On a fresh nutrient or Sabouraud agar plate, 100 µL of the prepared microbial suspension was evenly spread with the help of a glass spreader. A few 5 mm filter paper (Whatmann no. 1) disks were placed on agar plates which were later loaded with 5 µg (5 µl) of the standard antimicrobials. Following incubation at 37 °C for 24 hours and 28 °C for 48 hours for bacterial and fungal pathogens, respectively clear zones of growth inhibition were measured in millimetres. The mean of inhibition zone were determined in triplicates. Similarly, 30 µl of essential oil per 5 mm disc was applied and zone of inhibition was determined in triplicate.

The MIC for all the bacterial and fungal pathogens was achieved by the broth dilution method in a 96-well microtiter plate. Broth cultures were prepared for all pathogens in their respective media which were adjusted to 0.5 McFarland standard turbidity to obtain 1 × 10⁸ Colony Forming Units (CFU)/ml. In order to achieve a concentration of 50 µg/ml, 15 µL of the selected antimicrobial drugs from the stock solution of 1 mg/mL were used for MIC determination. The well volume was then made up to 300 µL. Following a two-fold dilution series, 10 µL of the microbial suspension was added to each well except the negative control. The plates were then incubated at 37 °C for 24 hours (bacterial pathogens) or 28 °C for 48 hours (fungal pathogens). After the incubation period, the visual representation of MIC was done by a redox indicator dye that changed its color from purple to pink in the presence of the cellular reductases. The experiment was repeated thrice in duplicates and mean values of MIC were determined.

The in vitro antimicrobial activity of essential oils from lemongrass (C. flexuosus), palmorosa (C. martinii), and peppermint (M. piperita), essential oils was assessed by evaluating the in vitro inhibition of the selected bacterial and fungal pathogens following the method described previously by other authors with some modifications [38]. All the oils were dissolved in tween 80 with 3% (v/v oil). MIC was determined by the broth microdilution assay. Broth cultures of all the pathogens were prepared in their respective media and their concentrations were adjusted to 0.5 McFarland standard turbidity for obtaining 1 × 10⁸ Colony Forming Units (CFU)/ml. The plates were then incubated at their respective temperatures and time as described earlier. Each experiment was carried out in duplicates.

The MBC/MFC for all the bacterial and fungal pathogens was deduced via spot inoculation onto a fresh agar plate. The last three wells of each row of antibiotic/antifungal/essential oil from the MIC plate were chosen for this process. The plates were divided into quadrants and each section corresponded with the well coordinates. Following incubation for the respective bacterial and fungal pathogens, the MBC/MFC was allocated to the quadrant showing no bacterial/fungal growth.

All the test were performed in triplicates and the obtained data was statistically analysed by a statistical software Minitab™ version 20 (Minitab, LLC; PA, USA). The results were presented as the mean of the triplicate values along with standard deviation and 5% statistical significance (p < 0.05) through Fisher’s test.

The study evaluated the antimicrobial efficacy of commercially available drugs and essential oils against the listed bacterial and fungal strains that included Gram-positive bacteria S. aureus, S. epidermidis, M. smegmatis, E. faecalis, and fungi M. furfur and C. albicans (K4-1), respectively. The selected bacteria are particularly known for causing infectious diseases like folliculitis, cellulitis, impetigo, bacteraemia, and endocarditis whereas the fungal pathogens are linked to skin conditions like dandruff, seborrheic dermatitis, pityriasis versicolor, etc. (Van Tyne et al. 2013). Our results indicated that none of the antibiotics was commonly effective against all the tested bacterial pathogens but inhibited only S. aureus and E. faecalis. Penicillin maximum zone of inhibition (ZOI) in S. aureus and E. faecalis as compared to gentamicin, rifampin, kanamycin, vancomycin, and ampicillin (Table 1). The growth of S. aureus was highly suppressed by penicillin (40.0 mm), rifampicin (32.0 mm) and ampicillin (32.3 mm). Similarly, the growth inhibition of E. faecalis was found highest towards penicillin (30 mm) (Fig. 1). The order of inhibition for S. epidermidis was gentamicin > kanamycin > rifampicin. However, S. epidermidis exhibited no activity with penicillin, vancomycin as well as ampicillin. Furthermore, penicillin and ampicillin were found inactive against M. smegmatis. Two sets of pathogens, S. aureus and E. faecalis, as well as S. epidermidis and M. smegmatis exhibited nearly close ZOI to each other with gentamicin (23.7 mm and 24.0 mm, respectively). A similar trend was also observed with the pathogens S. aureus and S. epidermidis (15.0 mm and 15.3 mm, respectively) as well as the pathogens E. faecalis and M. smegmatis (19.0 mm for both) with the antibiotic kanamycin. The antibiotic vancomycin showed its activity against three of the four tested pathogens but proved ineffective against S. epidermidis. Among the fungal pathogens, M. furfur and C. albicans, clotrimazole proved to be highly effective against both the selected pathogens giving nearby ZOI values for each (34.0 mm and 32.0 mm, respectively). Similarly, amphotericin-B exhibited a similar trend in the ZOI values against these two pathogens (17.5 mm and 18.0 mm, respectively) (Fig. 2).

Figure 1. 

Zone of inhibition by antibiotics: (Pen: Penicillin, Van: Vancomycin, Gen: Gentamicin, Rif: Rifampicin, Kan: Kanamycin: Amp: Ampicillin) against pathogenic bacteria. A. SA (S. aureus); B. SE (S. epidermidis); C. EF (E. faecalis); D. MS (M. smegmatis); E. MS (M. smegmatis); F. CA (C. albicans).

Figure 2. 

Zone of inhibition by antifungals: (Clot: Clotrimazole, Amp-B: Amphotericin-B) against pathogenic fungi. A. MF (M. furfur); B. CA (C. albicans).

Parallelly, the disc diffusion assay results were found to be significant in the case of C. flexuosus giving prominent ZOI for each of the selected bacterial and fungal pathogens (Fig. 3). Prominent zones of inhibition (27.0 mm and 28.3 mm) were noted for M. furfur and C. albicans. Similar findings have been reported by Van Vuuren and Holl (2017) who evaluated antimicrobial activity of Cymbopogon oil against three Gram negative and positive bacteria and two fungi. The essential oil of C. martinii displayed highest zone of inhibition activity against M. furfur (16.7 mm) as compared to all other pathogens and produced smaller zones of inhibition, measuring 8.0 mm, 10.0 mm, and 7.3 mm against S. aureus, E. faecalis, and M. smegmatis, respectively. The inhibitory effect of M. piperita essential oil was maximum against M. smegmatis (31.7 mm), whereas minimum inhibitory activity (10 mm) was observed against S. aureus and E. faecalis. Our findings were found similar to a study reported by Verma et al. (2011).

Figure 3. 

Zone of inhibition showing antimicrobial activity of essential oils: (LG: Lemongrass, PR: Palmarosa, PM: Peppermint) against human pathogenic bacteria and fungi. A. SA (S. aureus); B. SE (S. epidermidis); C. EF (E. faecalis); D. MS (M. smegmatis); E. MF (M. furfur); F. CA (C. albicans).

Taking 50 µg/ml of each drug and following a two-fold dilution series, the most efficacious drugs required in minimum quantity Our results showed that at MIC 0.10 µg/ml, penicillin and rifampicin inhibited the growth of S. aureus and E. faecalis. Similarly, gentamicin suppressed the growth of S. epidermidis and M. smegmatis at MIC 0.10 µg/ml. Kanamycin had a higher MIC (0.39 µg/ml) than penicillin (0.10 µg/ml) to restrict the growth of S. epidermidis. Vancomycin was effective at a slightly higher concentration for all the pathogens except E. faecalis. MIC values were found to be at par for rifampicin (0.10 µg/ml) in case of S. aureus, S. epidermidis and E. faecalis. Similarly, S. aureus, and M. smegmatis had equal MIC values for kanamycin (1.56 µg/ml) also. The favourable drug to suppress the growth of M. smegmatis was found to be gentamicin (MIC 0.10 µg/ml) in this study.

Likewise, clotrimazole showed antimicrobial activity at (MIC 0.10 µg/ml for M. furfur and C. albicans, while amphotericin-B was required at a higher concentration for growth inhibition and death of both the fungal pathogens (MIC 3.12 & 0.10 µg/ml respectively).

In a parallel investigation, when assessing the antimicrobial efficacy of essential oils, specifically C. martini, yielded the most favorable results against a variety of tested bacterial pathogens. The MIC for this oil ranged from 0.07% to 0.09% v/v. Our result was consistent with those of previous studies done by Vuong and Otto (2002). Similarly, the essential oil derived from M piperita demonstrated significant effectiveness, particularly against S. aureus, with an MIC value of 0.07% v/v. It also exhibited positive results against S. epidermidis (MIC 0.09% v/v), E. faecalis (MIC 0.09% v/v), and M. smegmatis (MIC 0.19% v/v). Almost similar efficacy was reported in a study reported by Wang et al. (2022). On the contrary the essential oil from C. flexuosus required slightly higher concentrations, to inhibit the growth of all the tested pathogens (MIC 0.19% v/v). Notably, the MBC values aligned with the MIC values for these bacterial pathogens. Moreover, the investigation extended to two fungal pathogens, and the essential oils from C. martinii and M. piperita demonstrated their capability to inhibit both pathogens at a concentration of 0.3% v/v of MIC/MFC, while C. flexuosus achieved similar inhibition at a concentration of 0.12% v/v of MIC/MFC (Table 2). These values almost coincide with the results reported by Vuong and Otto (2022).

The comparison of essential oils with antibiotics in the context of antimicrobial activity is a subject of growing interest in the field of microbiology as well as medical research. As essential oils are natural extracts from plants that contain a wide variety of bioactive compounds, including terpenes and phenolic compounds, that can have antimicrobial properties (Watts et al. 2018). In contrast, antibiotics are either synthetic or semi-synthetic compounds designed specifically to target and kill or inhibit the growth of bacteria which in due course of time can develop resistance by altering the target of the antibiotic or by developing efflux pumps to expel the drug (Wisplinghoff et al. 2004).