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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 1  |  Page : 98-104

Antibacterial and antibiofilm properties of Azadirachta indica (Neem), Aloe vera (Aloe vera), and Mentha piperita (Peppermint) against multidrug-resistant clinical isolates


1 Department of Microbiology, SGT Medical College, Hospital and Research Institute, SGT University, Gurugram, Haryana, India
2 Department of Microbiology, ESIC Medical College and Hospital, Faridabad, Haryana, India
3 Department of Microbiology, Maharishi Markandeshwar Medical College and Hospital, Maharishi Markandeshwar University, Solan, Himachal Pradesh, India

Date of Submission06-Aug-2021
Date of Acceptance13-Oct-2021
Date of Web Publication11-Mar-2022

Correspondence Address:
Priti Agarwal
Department of Microbiology, ESIC Medical College and Hospital, Faridabad - 121 001, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_178_21

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  Abstract 


Background: Misuse of antibiotics globally has resulted in the development of resistant bacterial strains. One of the sole reasons for bacteria being resistant to antibiotics is the production of biofilm. Biofilms are microbial communities which get adhere to solid surfaces easily and pose an important virulence factor for causing many chronic infections. Therefore, there is an urge to find out new potential sources which can be used as an alternative to the existing antibiotics. Methods: The present study was conducted on three medicinal plant extracts Azadirachta indica, Aloe vera, and Mentha piperita to assess their antibacterial and antibiofilm properties against 58 multidrug-resistant clinical isolates using agar well diffusion method, minimum bactericidal concentration (MBC), and crystal violet modified assay at 50, 25, 12.5, and 6.25 mg/ml concentration. Results: A. indica showed a maximum zone of inhibition of (17.8 ± 1.52 mm) and (18.1 ± 1.45 mm) at 50 and 25 mg/ml concentration. Biofilm inhibition was more than 80% for Staphylococcus aureus and Pseudomonas aeruginosa and MBC came out to be 6.25 ± 2.96–6.25 ± 4.91 mg/ml (mean range). A. vera showed the highest zone of inhibition for S. aureus (18.2 ± 1.48 mm) at 50 mg/ml concentration followed by Staphylococcus saprophyticus (17.8 ± 1.48 mm) and Staphylococcus epidermidis (18.0 ± 1.60 mm). Biofilm inhibition was seen more than 50% and MBC was 50 ± 23.14–50 ± 25.72 mg/ml (mean range). Conclusion: All the three plant extracts were effective, but A. indica and A. vera were found to be more potent than M. piperita.

Keywords: Antibacterial, antibiofilm activity, medicinal plant extracts, multidrug resistant


How to cite this article:
Mehrishi P, Agarwal P, Broor S, Sharma A. Antibacterial and antibiofilm properties of Azadirachta indica (Neem), Aloe vera (Aloe vera), and Mentha piperita (Peppermint) against multidrug-resistant clinical isolates. Biomed Biotechnol Res J 2022;6:98-104

How to cite this URL:
Mehrishi P, Agarwal P, Broor S, Sharma A. Antibacterial and antibiofilm properties of Azadirachta indica (Neem), Aloe vera (Aloe vera), and Mentha piperita (Peppermint) against multidrug-resistant clinical isolates. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 May 22];6:98-104. Available from: https://www.bmbtrj.org/text.asp?2022/6/1/98/339363




  Introduction Top


Misuse of antibiotics globally has resulted in the development of resistant bacterial strains. One of the sole reasons for bacteria being resistant to antibiotics is the production of biofilm. Biofilms are microbial communities which get adhere to solid surfaces easily and pose an important virulence factor for causing many chronic infections. Bacteria get protected inside the biofilm exo-polysaccharide layer due to which it is resistant to the effect of antibiotics.[1],[2] Therefore, there is an urge to find out new potential sources which can be used as an alternative to the existing antibiotics. Research on medicinal plants as new antimicrobial sources has been well-documented.[3] New technological science is focusing more on the development of such therapeutic activities which have no harmful side effects. Thus, the interest has shifted toward the medicinal plants which have known bioactive compounds such as flavanoids, tannins, alkaloids, steroids, and phenol compounds.[4],[5] Medicinal plants such as Azadirachta indica (Neem) are known since ancient times. Activity of A. indica with its different fractions such as root, leaves, bark, oil, and stem has been reported to have antimicrobial properties. Leaves of A. indica have some important antibaterial and antifungal components such as B-sitosterol, quercetin and polyphenolic flavonoids whereas seed part contains geduninin component.[6],[7] The active compounds present in Aloe vera are anthraquinone aloin, aloe-emodin, acemannan amino acids, sterols, and vitamins.[8] Mentha piperita (Peppermint) contains phenolic compounds such as α-pinene, citronellol, and major compounds such as menthol and carvone with minute amounts of menthone and men-thylacetate which possess antimicrobial activity.[9] Therefore, the aim of our study is to demonstrate the antibacterial and antibiofilm activity of methanolic extract of A. indica (Neem), A. vera, and M. piperita (Peppermint) against multidrug-resistant clinical isolates.


  Methods Top


The present study was conducted in the Microbiology Department of SGT Medical College, Hospital and Research Institute, Gurugram. All clinical specimens sent to microbiology laboratory were screened for isolation of Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis strains which were further assessed for their multidrug resistance status.[10] All the bacterial isolates which showed multidrug-resistant patterns were further checked for their biofilm production and classified accordingly in three groups: strong, moderate, and weak biofilm producers.[11] The present study was approved by the Institutional Ethical Clearance Committee of SGT University Reference Number SGTU/FMHS/MICRO/341/15 on August 22, 2016.

Collection of plants

A. indica (UHF herbarium: 13588), A. vera (UHF herbarium: 13589), and M. piperita (UHF herbarium: 13591) were certified from Department of Forestry, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh. Referral ATCC bacterial strains of the similar isolates were simultaneously tested and single testing for biofilm inhibition assay was performed in the laboratory for antibacterial activity.

Plant extract preparation

The methanolic extracts of the above-mentioned plants were prepared. Dried leaves of A. vera, A. indica, and M. piperita plants were crushed and soaked in methanol (50 ml). Further, the methanolic mixtures of plants were boiled continuously with an interval of few minutes between each boiling time. Clear supernatant was obtained after centrifugation at 5000 rpm was done for 5 min. 0.2 um (Micropore filters) was used to filter the supernatant and filtrate was further stored at 4°C.[12]

Antimicrobial activity by using agar well diffusion method

Sterile Petri plates containing Mueller–Hinton agar were prepared. Fresh culture suspensions (0.5 McFarland) of isolated bacteria were swabbed on the respective plates. Sterile gel puncher was used to make wells over the agar plates into which plant extracts were added at various concentrations of (50, 25, 12.5, and 6.25 mg/mL). These plates were further incubated for 24 h at 37°C. After incubation, the diameter of inhibitory zones around each well was measured in mm and recorded.[12],[13]

Detection of minimum bactericidal concentration

Minimum bactericidal concentration (MBC) is defined as the concentration producing a 99.9% reduction in colony-forming units number in the initial inoculums. It was determined by twofold dilutions of the plant extracts which were prepared at different concentrations of 50, 25, 12.5, and 6.25 mg/ml. Microorganism suspension of 100 μL was added to the above-mentioned concentration of extracts making the final density of the suspension to 105 cells/ml and was further incubated at 37°C for 24 h after which it was sub-cultured on MuellerHinton agar. On the next day, bacterial growth was observed. MBC was determined as the lowest concentration of plant extract that failed to show any bacterial growth in the subcultures.[14]

Detection of biofilm formation by bacterial isolate using modified crystal violet assay

Tissue culture plate of 96 wells was used. Mueller–Hinton broth (50 μl) was added to each well followed by 50 μl of fresh bacterial suspensions (1.0 McFarland) and was kept for incubation at 37°C for 48 h. To check for the formation of biofilm, contents in the wells were firstly washed with normal saline (200 μl) followed by 0.1% crystal violet stain (200 μl) and incubated for 20 min. After the incubation, each well was completely washed with the deionized water and fixed later with 96% ethanol (200 μl). ELISA reader was used to check for the optical density (OD) of bacterial adherence at 630 nm and biofilm formation was assessed using the formula.

OD of bacteria= [(OD growth control – OD sample)/OD growth control] × 100.

Strains were classified as follows:[15]

No biofilm producer: OD ucODc

Weak biofilm producer: ODc < OD ≤ 2 × ODc

Moderate biofilm producer: 2 × ODc < OD ≤ 4 × ODc

Strong biofilm producer: 4 × ODc < OD.

Determination of antibiofilm activity of plant extracts using modified crystal violet assay

Sterile tissue culture plates of 96 wells were used. Mueller–Hinton broth (50 μl) was added to each well. Twofold serial dilutions of plant extract at concentrations of 50, 25, 12.5, and 6.25 mg/ml were made in the tissue culture plates to which 50 μl of fresh bacterial suspensions (1.0 McFarland turbidity standard matched) was added. Growth control (bacteria without plant extract) was used. Modified crystal violet assay was performed again after 24 h of incubation as described above. Percentage of biofilm reduction was calculated using the following formula: [(OD growth control – OD sample)/OD growth control] ×100. The biofilm inhibition concentration (BIC50) was defined as the lowest concentration of extracts that showed 50% inhibition on the biofilm formation.[15]


  Results Top


A total of 58 MDR bacterial isolates were obtained from clinical specimens. Out of 58 MDR isolates, 49 moderate and 9 were strong biofilm producers. Extracts of A. indica (Neem), A. vera, and M. piperita (Peppermint) plants were tested against these 58 MDR isolates at four different concentrations of 50, 25, 12.5, and 6.25 mg/ml, as described in [Table 1].
Table 1: Distribution of bacterial isolates into strong, moderate, and weak biofilm producers

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Antibacterial and antibiofilm activity of Azadirachta indica (neem)

A. indica showed its best activity against S. aureus with the highest zone of inhibition of 17.8 ± 1.52 mm and 18.1 ± 1.45 mm at 50 mg/ml and 25 mg/ml concentration, respectively. Biofilm inhibition was more than 80% for S. aureus and P. aeruginosa and MBC came out to be 6.25 ± 2.96–6.25 ± 4.91 mg/ml (mean range), as described in [Table 2].
Table 2: Antibacterial and antibiofilm activity of Azadirachta indica

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Antibacterial and antibiofilm activity of Aloe vera

A. vera showed the highest zone of inhibition for S. aureus (18.2 ± 1.48 mm) at 50 mg/ml concentration followed by S. saprophyticus (17.8 ± 1.48 mm) and S. epidermidis (18.0 ± 1.60 mm). Biofilm inhibition was seen more than 50% and MBC was 50 ± 23.14–50 ± 25.72 mg/ml (mean range), as described in [Table 3].
Table 3: Antibacterial and antibiofilm activity of Aloe vera

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Antibacterial and antibiofilm activity of Mentha Piperita (peppermint)

M. piperita showed its best antibacterial activity for S. saprophyticus with zone of inhibition of 19.8 ± 1.79 mm and 18.8 ± 1.18 mm at 50, 25 mg/ml concentration. It did not show any antibacterial activity at 12.5 and 6.25 mg/ml concentration with no zone of inhibition against P. aeruginosa, S. epidermidis, and S. saprophyticus. Biofilm inhibition was <50% in case of S. aureus but was more than 50% for other bacterial isolates. MBC came out to be 50 ± 11.18–50 ± 25 mg/ml (mean range), as described in [Table 4].
Table 4: Antibacterial and antibiofilm activity of Mentha piperita

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  Discussion Top


Increasing multidrug resistance status in the bacteria has become a global concern due to the inadequate use of antibiotics. Therefore, finding an alternative for antibiotics is an urgent need which has focused attention on natural products. In our study, methanolic extract of A. indica (Neem) has shown great activity against biofilm-producing bacteria. More than 70% and 80% reduction in biofilm was seen against strong and moderate clinical isolates of S. aureus, P. aeruginosa, S. epidermidis, S. saprophyticus, and A. baumannii at all the concentrations (50, 25, 12.5, and 6.25 mg/ml). In concordance to our study, Geethashri et al.[16] have also reported the suppression in biofilm of E. feacalis using A. indica extract at 7.5 mg/ml concentration. Another study done by Jahan et al.[17] revealed strong antibiofilm activity by methanolic extract of A. indica at 2 mg/ml concentration against biofilm-producing P. aeruginosa. In our study, methanolic extract of A. indica gave maximum zone of inhibition for S. aureus (17.8 ± 1.52 mm), S. epidermidis (18.4 ± 1.51 mm), A. baumannii (14.6 ± 2.07 mm), P. aeruginosa (14.1 ± 1.41 mm), and S. saprophyticus (12.6 ± 1.52 mm) at 50 mg/ml concentration. Almost similar results were given by Tirumalasetty et al.[18] in which 50 mg/ml concentration of methanolic extract of A. indica has shown 20 mm zone of inhibition for S. aureus and 14 mm for P. aeruginosa. MBC for A. indica came out to be 6.25 ± 2.96 mg/ml–6.25 ± 4.91 mg/ml (mean range) in our study, whereas Arévalo-Híjar et al.[19] revealed 25ug/ml MBC for Enterococcus faecalis. Another study done by Abalaka et al.[20] revealed MBC of 50 mg/ml for S. aureus and P. aeruginosa which is quite high compared to our study.

In the present study, methanolic extract of A. vera did not reveal higher anti-biofilm activity as inhibition in biofilm was 52%at 50 mg/ml concentration for S. aureus. Biofilm inhibition ranged between 50% and 54% for isolates of S. saprophyticus, S. epidermidis, and A. baumannii. In contrast to our study, Abraham et al.[21] reported no effect of A. vera extract on biofilm inhibition, whereas a study by Sasirekha et al.[22] reported <50% reduction in biofilm using 200 μl/ml concentration of A. vera extract. In our study, A. vera gave a maximum zone of inhibition of 20.9 ± 1.96 mm for S. epidermidis and 20.6 ± 1.14 mm for S. saprophyticus at the concentration of 6.25 mg/ml, whereas Abakar et al.[23] revealed 19 and 25 mm zone of inhibition against P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. Another study by Malar et al.[24] revealed a 10.5 mm zone of inhibition for S. aureus. In our study, MBC was found to be 50 ± 23.14–50 ± 25.72 mg/ml (mean range). In contrast to this, Muhuha et al.[25] reported MBC of 180 mg/ml against multidrug-resistant S. aureus and P. aeruginosa using A. vera extract.

M. Piperita (Peppermint) extracts did not show any effect on biofilm formation as the reduction was <50%. In concordance to our study, Abraham et al.[21] presented similar results with M. piperita extract as did not have any effect on inhibiting the biofilm formation. In the current study, the best activity of M. piperita extract was seen for S. saprophyticus with the highest zone of inhibition of 19.8 ± 1.79 mm at 50 mg/ml concentration and 14.4 ± 1.62 mm zone of inhibition for S. aureus at 25 mg/ml concentration. In concordance to our study, 15 mm zone of inhibition for S. aureus and 24 mm zone of inhibition for P. aeruginosa has been reported by Mathur et al.[26] using 200 μg/ml extract of M. piperita. In our study, MBC of M. piperita came out to be 50 ± 11.18–50 ± 25 mg/ml (mean range), whereas Radaelli et al.[9] have mentioned MBC of 10 mg/ml for S. aureus using M. piperita oil extract which is comparatively lower to our study.


  Conclusion Top


All the three plant extracts exhibited good activity, but A. indica and A. vera plant extracts were found to be more potent than M. piperita as antibacterial and antibiofilm agents.

Limitation of study

No limitation in the study samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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