Antibacterial activity of Illicium verum essential oil against MRSA clinical isolates and determination of its phyto-chemical components

2.1 Materials used

MRSA clinical isolates of Staphylococcus aureus from nosocomial infected patients, Blood agar plates, Mueller Hinton agar, Peptone, Standard antibiotic e- test strip, Illicium verum essential oil from the e-commerce platform (Old tree essentials), Standard Biochemical reagents used. Analytical grade chemicals were used for this research studies.

2.2. Fourier transform infra-red spectra of Illicium verum EO carried out using FTIR, Bruker spectrophotometer. GC-MS analysis was performed on a Shimadzu GCMS-QP2010 SE Standard Gas Chromatograph-Mass Spectrometer (Shimadzu Corp., Kyoto, Japan) equipped with a GCMS solution software. The GC-MS analysis was performed as per standard procedure mentioned in literature (Sitaram et al., 2011; Tsugawa and Fukusaki, 2020). Methodology for Phyto-bio synthetic investigation of the Illicium verum EO performed using Mayer's, Fehling's, Iodine, Salkowski's, Ninhydrin, Ferric, Libermann Burchard's, Benedict's, Keller-Kilani, Ammonia test. The phytochemical properties investigation of the Illicium verum EO acquired from the e-commerce platform (Old tree essentials) was done as previously described standard procedure (Gouse et al., 2017; Muazzam et al., 2020; Maqbul et al., 2020).

2.2 Microbiological processing of MRSA clinical samples

The MRSA clinical isolates were collected from the nosocomial patients which included isolates from nasal samples, catheter test, urine test, crotch test, skin tests, canker test, ear test, throat swab tests, ulcer tests, wound sample, and from the surgical samples. The gathered samples were handled in the microbiology lab by following the standard aseptic microbiological procedure of streaking on Blood improved media at 37 °C for overnight incubation. The brooded detaches were distinguished and cleansed by performing Gram's staining and the necessary biochemical response tests, with the catalase and coagulase tests being the most recognizable proof test. (Muazzam et al., 2019; Gouse et al., 2017; Muazzam et al., 2020; Maqbul et al., 2020; Maqbul et al., 2020).

2.3 Antimicrobial vulnerability testing

The antimicrobial vulnerability test for the segregated clinical samples of MRSA against the standard antibiotics were done by utilizing the cutting edge fast e-test system where the confines were immunized on Mueller-Hinton agar independently & standard e-test strips for individual anti-infection agents inoculated, at 37 °C incubated for short-term to determine inhibition zone. The connection of the oval is perused as MIC whereas, the zone as the weakness of the antibiotic on-to the bacterium and results were organized for the translation. The customary ordinary standard anti-toxin measure strategies, for example, Kirby-Bauer circle dispersion strategy was utilized to notice the susceptibility of the clinical examples of MRSA standard plate ready from the Illicium verum essential oil, where the bacterium disengages were immunized independently on Mueller-Hinton agar plates alongside the impregnated circles at 37 °C for 24 h to notice the zone development deciding the affectability of the bacterium towards the plate. The outcomes were organized for the understanding. The MIC values alongside Minimum Bactericidal Concentration (MBC) values for Illicium verum towards the MRSA isolates was assessed by playing out the standard cylinder weakening strategy where the samples were inoculated independently in the various concentrations and incubated at 37 °C for 24 h. The outcomes were organized for the translation. The MBC was dictated by immunizing every weakening of MIC weakenings on different plates of agar for every disengages and weakening independently. The vaccinated were brooded at 37 °C for 24 h to notice the no development deciding the affectability of the bacterium. The outcomes were arranged for the understanding (Muazzam et al., 2019; Muazzam et al., 2020; Maqbul et al., 2020).

3.1 GC–MS analysis

Fifty compounds were confirmed by searching NIST-EPA-NIH Mass Spectral Library (NIST 17) in ChemData. NIST. GOV (Fig. 1). These included Anethole, Eucalyptol, D-Limonene, Linalool, Squalene, Sitosterol, Piperine, etc. The volatile components like anethole, levomenthol, eucalyptol and estragole responsible for the characteristic aroma of star anise essential oil were also identified. The GCMS analysis results for star anise essential oil corroborate with previous reports (Zhang et al., 2015; Cu et al., 1990).

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Fig. 1

GC–MS chromatogram of Illicium verum essential oil.

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The IR spectrum (Fig. 2) shows peaks at 3022 cm⁻¹ indicate presence of aromatic benzene, peaks at 1607 to 1509 cm⁻¹ indicates framework of benzene ring and C-O-C stretching vibration at 1305 cm⁻¹.

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Fig. 2

IR spectra of Illicium verum essential oil.

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The investigation was likewise directed to discover the potential phyto constituents that might be responsible for the antibacterial activity of Illicium verum essential oil. The phytochemical investigation revealed the presence of alkaloids, steroids, proteins, phenols, glycosides, cardenolides and amino acids (Table 1). Thus, the potent antimicrobial activity exhibited by Illicium verum EO can be attributed to the presence of phenolics and flavonoid content. Notably, an earlier study has proven that star anise ethanol fraction is higher in antioxidant activity compared to the non-polar petroleum ether fraction, which is due to the solubility of phenolic compounds in ethanol (Padmashree et al., 2007).

Medicinal plants have long been recognized as a source of compounds with medicinal promise, and they continue to be a valuable resource for finding new drug leads today, to treat several diseased conditions including nosocomial infections. MRSA and A. baumannii infections pose a major threat due to the rise of resistance to a variety of antibiotics. Because of their capacity to build biofilm on abiotic surfaces in hospitals, A. baumannii and MRSA have been linked to a number of nosocomial illnesses (Salem et al., 2021). They have been found to be resistant to a wide range of antimicrobials, prompting the World Health Organization (WHO) to identify them as one of the high priority microorganisms for the discovery and development of modern antimicrobials (Govindaraj Vaithinathan and Vanitha, 2018).

In the current study, Illicium verum essential oil showed encouraging antibacterial results (Table 2) against all the clinical isolates with a normal plate dissemination of 24.05 mm zone of inhibition with a MIC 0.70 & MBC of 0.97 µg/mL. The good clinical isolate MRSA vulnerability was seen in swab nasal test with inhibition zone of 28 mm with MIC of 0.25 µg/mL and MBC of 0.5 µg/mL. The clinical isolate test of MRSA which showed the least effectiveness was the surgical isolate with 18 mm zone of inhibition with MIC of 1 µ/mL and MBC of 1.25 µ/mL. However, these results were better compared to the standard synthetic antibiotics for similar isolates with the exception of vulnerability towards Vancomycin (Patra et al., 2020; De et al., 2002; Ohira et al., 2009; Maqbul et al., 2020). The other MRSA clinical isolates, for example, catheter test showed vulnerability towards the Illicium verum essential oil with zone width of 27 mm in circle dispersion strategy with MIC of 0.25 µg/mL and MBC of 0.5 µg/mL, while the urine test shown vulnerability towards the Illicium verum essential oil with zone width of 25 mm in plate dissemination technique with MIC of 0.25 µg/mL and MBC of 0.5 µg/mL, whereas, the crotch test shown vulnerability towards the Illicium verum essential oil with zone width of 24 mm in plate dissemination technique with MIC of 0.5 µg/mL and MBC of 0.75 µg/mL, and the skin tests, ear test, throat swab tests, ulcer tests, wound sample and surgical sample shown vulnerability towards the Illicium verum essential oil with zone breadth ranging from 18 mm to 23 mm in circle dispersion technique, with MIC of 0.5 µg/mL to 1 µg/ml and MBC of 0.75µg/mL to 1.25 µg/mL, which were better than that of the standard antibiotics results (Table 3 and Table 4). The vulnerability of MRSA clinical isolates towards the standard antibiotic Vancomycin showed consistent results, yet, relatively the antibacterial activity of Illicium verum essential oils towards the MRSA isolates showed all the more better and promising results (Table 2). The antibacterial activity against the clinical isolates displayed by Illicium verum essential oils was far more superior than other standard antibiotics employed in the assay (Chart 1). It was observed that majority of the standard antimicrobial agents with the exception of Vancomycin, showed conflicting vulnerability result, with only a few MRSA clinical isolates showing significant susceptibility against the standard antibiotics. We found that the impact of standard antibiotics towards the MRSA clinical segregates were restricted with higher MIC and MBC values going from 0.75 µg/mL up to 2.75 µg/mL. The higher MIC and MBC values deduce that, even as MRSA clinical isolates have shown vulnerability to standard antibiotics, it may still need a higher antibiotic dose to control the infection and/or completely eliminate the disease. However, using a higher dose of antibiotic is linked with antibiotic-associated adverse drug reactions (Tamma et al., 2017). Certain patient-related risk factors may increase the likelihood of infection as well as the morbidity and mortality associated with infection, notably mycoplasma pneumonia, pneumococcal pneumonia, urinary tract infections, influenza virus infections, and other infections caused by multi-drug resistant organisms.

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Chart 1

Comparative chart of MRSA isolate's susceptibility towards Illicium verum essential oil.

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The results from the current study showed that all the MRSA clinical isolates were susceptible to the tested extract (Illicium verum essential oil) with inhibition zone diameter ranging from 18 mm to 28 mm (Mean 24.05 mm). The MBC and MIC results confirmed that the tested extract was potent against the highly virulent MRSA clinical isolates with MBC and MIC of 0.97 µg/mL and 0.70 µg/mL, respectively, which was lower compared to MBC and MIC values of the standard antibiotics. The MIC and MBC values of the Illicium verum essential oil ran between 0.25 µg/mL upto1.25 µg/mL for all the MRSA clinical isolates, confirming that a lesser dose of the Illicium verum essential oil is adequate to hinder or annihilate the disease totally. Henceforth, the antibacterial rich Illicium verum essential oil showed much desired susceptibility results compared to the standard antibiotic agents towards the acquired MRSA clinical isolates (Muazzam et al., 2019; Muazzam et al., 2020; Maqbul et al., 2020).

Previous studies have demonstrated that plants rich in phenolic compounds possess potent antioxidant activity and therefore exhibited significant antibacterial activity (Shan et al., 2007). Even though the mechanism of the anti-bacterial potentials of polyphenols is not fully understood, there are several arguments that phenolic compounds can either affect the permeability of the cell membrane; induce intracellular enzymes hydrogen-binding and thus influence their proper function; produce permanent damage to the cytoplasmic membrane and cell contents coagulation, and/or loss of integrity of cellular membranes stiffness. Subsequently, this examination has shown that the phytochemicals present in the Illicium verum essential oil ends up being a powerful antibacterial components against the MRSA clinical isolates when contrasted with the standard antibiotics (Lenora et al., 2016; Maqbul et al., 2020; Maqbul et al., 2020).