Abstract: Background and Objective: Antibiotic resistance is becoming a critical concern for public health that has accelerated the search for new antimicrobial compounds from the natural resources. This study aim to evaluate the antibacterial activities of plant extract (Aloe Vera and Garlic) on multi-drug resistant (MDR) bacteria isolated from polluted water samples of clinical importance by molecular docking and its respective relationship corresponding to the multi-drug efflux pump PBD ID 5YIL protein of E. coli. Methodology: Aloe vera extracted with methanol PBS shown maximum inhibition (28 mm) against MDR strain of Escherichia coli using agar well diffusion. The natural compounds of plant extract responsible for antibacterial activity were screened on the basis of using computational approaches such as molecular docking based validation analysis. The 22 compounds were selected as legend and multi-drug efflux pump (MDEP) transport protein (5 YIL) of E. coli which plays an important role in actively transport of many antibiotics out of the cell was selected as receptors for molecular docking analysis. Results: In silico analysis showed that Aloin compound has strong binding affinity for selected target protein receptors. Hence, results of study showed the ability of Aloin to bind with active sites of MDEP protein targets to disrupt the mechanism of MDR in bacterial cell and allow the drug towards the treatment of diseases. Conclusion: In silico and in vitro methods help us to explore and investigate anti-MDR properties of selected medicinal plants extracts and their overall 22 phytoconstituents.
INTRODUCTION
The launch of antibiotics for the treatment of infectious diseases was the hallmarks of the 20th century medicine. However, shortly after their introduction into the clinical practice, the first bacteria showing antibiotic resistance were also found1,2. Since then, the development of new antibiotics has been accompanied due to the steady increase of antibiotic-resistant bacterial strains and the diversity of mechanisms used by bacteria to surpass the lethal effect of these compounds. Antibiotics are used widely on a day to day basis to control the microbial growth. Thus, it becomes quite alarming to raise the effect of its persistence in the environment3. Not only overuse but exposure to sub-lethal concentrations of antibiotics associated with development of antibacterial resistance4. In the present deadly scenario of this phenomenon, approximately 30% deaths were accompanied with bacterial infection in this era5. Drug resistant infectious microbes have become an important public health concern warranting organizations in public and private sectors worldwide to work together6.
Mainly, antibiotics producing microbes interrupt the microbial metabolism by various mechanisms7. Over the period, synthetic derivatives of naturally produced antibiotics were overused and fueled the adaptation in bacteria to become drug resistant8. It has been seen that not only antibiotics but biocides and dyes commonly used in hospital and laboratory are also responsible for over expression of efflux pump system of bacteria and make it resistant to antibiotics9,10. Bacteria have developed a variety of efficient resistance mechanisms such as efflux pumps, secreted proteases and alterations of the bacterial cell surface composition against antimicrobial peptides (AMP)11,12. These AMP are related to resistance mechanisms, modification of xenobiotics or drug transport, stress response, porins, outer membrane proteins, transporters and secretion, cell wall-related proteins, lipoproteins and DNA or plasmid-related proteins13.
In recent years, various researchers are looking to discover new bioactive compounds from plant origin with the hope to control MDR micro-organisms. Aloe vera and garlic are already well proved for its anti-inflammatory, anti-microbial, anti-diabetic and immune-boosting properties and regularly used over the periods14. Aloe vera extract is boon against the hazardous effect of multidrug-resistance bacteria isolated from polluted water without any side effect like available anti-microbial chemical drugs15. Natural products remain the most propitious source of novel antibiotics16. Particular attention was paid to test the therapeutic potential of Aloe vera gel and garlic extract against MDR bacteria and majorly involved AcrAB efflux pump in resistance17. As the electronegative entrance is widely conserved in the TolC family, it may be a useful target for the development of inhibitors against multidrug-resistant pathogenic bacteria18.
The frequent cause of multidrug resistant in gram-negative bacteria is the overexpression of RND multidrug pump such as AcrB transporter19 which make a major threat for public health nowadays. In another side, there are very few compounds that are successfully crystallized with AcrB. So, in silico approaches are emerged as a boon to enlighten the interaction between the ligand and protein20. Molecular docking is one of the most frequently used methods for the prediction ability in structure-based drug designing with a substantial degree of accuracy, the confirmation of small-molecule ligands within the appropriate target binding site21. Molecular docking and structure-based drug design strategies accomplishes computer simulations and thus suggested direct and stable tip to tip interaction between the outer membrane channel TolC and the isolated docking domain of the multidrug RND efflux transporter AcrB22.
Hence, the present study was focused on the in silico and in vitro analysis of two medicinal plants (Aloe vera and garlic) extracts against selected MDR microbial strains. Docking study was performed to analyze and identify the interaction of an obtained bioactive compound of selected plants (served as ligands) with the MDR pathogens inhibitory targets of transporter proteins.
MATERIALS AND METHODS
Media and chemicals: Minimal broth and nutrient agar media (Hi-media, India; final pH 7.2±0.2 at 25°C), amoxicillin, ciprofloxacin ofloxacin, levofloxacin, azithromycin and cefixime of 500 mg procured from the department for testing of multidrug resistivity. All other chemicals were of highest purity (>99%) ethanol, methanol, DMSO, conc. H2SO4, chloroform, NaNO2, Molisch’s reagent, dilute ammonia solution, glacial acetic acid, ethanolic ferric chloride solution etc. The rest of the chemicals utilized in this study were of analytical reagent grade and were procured from reputed suppliers within India.
In silico molecular docking analysis: Molecular docking is popular for screening active compounds from medicinal plants for inhibition against pharmacological receptors23-25. To have a better understanding about the inhibitory mechanism as well as the mode of interactions of the phytochemical compounds of the crude extract, docking analysis was accomplished using the DruLito and MVD tools. Drug target pathway has been accomplished by the E. coli transporter protein AcrB to be targeted with plant bioactive compounds.
Selection and preparation of natural compounds: Compounds of aloe vera and garlic with reported medicinal properties are taken from zinc database (Table S1-S3)26,27. Molecular identification of the compounds also searched from Chebi for further detailed validation28. Toxicity and drug-likeness property of the selected compounds has been checked with the DruLiToon the basis of Lipinski rule (Table S4, S5)29. The compounds fit to Lipinski rule are further used as a ligand in sdf format for docking. In this study, the 22 selected constituent compounds having traditional health importance is summarized in tabulated form (Table S1).
Protein preparation: The MDR transporter protein AcrB of E. coli is selected as a target protein for the docking with the prepared natural compounds of medicinal plants. X-ray structure with 3 A resolution of the protein bacterial multidrug exporter AcrB PDB ID 5YIL19 retrieved from RCSB PDB. The protein is taken in pdb30. The downloaded pdb structure of the selected protein was viewed and prepared by removing water molecules and extra ligands by Pymol version 1.7.4.5. Pymol is software for molecular visualization and producing high quality of 3D images of biological molecules31.
Binding site prediction: For the identification of binding site, pre-edited protein 5YIL is uploaded in COACH meta server32. For the better selection of ligand and target BioLip database is used for high quality biologically relevant protein-ligand binding interaction33. The COACH result was downloaded and again protein was post-edited and prepared for docking.
Molecular docking: Initially, BioLip database was used for the confirmation about the protein function and ligand check and the minimum identified cavity in the AcrB transporter protein in hosting loop region for potential binding site with ligands was set for each ligand were docked with protein by setting up grid one by one after several docking runs. Visualization of results by analyzing drug interaction sites by using MVD. Prepared ligand and protein was docked with Molegro Virtual Docker (MVD) (Version 2013.6.0.0) due to its higher docking accuracy which handles all aspects of the docking process from preparation of the molecules to determination of the potential binding sites of the target protein and prediction of the binding modes of the ligands34-36. Finally, results of docking were compared for the better tendency of inhibition on the basis of binding affinity, MolDock score and re-ranking. The MolDock scoring function calculated by involved hydrogen bonding and charges during protein ligand interaction. The docking scoring function, E score defined the energy and re-rank-score provides an estimate for interactions.
Isolation of MDR bacterial cultures: The water sample was collected on the basis of hazardous properties of polluted water and analyzed using standard microbiological techniques for the isolation and identification of bacterial pathogens. From the initial minimal media, pure cultures was obtained and these obtained selective colonies were grown on nutrient agar plated and tested against different antibiotics namely, amoxicillin, ciprofloxacin, ofloxacin, levofloxacin, azithromycin and cefixime using agar well diffusion method at 37°C for 24 h. Their resistivity or sensitivity against used anti-biotics determines multidrug resistant properties of isolated pathogens. Further, isolates were characterized and analyzed based on its cultural, morphological, biochemical methods according to the guideline of the Bergey's manual of determinative bacteriology37. The bactericidal activities of plant extract were carried out against Gram-positive MDR Staphylococcus aureus (ATCC 25923), Gram-negative MDR Escherichia coli (ATCC 25922). All the standard bacterial strains were obtained from MTCC, Chandigarh and preserved culture extracted from MRD Life Sciences lab, Lucknow. Active cultures for experiment were prepared by transferring A600 = 1 OD cell of culture from the stock culture to eppendorf tubes which contained 1 mL of nutrient broth (NB).
| Table S1: | The 22 constituents of the 2 medicinal herbs |
| Table S2: | Physical representation of selected natural compounds from garlic |
| Table S3: | Physical representation of selected natural compounds from aloe vera |
| Table S4: | Drug likeness properties of selected natural compounds from garlic |
| Table S5: | Drug likeness properties of selected natural compounds from aloe vera |
Preparation of the plant extract
Aloe vera extract: The fresh leaves of THE aloe vera were collected and rinsed with sterile distilled water and cut at base using sterile knife. Aloe vera gel was scooped out from fresh leaves with removed spikes without the fibers and dried in shade at the 32°C. Thereafter, the gel was extracted with a sample to solution ratio of 1:10 using ethanol (70%) and methanol (80%) at room temperature for 72 h with occasional stirring. The extracted solvent were filtered and dried in an oven at the temperature of 40°C. The aliquots were mixed with 5 mL DMSO and 5 mL PBS separately for methanol and ethanol extracted solvent, respectively. Finally, the prepared four types (Ethanol PBS, Ethanol DMSO, Methanol PBS and Methanol DMSO) of aloe vera plant extract were stored at 0°C for further experiments38.
Garlic extract: Whole garlic plant extract was prepared same as the above procedure38-40.
Anti-bacterial activity assay: Four bacterial pathogens were used to evaluate the antimicrobial properties, including strains isolated from polluted water and tested against different antibiotics and showed multidrug resistant properties. The antibacterial assays were performed by the agar well diffusion method and broth microdilution method41,42. Bioactive compounds of plant extracts were investigated by the agar well diffusion method. Nutrient agar plates were prepared and overnight grown on each wild-type and MDR cultures (15 μL) and were plated by spread plate technique. The uniform diameter (0.5 cm) wells were created in the nutrient-agar plates with a sterile borer. The plant extracts was poured at different concentrations into each of the wells using an auto-pipette. Thereafter, the plates were incubated at 37±1°C for 28 h to observe the bacterial growth and zone of clearance. Microbial inhibition zone was visualized and measured the diameter of the zones surrounding the well and recorded in millimeter. All tests were recorded in triplicates for each combination of plant extracts and microbial strains.
Determination of minimum inhibitory concentration: Minimum inhibitory concentrations (MIC) of different plant extracts were performed against various MDR strains using microbroth dilution method according to the approved standard M7-A09 as recommended by the clinical and laboratory standards institute. The MIC test was done for aloe vera plant extract (in Ethanol PBS solution) against E. coli wild-type strain. Second MIC test against PA MDR strain (in Ethanol DMSO and ethanol PBS plant extract solution) and third, against GC MDR bacterial strain (in Aloe vera ethanol PBS, methanol PBS plant extract solution). For MIC to be performed each test tube was having 3 mL mixture of 2.8 mL nutrient broth (100 μL of bacterial culture) and 20 mg/20 μL to 4 mg/20 μL sets of test tubes having different plant extract dissolved in various solvents and maintained at the final volume for 3 mL by using normal saline. The MIC readings were taken after 24 h on the basis of the turbidity due to the growth of the bacteria43.
Phytochemical screening of aloe vera plant extracts: The extracts prepared were used for screening of phytochemicals and other biologically active compounds. Phytochemical analysis was carried out according to the standard methods44. The extracts were screened for the presence of terpenoids, flavonoids, carbohydrates and cardiac glycosides45-48.
Statistical analysis: The obtained results of the zone of inhibition were expressed as the mean value±standard error of the mean (SEM) for each group49.
RESULTS
In silico analysis
Molecular docking studies of natural compounds with the transporter protein: Molecular docking were carried out with each selected natural compounds of aloe vera and garlic at the specific sites of interaction predicted from MVD. For each compound, out of the many docking poses, only those which possess the highest MolDock score (Table 1) and relatively good hydrogen bond interaction were chosen. The best five docking result was seen in DiallylTrisulfide (Zinc_01633229), Allyl Sulfide (Zinc_01531083), Alliin (Zinc_01531038), Aloin (Zinc_04214775) and 2-Bromo-5-Hydroxy-4-Methoxy Benzoic Acid (Zinc_64033873) with the total complex binding energy in kcal/jule -63.1319, -51.1096, -79.1436, -103.831 and -66.7682, respectively.
| Table 1: | RMSD value and MolDock score of natural compounds of Aloe vera plant (A comparative analysis of selected natural compounds) |
Docking evaluation for the best docked natural compound: From the all 22 natural compounds showing better drug-likeness property after docking five compounds showed a good tendency to bind with MDR transporter protein to inhibit its expression. These top five best docked compounds were selected on the basis of predicted binding energy and other binding parameters like hydrogen bond interaction electrostatic interaction (Table 2) and binding energy (Table 3). The best docked natural compound Aloin interacted with protein 5YIL having binding energy -103.831 kcal/mol with active site Ala33, Ala39, Ala297, Ala299, Asn298, Gln34, Glu673, Gly296, Ile38, Ile671, Leu137, Leu293, Lys292, Phe136, Pro36, Ser133, Ser135, Thr37, Thr295, Thr329, Tyr35, Val32, Val333 is minimum and hence, can be useful to inhibit the expression of 5YIL.
Thus, in silico approach to be particular about the responsible natural compound for antimicrobial property against the targeted protein 5YIL present in Aloe vera and Garlic. On the basis of different parameters and their comparative analysis, Aloin zinc id 04214775 and ChEBI ID 73222 come up as a best potential drug with least binding energy.
Anti-MDR activity assay: It has been seen from the results that E. coli wild-type and P. aeruginosa MDR strain showed sensitivity to all type of Aloe vera plant extracts with both solvents and mediums. But the best result was seen for aloe vera methanol PBS with 28±1.15 zone of inhibition in mm±SEM against E. coli strain (Table 4). Also, better zones were obtained against S. aureus for aloe vera extracts. S. aureus wild-type and GC MDR (isolated from Gomti river in Lucknow) shown sensitivity only for aloe vera ethanol PBS and methanol PBS. Thus, all extracts of aloe vera showed promising zones against MDR strains with only PA MDR strain observed as least zones and Aloe vera DMSO extracts could not be able to inhibit the growth of S. aureus as an exception. Garlic extract shown very narrow zones against E. coli wild-type which could not be measured while no zone of inhibition were observed for other cultures like S. aureus, P. aeruginosa and MDR strains P. aeruginosa and GC. Also, none of the extracts showed any inhibition in growth of P. aeroginosa wild type culture. The PBS and methanol could be better medium and solvents in plant extract preparation. Aloe vera extract was unable to inhibit P. aeruginosa strain but could be able to inhibit some MDR strain. As garlic extract did not show any inhibition. It was observed that P. aeruginosa showed best resistance against 5 antibiotics azithromycin, cefixime, amoxicillin, ciprofloxacin and ofloxacin and GC against 4 antibiotics amoxicillin, ciprofloxacin, azithromycin and cefixime).
Throughout the study, samples from the polluted water were collected and grown in antibiotic rich media as a carbon source for their utilization and checked for their resistant and sensitivity against different antibiotics. Plant extracts which were prepared have an initial concentration of 1 g/5 mL for Aloe vera and 750 mg/5 mL for Garlic. In our study, it have checked the effect of plant extracts against these types of notorious opportunistic multidrug-resistant bacterial strains (P. aeruginosa strain isolated from pond water) showed the best resistance against 5 antibiotics azithromycin, cefixime, amoxicillin, ciprofloxacin and ofloxacin, GC from Gomti river Lucknow against 4 antibiotics amoxicillin, ciprofloxacin, azithromycin and cefixine. Thus further species identification was carried out by Gram staining and standard biochemical characterization tests. In this study, P. aeruginosa strain was Gram-positive and of Bacillus family and GC strain was Gram-positive and catalase negative. While E. coli and S. aureus of wild-type strains collected from the MRD lab. Aloe vera showed the best result against E. coli and obtained a maximum zone of inhibition.
| Table 2: | Hydrogen bond and electrostatic interactions of best docked natural compounds screened from the aloe vera plant |
| Table 3: | Docked amino acid residues and their respective binding energy and total binding energy of best docked natural compounds from aloe vera plant extract |
| Table 4: | Anti-bacterial activity assay of the aloe vera extract prepared in different solvents and their zone of inhibition against various bacterial strains |
MIC of aloe vera extract against selected strains: The MIC value of the plant extract against the MDR strains showing promising results and were determined for quantitative evaluation of anti-MDR potential. The MIC results for various extracts of Aloe vera (initial concentration starting from 1 g/5 mL and diluted at ranging from 20 mg/20 μL to 4 mg/20 μL) at 660 nm absorbance were recorded. The lowest MIC value was 0.4 mg μL–1 (Aloe vera extract in ethanol PBS) for E. coli whereas highest was 0.1 mg μL–1 (Aloe vera extract in methanol PBS) against GC MDR strain. Because no satisfactory result was seen by garlic plant extract, so MIC was done diluted at ranging from 20 mg/20 μL to 4 mg/20 μL at 660 nm absorbance only with all four type Aloe vera extract against E. coli wild-type strain and P. aeruginosa, GC MDR strains. In the study, garlic extract as found to have little or no activity against selected bacterial strains at all tested concentration. The better MIC result has been seen in ethanol extract than aqueous extract and methanol extract on E. coli.
| Fig. S1: | 5YIL Protein 3D Structure: 5YIL Function: AcrA-AcrB-AcrZ-TolC is a drug efflux protein complex with broad substrate specificity that uses the proton motive force to export substrates |
| Table 5: | Studies on the phytochemical profile of aloe vera and garlic extract prepared in different solvents |
Phytochemical studies: Preliminary screening of phytoconstituents from the leaves of aloe vera revealed that the extracts contain terpenoids, flavonoids, carbohydrates and cardiac glycosides. Both plant extract showed contains the various amount of phytoconstituents. The maximum amount were observed in Aloe vera methanol PBS for terpenoids, methanol DMSO for carbohydrate as well as cardiac glycoside (Table 5) and promising amount for carbohydrate in garlic ethanol PBS extract.
DISCUSSION
Ethno-botanical survey reveals that plants are important sources of potentially useful compounds for the development of chemotherapeutics agents. Most important aspects will be the in vitro antibacterial activity assay50. Many studies and reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants51. On the other hand, antibiotic resistance is a major clinical problem in treating infections caused by several pathogenic micro-organisms. The resistance to the antimicrobials has increased over the years and normal intestinal microbial flora became a reservoir for resistant genes52.
Throughout the study, there is urgency for the establishment of a new antibiotic intake policy that should be strictly followed by all the concerned authorities. Docking was performed of Efflux protein having PDB ID 5YIL (Fig. S1) the pocket domain in a complex of E. coli among the known inhibitors Diallyl Trisulfide, Allyl Sulfide, Alliin, Aloin and 2-Bromo-5-Hydroxy-4-Methoxy Benzoic Acid has shown quite good scores for PDB ID 5YIL of efflux protein of MDR E. coli. Thus, the natural compound Aloin (Fig. S2) can be a potent ligand for the drug target and in the future can be optimized to form improved drug for treatment of MDR caused diseases. Being an anthracene, it has a nuclease property. It also shows a cyclic ketone and c glycosyl property make it more prone to inhibit the target protein.
In general, Gram-negative bacteria are found to be more resistant towards antimicrobial agents as compared to Gram-positive bacteria. The reasons include covering of phospholipids membrane carrying the structural lipopolysaccharides components which are responsible to allow impermeability against these antimicrobial substances53.
| Fig. S2: | Aloin (Zinc Id04214775) used as a laxative and play important role in increasing prostaglanding synthesis. Na+, K(+)- adenosine triphosphatase (ATPase) Molwt 418 |
The lowest concentration of an antimicrobial agent that inhibits the visible growth of micro-organisms known as the MIC54. Because no satisfactory result was seen by garlic plant extract, this may be due to presence of some diverse sub-chemical types in the extracts55,56. Aloe vera extract were found to have significant amount of secondary metabolites such as terpenoids, flavonoids, carbohydrates, cardiac glycosides, alkaloids and phenolic compounds. Their presence might be responsible for antimicrobial activities of extracts55.
SIGNIFICANCE STATEMENT
Docking output of in silico data demonstrated various molecular interactions in different levels and showed that Aloin has more specificity towards the MDR protein targets and could be a potent antimicrobial compound. As the search for new antimicrobial agents intensifies, these plant extracts may provide attractive alternate sources of molecules for consideration.
ACKNOWLEDGMENTS
This work was supported in part by the DBT-Centre of Excellence in Department of Zoology and BCIL-MRD Life Sciences lab partnership programme. Authors acknowledge infrastructure support from the BCIL-DBT Partnership Programme. Authors thank Mr. Manoj Verma for technical help, Mr. Gaurav Rai for help in guidance and support. Also acknowledges BIFC Staff of D.G.P.G. College and MRD Lab.