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Antibacterial potential of indigenous plant extracts against multidrug-resistant bacterial strains isolated from New Delhi region

The extensive use of antibiotics to treat bacterial infections has led to the widespread emergence of multidrug-resistant (MDR) pathogens, becoming increasingly difficult to treat with currently available antibacterial agents. The present study is based on prospecting the ethnomedicinal potential of Indian plant varieties for the treatment of MDR bacteria. Plants produce an array of diverse pharmacological compounds in defence against microbial pathogens which may be employed as a novel intervention strategy to combat MDR human pathogens. In the present study, the antimicrobial activity of extracts of four common Indian plants: Azadirachta indica (Neem), Murraya koenigii (Kadipatta), Phyllanthus emblica (Amla), and Ocimum sanctum (Tulsi) prepared in four solvents, water, methanol, ethanol, and chloroform was tested against nine MDR bacterial isolates. Kirby-Bauer well diffusion assays were adopted to assess the antimicrobial activity of plant extracts against the MDR strains. The potency of plant extracts was examined by determining the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). All MDR isolates including Staphylococcus haemolyticus, Bacillus subtilis, B. thuringiensis, B. cereus, Enterobacter xiangfangensis, Klebsiella pneumoniae, and Pseudomonas aeruginosa were significantly inhibited by the plant extracts. Test extracts showed promising antibacterial potential against MDR P. aeruginosa and Bacillus sp. with low MIC values ranging between 0.021.56 mg/ml, while most plant extracts exhibited either moderate MBC values or bacteriostatic effects. To the best of our knowledge, this is the first study that demonstrates the potential use of endemic A. indica, M. koenigii, P. emblica, and O. sanctum as therapeutic agents against circulating MDR human pathogens in the national capital.


Introduction
Antibiotics are small antimicrobial drugs administered to cure bacterial infections in patients. They revolutionized medical science in the 20th century and emerged as wonder drugs, especially prolonging life expectancy and decreasing mortality due to microbial infections. However, their rampant and irrational use has led to dissemination in environmental ecosystems escalating the problem of Antimicrobial Resistance (AMR) [1,2]. The emergence of AMR is a natural evolutionary process used by microorganisms in response to selective pressure against antimicrobial compounds in their surroundings [3]. Sir Alexander Fleming spoke about the emergence of AMR in his Nobel lecture. AMR to Penicillin was first reported in several strains of Staphylococcus aureus [4]. With the evolution of "superbugs" such as 'ESKAPE' pathogens: Enterococcus sp., S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter sp., AMR has now manifested in the form of a global health crisis. The problem of AMR has become more serious today due to the lack of newer antimicrobial scaffolds [1]. India has been called the "AMR capital of the world" due to the rise of MDR pathogens along with indiscriminate consumption of antibiotics [5]. It is estimated that by 2050, the world economy might have to face an additional cumulative burden of US$100 trillion and 10 million deaths a year because of AMR, which at present is claiming as many as 35,000 lives annually infecting more than 2.8 million people in USA alone [6]. We are entering the post-antibiotic era where common infections and minor injuries can be fatal. Realizing this, the United Nations General Assembly at New York (2016) conducted a high-level meeting to address the AMR issue [7]. In this scenario, there is a global need to look for alternative treatment strategies against the rising burden of AMR. To the rescue, In recent times, medicinal plants and herbal products have regained prominence amongst researchers and the scientific community.
The origin of Ayurveda, Siddha, and Unani, the ancient healthcare systems of Indian medicine can be traced back to the "Vedas" which are now being revived and modernized [8]. The antimicrobial properties of plants have found favor worldwide due to their abundant natural availability, pharmacological properties, and negligible cytotoxicity as compared to antibiotics [9]. Research on traditional medicines has proven to be a cynosure for pharmaceutical industries as plants are rich repository of secondary metabolites such as alkaloids, flavonoids, terpenoids, tannins, saponins, and polyphenols which have been shown to resist pathogens, and boost immunity [10]. The anti-malarial drug Artemisinin obtained from Artemesia annua has been used in traditional Chinese medicine for over two thousand years [11]. Other popular examples include Morphine, obtained from Papaver somniferum, Tiotropium from Atropa belladonna, Dronabinol and Cannabidiol from Cannabis sativa, Quinine and Quinidine from Cinchona sp., etc. [12]. The numbers of bioactive molecules derived from medicinal plants have been estimated to be more than 200,000, which still accounts for only a fraction of all the compounds produced by plant species [13]. Many plants have been found to cure respiratory diseases, urinary tract infections, cutaneous ailments and, even gastrointestinal disorders [14,15]. Therefore, the problem of AMR may be tackled using plant bioactives as traditional remedies based on Ayurveda.   [27] Oleanolic acid, Rosmarinic acid, Ursolic acid, Eugenol, Linalool, Carvacrol, β-elemene, βcaryophyllene, Germacrene [28] Considering the rich reserves of flora and medicinal plants in developing countries like India, their bioactive phytochemicals can act as game-changers in controlling the national medical burden. Among the thousands of Indian medicinal plants, four were selected for this study against 9 MDR bacterial isolates on the basis of their therapeutic properties and traditional use as given in Table 1. Previous studies have highlighted the prevalence of various antibioticresistant bacteria in numerous water sources such as wastewater [16], hospital wastewater [17], surface and groundwater [18], etc. which may eventually find their way into drinking water [19]. Therefore, this study aims to investigate the antimicrobial activities of 4 Indian plant varieties in different solvent formulations against 9 MDR human pathogenic bacterial strains isolated in a previous study from different sources across Delhi, India.

Bacterial cultures
Nine pre-isolated bacterial strains from our previous study were employed for the study [19]. Their antibiotic resistance profiles have been listed in Table 2. Bacterial cultures were grown till mid-log phase (OD600: 0.8-1.0); mixed with 40% glycerol and frozen at -20°C till further use. Cultures were revived in Mueller-Hinton broth (HiMedia Labs, Mumbai, India) and maintained on LB agar, unless indicated otherwise. Table 2 Gram characteristics and resistance profiles of the bacterial strains used in the study. *Antibiotics: Ampicillin (AMP); Cefuroxime (CXM); Nitrofurantoin (NIT); Co-trimoxazole (COT); Meropenem (MRP).

Preparation of plant extracts
Plant parts were collected from locations of in New Delhi, India and verified at the Department of Botany, University of Delhi with voucher numbers (Table 1). Plant parts were washed with distilled water, air-dried in shade at room temperature (RT) for 7 days and stored at 4°C until further use. Plant extracts were prepared as described previously with minor modifications [29]. 100 g of dried plant material was subjected to Soxhlet extraction for 4 days using the following solvents (400ml): chloroform, ethanol, methanol, and water. The extracts were filtered using Whatman filter paper No.1 and concentrated by drying at RT or using a hot-plate magnetic stirrer. The plant concentrates were stored at 4°C. For further experiments, plant extracts were prepared in 10% DMSO.

Evaluating the antimicrobial activity of plant extracts
Well-diffusion assays were performed by Kirby-Bauer method for primary screening of plant extracts against the 9 MDR strains [30]. Bacterial cultures with turbidity comparable to 0.5 McFarland Standard were swabbed onto MH agar plates and 6 wells of diameter 7 mm each were punched, 4 of which were loaded with 50μl of appropriate concentrations (25 mg/ml, 50 mg/ml, 75 mg/ml, 100 mg/ml) of plant extract. One well was loaded with 50μl of 50% solvent (Control 1) and the final one with 50μl of 10% DMSO (Control 2). Following incubation at 37°C for 18 h; zones of inhibition were measured and normalized to those of corresponding controls. All extracts were tested in triplicates to obtain statistically significant results.

Determining the MIC and MBC of plant extracts
The MICs of plant extracts were determined using 96-well microtiter plate-based cell viability assay for 3 selected MDR bacterial strains using previously established protocols [31]. These strains were selected based on their resistance profiles and prevalence rates in human infections. 100μl of double-strength MH broth was added to all the wells and 100μl of 2X plant extract (50 mg/ml) was dispensed in the first well. 1:1 serial dilutions of plant extracts were prepared by transferring 100μl suspension from the previous well and to the next well. This was repeated serially until desired lowest dilutions of plant extracts were achieved. 10μl of standardized bacterial inoculum was added to each well, barring the negative controls. Wells with only MH broth and MH broth-containing plant extracts served as negative controls, while those with inoculated MH broth lacking plant extracts served as positive growth controls. The plates were incubated for 18 h at 37°C. 20μl of p-INT solution (0.2 mg/ml stock) was added to each well and incubated at 37°C for 30 min. MIC was regarded as the lowest sample concentration which failed to produce a visible color change from colorless to pink after the addition of p-INT indicating complete inhibition of microbial growth. For the evaluation of MBCs, cell suspensions containing plant extracts that failed to show a color change in the MIC assays were dispensed in fresh MH broth and appropriate dilutions were plated onto LB agar and incubated at 37℃ for 24 h. MBC was defined as the minimum concentration of the plant extract that completely abolish inhibited colony formation in the candidate bacterial strains.

Statistical analysis
All assays were performed in triplicates, and the mean values ± standard deviations were calculated. Experiments were conducted in three independent events and a representative set of data has been reported.

Plant extracts inhibit the growth of MDR strains.
Previous reports have affirmed the antimicrobial efficacy of medicinal plant extracts against human MDR pathogens [32][33][34]. In view of the rapid global spread of MDR bacteria, there is a pressing requirement to discover new antimicrobial agents. Identifying the antimicrobial action of indigenous plant extracts and plant secondary metabolites is important for exploring their potential as natural antimicrobial agents [30] and alternative therapeutic agents [35,36] which can be used alone or in combination with antibiotics [37,38]. To extend these findings, we assessed the antimicrobial properties of plant extracts obtained from endemic Neem, Amla, Tulsi, and Kadipatta leaves using various solvents against 9 MDR bacterial strains. The resistance profile of MDR strains procured was confirmed and found to show resistance against 2nd generation antibiotics including Cefuroxime, Co-trimoxazole, Nitrofurantoin, and Ampicillin. Zone of inhibition diameters for plant extracts varied from no inhibition (0 mm) to 15.67 mm. From the well diffusion assays, it was evident that aqueous plant extracts significantly inhibited the growth across all MDR genera. Tulsi, Amla, and Kadipatta extracts proved to be more promising in terms of antimicrobial activity as compared to Neem extracts. Considering the notorious nature of P. aeruginosa and its implications in nosocomial infections, the tested plant extracts can prove to be worthwhile in devising treatment strategies. R A representative set of images has ve been shown in Figure 1. Data obtained in the form of inhibition zones (maximum 15.67 mm) has been summarized in the form of heat maps in Figure 2. We found that all plant extracts exhibited promising antimicrobial activity against both g Gram-positive and g Gram-negative MDR pathogens.

Evaluating MIC of plant extracts against candidate MDR strains
The MIC of a compound serves as a pharmacological parameter for the evaluation of its antimicrobial potency. Several investigations have reported plant extracts to exhibit MIC values on a higher scale, raising concerns over their dosage and efficacy [39,40]. Therefore, in an attempt to resolve this, MIC of the plant extracts was determined using a rapid microdilution assay by examining the reduction of p-INT in a colorimetric assay. As per the results, our plant extracts indicated high efficacy with comparatively lower MIC values for three selected MDR isolates, B. thuringiensis, B. cereus, and P. aeruginosa than reported in previous studies [41]. The MIC values ranged from 0.02 mg/ml to 6.25 mg/ml in the current investigation for the mentioned extracts. Amla and Kadipatta extracts showed promisingly lower MIC values (0.02 mg/ml), indicating their prolific antimicrobial activities. The Amla fruits have been reported to exhibit antimicrobial activities owing to tannins: Emblicanin A and B [42]. The results of the present investigation are in consonance with previous studies which also reported E. officinalis to have a broad-spectrum antibacterial potential against B. subtilis, K. pneumoniae and P. aeruginosa [43,44]. Aqueous, ethanolic, and acetonic extracts of Amla and Neem have previously been shown to possess antimicrobial properties against Enterococcus faecalis, Candida albicans and Streptococcus mutans [45]. E. officinalis has also been reported to be more effective against Gram-positive than Gramnegative bacteria, owing to differences in structure and composition of the cell wall [24]. The leaves of M. koenigii contain numerous bioactives including murrayastine, murrayaline, pyrayafoline carbazole alkaloids, and glycosides [21]. These constituents have been demonstrated to exhibit antibacterial properties, even better than Amikacin and Neem is widely regarded for its therapeutic potential as it contains numerous bioactives including azadirachtin, nimbolinin, nimbin, nimbidin, nimbidol, salannin, and quercetin [48]. Alzohairy et al. reported antimicrobial activity of neem due to inhibition of microbial growth and cell wall development [24]. Our Neem aqueous formulations showed a lower MIC value of 190 µg/ml [0.09 mg/ml] against MDR P. aeruginosa as compared to an MIC value of 500 μg/ml reported by a previous study [49]. However, MIC values for its ethanolic extract were on the higher end (3.125-6.25 g/ml). Indian Tulsi varieties are known to possess antimicrobial phytochemicals like eugenol, cyclooctene, methyl eugenol, camphor, and β-caryophyllene [50]. Previous studies have shown Tulsi extracts to possess extremely high MIC values of 200 mg/ml against E. coli and S. aureus [51]. On the contrary, our Tulsi extracts inhibited the growth of notorious human pathogens at extremely low MIC values ranging between 0.09 mg/ml and 0.78 mg/ml. As is evident from Figure 3, the order of potency was Kadipatta, Tulsi, Amla, and Neem. Kadipatta extracts were most potent at low concentrations, exhibiting low MIC values (mean 0.045 mg/ml) against the 3 test strains. While Amla and Tulsi extracts showed MIC values in the moderate range averaging 1.56 and 0.39 mg/ml. This accounts for the prolific antimicrobial activities of these endemic plant extracts against the commonly circulating MDR pathogens in the Delhi region.

Plant extracts exhibit both bacteriostatic and bactericidal properties
The Minimum Bactericidal concentration (MBC) of the extracts was determined by appropriately diluting the cell suspensions from plant extract containing-wells which did not show any growth after incubation during MIC assays in fresh broth. The test MBC values ranged from 0.045 to 25 mg/ml, while extracts for which MBCs could not be estimated in the tested range appeared to be bacteriostatic in nature. Kadipatta extracts exhibited strong bactericidal activity against all the test strains at extremely low concentrations ranging between 0.09 mg/ml and 1.56 mg/ml. While Amla and Tulsi extracts did not exert killing effects but rather exhibited bacteriostatic properties. Neem extracts on the other hand showed both bacteriostatic as well as bactericidal effects with varying solvents. Chloformic and ethanolic extracts of Neem showed bactericidal properties against B. cereus and B. thuringiensis within the range of 0.78-6.25 mg/ml. We observed a significant variation in the antibacterial potential of plant extracts prepared in water, chloroform, methanol, and ethanol. The differences in the bactericidal properties of plant extracts can be accounted for the use of different solvents. Solvents with different polarities including water, ethanol, methanol, cyclohexane, and chloroform have been reported to exert varying antibacterial effects [52]. Salem et al. also reported a difference in antibacterial activities of plant extracts with the solvents used for extraction [53]. Our results indicate that aqueous, chloroformic, and methanolic plant extracts possess strong antimicrobial activity corresponding to lower MIC and MBC values against the MDR strains tested (Table 3)]. However, ethanolic extracts exhibited moderate or higher MIC and MBC values as compared to other solvents. Generally, organic solvents have been shown to exhibit greater antimicrobial potential due to greater solubility of phytoconstituents. In a recent study by Samman et al., aqueous and methanolic extracts of Amla fruits have been shown to exert varying antibacterial activity against pathogenic bacteria [54]. Raghu and Ravindra reported that the MICs exhibited by methanolic extract of Amla against the tested organisms ranged between 0.261-0.342 mg/ml and were more potent than chloroform and diethyl ether extracts [55]. These justify the varying bactericidal and bacteriostatic potential of the plant extracts tested. Our results are also in agreement with Fatima et al. showing prolific antibacterial potential of Neem against UTI pathogens like S. aureus, E. coli, and Pseudomonas sp. with ethanolic extract [23]. At a glance, our findings reinforce the potential of natural plant extracts as effective therapeutic formulations against MDR bacteria.

Conclusion
In summary, the ever-evolving threat of AMR is at an unprecedented juncture today. The presently available drugs have become ineffective and compromised our fight against MDR pathogens. To the rescue, p Plant extracts and their bioactive phytochemicals have shined offer a ray of hope as emerging antimicrobials to combat the deadly bacterial manifestations. This study affirmatively establishes the affirmative role antimicrobial potential of Indian medicinal plants: Amla, Neem, Tulsi, and Kadipatta harboring extensive antimicrobial activity. Amla, Tulsi, and Kadipatta extracts demonstrated prolific antibacterial activities even at low doses, indicative of their high potency and therapeutic efficacy. Neem extracts also exhibited significant antimicrobial responses, but at comparably high doses. Kadipatta extracts exhibited remarkable bactericidal properties, while Amla, Neem, and Tulsi extracts were bacteriostatic in nature.
Considering the common usage of these plants among the Indian population, their phytoconstituents can be repurposed and exploited to serve as "life-saving elixirs". A better understanding in this direction through systematic modifications and medicinal chemistry can be undertaken for lead identification to develop novel intervention strategies against emerging microbial pathogens and superbugs. As a future medicine and alternative to antibiotics, this can help in reducing the incidence of AMR with improved quality of patient life. Overall, this can reduce the global medical burden and provide substantial societal and economic returns, essential to improve life today and for future generations.

Compliance with ethical standards
We also thank Dr. Rakesh Kumar Gupta and Dr. Kusum Gupta, faculty at the Department of Microbiology for their support in this work.

Disclosure of conflict of interest
The authors declare no conflict of interest.