CN112961216A

CN112961216A - Streptococcus mutans specific targeting antibacterial peptide and application thereof

Info

Publication number: CN112961216A
Application number: CN202110226901.2A
Authority: CN
Inventors: 涂欢芯; 邱莉莉; 张学佳; 潘乙怀; 任佩琪
Original assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Current assignee: SCHOOL & HOSPITAL OF STOMATOLOGY WENZHOU MEDICAL UNIVERSITY
Priority date: 2021-03-01
Filing date: 2021-03-01
Publication date: 2021-06-15
Anticipated expiration: 2041-03-01
Also published as: CN112961216B

Abstract

The invention discloses a streptococcus mutans specific targeted antibacterial peptide and application thereof, and belongs to the technical field of biological engineering, wherein the amino acid sequence of the streptococcus mutans specific targeted antibacterial peptide is TFFRLFNRGGGRRWWRF, and the streptococcus mutans specific targeted antibacterial peptide is formed by combining a CSP fragment and a short-chain artificial antibacterial peptide. The narrow-spectrum and high-efficiency antibacterial characteristics of the heterosexual targeting antibacterial peptide are adopted to prompt that the streptococcus mutans specific targeting antibacterial peptide obtained by connecting the targeting peptide fragment capable of being specifically combined with the surface of streptococcus mutans with the broad-spectrum antibacterial peptide fragment has the application potential of preventing and treating caries, the narrow-spectrum antibacterial medicament is used for selectively reducing the content of main cariogenic bacteria (such as streptococcus mutans) in dental plaque, so that the non-cariogenic bacteria are converted into dominant bacteria, the re-planting of the cariogenic bacteria can be effectively inhibited, and finally the purpose of preventing the caries for a long time is achieved.

Description

Streptococcus mutans specific targeting antibacterial peptide and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to a streptococcus mutans specific targeted antibacterial peptide and application thereof.

Background

Caries is the most common oral disease in humans and has a wide prevalence worldwide. By 2010, the number of children who had milk caries but were untreated worldwide was 6.21 million people, while untreated permanent caries patients were as many as 24 million people, accounting for 35% of the worldwide population. In China, the prevention and treatment of caries is also very serious. According to the third and fourth oral health epidemiological survey reports in China, the caries condition of children in China is in an ascending situation, the caries rate of 5-year-old children is 70.9 percent, and the caries rate is 5.8 percent higher than that of the children ten years ago; the permanent tooth caries rate of the children aged 12 years is 34.5 percent, which is 7.8 percent higher than that of the children aged ten years. While treatment of caries requires a significant medical expenditure, the total amount of direct medical expenditure in maintaining oral health worldwide in 2010 is $ 2976.7 billion dollars, with a total cost of $ 530 billion for treatment of caries and tooth loss resulting from caries. Therefore, early prevention and treatment of caries are of great significance to improve quality of life and save medical resources.

The species of bacteria in human oral cavity is over 700, the bacteria and host are in harmonious and concurrent relationship most of the time, and the health of oral environment is maintained through interaction. When this harmonious relationship is disturbed, opportunistic pathogens may be caused to develop the disease. Dental caries is a chronic infectious disease, and oral microorganisms adhere to and colonize the tooth surface and metabolize carbohydrates to produce acid, so that local hard tissue demineralization is larger than remineralization, which is the most direct cause of dental caries. Mutans streptococci are well-established major cariogenic bacteria due to: firstly, streptococcus mutans can be separated from the caries; secondly, the streptococcus mutans can successfully induce the formation of caries in an animal model through high-sugar diet; thirdly, the streptococcus mutans has high acid production and acid resistance; s. mutans produces type I/II surface antigens and water insoluble glucan for improved adhesion to dental surfaces and other bacteria. But the content of streptococcus mutans in the healthy dental plaque on the surface of normal enamel is very low, and the dominant bacteria mainly comprise non-streptococcus mutans (such as streptococcus sanguis, streptococcus grignard and the like) and actinomycetes and can resist the colonization of external cariogenic bacteria; when the oral hygiene of an individual is poor and the individual is on a high-sugar diet for a long time, cariogenic bacteria in dental plaque are gradually converted into dominant bacteria, and the content of streptococcus mutans is obviously increased.

The broad-spectrum antibacterial drug can reduce the bacterial content in saliva and dental plaque, and theoretically can effectively prevent the occurrence and development of caries. At present, in vitro researches show that various plant extracts and derivatives thereof, and natural or artificial broad-spectrum antibacterial peptides have certain broad-spectrum bactericidal effects on oral cariogenic bacteria and non-cariogenic bacteria including streptococcus mutans. However, no clinical test at present proves the effectiveness of broad-spectrum antibacterial drugs in reducing the occurrence of caries, and the broad-spectrum antibacterial drugs have numerous disadvantages in the application of caries prevention and treatment: after the broad-spectrum antibacterial agent kills bacteria in a non-specific way, the broad-spectrum antibacterial agent still provides an opportunity of equal competition for the re-planting of cariogenic bacteria and non-cariogenic bacteria, so theoretically, the broad-spectrum antibacterial agent cannot prevent the re-infection of the cariogenic bacteria such as streptococcus mutans for a long time; the long-term use can cause the imbalance of flora in the oral cavity and the intestinal tract and generate side effects of bacterial drug resistance, vomit, diarrhea, tooth staining and the like.

Disclosure of Invention

1. Technical problem to be solved

Aiming at the antibacterial peptide fragment of the existing streptococcus mutans targeted antibacterial peptide, natural antibacterial peptide or derivatives thereof are mostly adopted. The natural antibacterial peptide has the defects of long peptide chain, weak antibacterial activity, high cytotoxicity and the like, and the artificially designed and synthesized novel antibacterial peptide has greater potential in the aspect of anticarious application because the characteristics of the artificially designed and synthesized novel antibacterial peptide can be artificially determined. Such as KSL and RW synthesized by a chemical combined library method and WLBU2 and GH12 obtained by a spiral wheel method, and the like can effectively inhibit streptococcus mutans or other common cariogenic bacteria. The invention aims to provide the streptococcus mutans specific targeting antibacterial peptide formed by combining the CSP fragment and the short-chain artificial antibacterial peptide, which has the advantages of shorter peptide chain, lower synthesis difficulty, lower cost and greater application potential.

2. Technical scheme

In order to solve the problems, the invention adopts the following technical scheme:

the invention discloses a streptococcus mutans specific targeting antibacterial peptide M8RW, which is characterized in that the amino acid sequence of the streptococcus mutans specific targeting antibacterial peptide is TFFRLFNRGGGRRWWRF.

As a preferred scheme of the invention, the preparation method of the streptococcus mutans specific targeting antibacterial peptide comprises the following steps:

S1A suitable amount of Wang resin was weighed into the reaction column and Dichloromethane) (DCM) was added to swell the resin sufficiently. After washing with Dimethylformamide (DMF), DCM was aspirated, the Fmoc protecting group on the resin was removed with a formulated deprotecting agent (decapping) for 20 minutes, followed by 3 washes with DMF, and the blue color was verified, where the Fmoc protecting group was successfully removed, and if colorless, it was not reacted, and it was necessary to recap

S2, adding a second amino acid arginine according to the amino acid sequence of the polypeptide, and adding a condensing agent HBTU (benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate) and a catalyst NMM (N-methylmorpholine) or DIEA (N, N diisopropylethylamine) for condensation reaction; after the reaction is finished, DMF is added for repeated washing to remove unreacted amino acid

And S3, detecting whether the connection is complete by ninhydrin, wherein the detection result is that the solution is bright yellow, and the resin is transparent and has no impurity color.

And S4, uncapping and washing a product obtained after washing according to the same method of the step 1, sequentially adding amino acids according to the amino acid sequence in the step 2 for condensation reaction and washing, removing the final Fmoc-protecting group by adopting a method S1 after all the amino acids are connected, and washing with DMF.

S5, washing and shrinking by respectively using DCM and methanol (MeOH) after the reaction is finished, adding trifluoroacetic acid TFA for cleavage, and filtering the cleaved product to obtain filtrate

S6: adding 3 times of glacial ethyl ether (4 ℃) to the filtrate obtained according to the step S6 to precipitate the polypeptide, sealing, reversing and uniformly mixing, and recovering the precipitate; re-suspending the precipitate with cold ether, repeating the above steps for three times, and drying the obtained precipitate to obtain a crude polypeptide product;

s7 purification of crude synthetic peptide: purifying the crude product by reverse phase high performance liquid phase method, and freeze drying

S8, purifying the dried antibacterial peptide, determining the purity by using High Performance Liquid Chromatography (HPLC) and detecting the molecular mass by using a Mass Spectrum (MS) to be consistent with the molecular weight calculated by theory.

As a preferable scheme of the invention, the streptococcus mutans specific targeting antibacterial peptide is formed by combining a CSP fragment with a short-chain artificial antibacterial peptide, and the peptide chain is shorter.

As a preferred embodiment of the present invention, the method for determining the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the Streptococcus mutans specific targeting antibacterial peptide comprises the following steps:

(1) single colonies were picked up in 10mLBHI medium and placed in a thermostatted anaerobic jar for anaerobic culture overnight at 37 ℃ (80% N)₂，10％H₂,10％CO₂)；

(2) 100 mul of overnight bacterial liquid is absorbed and added into 10mL of culture medium, and the mixture is placed in a constant-temperature anaerobic culture tank for overnight anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) Diluting the bacterial liquid to 2X 10⁶CFU/mL for standby;

(3) adding 20 mu L of polypeptide solution, 80 mu L of BHI culture medium and 100 mu L of standby bacterial liquid into a 96-well plate by adopting a 2-fold dilution method to ensure that the final concentration of the polypeptide is 512 mu g/mL-0.5 mu g/mL, placing the well plate into a constant-temperature anaerobic culture tank for anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂)24 hours;

(4) MIC is the lowest clarified polypeptide concentration in a 96-well plate;

(5) sucking 100 μ L bacterial liquid from the clear well, spreading on BHI agar plate, placing in constant temperature anaerobic culture tank, and anaerobic culture at 37 deg.C overnight (80% N)₂，10％H₂,10％CO₂)48 hours;

(6) MBC is the lowest polypeptide concentration for plate sterile colony formation;

(7) chlorhexidine is used as a positive control, and BHI culture medium is used as a negative control;

(8) the experiment was repeated 3 times, with 3 replicates per group.

3. Advantageous effects

Compared with the prior art, the invention has the advantages that:

(1) the streptococcus mutans specific targeting antibacterial peptide has the advantages of short peptide chain, small molecular weight, good bactericidal effect on streptococcus mutans and good stability in saliva.

(2) The streptococcus mutans specific targeting antibacterial peptide is convenient to synthesize, has low cost, can effectively kill streptococcus mutans, and has huge application potential in the direction of anticarious antibacterial agents.

Drawings

FIG. 1 shows the result of the analysis of the ratio of the dead bacteria to the live bacteria staining and the dead bacteria ratio of the Streptococcus mutans biofilm after the M8RW action;

FIG. 2 shows the inhibition loop of M8RW for 3 bacteria according to the present invention;

FIG. 3 shows the statistics of the bacteriostatic ring diameter of M8RW against Streptococcus mutans in the invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.

Example 1: a preparation method of streptococcus mutans specific targeting antibacterial peptide comprises the following steps:

Example 2: minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination of streptococcus mutans specific targeting antibacterial peptide

The minimum inhibitory concentration refers to the lowest concentration of the antibacterial drug capable of inhibiting the growth of pathogenic bacteria in the culture medium in an in vitro drug sensitivity test. The minimum bactericidal concentration refers to the minimum drug concentration at which the antibacterial drug can reduce the growth of pathogenic bacteria in the culture medium by more than 99.9% in an in vitro drug sensitivity test. MIC and MBC are gold indicators of the antibacterial activity of the response drugs. The bacteria used in the experiment are Streptococcus mutans UA159, Streptococcus sanguinis ATCC10556, and Streptococcus gordonii DL 1. The positive control is chlorhexidine, which is a powerful broad-spectrum antibacterial drug and is clinically used for adjuvant treatment of gingivitis, periodontitis, oral mucosa ulcer and the like.

(4) MIC is the lowest clarified polypeptide concentration in a 96-well plate;

(8) the experiment was repeated 3 times, with 3 replicates per group.

Table 1 MIC and MBC of M8RW against 3 streptococcus oralis (mean ± standard deviation, n ═ 3) (μ g/mL)

Result data:

the MIC and MBC results for M8RW against 3 S.oralis are shown in Table 1. The MIC and MBC values of M8RW for S.mutans were only 16. mu.g/mL. The MIC and MBC values of M8RW to Streptococcus sanguis and Streptococcus Grignard were significantly increased compared with Streptococcus mutans (P < 0.05), and neither the MIC nor MBC values were lower than 64 μ g/mL. The antibacterial activity of the chlorhexidine on 3 bacteria is stronger than that of M8RW (P is less than 0.05), but the sensitivity of the 3 bacteria on the chlorhexidine is not significantly different (P is more than 0.05).

Example 3: minimum biofilm formation inhibitory concentration (MBIC) assay

The minimum biofilm formation inhibiting concentration reflects the ability of the antibacterial agent to inhibit biofilm formation, MBIC₅₀Minimum drug concentration, MBIC, to inhibit biofilm formation by at least 50%₉₀Is the lowest drug concentration that inhibits at least 90% of biofilm formation. The bacteria used in this experiment were Streptococcus mutans, Streptococcus sanguis, and Streptococcus grignard. The bacteria used in the experiment are Streptococcus mutans UA159, Streptococcus sanguinis ATCC10556, and Streptococcus gordonii DL 1.

1. Single colonies were picked up in 10mLBHI medium and placed in a thermostatted anaerobic jar for anaerobic culture overnight at 37 ℃ (80% N)₂，10％H₂,10％CO₂)。

2. 100 mul of overnight bacterial liquid is absorbed and added into 10mL of culture medium, and the mixture is placed in a constant-temperature anaerobic culture tank for overnight anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) The bacterial suspension was diluted to about 2.0X 10 with BHI medium (BHI-S) containing 1% sucrose⁶CFU/mL is ready for use.

3. Adding 20 mu L of polypeptide solution, 80 mu L of BHI-S culture medium and 100 mu L of standby bacterial liquid into a 96-well plate by adopting a 2-fold dilution method to ensure that the final concentration of the polypeptide is 512 mu g/mL-0.5 mu g/mL, and placing the well plate into a constant-temperature anaerobic culture tank for anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) For 24 hours.

4. The supernatant in the well plate was removed and washed with PBS buffer to remove planktonic bacteria.

5. The biofilm was fixed with methanol for 15min, stained with 0.1% crystal violet for 5min, then rinsed gently under running water and dried for 1 hour.

6. 200ul of 95% ethanol was added to the well plate and placed on a shaker for half an hour, the solution was transferred to a new 96 well plate and the A595 value was measured with a microplate reader. Inhibition rate of (1-A)₅₉₅Experimental group/A₅₉₅Negative control group) × 100%.

7. Recording MBIC₅₀And MBIC₉₀。

8. Chlorhexine was used as a positive control and BHI-S medium was used as a negative control.

9. The experiment was repeated 3 times, with 3 replicates per group.

TABLE 2M 8RW MBIC against 3 Streptococcus oralis₅₀And MBIC₉₀(mean. + -. standard deviation, n. RTM.3) (μ g/mL)

Result data:

the MBIC results of M8RW against 3 S.oralis are shown in Table 2. MBIC50 and MBIC of M8RW against S.mutans₉₀Both were only 16. mu.g/mL, identical to the MIC and MBC values. M8RW MBIC against Streptococcus sanguis and Streptococcus grignard₅₀And MBIC₉₀The value is obviously increased compared with the streptococcus mutans (P is less than 0.05), and the 90 percent of streptococcus sanguis biofilm formation can be inhibited when the concentration of M8RW reaches 64 mu g/ml; can inhibit 90 percent of streptococcus sanguis biofilm formation when reaching 256 mug/mL. Chlorhexidine has a stronger ability to inhibit biofilm formation of 3 bacteria than M8RW (P < 0.05), and at a concentration of 4. mu.g/mL, it was able to inhibit biofilm formation of 3 bacteria at the same time.

Example 4: the antibacterial activity of M8RW on a streptococcus mutans biological membrane is observed by a fluorescence microscope

In the experiment, a fluorescence microscope is adopted to observe the short-time sterilization effect of M8RW on the streptococcus mutans biofilm. The bacterium used in this experiment was Streptococcus mutans UA 159.

2、100 mul of overnight bacterial liquid is absorbed and added into 10mL of culture medium, and the mixture is placed in a constant-temperature anaerobic culture tank for overnight anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) The bacterial suspension was diluted to about 1.0X 10 with BHI medium (BHI-S) containing 1% sucrose⁷CFU/mL is ready for use.

3. Placing a round slide at the bottom of a 24-well plate, adding 2mL of standby bacterial liquid, and carrying out anaerobic treatment (80% N) at 37 DEG C₂，10％H₂,10％CO₂) After culturing for 6h, the bacterial suspension is transferred, and the slide is washed 2 times by physiological saline.

4. The wells of the experimental group were filled with 200. mu.L of M8RW solution at a concentration of 64. mu.g/mL, incubated at room temperature for 5min, and then washed 2 times with physiological saline.

5. Mixing SYTO 9 and Propidium iodide in LIVE/DEAD bacterial activity kit at a ratio of 1:1, diluting with normal saline, sucking 200 μ L of diluted normal saline, adding into a pore plate, culturing in dark for 15min, washing with normal saline, sucking water from slide, sealing, and observing with fluorescence microscope.

6. And (3) randomly selecting three visual fields for each sample, and then collecting images, and respectively calculating red and green fluorescence areas by using Image J6.0 so as to obtain the proportion of dead bacteria.

7. Chlorhexidine (final concentration of 0.12%) was used as a positive control and physiological saline solution as a negative control.

8. Each set was set with 3 replicates.

Result data: the results of the staining of the viable and dead Streptococcus mutans biofilm are shown in FIG. 1. The green fluorescence in FIG. 1 indicates viable bacteria, and the red fluorescence indicates dead bacteria. The negative control group (FIG. 1A) was not treated with M8RW or chlorhexidine and only a few bacteria in the S.mutans biofilm fluoresced red. After the positive control group (fig. 1C) was exposed to 0.12% chlorhexidine for 5min, it was observed that most of the bacteria exhibited red fluorescence, and the ratio of dead bacteria was more than 90% (fig. 1D). After the biofilm is acted by M8RW with the concentration of 64 mu g/mL for 5min (figure 1B), the red fluorescence bacteria are obviously increased, the ratio of dead bacteria reaches more than 70 percent (figure 1D), and the ratio is obviously higher than that of a negative control group (P is less than 0.05).

Example 5: experiment of bacteriostatic Ring

In the experiment, the stability of the antibacterial effect of M8RW in saliva is observed by measuring the bacteriostatic rings formed when M8RW is dissolved in water and saliva respectively. The bacteria used in the experiment are Streptococcus mutans UA159, Streptococcus sanguinis ATCC10556, and Streptococcus gordonii DL 1.

1. Non-irritant saliva was collected from 3 healthy volunteers, mixed and centrifuged at 10,000rpm for 20min at 4 ℃ and the supernatant was sterilized by filtration through a 0.22 μm filter.

2. Single colonies were picked up in 10mLBHI medium and placed in a thermostatted anaerobic jar for anaerobic culture overnight at 37 ℃ (80% N)₂，10％H₂,10％CO₂)。

3. 100 mul of overnight bacterial liquid is absorbed and added into 10mL of culture medium, and the mixture is placed in a constant-temperature anaerobic culture tank for overnight anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) Diluting the bacterial liquid to 1 × 10⁹CFU/mL is ready for use.

4. M8RW was dissolved in water and saliva, respectively, to give polypeptide concentrations of 1280. mu.g/mL and 640. mu.g/mL.

5. Will 10⁹Adding CFU/ml of the standby bacterial liquid into soft agar culture medium (0.75% agar), mixing uniformly and diluting to 10%⁸CFU/mL, spread on BHI agar medium and cooled for 1 hour at room temperature to obtain agar plate containing bacteria.

6. M8RW dissolved in water and saliva was pipetted 5. mu.L each sequentially onto agar plates containing bacteria, incubated at room temperature for 1h, anaerobically cultured at 37 ℃ for 48h, and the diameter of the zone of inhibition was measured using a vernier caliper.

7. Saliva without polypeptide served as a negative control.

8. The experiment was repeated 3 times, with 3 replicates per group.

Result data:

the formation of the zone of inhibition and the diameter measurement of M8RW on S.mutans are shown in FIGS. 2 and 3. As shown in FIG. 2, M8RW dissolved in water at a concentration of 640. mu.g/mL formed well-defined zones on agar plates containing S.mutans, whereas M8RW formed zones after dissolution in saliva had reduced resolution and reduced zone diameters, but the difference in diameters was not statistically significant (P > 0.05). When the concentration of M8RW was increased to 1280. mu.g/mL, the diameter of the inhibition loop of Streptococcus mutans formed by water-soluble or saliva-soluble M8RW was significantly increased (P < 0.05), and the difference was not statistically significant (P > 0.05).

Whereas M8RW at a concentration of 640. mu.g/mL failed to form a significant zone of inhibition on agar plates containing Streptococcus sanguis or Streptococcus formatted.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the equivalent replacement or change according to the technical solution and the modified concept of the present invention should be covered by the scope of the present invention.

Claims

1. The streptococcus mutans specific targeting antibacterial peptide is characterized in that the amino acid sequence of the streptococcus mutans specific targeting antibacterial peptide is TFFRLFNRGGGRRWWRF.

2. The method for preparing the streptococcus mutans specific targeting antibacterial peptide according to claim 1, comprising the steps of:

3. The streptococcus mutans-specific targeting antimicrobial peptide of claim 2, wherein: the streptococcus mutans specific targeting antibacterial peptide is formed by combining a CSP fragment with a short-chain artificial antibacterial peptide, and the peptide chain is shorter.

4. The streptococcus mutans specific targeting antimicrobial peptide Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assay of claim 1 or 2, wherein:

(1) single colonies were picked up in 10mLBHI medium and placed in a thermostatted anaerobic jar for anaerobic culture overnight at 37 ℃ (80% N)₂，10％H₂,10％CO₂)。

(2) 100 mul of overnight bacterial liquid is absorbed and added into 10mL of culture medium, and the mixture is placed in a constant-temperature anaerobic culture tank for overnight anaerobic culture at 37 ℃ (80% N)₂，10％H₂,10％CO₂) Diluting the bacterial liquid to 2X 10⁶CFU/mL is ready for use.

(3) Adding 20 mu L of polypeptide solution, 80 mu L of BHI culture medium and 100 mu L of standby bacterial liquid into a 96-well plate by adopting a 2-fold dilution method to ensure that the final concentration of the polypeptide is 512 mu g/mL-0.5 mu g/mL, placing the well plate in a constant volumeAnaerobic culture at 37 deg.C (80% N) in a warm anaerobic culture tank₂，10％H₂,10％CO₂) For 24 hours.

(4) MIC is the lowest clarified polypeptide concentration in a 96-well plate.

(5) Sucking 100 μ L bacterial liquid from the clear well, spreading on BHI agar plate, placing in constant temperature anaerobic culture tank, and anaerobic culture at 37 deg.C overnight (80% N)₂，10％H₂,10％CO₂) For 48 hours.

(6) MBC is the lowest polypeptide concentration for plate sterile colony formation.

(7) Chlorhexidine was used as a positive control and BHI medium as a negative control.

(8) The experiment was repeated 3 times, with 3 replicates per group.