Antibacterial And Antibiotics Resistance Modifying Activity of Oil Extract of Myristica Fragrans’s Mace (Aril) Against Multi-Antibiotic Resistant Phenotypes with A Comparative Study Against Cymbopogon Citratus

This study systematically inspects the potent antimicrobial properties inherent in Myristica fragrance (Javitri) and Cymbopogon citratus (Lemongrass) essential oils. The core point is the targeted action against multiantibiotic-resistant (MAR) bacteria, a critical concern amidst the rising tide of antibiotic resistance. Notably, herbal alternatives are gaining traction as promising solutions. The research involves methodical extraction and assessment of these oils against formidable MAR strains, involving Escherichia coli and Pseudomonas sp . and through GC-MS analysis, we were able to find out the phytochemicals responsible for outstanding antimicrobial property of essential oil extracted from the mace of Myristica fragrans (Javitri). A significant part of this investigation delves into the synergistic interplay of Myristica fragrans oil with antibiotics. Through our research, we intend to re-establish the application of traditional herbal components for the purpose of medication and also in synergism with ineffective antibiotics and therapeutic strategies to face the antibiotic resistance crisis.


Introduction
The expanding gene pool across various species signifies both evolutionary growth and potential threats, as the increase in genetic variations within species may lead to significant consequences, including the possible extinction of one or more species.While biodiversity is generally considered a positive symbol of evolution, the challenges associated with it could pose a threat to the stability of ecosystems.This intricate relationship between genomic diversity and ecological balance constitutes the broader perspective of micro and macrobiota dynamics (Flandroy et al., 2018).As humanity focuses on resolving issues related to macroorganisms such as animals, birds, and plants, it is crucial not to overlook the threats that microbiota pose to human health.Microbiota, encompassing a wide range of microorganisms, has the potential to pose a substantial risk to the global ecosystem.The continuous evolution and adaptation of microbiota make them increasingly resistant to the antimicrobial drugs currently available in the market (Dhama et al., 2014).The cornerstone of treating microbial infections, both bacterial and fungal, has traditionally been antibiotics.The discovery of antibiotics was initially celebrated within the medical community, with the anticipation that these drugs would lead to the eventual eradication of infectious diseases.However, the stark reality is the emergence and rapid proliferation of antibiotic resistance, which has become a severe and pervasive issue, particularly in developing nations.This predicament affects not only hospitals but also the general public, resulting in alarming mortality rates annually (Gavazzi et al., 2004).Multiantibiotic resistance microorganisms (MAR), also known as multidrug-resistant microorganisms (MDR), represent strains of bacteria and fungi that have developed resistance to multiple types of antibiotics.The mechanisms driving this resistance are diverse and can include genetic mutations or the acquisition of resistance genes from other bacteria.Importantly, antibiotic misuse has become a primary contributor to the emergence and dissemination of multi-drug resistance strains across various categories of bacteria (Fernández et al., 2016).A significant therapeutic challenge has arisen due to the widespread prevalence of ß-lactamase producers, such as E. coli, Pseudomonas sps., Klebsiella pneumoniae (K.pneumoniae), Haemophilus, and other pathogens commonly found in healthcare settings (Sanders et al.,1989).Hospitals are now home to several multidrug-resistant strains of E. coli and Pseudomonas species, which exhibit resistance to antibiotics such as Ceprofloxacin, Penicillin, and Aminopenicillins.These resistant strains are increasingly isolated not only from hospital-acquired infections but also from diseases contracted in the community (Acar et al., 1997).The challenge of antibiotic resistance necessitates the exploration of alternative medicinal agents derived from plants that exhibit efficiency against bacteria resistant to antibiotics.Such alternatives should not only be effective but also secure and affordable (Kebede et al., 2021).In response to the urgent need for innovative treatments for multidrug-resistant microbes, researchers are increasingly turning to herbal products.This shift is underscored by the growing evidence of the rapid global expansion of resistant clinical isolates.Certain plants have undergone successful evaluations for their direct antibacterial action and their ability to modulate the effects of antibiotics (Cheeseman et al., 2017).Essential oils (EOs), also known as phytochemicals, emerge as promising candidates in the quest for novel antimicrobial agents.In the historical context, the use of spices in India, prized for enhancing food flavour and preservation, can be attributed to the presence of essential oils in these plants (Sharma et al., 2023).The antimicrobial efficacy of various essential oils has been well-documented over many years.Notable examples include Syzygium aromaticum (clove), Trachyspermum ammi (ajwain), Myristica fragrance (nutmeg), Cinnamomum verum (dalchini), Foeniculum vulgare (saunf), Eucalyptus camaldulensis (Nilgiri), and Cymbopogon citratus (lemongrass) (Bajwa et al., 2020).These essential oils, derived from herbs, spices, and plants, present an opportunity to develop antifungal and antibacterial medications that can effectively combat MDR microorganisms.This can be achieved either by enhancing the potency of existing antimicrobial medications or by creating novel medications using various concentrations of essential oils (Chouhan et al., 2017).The focus of this study is to investigate the in vitro antimicrobial and antibiotic resistance-modifying activities of essential oils from Cymbopogon citratus and Myristica fragrance against multidrug-resistant bacteria and various fungal species isolated from bread.Myristica fragrance, an aromatic evergreen plant belonging to the family Myristicaceae, produces seeds known as nutmeg and arils known as mace.These components are utilized as spices in a variety of cuisines and hold significance in Asian Ayurvedic medicine.On the other hand, Cymbopogon citratus, commonly known as lemongrass, emits a tropical citrus aroma and possesses various therapeutic properties, including antiamoebic, antibacterial, antifungal, antiinflammatory, antimalarial, antimutagenic, and antioxidant activities (SE Atawodi et al., 2014).Previous research has demonstrated the efficacy of essential oils in inhibiting multi-resistant bacteria and Candida species.For example, essential oils from nutmeg leaves showed inhibitory activity against K. pneumoniae, Acinetobacter species, Enterobacter cloacae, and group A beta-haemolytic streptococcus, as well as Candida species.Similarly, lemongrass essential oil exhibited higher antibacterial activity than tetracycline against Streptococcus mutans and Staphylococcus epidermis.This study aims to expand on existing knowledge by investigating the antifungal and antibacterial activity of essential oils extracted from the dried mace of Myristica fragrance and the leaves of Cymbopogon citratus.The experimental extraction of essential oils will be coupled with an assessment of their antimicrobial potential against multidrug-resistant bacteria, including E. coli and Pseudomonas, as well as various fungal variants.In addition to assessing the antimicrobial properties of these essential oils, this study will conduct qualitative and semi-quantitative chemical characterization using GC-MS analysis.This analysis aims to elucidate the phytoconstituents present in the essential oils, providing valuable insights into their chemical composition and potential pharmacological properties.

MATERIALS AND METHOD: PLANT SAMPLE COLLECTION:
The dried mace of (Myristica fragrans) was procured from a local Ayurvedic store situated in Bilaspur city, within the Chhattisgarh state of India.Fresh samples of Lemongrass (Cymbopogon citratus) were meticulously gathered from 'The Department of Forestry' at Guru Ghasidas Vishwavidyalaya in Bilaspur, Chhattisgarh.This strategic acquisition and collection process ensured the authenticity and quality of the plant materials, laying a robust foundation for subsequent analyses and investigations in our research study.

EXTRACTION OF ESSENTIAL OIL:
The Myristica fragrans, weighing approximately 150 grams, underwent an initial rinsing with distilled water before being placed in a Clevenger apparatus.This apparatus facilitated a hydro-distillation process involving boiling, condensation, and evaporation.Simultaneously, fresh leaves of Cymbopogon citratus (approx.150 grams) were precisely cut into 2-4 cm pieces and introduced into the Clevenger apparatus.The distillation process, operating individually for each sample over 6-8 hours, involved initiating boiling at 60% temperature, followed by regulated heating at 25-30%.Essential oils from both samples were then collected in transparent glass tubes above the water level within the condenser tube.The extracted oils were stored at 4℃ for subsequent detailed analysis (Schmidt et al., 2020).After that, the concentrated essential oils were collected and transferred into their respective transparent vials and were stored at about 4°C until used for further experimental process.

Test organisms and isolation
The bacterial strains, specifically Escherichia coli (E.coli) and Pseudomonas sp., were acquired from the Microbiology laboratory located in the Department of Biotechnology, with the sole purpose of conducting in-depth scientific research and analysis.

Bacterial Culture
Bacterial specimens, such as E. coli and Pseudomonas sp., were intricately isolated from their respective cultures, employing aseptic techniques.These specimens were then introduced into freshly prepared Nutrient Agar Medium (NAM) using a sterile inoculating loop, with rigorous heat sterilization before each transfer to ensure a sterile environment.The application of the streak plate method facilitated an even distribution of bacterial samples on NAM plates.Subsequently, the inoculated plates underwent incubation in a BOD incubator set at 33ºC for 24-48 hours, fostering optimal conditions for bacterial growth before undergoing meticulous observation.

Selection of Modern antibiotics
Novel antibiotics such as Cefixime (CFM,5 mcg), Levofloxacin (LE,5 mcg), Ampicillin (AMP ,10 mcg), Rifampicin (RIF,5 mcg), Penicillin-G (P, 10 mcg), Gentamicin (GEN,10 mcg) were implemented for the identification of multidrug-resistance in the isolated bacterial strains according to (Polsfuss et al., 2012).Screening For Susceptibility of Bacteria to antibiotics: Through the implementation of disc diffusion method the susceptibility of bacterial strains against antibiotics (gentamicin, penicillin-G, rifampicin, levofloxacin, cefixime, and ampicillin) were evaluated, prior to it successful introduction of bacterial culture uniformly throughout the agar plate was taken into consideration through a spreader.After all the processes were complete, the petriplates were stored in an incubator for 2-3 days until the evaluation of the result This provided insights into antimicrobial efficacy, guiding potential therapeutic options for infections caused by these strains (McLain et al., 2016).

Screening for efficiency of essential oil against MAR bacteria
The antimicrobial susceptibility test followed the well diffusion assay protocol outlined by Magaldi et al. (2004).NAM plates were utilized, and 0.5 ml of bacterial culture was evenly spread on each plate using a sterilized micropipette and spreader.After 10-15 minutes, wells (6mm in diameter) were created with a sterilized cork borer.Different concentrations (25%, 50%, 75%, and 100%) of ethanol extracts from M. fragrans mace and C. citratus leaves were prepared and added (100µl) to respective wells.The plates were then incubated undisturbed in a BOD incubator at 36ºC for 24 hours for observing inhibition zones.

Synergistic Antimicrobial Effects of Essential Oil
The potential synergistic effects of oil extracts in combination with the antibiotic three different antibiotics cefixime (5 mcg), penicillin-G (10 units), and ampicillin (10 mcg) against two common pathogenic bacteria, E. coli and Pseudomonas sp.The disc method was utilized to evaluate the antibiotic resistance modifying activities of different concentrations of the oil extract and antibiotics.The discs of the above mentioned antibiotics were dissolved into some selectively different concentrations of the essential oil of Myristics fragrans during a comparative analysis of oil against the antibiotics mentioned above (50% oil and 75% oil), resulting in the demonstration of varying degrees of synergistic effects, suggesting the potential of this oil extracts to enhance the antibiotic efficacy.Two reading were considered for the checking the synergism of essential oil with the above mentioned antibiotics.

GC-MS ANALYSIS
The GC-MS analysis of the essential oil sample (Sample ID: ABJ-01) was conducted utilizing 1.00 mL injection volume with a dilution factor of 1.The analysis was performed on Vial #26, using the n-Alkanes method file.The analysis was executed with an AOC-20i+s instrument, and the method involved multiple rinses and specific settings for plunger and syringe speeds.The GC-2010 parameters included a column oven temperature of 40.0 °C, injection temperature of 250.00 °C, and a split injection mode.The temperature program involved ramping from 40.0 °C to 220.0 °C in 4.00 minutes, and further to 250.0 °C in 15.00 minutes.The GCMS-QP2010 Ultra parameters included an ion source temperature of 200.00 °C and interface temperature of 260.00 °C.The qualitative analysis report from the Central University of Punjab, Bathinda.The analysis involved scanning in the m/z range of 40.00 to 800.00 over 60 minutes, with a solvent cut time of 4.50 minutes.The detector gain mode was set to Relative, with a detector gain of 1.26 kV.The analysis provided insights into the composition of the essential oil.

RESULT Extraction yield of Essential oil
Extraction of essential oil of dried mace of Myristica fragrans and fresh leaves of Cymbopogon citratus were obtained and the total amount of oil extracted from 250g of M. fragrans was about 12 ml having colour appearance of transparent soft green hue.The 250g of Cymbopogon citratus oil extract was measured to be about 1.7 ml and the appearance was found to be of vibrant mustard yellow colour.

Synergistic effect of Essential Oil
In this study, the potential synergistic effects of essential oil extracts from Myristica fragrans (M.fragrans) was explored in combination with three antibiotics (cefexime, penicillin, and ampicillin) against multiantibiotic resistant strains of Escherichia coli and Pseudomonas sp.The disc method was employed to assess antibiotic resistance modification at different concentrations.The results revealed varying degrees of synergistic effects, demonstrating the capability of these oil extracts to enhance antibiotic efficacy.For M. fragrans oil extract combined with cefixime against E. coli, concentrations of 25% and 75% exhibited enhanced inhibitory effects yielding (7.75±0.3)and (11.2±0.3)zones of inhibition, respectively (Figure 3).However, at 100% oil concentration, the inhibitory activity decreased to (7.3±0.4)(Table 7).Similar synergistic effects were observed against Pseudomonas sp., with 25% concentrations resulting in cloudy zone of inhibition and and 75% oil resulting in (9.7±0.3),respectively.Notably, 100% oil concentration led to a decrease in the inhibitory zone to (7.2±0.3) and the antibacterial disc of CFM individually (without synergism) formed hazy/cloudy zone of inhibition, Pseudomonas (Table 8).Combining Myristica fragrans oil with penicillin against E. coli showed enhanced inhibitory effects, with concentrations of 25% and 75% resulting in zones of inhibition of (9.2±0.3) and (9.2±0.3)mm, respectively.However, at 100% oil concentration, the inhibitory zone decreased to (6.7±0.3) and the penicillin disc individually did yield any zone of inhibition of (7.2±0.3)(Table 9).Similar effects were observed against Pseudomonas sp., where 25% and 75% oil concentrations with synergism to the ampicillin disc led to zones of inhibition of (10.2±0.3) and (11±0), respectively (Figure 4), while 100% oil concentration resulted in a (7.2±0.3)mm inhibitory zone (Table 10).The synergistic effect of Myristica fragrans oil with ampicillin against E. coli showed increased antibacterial efficiency, with 25% and 75% oil concentrations resulting in zones of inhibition of (9.2±0.3)mm and (9.2±0.3)mm, respectively.However, at 100% oil concentration, the inhibitory zone decreased to (7±0) and the same synergism of oil extract of M. fragrans with ampicillin antibacterial disc (Table 11), in case of pseudomonas was observed to form a zone of inhibition (11.2±0.3) at 25% and (10.2±0.3) for 75%, respectively.Adding on to it, 100% oil resulted in the zone formation of (7.2±0.3) and a (8.2±0.3)(Figure 5) for the disc without synergism (Table 12).These findings suggest that the combination of essential oil extracts of Myristica fragrans with antibiotics has the potential to address antibiotic resistance in both E. coli and Pseudomonas sp., offering new avenues for therapeutic strategies.

PHYTO-CHEMICAL COMPOSITION OF Myristica fragrans
The GC-MS analysis outcomes provide an intricate portrayal of the composition of the oil sample, depicted in the chromatogram.Thirty-one distinct compounds were identified, each characterized by specific retention times (RT), peak heights, and areas (Figure 6 and 7).Terpinen-4-ol predominated, constituting 21.06% of the peak area.Other notable compounds included Methyleugenol (13.01%), 1,3-Benzodioxole, 4-methoxy-6-(2-propenyl)-(10.70%), and alpha-Pinene (8.97%).This diversity underscores the myriad bioactive compounds within the sample.Compounds like alpha-Pinene, beta-Pinene, and Terpinen-4-ol, recognized for antimicrobial and pest-repelling properties, bear agricultural significance.Additionally, compounds like Linalool and gamma-Terpinene, known for pleasant aromatic qualities, hold potential for applications in fragrance and cosmetics.The intricate GC-MS analysis underscores the complexity and richness of the sample, presenting opportunities for diverse applications, including natural product formulation, agrochemical development, and the exploration of novel bioactive compounds for various industrial purposes.Subsequent research and validation studies are imperative to effectively harness the potential of these compounds.

DISCUSSION
Our comprehensive investigation focused on assessing the antibacterial properties of essential oils derived from dried mace of M. fragrans and C. citratus.The bacterial strains under scrutiny displayed multidrug resistance, exhibiting resilience against commonly prescribed antibiotics such as penicillin, CFM, and AMP, as confirmed through rigorous antibiotic susceptibility testing.
The well diffusion assay was meticulously conducted to validate the antibacterial efficacy of M. fragrans essential oil.Significantly, this oil demonstrated pronounced effectiveness, particularly against E. coli and Pseudomonas, as further confirmed by a meticulous disc diffusion setup.Conversely, the essential oil from C. citratus did not yield satisfactory results in the well diffusion assay, leading to the decision to forego further antimicrobial testing for this specific oil.To explore the potential synergistic effects of the essential oil from M. fragrans with antibiotic drugs, various concentrations of antibiotics to which the bacteria were initially resistant were combined with the oil.Surprisingly, this combination yielded enhanced results, surpassing the efficacy of the oil or antibiotics used individually.Additionally, the essential oil showcased the ability to augment the activity of previously ineffective drugs against opportunistic bacterial strains.Furthermore, we conducted disc diffusion assays to evaluate the antifungal properties of M. fragrans essential oil against self-cultured fungal strains, namely BFG-01AJ and BGF-01AJ.The oil exhibited significant and effective results against these isolated fungal strains, underscoring its potential in combating antimicrobial resistance in both bacterial and fungal domains.In comparison to the study conducted by Nikolic et al. (2021), our findings regarding the antibacterial effects of M. fragrans oil deviated.Our results indicated potent effectiveness against E. coli and Pseudomonas, highlighting the inherent variability in essential oil effects on different bacterial strains.This variability emphasizes the importance of considering specific bacterial strains and their unique responses when evaluating the potential applications of essential oils in combating multidrug resistance.In our comprehensive investigation, we expanded the scope beyond the antibacterial and antifungal activities explored in the study to acknowledge the diverse antimicrobial properties found in essential oils from various plants.Tea tree oil (Melaleuca alternifolia), for instance, has been extensively studied for its potent antimicrobial effects against bacteria, fungi, and viruses (Carson et al., 2006).Eucalyptus oil, derived from Eucalyptus species, emerged as another essential oil with notable antimicrobial properties.Research indicates its effectiveness against various bacterial strains, including Staphylococcus aureus and Escherichia coli, with 1,8-cineole and alpha-pinene identified as key constituents responsible for its antimicrobial effects (Juergens et al., 2011).Furthermore, lavender oil (Lavandula angustifolia) showcased broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi.The antimicrobial prowess of lavender oil is attributed to major components like linalool and linalyl acetate (Cavanagh et al., 2002).These examples collectively underscore the diverse and potent antimicrobial activities present in essential oils from different plants, suggesting their broad potential applications in combating various microbial challenges.The multifaceted nature of essential oils offers a promising avenue for future research and applications in the field of antimicrobial therapy.

Conclusion
The experimented Javitri sample was found potent not only against MAR strains of bacterias i.e, E.coli and Pseudomonas sp. but also showed efficacy against cultured strains of Aspergillus strains.Phytochemicals present in the aril are confirms the reason behind the aroma and efficiency against microbes which were considered for the experiment.In addition to sabinene, the essential oil also exhibited notable concentrations of methyl eugenol, β-pinene, terpinen-4-ol, and α-pinene.These constituents contribute to the overall aromatic profile and therapeutic properties of the oil, enhancing its efficiency which was confirmed through the GC-MS.Observably the yield of the essential oil obtained through the hydrodistillation process was highly distinguishable in comparison with Cymbopogon citritus and comparatively and, the latter showed less effective result against bacterias and self cultured fungal strains.Variety of healthcare products and hygene utilities can be produced using the crude oil extracts of M. fragrans and through this research we can conclude its usage in formulating drugs in different pharmaceutical industries.

Figure 1 -
Figure 1-Multiantibiotic resistance along with antibiotic susceptibility test of (A) E. coli and (B)Pseudomonas sp.

Figure 5 .
Figure 5. Synergistic effect of Ampicillin along with oil against (A) E. coli (B) Pseudomonas sp.

Figure 7 .
Figure 7.-Tabular representation of the data output through GC-MS analysis.