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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 3  |  Page : 372-381

Antibacterial activity of some medicinal plants and antibiotics against Staphylococcus aureus and Mammaliicoccus sciuri isolated from acne


Department of Biotechnology, Gurucharan College, Silchar, Assam, India

Date of Submission28-May-2022
Date of Decision28-Jun-2022
Date of Acceptance29-Jul-2022
Date of Web Publication17-Sep-2022

Correspondence Address:
Meghali Goswami
Department of Biotechnology, Gurucharan College, Silchar - 788 004, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_135_22

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  Abstract 


Background: Acne vulgaris is a distressing condition that affects most adolescents, causing scarring and reducing the quality of life. Among all the available therapeutic options, antibiotics are routinely used to treat moderate acne. In some cases, the clinical symptoms temporarily disappear after applying the prescribed medications, but its reoccurrence along with the misuse and overuse of the prescribed antibiotics may result in the emergence of multidrug-resistant strains. Methods: The present study aims to isolate and identify acne-causing bacteria from two individuals, who developed chronic or recurrent papules or pustules on their face and neck. The effect of different physiological factors on the growth of these bacteria was evaluated, followed by an evaluation of microbial adhesion, biofilm formation, hemolytic activity, extracellular enzyme production, and antibacterial activity of some medicinal plants and antibiotics. Results: The predominant bacteria in acne samples were Gram-positive cocci, which were identified as Staphylococcus aureus strain GCC20_MS and Mammaliicoccus sciuri strain GCC20_MS. Both the isolates showed maximum viability at pH 7.0 and significant growth up to 10% NaCl concentration. A decreased viable count with the increase in Lysozyme concentration was also observed. The cell-surface hydrophobicity and auto-aggregation of both the tested isolates were very low; however, the strains were moderate biofilm producers. S. aureus strain GCC_20MS exhibited β-hemolysis, whereas M. sciuri strain GCC_20RS showed γ-hemolytic activity (no-hemolysis). Antibiotic-sensitivity test showed resistance of both the strains toward penicillin and sensitivity toward tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, co-Trimoxazole, gentamicin, and ofloxacin. The aqueous extracts of Citrus limon and Psidium guajava significantly inhibit the growth of the isolated strains. Moderate growth inhibition was observed by the aqueous extracts of Mentha sachalinensis and Punica granatum. Conclusion: The study suggests the efficacy of topical anti-acne formulations using plant extracts that may target the early processes of acne development and combat the emergence of multidrug-resistant strains.

Keywords: Acne, antibacterial activity, antibiotics, aqueous plant extracts, extracellular enzymes


How to cite this article:
Goswami M. Antibacterial activity of some medicinal plants and antibiotics against Staphylococcus aureus and Mammaliicoccus sciuri isolated from acne. Biomed Biotechnol Res J 2022;6:372-81

How to cite this URL:
Goswami M. Antibacterial activity of some medicinal plants and antibiotics against Staphylococcus aureus and Mammaliicoccus sciuri isolated from acne. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Dec 8];6:372-81. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/372/356143




  Introduction Top


The composition of microbial communities in the skin varies according to the physiology of the site, and their relative abundance is associated with dry, moist, and sebaceous microenvironments. The lipophilic bacteria such as Propionibacterium species are mostly dominated in the sebaceous sites. In contrast, bacteria such as Corynebacterium and Staphylococcus species are found in moist areas, including the bends of the feet and elbows.[1] Bacterial infections of the skin are mostly associated with the epidermis, dermis, subcutis, and hair follicles. Some skin flora such as P. acnes, Streptococcus pyogenes, S. aureus, Klebsiella, and Clostridium cause many skin diseases, which include acne vulgaris, cellulitis, erysipelas, erythema nodosum, impetigo, and necrotizing fasciitis.[2] Other diseases such as chronic ulcers caused by Streptococcus, Clostridium and Bacteroides, staphylococcal scaled skin syndrome by S. aureus, and chronic wounds by P. aeruginosa are also observed in some cases.[2]



Acne vulgaris is related to the pilosebaceous unit of the skin, which is fundamentally composed of hair follicles and the sebaceous gland. It is generally characterized by some changes in the skin, such as papules (pinheads), comedones (blackheads and whiteheads), seborrhea (scaly red skin), and nodules (large papules).[3] It commonly occurs during the adolescence phase, where about 80%–90% of teenagers have acne starting at 12 years, and half of them continue to experience symptoms as adults up to an average age of 45 years.[4] The physical consequences of acne include discoloration, scarring, and psychological effects such as low self-esteem, depression, and anxiety.[5] Acne lesions can affect an individual's personal, social, vocational, and academic life. Studies have shown that acne profoundly impacts emotions (self-esteem, self-embarrassment, and feelings of unworthiness), experience social anxiety and shame, annoyance by physical symptoms (itch and pain), and discomfort during the treatment procedure.[6] Individuals with acne usually grow long hair to cover their face, avoid eye contact, select a specific clothing style and use makeup to hide acne lesions. Acne lesions are of two types-inflammatory and noninflammatory. The noninflammatory lesions are blackheads and whiteheads comedones, while the inflammatory lesions of acne include pustules and nodules/nodulocystic lesions.[7] The major factors contributing to acne pathogenesis are increased sebum production, keratinization of the middle infundibulum, bacterial colonization of the follicle, and inflammation of the immune system.[6] Other factors such as hygiene, diet, stress, and hormones can contribute to the multi-factorial process of microbial pathogenesis.[8]

The production of some virulence factors such as protease, DNase, lipase, lecithinase, and hemolysin by Staphylococcus species has been reported.[9] The major factors in the bacteria which are responsible for virulence are enzymes, peptidoglycan, and toxins. Other factors such as protein A and clumping factors lead to tissue damage. In the human body, hemolysin disrupts red blood cells, and Panton-Valentine leukocidin causes the killing of white blood cells, whereas enzymes such as lipases, proteases, DNase, coagulase, and fibrinolysin enable the spread of the toxins.[10]

In modern medical practices, acne can be treated with antibiotics, topical retinoids, isotretinoin, and hormonal therapy.[11] Among all the available therapeutic options, antibiotics such as macrolides, tetracyclines, and trimethoprim are routinely used to treat moderate acne.[12] They exert an antibacterial effect, minimum toxicity, and easy diffusion through tissues. Since the discovery of these antibiotics, there has been a belief in the medical fraternity that the use of antibiotics as chemotherapeutic agents would eradicate infectious diseases. However, the misuse and overuse of antibiotics result in the emergence of multi-drug resistant strains of several groups of microorganisms. These infections are difficult to treat by available antibiotics and sometimes lead to treatment failure. Antimicrobial agents such as topical retinoids also inhibit the formation of microcomedones and help in reducing the quantity of noninflammatory acne. The commonly used topical retinoids in acne treatment are tretinoin, adapalene, and tazarotene, which are derivatives of Vitamin A (retinol).[13] The chemical peels of salicylic acid and mandelic acid are also effective in the treatment both inflammatory and noninflammatory acne.[14]

The present study aims to identify acne-causing bacteria in two individuals. The evaluation of some physiological factors favoring bacterial growth, exoenzyme production, and sensitivity toward a group of antibiotics was studied. The study also reports the efficacy of locally available medicinal plants as an alternative treatment procedure to prevent the growth of acne-causing bacteria.


  Methods Top


Clinical diagnosis, isolation, and identification of bacteria from acne

Clinical diagnosis of acne

The present study evidenced cutaneous disorder among two participants (one male and one female) of age 20 years. Both of them developed chronic or recurrent papules or pustules on their face and neck. A papule is a raised lesion on the skin <1 cm in diameter, while a pustule looks like a papule but is inflamed and filled with pus. The cutaneous disorder was clinically diagnosed by dermatologists and general practitioners and characterized as acne vulgaris. The clinical symptoms temporarily disappeared after applying the prescribed medications. A survey was conducted on eating habits, medical records, self-hygiene, cosmetic use, and skin type of the participants. To perform the microbiological studies, formal approval was obtained from the institutional ethical research committee. A formal approval was obtained from the institutional ethical research committee of Gurucharan College, Silchar.

Isolation of bacteria

In order to collect acne samples from the participants, the surface of pustules was disinfected with cotton soaked in 75% alcohol. The pustule was gently pressed to ooze out pus samples and collected using a sterile cotton swab. The pus samples were inoculated onto freshly prepared nutrient agar (NA) plates and incubated at 37°C for 24 h. The distinct individual colonies were subcultured to obtain the pure culture of the isolate.

Morphological, biochemical, and physiological characteristics of bacteria

Cultural characteristics and colony morphology of the isolates were noted, and Gram's staining was performed. The biochemical tests conducted in the present study include indole test, methyl red test, Voges–Proskauer test, citrate utilization test, oxidase test, catalase test, nitrate reduction test, and starch hydrolysis test were performed for preliminary identification of bacteria. The isolates were tested for anaerobic growth with an anaerobic gas pack system (HiMedia, Mumbai, India).

Carbohydrate utilization test

The carbohydrate utilization test was carried out by Hi-IMViC Biochemical Test Kit (Himedia, Mumbai, India), which inspects the bacterial ability to utilize selected sugars. The wells of the test kit were inoculated with 50 μL of the test bacteria by surface inoculation method and incubated at 37°C for 24 h. After the incubation period, the wells were visually inspected for color change and compared with the color chart that was supplied with the test kit.

Molecular identification

Genomic DNA of the bacteria was isolated by following the protocol of Green et al.,[15] and the 16S rDNA region of the isolated DNA was amplified using forward primer (5'-GGATGAGCCCGCGGCCTA-3') and reverse primer (5'-CGGTGTGTACAAGGCCCGG-3'). Sequencing of the amplified gene was carried out at Theomics International Private Limited, Bengaluru, India, using ABI 3130 × l96 capillary system using Big Dye Terminator version 3.1 kits. The sequences obtained by sequencing were assembled using Geneious R8 software package (Biomatters Ltd., Auckland, New Zealand) to generate a consensus sequence of 16s rDNA, followed by BLAST analysis to find the closest homologous sequence that are present in the nonredundant database. The first ten sequences in the database that showed the highest similarity and maximum identity score were selected. All the sequences were aligned using Clustal-W, and a Neighbor-Joining tree was constructed using the Geneious R8 software package (Biomatters Ltd., Auckland, New Zealand).[16]

Physiological conditions and bacterial growth

Effect of pH on bacterial growth

Fifty microliters of test bacterial broth were spread onto NA plates of varying pH, ranging from pH 2 to 8, which was adjusted with 1 N hydrochloric (HCl) and 1 N NaOH. The plates were incubated at 37°C for 24 h, and the growth pattern in extremely acidic and basic environments was noted by counting the viable colonies.

NaCl tolerance test

NA plates supplemented with different concentrations of NaCl were prepared. NaCl concentration ranges from 0.5% to 20% (w/v), and the plates without NaCl amendment were used as control. The test isolate was streaked onto the surface of NA plates and incubated at 37°C for 24 h. After the incubation period, the growth of bacteria in each plate was recorded.

Lysozyme tolerance

Lysozyme tolerance test was performed in nutrient broth supplemented with different concentrations of lysozyme. The lysozyme concentration in the broth was maintained at 100, 200, and 300 mg/L. The broth without lysozyme was used as a control. Thereafter, 1 mL of test isolate was inoculated in 10 mL of the broth and incubated at 37°C for 3 h. The viability of bacteria was evaluated by spreading 100 μl of the bacterial sample onto the freshly prepared NA plates.

Microbial adhesion and hemolytic activity

Cell surface hydrophobicity

Overnight grown broth culture was taken and centrifuged at 5000 rpm for 10 min at 4°C to harvest the cell pellets. The cell pellets were washed twice with phosphate buffer saline (PBS) solution and resuspended in 6 mL PBS solution. The initial absorbance (ODinitial) was recorded at 600 nm. Thereafter, 3 mL of the bacterial suspension was added with 1 mL of hydrocarbons (n-Hexadecane and Toluene), and vortexed for 2 min. The suspension was left undisturbed for 1 h for phase separation, following which the aqueous phase was carefully removed. The remaining volume was used to record the final absorbance (ODfinal). The initial and final absorbance was used to measure the percentage of cell surface hydrophobicity by the formula:



Cellular autoaggregation

Overnight grown broth culture was centrifuged at 5000 rpm for 10 min at 4°C to harvest the cell pellets. The pellets were washed twice with PBS solution and resuspended in 6 mL PBS solution. The initial absorbance (ODinitial) was recorded at 600 nm. Thereafteṛ, the cell suspension was incubated at 37°C for 2 h, and the final absorbance (ODfinal) was measured to determine the percentage of cellular autoaggregation using the formula:



Test for biofilm formation

Biofilm formation by the test isolates was determined by the modified crystal violet staining method.[17] Briefly, 50 μL of overnight grown bacterial suspension was diluted (1:20) in 950 μL of Luria-Bertani medium in an Eppendorf tube. The tubes were incubated at 37°C for 24 h, following which the planktonic bacteria were removed. The tubes were washed thrice with PBS, air-dried for 1 h in an airflow cabinet, and stained with 200 μL of 0.1% crystal violet for 20 min. The excess stains were removed by washing the tubes with distilled water. The remaining crystal violet was solubilized with 200 μL of 95% ethanol and incubated for 20 min. Finally, the optical density was measured at 550 nm to determine the biofilm formation capacity of the test isolate. An un-inoculated Luria-Bertani medium was used as a negative control. The experiment was carried out in triplicate. An OD of more than 0.500 is recognized as a high biofilm producer, OD lower than 0.100 is considered a poor biofilm producer, and an OD between 0.500 and 0.100 is a moderate biofilm producer.

Hemolytic activity

Hemolytic test was performed on blood agar plates, which was prepared by supplementing 5% sheep blood (obtained from a local butcher shop) on brain heart infusion agar. The test isolates were streaked onto the surface of freshly prepared blood agar plates. The plates were incubated at 35°C for 48 h, and the hemolysis pattern (α, β, and γ) was noted.

Exoenzyme production

Proteolytic activity

Proteolytic activity was performed on an agar medium composed of skim milk powder (10% w/v) and agar (2% w/v). The overnight grown test isolates were spotted on the surface of freshly prepared agar medium, followed by incubation at 30°C for 4 days. The plates were then observed for a clear zone around the colonies, indicating a positive result for the proteolytic activity.

Lipolytic activity

The lipolytic activity was performed by streaking the test isolates on tributyrin agar plates. The formation of clear halos around the colonies after 4 days of incubation at 30°C indicates positive results for lipolytic activity.

Amylolytic activity

Luria-Bertani agar plates containing 20 g/L of soluble starch were prepared, onto which the overnight grown test isolates were spotted. The plates were incubated at 37°C for 72 h. After the incubation period, the plates were then flooded with 1% iodine solution. The appearance of a halo zone around the colonies indicates amylolytic activity/starch hydrolysis.

DNase activity

The DNase-producing ability of test isolates was assessed by streaking the test isolates on DNase agar plates, followed by incubation at 37°C for 72 h. The plates were then flooded with 3% HCl for 8 min. The appearance of a clear zone around colonies indicates a positive result for the DNase test.

Antibacterial activity of plant extracts and antibiotics against the test isolates

Collection of plant material and preparation of aqueous extract of plant parts

Fresh leaves of Eclipta prostrata (bhringraj), Ocimum tenuiflorum (krishna tulsi), Ocimum sanctum (rama tulsi), Coriandrum sativum (coriander), Azadirachta indica (neem), Citrus limon (lemon), Mimosa pudica (touch-me-not), Psidium guajava (guava), Strobilanthes alternate (red-flame ivy), Kalanchoe pinnata (Bryophyllum pinnatum), Murray koenigii (curry), Mentha sachalinensis (mint), Aloe barbadensis Miller (Aloe vera), Tagetes erecta (Marygold). Brassica juncea (mustard), Punica granatum (pomegranate), and Zingiber officinale (ginger); fruits of Carica papaya (papaya); roots of Curcuma longa (turmeric) and Zingiber officinale (ginger), and peel of Solanum lycopersicum (tomato) were collected from various locations of Cachar district, Assam, India [Figure 1]. The plant materials were immediately brought to the laboratory, thoroughly washed, and shade dried. Afterward, 25 g of each plant material was taken, cut into small pieces, and soaked in distilled water for 24 h. The mixture was homogenized, filtered through a double layer of cheesecloth, and the homogenate was centrifuged at 3000 rpm for 10 min at 4°C to collect the supernatant. Finally, the supernatant was filtered through a sterile filter (0.22 μm) and stored at –20°C for later experiments.[18]
Figure 1: Plant parts used for preparing the aqueous extract

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Antibacterial activity of the aqueous plant extracts

Mueller Hinton Agar media plates were prepared in which wells were made by a 6 mm sterilized cork borer.[19] One hundred microliters of test isolates were spread onto the surface of Muller Hinton Agar plates using a glass spreader, and the wells were filled with 50 μL of aqueous plant extract. The test plates were incubated at 37°C for 24–48 h. Dimethyl sulfoxide was used as a negative control. The antibacterial activity of the aqueous plant extracts was evaluated by measuring the inhibition zone in millimeters.

Antibiotic susceptibility test

The antibiotic sensitivity test was performed by the Kirby–Bauer disk diffusion method.[20] The standard antibiotic discs were procured from HiMedia, Mumbai, India, which include azithromycin, erythromycin, clindamycin, penicillin, tetracycline, vancomycin, rifampin, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, co-trimoxazole, gentamicin, oxacillin, norfloxacin, ofloxacin, methicillin, and cefoxitin. The antibiotic discs were placed onto the freshly prepared lawns of each isolate on Muller Hinton Agar plates. The plates were incubated at 37°C for 24–48 h, and the diameter of the zone of inhibition was measured in millimeters.[21] The strains were classified in accordance with clinical and laboratory standards Institute,[22] following the standard antibiotic disc chart.


  Results Top


Identification of bacteria from acne

The culture of pus samples collected from acne samples showed the presence of Gram-positive cocci that tends to be arranged in clusters. The isolate designated as GCC20_MS showed golden or yellow colonies with a sticky and jelly-like appearance on NA medium, whereas the isolate GCC20_RS appears in white and creamy colonies with sticky surfaces. Both the isolates were able to can grow in aerobic and anaerobic conditions [Table 1]. Both the test isolates exhibited positive results in the catalase and nitrate reduction tests and negative results in the indole and starch hydrolysis tests. The isolate GCC_20MS showed positive results for the methyl red test, Voges-Proskauer test, and citrate utilization test. Both the test isolates were able to utilize glucose, lactose, sorbitol, mannitol, and sucrose [Table 1], which were visually inspected by a change in color of the wells of the Hi-IMViC Biochemical Test Kit. The 16S rDNA sequence of the isolated strains was aligned with database sequences, followed by the construction of a Neighbor-Joining tree [Figure 2]. Based on biochemical test results and 16S rDNA sequencing, the isolates GCC_20MS and GCC_20RS were identified as Staphylococcus aureus and Staphylococcus sciuri. However, recent phylogenomic analysis suggests the reclassification of S. sciuri within the genus Staphylococcus as heterotypic synonyms and taxonomically reassigned as Mammaliicoccus species.[23] The 16S rDNA sequence of both isolates was submitted to NCBI, archiving GenBank accession numbers MZ305087 and MZ305085 for S. aureus and Mammaliicoccus sciuri, respectively.
Table 1: Growth parameters and biochemical characteristics of isolates GCC_20MS and GCC_20RS

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Figure 2: Phylogenetic tree of isolate GCC_20MS and GCC_20RS, constructed by Neighbor-Joining method. (a) The isolate GCC_20MS was identified as Staphylococcus aureus, and (b) The isolate GCC_20RS was identified as Staphylococcus sciuri, which is taxonomically reassigned as Mammaliicoccus sciuri

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Physiological conditions and bacterial growth

Growth of bacteria at varying pH

Isolated strains of S. aureus and M. sciuri showed significant growth and maximum viability at pH 7.0. Our study evidenced that a lower pH negatively affects the viable growth of S. aureus and M. sciuri. Similarly, at alkaline pH (pH ≥8.0), a significant reduction of viability was observed by both the isolates.

NaCl tolerance

S. aureus strain GCC_20MS and M. sciuri strain GCC_20RS showed significant growth at 10% NaCl concentration. However, upon increasing the concentration to 13%, the growth of S. aureus strain GCC_20MS significantly decreases. M. sciuri strain GCC_20RS, however, exhibited tolerance up to 18% NaCl concentrations.

Lysozyme tolerance

Both the test isolates showed a decreased viable count with the increase in Lysozyme concentration. S. aureus strain GCC_20MS, however, showed a marginal increase in the viable count at a concentration of <200 mg/L Lysozyme [Figure 3]. M. sciuri strain GCC_20RS exhibits a significantly decreased colony count in all tested Lysozyme concentrations [Figure 4].
Figure 3: Viable plate count of S. aureus strain GCC_20MS at different concentrations of lysozymes. '0 h' represents initial plate count, whereas '3 h' represents final plate count after 3 h of incubation. The results are expressed in log10 CFU/mL, CFU: Colony-forming units

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Figure 4: Viable plate count of M. sciuri strain GCC_20RS at different concentrations of lysozymes. “0 h” represents initial plate count, whereas “3 h” represents final plate count after “3 h” of incubation. The results are expressed in log10 CFU/mL, CFU: Colony-forming units

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Microbial adhesion and hemolytic activity

Cell surface hydrophobicity

The cell surface hydrophobicity of S. aureus strain GCC_20MS was 10.20% and 24.36% for the hydrocarbons n-hexadecane and toluene, respectively. The M. sciuri strain GCC_20RS showed 10.62% and 20.68% hydrophobicity for n-hexadecane and toluene, respectively.

Cellular auto-aggregation

Autoaggregation is capable of establishing ecological niches and adhering to membranes within the host cells. The auto-aggregation result was very low, exhibiting 15.72% and 13.21% for S. aureus strain GCC_20MS and M. sciuri strain GCC_20RS, respectively.

Biofilm formation

In the present study, both the reported strains were found to be moderate biofilm producers.

Haemolytic activity

S. aureus strain GCC_20MS exhibited β-hemolysis (clear zone), whereas M. sciuri strain GCC_20RS showed γ-hemolytic activity (no hemolysis).

Exoenzyme production

Proteolytic activity

The isolate M. sciuri strain GCC_20RS showed a clear zone around the colonies after incubation in skimmed milk agar medium, indicating positive results for proteolytic activity. The isolate S. aureus strain GCC_20MS showed negative results for proteolytic activity.

Lipolytic activity

S. aureus strain GCC_20MS showed a positive result for the lipolytic test, evidenced by a clear halo zone around the colonies. The isolate M. sciuri strain GCC_20RS showed negative results for the lipolytic test.

Amylolytic activity

Both the tested isolates showed negative results for the amylolytic test.

DNase activity

The test isolates showed a clear zone around the colonies after flooding the DNase agar plates with 3% HCl, indicating positive results for DNase activity.

Antibacterial activity of plant extracts and antibiotics against the test isolates

Antibacterial activity of the aqueous plant extracts

The aqueous plant extracts differ significantly in their activity against test microorganisms. The growth of S. aureus in culture plates was inhibited considerably by aqueous extracts of C. limon and P. guajava, exhibiting a zone of inhibition of 15 mm and 17 mm, respectively. Both the plant extracts (C. limon and P. guajava) showed antibacterial activity against M. sciuri. C. limon also demonstrates the highest inhibition zone of 18 mm against M. sciuri [Figure 5]. The aqueous extract of M. sachalinensis and P. granatum showed moderate growth inhibition against both the tested strains. The extracts of Strobilanthes alternata, M. koenigii, Curcuma longa, and Carica papaya inhibited the growth of S. aureus, but failed to demonstrate any antibacterial activity against M. sciuri [Table 2]. The aqueous extracts of Eclipta prostrata, Ocimum tenuiflorum, Ocimum sanctum, Coriandrum sativum, Azadirachta indica, Zingiber officinale, Mimosa pudica, Kalanchoe pinnata, Aloebarbadensis miller, T. erecta, Solanum lycopersicum, and Brassica juncea did not show any antibacterial activity against both the tested bacterial strains.
Table 2: Antimicrobial activity of selected plant extract against Staphylococcus aureus strain GCC_20MS and Mammaliicoccus sciuri strain GCC_20RS

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Figure 5: Antibacterial activity of the aqueous plant extracts on Staphylococcus aureus strain GCC_20MS and Mammaliicoccus sciuri strain GCC_20RS. The zone of inhibition was measured in mm, and the codes on each well represent the plant name. EP: Eclipta prostrata (leaf), OS: Ocimum sanctum (leaf), OT: Ocimum tenuiflorum (leaf), CS: Coriandrum sativum (leaf), AI: Azadirachta indica (leaf), ZR: Zingiber officinale (root), CL: Citrus limon (leaf), MP: Mimosa pudica (leaf), PS: Psidium guajava (leaf), SA: Strobilanthes alternata (leaf), KP: Kalanchoe pinnata (leaf), MK: Murray koenigii (leaf), CT: Curcuma longa (root), CP: Carica papaya (fruit), MS: Mentha sachalinensis (leaf), AM: Aloebarbadensis miller (leaf), TE: Tagetes erectaI (leaf), SL: Solanum lycopersicum (Tomato peel), BJ: Brassica juncea (leaf), PG: Punica granatum (leaf), ZL: Zingiber officinale (leaf) and DMSO: Dimethyl sulfoxide (control)

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Antibiotic susceptibility test

The isolated strain of S. aureus and M. sciuri were sensitive to a group of antibiotics which includes tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, co-Trimoxazol, gentamicin, and ofloxacin. Methicillin-resistant staphylococci were not detected, which was confirmed by oxacillin/methicillin sensitivity test results. The isolates also showed a significant inhibition zone against azithromycin, moxifloxacin, and norfloxacin [Table 3]. Both the isolates exhibited intermediate results against erythromycin and clindamycin; however, they failed to show any inhibition zone against penicillin.
Table 3: Antibiotic sensitivity test

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  Discussion Top


Acne vulgaris is a condition that is characterized by chronic inflammation of the skin. It is commonly associated with nutrition, medication, climatic conditions, pollutants, psychological factors, and occupational exposures. Acne is primarily caused by excess sebum production, increased proliferation of keratinocytes, and a reduction in desquamation of keratinocytes in the pilosebaceous unit. The conjunction of sebum and keratinocytes causes keratotic plugs that block the pilosebaceous ducts, eventually forming microcomedones.[24] The present study reports the presence of follicular papules and pustules on the face of the studied individuals. The culture of pus samples from pustules of facial lesions showed the presence of Gram-positive cocci that were identified as S. aureus and M. sciuri. S. aureus is a common skin colonizer but is sometimes associated with local and systemic infections.[25] M. sciuri has been implicated in the case of endophthalmitis, endocarditis, scalded skin syndrome, urinary tract infection, pelvic inflammatory disease, catheter-associated bacteremia and septic shock. M. sciuri has been cultured from the pharynxes, axillae, nares, vagina, and human genitourinary tract of hospitalized patients as well as healthy controls, and found in hospital settings and other surfaces.[26]

The growth responses of bacteria are greatly affected by pH and NaCl concentration. A higher concentration of NaCl (above 13%) inhibits the growth of S. aureus strain GCC_20MS, which is also evidenced by other studies.[27] M. sciuri strain GCC_20RS showed significant growth up to 18% NaCl concentration. A study on Staphylococcus sciuri strain LCHXain showed optimum growth at 20–37°C and pH 6–8. In addition, the specific growth rate was reduced by 50% in the presence of 1 M NaCl.[28] Lysozyme is a naturally occurring antimicrobial enzyme found in tears, saliva, and milk. It destroys the peptidoglycan component of bacterial cell walls, resulting in the death of the cell. The present study reported a decreased viable count of the test bacteria with the increase in Lysozyme concentration.

The colonization of S. aureus causes various skin diseases such as abscesses, impetigo, psoriasis, and atopic dermatitis.[29] On the other hand, M. sciuri rarely colonizes human skin, but the presence of transient carriage is relatively common, particularly in the mucous membranes of the nasal vestibule.[30] The present study reported poor results for cell surface hydrophobicity and cellular auto-aggregation by S. aureus strain GCC_20MS and M. sciuri strain GCC_20RS. Although both isolates produced moderate biofilm, the ability to form biofilm in the host could result in chronic and recalcitrant disease in the host. The production of biofilms and virulence factors by S. aureus and M. sciuri has been documented in other studies.[31],[32] In the present study, S. aureus strain GCC_20MS showed lipolytic activity, while M. sciuri strain GCC_20RS showed proteolytic activity. Both the test isolates also evidenced positive results in the DNase test and negative results in the amylolytic test. It has been reported that S. aureus and M. sciuri secret extracellular enzymes, that are responsible for disrupting host tissues and inactivating host antimicrobial mechanisms to acquire nutrients and facilitate bacterial propagation.[33] S. aureus strain GCC_20MS showed lysis of red blood cells surrounding bacterial colonies, indicating β-hemolysis. M. sciuri strain GCC_20RS showed γ-hemolysis, with no notable zones around the colonies. Similar results were also evidenced in other studies.[9],[34]

The adherence to oral antibiotic therapy for skin infections and its relationship to clinical are poorly understood. Following an initial S. aureus skin infection, recurrent or relapsed infections are often reported, and in some populations, the rate of reoccurrence has exceeded 50%.[35] Our in vitro investigation showed significant sensitivity against a group of antibiotics. The patient's condition improves after taking oral antibiotics or ointment application in affected areas. However, frequent recurrence of the same infection has been observed, likely due to poor adherence to oral antibiotics. The poor adherence and recurrence of skin infection are very likely to be associated with the choice of antibiotics, misuse and overuse of antibiotics, patient's age, treatment duration, and many others.[36],[37]

An excellent alternative to antibiotics and synthetic chemical substances are phytochemicals that are in use to treat a variety of skin infections. Our data demonstrated that the leaf extract of C. limon, P. guajava, M. sachalinensis and P. granatum significantly inhibits the growth of S. aureus and M. sciuri. Many studies also reported similar results, signifying the efficacy of these plant extracts against drug-resistant bacteria. The phytochemical constituents of C. limon leave oil are sabinene, 3-carene, limonene, and β-ocimene. These active phytochemicals exhibit remarkable inhibition against a wide group of Gram-negative and Gram-positive bacteria.[38],[39] C. limon leave oil irreversibly damages the bacterial membrane, resulting in cytoplasmic losses causing ions leakage, bacterial lysis, and death. It also greatly impacts protein and nucleic acid biosynthesis in the Staphylococcus sp.[38] The leaf extract of P. guajava contains flavonoids, mainly quercetin derivatives,[40] which exhibited antibacterial activity against S. aureus and M. sciuri. Several studies documented the therapeutic and antimicrobial phytochemical ingredients of M. sachalinensis, which comprises menthol, carvone, limonene, 1, 8-cineole, linalool, menthone, and isomenthone.[41] The P. granatum leaf contains flavonoids (luteolin, rutin, apigenin, etc.), phenolic acids (ellagic acid and gallic acid), and hydrolyzable tannins (granatin, corilagin, punicafolin, etc).[42] Many studies have well documented the antibacterial activity and therapeutic effects of these active compounds.[38],[41] The present study also reported significant activity by the aqueous extracts of Strobilanthes alternata and M. koenigii leaf against S. aureus strain GCC_20MS, which might be due to phenolics flavanoids and coumarin.

Curcuma longa, a dietary plant with antibacterial, anti-inflammatory, antioxidant, anticancer, and anti-clotting properties, is another essential dietary plant.[43],[44] Alkaloids and flavonoids found in Curcuma longa have been linked to antibacterial activity against many bacterial species, including E. coli, S. aureus, methicillin-resistant S. aureus, Vibrio cholera, E. faecalis, S. pyogenes, and S. agalactiae.[43] Carica papaya is an excellent source of vitamins and nutrients and contains essential phytochemicals such as alkaloids, terpenoids, phenol, tannins, saponins, proteins, steroids, and fixed oils.[45] The antibacterial activity of C. papaya was consistent with previous studies, which showed that the aqueous extract exhibits antibacterial activity against a wide range of bacterial species.[45]


  Conclusion Top


Acne vulgaris is a distressing condition that affects most adolescents, causing scarring, and reducing the quality of life. The findings of the present study demonstrated that the aqueous extracts of C. limon, P. guajava, M. sachalinensis, and P. granatum possess natural antimicrobial agents that significantly stop the growth of S. aureus and M. sciuri. Thus, these plant extracts can be used individually or in combination with antibiotics to develop topical anti-acne formulations.

Acknowledgments

The author extends their thanks to DBT, New Delhi for establishing Institutional Biotech Hub and Bioinformatics Centre in Gurucharan College, Silchar, to carry out the research activity. Special thanks to all the faculty members of the Department of Biotechnology, Gurucharan College, Silchar for providing the necessary arrangements to conduct the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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