biosurfactant produced by Bacillus pumilus MMLN01 Fourier-transform infrared spectroscopy (FTIR) Analysis

Significant absorption bands were observed in the spectral range of 3293 to 1061 cm⁻¹ during the analysis of functional groups present in the crude biosurfactant produced by Bacillus pumilus MMLN01 (Fig. 8). Characterization of the biosurfactant via FTIR revealed the several significant vibrational modes. The presence of an S = O stretching band suggests the occurrence of sulfoxide or sulfone functional groups. The C-N stretching band indicates the presence of amine, nitrile, or imine moieties within the biosurfactant. Furthermore, the O–H bending band is indicative of alcohol, carboxylic acid, or phenol groups. The N–O stretching band suggests the presence of either nitro or nitroso groups. Additionally, the appearance of a C = C stretching band suggests the presence of carbon–carbon double bonds, as seen in alkenes and aromatic compounds. Finally, the C-H stretching band confirms the presence of carbon-hydrogen bonds.

Fig. 8.

FTIR analysis of biosurfactant produced by Bacillus pumilus MMLN01

Statistical Analysis

All experiments were performed in triplicate, and graphs were plotted using mean values with standard deviation error bars. Data were normally distributed. Independent sample t-test was performed for cell permeabilization and F₁F₀ ATPase activity, Biofim inhibition assay, and synthesized glucan assays. Correlations were performed for Minimum Inhibitory Concentration and Time-Killing assays, with a significant level of P < 0.0001.

Discussion

This study effectively isolated and characterized oral bacterial strains with potential inhibitory effects against S. mutans MTCC 497, a primary pathogen in dental caries. The diversity of the oral microbiota was evidenced by the significant number of bacterial isolates demonstrated antagonistic activity against S. mutans MTCC 497. A critical finding was the identification of 17 isolates capable of biosurfactant production, which was corroborated by various screening assays. The ability to collapse oil droplets and form clear zones in the CTAB methylene blue agar assay indicated the presence of biosurfactants. These results align with previous studies where similar methods have effectively identified biosurfactant producing bacteria[32]. The production of biosurfactants by oral bacteria is particularly significant, as these compounds play a crucial role in disrupting biofilms and inhibiting pathogenic bacteria. Most of the isolated strains were non-pathogenic, as indicated by the lack of hemolysis on blood agar plates, except for DMTMMBA20. This suggests that the majority of these bacteria could be considered safe for potential therapeutic use. Similar non-pathogenic profiles have been observed in other studies, investigating biosurfactant producing bacteria from various environments. For example, a previous report [33] indicated that Proteus mirabilis strain SLM-B52 did not cause any hemolytic activity, even though it exhibited the highest surface tension reduction capacity. The minimum inhibitory concentration (MIC) studies revealed that biosurfactants were effective at 75 µg/mL, with complete inhibition at 90 µg/mL. This finding underscores the potential of these biosurfactants as potent antimicrobial agents against S. mutans MTCC 497. Prior research highlighted that the inhibition rate of pathogens at sub-MIC levels, such as 2.5 mg/mL of biosurfactants, reinforcing the current study’s findings [34]. The adhesion of surface adhesives like SpaP, plays a significant role in bacterial adherence when sucrose is absent. However, in the presence of sucrose, the enzyme such as glucosyltransferases become crucial, enhancing bacterial adhesion and contributing to the pathogenesis [35]. The anti-adherence assays demonstrated that the biosurfactants, particularly DMTMMBP46, significantly inhibited the adherence of S. mutans MTCC 497 to glass surfaces, both in the presence and absence of sucrose. This dual-action capability is essential for preventing dental plaque formation, as S. mutans adheres to teeth via sucrose-dependent and independent biofilm formation, this further validates its potential application in oral hygiene products to prevent dental caries. Time killing assays confirmed the time-dependent bactericidal activity of the biosurfactants. The decline in pathogen viability over 24 h suggests that these biosurfactants could provide sustained antimicrobial effects. Additionally, the reduction in ATPase activity indicates effective disruption of bacterial cell membrane integrity, which is critical for bacterial survival and biofilm formation. The ability of the biosurfactants to inhibit glucan synthesis, a key factor in biofilm formation, highlights their therapeutic potential. By reducing both water-soluble and insoluble glucan production, the biosurfactants effectively prevent the establishment and maintenance of biofilms by S. mutans [36]. The identification of DMTMMBP46 as Bacillus pumilus, based on 16SrRNA sequencing, and its subsequent characterization by FTIR, provided valuable insights into the structural components of the biosurfactants. The presence of functional groups such as sulfoxides, amines, and carboxylic acids suggests a complex molecular structure that could interact with microbial membranes and disrupt biofilm formation[37].

This study presents a comprehensive analysis of the antimicrobial properties of biosurfactants produced by oral bacteria, with Bacillus pumilus MMLN01 (DMTMMBP46) emerging as a particularly potent strain. These findings support the potential use of these biosurfactants in dental care products aimed at preventing and treating dental caries. Further research should explore the formulation of these biosurfactants into practical applications and their efficacy in clinical settings.

Conclusion

Streptococcus mutans is a cariogenic bacterium that metabolizes sugars to synthesize acids, leading to tooth demineralization and the widespread chronic oral disease known as dental caries. Key factors in the prognosis of dental caries include patient health, oral hygiene, and caries severity. This study aimed to analyze the effect of biosurfactants produced by the bacteria on enzymes GTFase and ATPase secreted by S. mutans MTCC 497. The biosurfactants extracted from the bacteria in oral swabs effectively inhibited these enzymes, reducing glucan synthesis and biofilm formation. Among the 10 selected bacterial biosurfactants, the biosurfactant from the bacteria DMTMMBP46, identified as Bacillus pumilus MMLN01, exhibited significant efficacy at 75 to 90 µg/mL. These findings underscore the importance of exploring biosurfactants as a cost-effective and preventive therapeutic application for dental care. The ability of these biosurfactants to target specific virulence factors of S. mutans MTCC 497 and their potential to be incorporated into oral care products presents a promising route for future research and clinical application.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This study was funded by DST–SERB (SB/-EEQ/2018/000961 Dt. 07.05.2019).

Declarations

Conflict of interest

None.

Ethical Approval.

Ethical clearance approved by Madurai Kamaraj University. Oral swabs were collected with their individual consent.