CC BY-NC-ND 4.0 · Dental Journal of Advance Studies 2020; 8(03): 109-114
DOI: 10.1055/s-0040-1715288
Original Article

Evaluation of Antimicrobial Activity of Different Herbal Products against Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis Using Agar Diffusion Test: An In Vitro Study

Rajinder Bansal
1  Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College and Research Institute, Sunam, Punjab, India
,
Manu Bansal
1  Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College and Research Institute, Sunam, Punjab, India
,
Nikhil Dev Wazir
2  Department of Conservative Dentistry and Endodontics, Institute of Dental Sciences, Sehora, Jammu, India
,
Mandeep Singh Matta
1  Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College and Research Institute, Sunam, Punjab, India
,
Sanjeev Jain
3  Department of Periodontics, Guru Nanak Dev Dental College and Research Institute, Patiala, Punjab, India
,
Jaspreet Kaur
1  Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College and Research Institute, Sunam, Punjab, India
› Author Affiliations
 

Abstract

Introduction The main aim of this study is in vitro evaluation of the antimicrobial efficacy of different herbal products, that is, propolis, garlic, neem, aloe vera, and rosemary, against Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis using agar diffusion test.

Materials and Methods In this study, total of 42 plates were prepared, 10 each of B. subtilis, S. aureus, and E. faecalis and 12 as test control (6 as positive and 6 as negative control). The effectiveness of five herbal products was ascertained by agar diffusion method against B. subtilis, S. aureus, and E. faecalis. Cultures of these test organisms were maintained on selective media slants in a test tube to collect sufficient number of microbial colonies for evaluation. The cultures were divided into three groups based upon microbes that were lawn cultured, respectively: In group A, 10 petri plates were having growth of B. subtilis; in group B, 10 petri plates were having growth of S. aureus; and in group C, 10 petri plates were having growth of E. faecalis. In all these 30 petri plates, five different herbal product discs were placed and these discs were designated as A (aloe vera), P (propolis), N (neem), R (rosemary), and G (garlic). Among remaining 12 petri plates, 4 petri plates were used as control (2 for positive and 2 for negative) for each of the bacteria.

Results Propolis and rosemary showed maximum zone of inhibition against B. subtilis. Garlic, neem, and aloe vera showed maximum zone of inhibition against S. aureus.

Conclusion All the herbal products showed zone of inhibition against S. aureus, B. subtilis, and E. faecalis.


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Introduction

The success of endodontic treatment depends on the complete eradication of microbes from root canal system and prevention of reinfection. Sterilization of root canal subsequent to its debridement and prior to its obturation has been considered as a prerequisite for successful endodontic therapy.[1]

Various measures to reduce the number of microorganisms in the root canal system include the use of various instrumentation techniques, irrigation regimens, and intracanal medicaments in the canal. Irrigation is complementary to instrumentation in facilitating the removal of pulpal tissues and microorganisms. Sodium hypochlorite is the most commonly used irrigant in endodontics due to its tissue dissolving capability and antimicrobial properties.[2]

However, sodium hypochlorite is toxic to vital tissues, resulting in hemolysis, skin ulceration, and necrosis. In case of contact with the patient’s or operator’s eye, it results in immediate pain, profuse watering of eyes, intense burning, and erythema.[3]

Gutta percha cones are usually decontaminated by submerging in 1% sodium hypochlorite for 1 minute (Milton’s solution) or 0.5% sodium hypochlorite for 5 minutes (Dakin’s solution). But sodium hypochlorite causes crystal deposition on the surface of gutta percha, which can impede the obturation.

Owing to the potential side effects and safety concerns of conventional antimicrobial agents, interest in the preparations from medicinal plants has increased over the last few decades. Herbal products have become more popular today due to their high antimicrobial activity, biocompatibility, anti-inflammatory, and antioxidant properties. The main advantages of using herbal alternatives are easy availability, cost-effectiveness, increased shelf life, low toxicity, and lack of microbial resistance.[4]

In endodontics, because of the cytotoxic reactions of the most of the commercial intracanal medicaments used and their inability to eliminate bacteria from dentinal tubules, there is a new trend to use biologic medication extracted from natural plants. Some of the commonly used herbal products in endodontics are propolis, garlic, neem, aloe vera, and rosemary.

Propolis (bee glue), a natural antibiotic, is a resinous substance that honey bees collect from trees of poplars. It possesses antimicrobial, anti-inflammatory, and antioxidant properties.[5] [6]

Garlic (Allium sativum) is antibacterial and has immune regulatory functions. Garlic extract has shown to have a wide spectrum of antibacterial activity, including effects on Staphylococcus, Streptococcus, Klebsiella, Escherichia, Salmonella, Proteus, Clostridium, Mycobacterium, and Helicobacter species.[7] [8]

Neem (Azadirachta indica) is a versatile medicinal plant having a wide spectrum of biological activity. Neem leaves, seeds, and bark possess a wide spectrum of antibacterial action against gram-negative and gram-positive microorganisms. Neem leaf extract is used to treat dental plaque and gingivitis. Its powerful antibacterial action makes it a useful ingredient in mouth wash and dentifrices.[9] [10]

Aloe vera (Aloe barbadensis) is a short succulent herb resembling a cactus. It has potent antibacterial, antifungal, and antiviral properties. The antimicrobial effects of aloe vera have been attributed to the plant’s natural anthraquinones that have demonstrated in vitro inhibition of Mycobacterium tuberculosis and Bacillus subtilis.[11] [12]

Rosemary (Rosmarinus officinalis) belongs to family Labiatae. It has antibacterial properties and is used in relieving toothache.[13]

Different species of microorganisms are commonly found in the infected root canal cases including Enterococcus, Streptococcus, and Staphylococcus species.

Enterococcus faecalis has been considered to be the most resistant species in the oral cavity. It is facultative anaerobic gram-positive cocci and is the most commonly implicated microorganism in asymptomatic persistent infections. The highly complex nature of the organism poses a great challenge for endodontists. Enterococcus faecalis is responsible for failed root canal treatment cases.[14] [15]

Staphylococcus aureus is a gram-positive coccal bacterium. It causes disease and destruction of tissue through the direct invasion and by production of toxins.[16]

Certain species of Bacillus produce special structures called spores when growth ceases owing to the exhaustion of an essential nutrient. No growth of B. subtilis subspecies is used for the assessment of proper sterilization.

This in vitro study was undertaken to compare the antimicrobial efficacy of five herbal products—aloe vera (Aloe barbadensis), neem (Azadirachta indica), garlic (Allium sativum), propolis (bee glue), and rosemary (Rosmarinus officinalis) against S. aureus, B. subtilis, and E. faecalis using agar diffusion disc test.


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Materials and Methods

Procurement of Microorganisms

The microorganisms, E. faecalis, S. aureus, and B. subtilis, used in this study were procured in freeze dried form in an air tight glass tube from Institute of Microbial Technology, Chandigarh.


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Procurement of the Herbal Products

Herbal products were procured in pure dry powder form from Sirmour Herbolife Private Limited Village & PO Surla, District Sirmour, Himachal Pradesh.


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Activation of Bacteria

Activation of Enterococcus faecalis

The freeze-dried form of E. faecalis was activated by dissolving E. faecalis in Brain Heart Infusion Broth. Ten milliliters of Brain Heart Infusion Broth were poured in each test tube. Then, five test tubes containing Brain Heart Infusion Broth and bacteria were heated on flame near open end of tube to avoid contamination. Then a tight, nonabsorbent sterile cotton plug was placed at the open end of tube to seal off the broth containing E. faecalis from external environment. After the inoculation, the test tubes were then kept in an incubator at 37°C for 24 hours. After 24 hours of incubation, turbidity was observed in all the tubes indicating bacterial growth.


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Activation of Staphylococcus aureus and Bacillus subtilis

Staphylococcus aureus and B. subtilis were activated by dissolving freeze-dried forms of these bacteria in nutrient broth. Twenty milliliters of nutrient broth were poured in each test tube. Then, five test tubes containing S. aureus and five test tubes containing B. subtilis were heated on flame near open end of tube to avoid contamination. Then a tight, nonabsorbent sterile cotton plug was placed at the open end of each tube to seal off the bacteria in broth from external environment. After the inoculation, the test tubes were then kept in an incubator at 37°C for 24 hours. After 24 hours of incubation, turbidity was observed in all the tubes indicating bacterial growth.


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Preparation of Test Plates for Enterococcus faecalis

In this study, a total of 14 culture plates containing Brain Heart Infusion Agar media were prepared. Ten plates were selected to test the antimicrobial efficacy of different herbal products against E. faecalis and four plates were kept as control group. In the control group, two served as positive and two served as negative control group. In positive control group, agar plates were tested for susceptibility of E. faecalis to tetracycline, whereas in negative control group, distilled water was used.

Each plate was inoculated with E. faecalis by evenly swiping the plate using heated glass spreader via lawn culture technique. Each plate was inoculated with E. faecalis by evenly swiping the plate using heated glass spreader via lawn culture technique.


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Preparation of Test Plates for Staphylococcus aureus

In this study, a total of 14 culture plates containing blood agar media were prepared. Ten plates were selected to test the antimicrobial efficacy of different herbal products against S. aureus. Four plates were kept as control group. In the control group, 2 served as positive and 2 served as negative control group. In positive control group, blood agar plates were used for testing the susceptibility of S. aureus to tetracycline, whereas in negative control group, distilled water was used.

Fourteen plates were inoculated with S. aureus by evenly swiping the plate using heated glass spreader via lawn technique. In each agar plate, five discs, that is, one for each herbal product were placed, at a safe distance from the edge and from each other to avoid overlapping of zones of inhibition around the discs.

In positive control group, tetracycline disc was placed in two test plates instead of herbal products. In negative control group, distilled water disc was placed in test plates instead of herbal products.


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Preparation of Test Plates for Bacillus subtilis

In this study, a total of 14 culture plates containing blood agar medium were prepared. Ten plates were selected to test the antimicrobial efficacy of different herbal products against B. subtilis. Four plates were kept as control group. In the control group, two served as positive and two served as negative control group. In positive control group, blood agar plates were tested for susceptibility B. subtilis to tetracycline, whereas in negative control group, instead of herbal product, distilled water was used.

Fourteen plates were inoculated with B. subtilis by evenly swiping the plate using heated glass spreader via lawn technique.

In positive control group, tetracycline disc was placed in two test plates instead of herbal products, and in negative control group, distilled water was placed in test plates.


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Introduction of Herbal Product in the Culture

In each agar plates, except control group, five discs, that is, one for each herbal product were placed, at a safe distance from the edge and from each other to avoid overlapping of zones of inhibition around the discs.


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Measurement of Inhibition Zone

All the plates were kept at room temperature for 2 hours for prediffusion of materials and then incubated at 37°C for 7 days. These plates were then observed for zones of inhibition after 24 hours, 48 hours, 72 hours, and 7 days.

The zones of inhibition formed around each disc were then examined and measured in millimeters using digital Vernier caliper.

The readings thus obtained from each plate were compared and put under statistically analysis.


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Data Analysis

The observations recorded in the present study were subjected to student’s t-test and Kruskal–Wallis one-way analysis of variance. The “p” value was taken as significant at “p” < 0.05, highly significant if < 0.001.


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Discussion

The present study shows that propolis had maximum mean zone of inhibition against B. subtilis, that is, 23.64 mm, whereas minimum mean zone of inhibition found against S. aureus was 11.92 mm. When compared statistically, this difference in mean zone of inhibition was found to be significant (p < 0.05) ([Table 1]).

Table 1

Propolis

Minimum

Maximum

Mean

± SD

SEM

Abbreviations: B. subtilis, Bacillus subtilis; E. faecalis, Enterococcus faecalis; S. aureus, Staphylococcus aureus; SD, standard deviation; SEM, standard error of mean.

Note: F = 8.527; p = 0.008; significant. Multiple comparisons using post-hoc Scheffe test.

a p < 0.05; significant.

S. aureus

09.50

14.67

11.92

2.52

01.26

B. subtilis

21.60

27.26

23.64

2.57

01.28

E. faecalis

13.36

26.44

17.63

5.94

02.97

Comparison

Mean difference

p-Value

S. aureus vs. B. subtilis

11.72

0.008a

S. aureus vs. E. faecalis

05.70

0.188

B. subtilis vs. E. faecalis

06.01

0.162

The mean zone of inhibition against E. faecalis was 17.63 mm. When compared statistically, this difference in mean zone of inhibition between S. aureus, B. subtilis, and E. faecalis was found nonsignificant (p > 0.05) ([Table 1]).

Akca et al suggested that antibacterial effect of propolis against microorganisms could be complex leading to the disintegration of the cytoplasmic membrane and cell wall, partial bacteriolysis, and inhibition of protein synthesis. They claimed that the pH and the concentration of propolis might alter due to solvents and acidic propolis solutions were more effective on bacteria. In addition, bacterial cell wall and their biofilm properties were excluded as adjunct factors, which determine bactericidal effect of propolis.[17]

Garlic had maximum mean zone of inhibition against S. aureus, that is, 39.64 mm, whereas minimum mean zone of inhibition was observed against E. faecalis, that is, 15.78 mm. When compared statistically, the difference in mean zone of inhibition was found to be significant (p < 0.05) ([Table 2]).

The mean zone of inhibition against B. subtilis was 27.98 mm. When compared statistically, the difference in mean zone of inhibition between S. aureus, B. subtilis, and E. faecalis was nonsignificant (p > 0.05) ([Table 2]).

Table 2

Garlic

Minimum

Maximum

Mean

± SD

SEM

Abbreviations: B. subtilis, Bacillus subtilis; E. faecalis, Enterococcus faecalis; S. aureus, Staphylococcus aureus; SD, standard deviation; SEM, standard error of mean.

Note: F = 15.542; p = 0.001; significant. Multiple comparisons using post-hoc Scheffe test.

a p<0.05; significant.

S. aureus

33.21

47.59

39.64

07.00

03.50

B. subtilis

24.63

32.74

27.98

03.51

01.75

E. faecalis

11.06

26.12

15.78

06.96

03.48

Comparison

Mean difference

p-Value

S. aureus vs. B. subtilis

11.66

0.067

S. aureus vs. E. faecalis

23.86

0.001a

B. subtilis vs. E. faecalis

12.19

0.055

Jose et al suggested that allicin present in garlic inhibits both germination of spores and growth of hyphae. The antimicrobial effect of allicin is due to its chemical reaction with thiol groups of various enzymes.[18]

Neem had maximum mean zone of inhibition against S. aureus, that is, 23.63 mm, whereas the minimum mean zone of inhibition observed against E. faecalis was 14.12 mm. The mean zone of inhibition against B. subtilis was found to be 18.38 mm.

When compared statistically, this difference in mean zone of inhibition between S. aureus, B. subtilis, and E. faecalis was nonsignificant (p > 0.05) ([Table 3]).

Table 3

Neem

Minimum

Maximum

Mean

± SD

SEM

Abbreviations: B. subtilis, Bacillus subtilis; E. faecalis, Enterococcus faecalis; S. aureus, Staphylococcus aureus; SD, standard deviation; SEM, standard error of mean.

Note: F = 1.907; p = 0.204; not significant. Multiple comparisons using post-hoc Scheffe test.

*p<0.05; significant.

S. aureus

09.67

32.61

23.63

09.96

04.98

B. subtilis

15.63

22.74

18.38

03.05

01.52

E. faecalis

09.67

22.68

14.12

05.84

02.92

Comparison

Mean difference

p-Value

S. aureus vs. B. subtilis

05.25

00.58

S. aureus vs. E. faecalis

09.51

00.20

B. subtilis vs. E. faecalis

04.25

00.69

Punetha and Srinivas showed that bactericidal effect of neem is due to the presence of chemical substances such as alkaloids, glycosides, saponins, flavonoids, steroids, anthraquinones, and tannic acid. Flavonoids act as antioxidants that provide protection against free radicals that damage cells and tissues. Moreover, it prevents colonization of organisms due to its antiadherence activity.[19]

Aloe vera had maximum mean zone of inhibition against S. aureus,that is, 23.78 mm, whereas minimum mean zone of inhibition observed against E. faecalis was 14.17 mm. When compared statistically, the difference in mean zone of inhibition was found to be significant (p < 0.05) ([Table 4]).

The mean zone of inhibition against B. subtilis was 17.27 mm. When compared statistically, the difference in mean zone of inhibition between S. aureus, B. subtilis, and E. faecalis was nonsignificant (p > 0.05) ([Table 4]).

Table 4

Aloe vera

Minimum

Maximum

Mean

± SD

SEM

Abbreviations: B. subtilis, Bacillus subtilis; E. faecalis, Enterococcus faecalis; S. aureus, Staphylococcus aureus; SD, standard deviation; SEM, standard error of mean.

Note: F = 6.310; p = 0.019; significant. Multiple comparisons using post-hoc Scheffe test.

a p<0.05; significant.

S. aureus

22.02

32.25

23.78

02.47

01.23

B. subtilis

14.63

20.87

17.27

02.64

01.32

E. faecalis

10.65

22.69

14.17

05.71

02.85

Comparison

Mean difference

p-Value

S. aureus vs. B. subtilis

06.50

0.115

S. aureus vs. E. faecalis

09.61

0.022a

B. subtilis vs. E. faecalis

03.10

0.554

Athiban et al also showed that aloe vera is more effective against S. aureus as compared with E. faecalis.[20]

The results of our study found that rosemary had maximum zone of inhibition against B. subtilis, that is, 13.80 mm and E. faecalis, that is, 13.73 mm, whereas minimum zone of inhibition was observed against S. aureus, that is, 13.49 mm.

When compared statistically, the difference in mean zone of inhibition was found to be nonsignificant (p > 0.05) ([Table 5]).

Table 5

Rosemary

Minimum

Maximum

Mean

± SD

SEM

Abbreviations: B. subtilis, Bacillus subtilis; E. faecalis, Enterococcus faecalis; S. aureus, Staphylococcus aureus; SD, standard deviation; SEM, standard error of mean.

Note: F = 0.011; p = 0.989; not significant. Multiple comparisons using post-hoc Scheffe test.

*p<0.05; significant.

S. aureus

09.59

17.68

13.49

03.62

01.81

B. subtilis

11.49

16.55

13.80

02.10

01.05

E. faecalis

10.40

17.68

13.73

03.31

01.65

Comparison

Mean difference

p-Value

S. aureus vs. B. subtilis

0.31

0.99

S. aureus vs. E. faecalis

0.24

0.99

B. subtilis vs. E. faecalis

0.07

0.99

The antimicrobial effect of rosemary is due to the presence of phenolic compounds: augustic acid, benthamic acid, salvianic acid, sage coumarin, sagerinic acid, and other flavonoids.[21]

Propolis, garlic, neem, aloe vera, and rosemary showed maximum zone of inhibition after 24 hours against E. faecalis, S. aureus, and B. subtilis. The minimum zone of inhibition was found after 7 days ([Figs. 1] [2] [3]).

Zoom Image
Fig. 1 Overall mean zone of inhibition of herbal products against Enterococcus faecalis.
Zoom Image
Fig. 2 Overall mean zone of inhibition of herbal products against Staphylococcus aureus.
Zoom Image
Fig. 3 Overall mean zone of inhibition of herbal products against Bacillus subtilis.

Shahzad et al compared antibacterial efficacy of different aloe vera products after 48 and 96 hours. They found significant decrease in antibacterial efficacy after 48 hours.[22]


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Conclusion

From the results of present study, it can be concluded that all the herbal products showed zone of inhibition against S. aureus, B. subtilis, and E. faecalis. Propolis showed maximum zone of inhibition against B. subtilis and minimum zone of inhibition against S. aureus. Garlic showed maximum zone of inhibition against S. aureus and minimum zone of inhibition against E. faecalis. Neem showed maximum zone of inhibition against S. aureus and minimum zone of inhibition against E. faecalis. Aloe vera showed maximum zone of inhibition against S. aureus and minimum zone of inhibition against E. faecalis.


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Conflict of Interest

None declared.


Address for correspondence

Rajinder Bansal, MDS
Department of Conservative Dentistry and Endodontics, Guru Nanak Dev Dental College and Research Institute
Sunam, Bathinda Road, Patiala 148028, Punjab

Publication History

Publication Date:
12 August 2020 (online)

© .

Thieme Medical and Scientific Publishers Private Ltd.
A-12, Second Floor, Sector -2, NOIDA -201301, India


Zoom Image
Fig. 1 Overall mean zone of inhibition of herbal products against Enterococcus faecalis.
Zoom Image
Fig. 2 Overall mean zone of inhibition of herbal products against Staphylococcus aureus.
Zoom Image
Fig. 3 Overall mean zone of inhibition of herbal products against Bacillus subtilis.