Photocatalytic TiO₂/CdS/ZnS nanocomposite induces Bacillus subtilis cell death by disrupting its metabolism and membrane integrity

Abstract

Titanium dioxide (TiO₂) is widely characterized for its application in clinical diagnostics, therapeutics, cosmetics, nutrition, and environment management. Despite enormous potential, its dependence on ultraviolet (UV) light for photocatalytic activity limits its commercialization. Accordingly in the present study, a photo catalytically superior ternary complex of TiO₂ with Cadmium sulfide/Zinc sulfide (CdS/ZnS) has been synthesized, as well as, characterized for photo-induced antimicrobial activity. The band gap of crystalline TiO₂/CdS/ZnS nanocomposite has been reduced (2.26 eV) and nanocomposite has shown the optimal photo-activation at 590 nm. TiO₂ nanocomposite has significant bactericidal activity in visible light (P < 0.01). Exposure of the TiO₂ nanocomposite affected the cellular metabolism by altering the 1681 metabolic features (P < 0.001) culminating in poor cellular survivability. Additionally, photo-induced reactive oxygen species generation through nanocomposite disrupts the microbial cellular structure. The present study synthesized photocatalytic nanocomposite as well as unveiled the holistic cellular effect of theTiO₂/CdS/ZnS nanocomposite. Additionally, the present study also indicated the potential application of TiO₂/CdS/ZnS nanocomposite for sustainable environment management, therapeutics, and various industries.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-021-00973-z.

Introduction

In the past few decades, the biotechnological sectors are playing a significant role in sustainable development. Multidisciplinary approaches have been employed for efficient biocatalysts and biotransformation processes [1–7]. Engineering of bioprocess and biocatalysts are largely used to improve the production of biomolecules [8–11]. Particularly, the nanotechnology sector is gaining a vital role in developing nano-biocatalyst for various biotechnological applications due to their unique features [12–16]. Multiple materials, bioactive molecules, and microbes have been widely used as therapeutic, antimicrobials, or pathogen inhibition applications [17–22]. Due to higher selectivity and specificity, nanomaterials are more beneficial for antimicrobials' purposes than others [23, 24].

Titanium dioxide nanoparticles were characterized for their potential in a wide range of industrial applications [25]. Photo-excitation of TiO₂ nanoparticles generates reactive oxygen species that could potentially kill microbes and degrade organic pollutants, industrial dyes, etc. These properties augmented its application potential in the area of therapeutics and environment management [25]. TiO₂ has a bandgap between 3.0 and 3.2 eV that allows electron excitation only in the UV range of the electromagnetic spectrum. Limited visible light absorption does not allow its photo-activation to achieve the desired photo-catalytic properties, limiting its augmentation for industrial applications [26]. Efforts are being made for TiO₂ bandgap engineering through various extrinsic modifications. It includes doping with metal and non-metals, developing metal oxide nanoconjugates, etc. [27]. In this trend, our previous report showed TiO₂/CdS/ZnS photocatalyst with varied concentrations of CdS and ZnS that absorbs up to 590 nm and shifts the band from 3.18 eV in TiO₂ to 2.26 eV in TiO₂/CdS (20%)/ZnS (10%) [28]. This composition showed optimal photocatalytic activity for the degradation of an organic moiety. An optimal nanomaterial for industrial, environmental, and therapeutic applications should be stable, self-sustainable, inexpensive, and should possess bactericidal properties [23, 24]. The antimicrobial activity of TiO₂ nanoparticles has been reported in recent studies [29]. TiO₂ nanoparticles induce antimicrobial activity either by altering microbial cell physiology [29] or through Reactive Oxygen Species (ROS)-induced microbial cell rupture [30]. These studies were performed in isolation, and no significant effort was made to report the holistic mechanism of action of the nanoparticles as antimicrobial agents. The antimicrobial property of the photocatalytic nanocomposite needs to be assessed by exploring their effect on the microbial cell structure and metabolism [23, 24, 29, 30]. The current study was designed with two broader objectives: the synthesis of photocatalytic nanocomposite and the exploration of their holistic mechanism of antimicrobial action.

Materials and Methods

Materials

The reagents titanium isopropoxide, cadmium sulfate (3CdSO₄.8H₂O), thiourea, and zinc nitrate were supplied by Sigma Aldrich. The sodium sulfide was purchased from Central Drug House (CDH) Ltd (India). Analytical grade chemicals were used without purification for the future process.

Synthesis and Characterization of TiO₂/CdS/ZnS Nanocomposites

The synthesis of TiO₂/CdS/ZnS ternary nanocomposite was done using the hydrothermal approach described in our earlier report [28]. The crystallinity and phase of the nanocomposite were analyzed using Rigaku Ultima IV powder X-ray diffractometer in the 2θ range of 20° to 80°. UV–visible diffuse reflectance spectrophotometer (Shimadzu 3600 Plus) was used to determine the optical characteristics. A Field Emission Scanning Electron Microscope determined the morphology of the synthesized sample (FESEM) operated at 30 kV. Transmission Electron Microscope (TEM) (Tecnai G2 20) operated at 200 kV was used to determine the particle size of the nanocomposite.

Antimicrobial Activity

The antimicrobial activity of nanoparticles was assessed against Bacillus subtilis (MTCC No. 2057) with standard approaches like disc diffusion assay and Micro broth dilution assay (Supplementary method SM1). All the experiments were performed in biological triplicates.

Estimation of Reactive Oxygen Species and Lipid Peroxidation

TiO₂/CdS/ZnS nanocomposite induced cellular generation of reactive oxygen species (ROS) was analyzed [31]. To check the oxidative degradation of bacterial lipids due to nanoparticles, a lipid peroxidation assay was performed [31]. Tests were performed in triplicates.

Analysis of Microbial Cell Morphology

Field Emission Scanning Electron Microscopy (FESEM) of the samples was performed at the central instrumentation facility at NIMS, Jaipur, Rajasthan, India. Untreated microbial cells were used as a control. All the experiments were performed in biological triplicates.

Metabolomic Analysis

The effect of nanocomposite on cellular metabolism was assessed after analyzing whole-cell metabolites with Liquid Chromatography with tandem mass spectrometry (LC–MS) (Supplementary method SM2). LC/MS-based metabolic profiles generated with both negative and positive mode Electrospray Ionization (ESI) were analyzed with XCMS software running under R environment version 3.7.1 for metabolic feature detection, chromatographic matching, and metabolite mapping using Bacillus subtilis metabolomic profile as a reference library by following its default settings.

Statistical Analysis

All experiments were carried out in triplicate. Statistical tests were performed to calculate P-value, standard deviations, etc.

Results

Characterization of the Synthesized Samples

The X-ray diffraction (XRD) pattern of TiO₂/CdS/ZnS nanocomposite (TCZ-1) shows the major peaks at 2θ 25.38°, 37.86°, 48.15°, 54.13°, 55.21°, and 62.79° that correspond to the anatase phase of TiO₂ and are well-matched with the Joint Committee on Powder Diffraction Standards (JCPDS) card No. 21-127 (Fig. 1a). The peak corresponding to anatase TiO₂ did not show any shift indicating that CdS and ZnS do not affect the basic structure of the lattice. The peaks other than anatase TiO₂ at 2θ 26.53°, 28.53°, 43.91°, and 51.98° indicated the presence of cubic and hexagonal phase of CdS (JCPDS card no. 41-1049), and at 2θ 28.53°, 48.17°, and 56.4° indicated the presence of cubic ZnS(ICDD PDF 65-1691). The peaks corresponding to CdS at 2θ 28.21° and ZnS at 2θ 28.53° superimposed [28]. Debye–Scherrer equation was used to calculate the average crystallite size and found that the size of TiO₂ is about 10 nm while that of CdS and ZnS is 30–40 nm. The optical properties are also studied using a UV visible spectrophotometer (Fig. 1b). The absorbance of the compound over a range of wavelengths is shown (Fig. 1b). The band gap is calculated using the Kubelka–Munk function where reflectance (R) at any wavelength is given as

$$F\left(R\right)=\frac{{(1-R)}^2}{2R}$$

Fig. 1.

Characterization of the synthesized sample. TiO₂/CdS/ZnS nanocomposite (TCZ1) was characterized by observing its XRD pattern (a) and Kubelkamunk spectra (b), FESEM imaging (c), and TEM imaging (d)

The graph is plotted for indirect bandgap semiconductor materials by taking hν as energy along the x-axis and [F(R) hν]^1/2 along the y-axis. It is well known that pure TiO₂ shows absorption in the UV region but it is seen from the spectra that the synthesized compounds showed absorbance near 590 nm corresponding to a bandgap of 2.26 eV. The shift in absorption and lowering of the bandgap is due to the CdS and ZnS in the composite. The enhanced absorption towards the visible region assists the high activity of the composite under visible light exposure. The morphological characteristics were uncovered using FESEM and TEM techniques. The FESEM image of the TCZ-1 sample showed the formation of spherical nanoparticles in the ternary composites (Fig. 1c). The TEM image of the ternary composites shows well-dispersed and uniform particles without any aggregation (Fig. 1d). The particle size estimated from the TEM image is in the nano range and in good agreement with that calculated from the XRD data.

Antimicrobial Activity of the Nanoparticles

Disc diffusion assay indicates antibacterial property against Bacillus subtilis (Supplementary Fig. S1). Microbroth dilution assay showed good growth inhibition efficiency of TiO₂/CdS/ZnS towards Bacillus subtilis. The minimum inhibitory concentration for Bacillus subtilis was 0.1953 mg ml⁻¹ in light conditions and 3.125 mg ml⁻¹ in dark conditions. Time Kill kinetics assay also indicates an optimum antibacterial activity after incubating the nanoparticle mixture with microbes for 25 min (Supplementary Fig. S2). Bactericidal activity of the nanocomposite was significantly higher (P < 0.01) in presence of light as compared to dark conditions (Supplementary Fig. S3).

Mechanism of Antibacterial Activity of Nanocomposite

The average reactive oxygen species (ROS) concentration in nanocomposite treated cells was 471 folds higher than the untreated cells. Even a ~ 273 fold higher membrane lipid oxidation was observed in treated cells compared to untreated cells. Scanning electron micrographs showed adherence of nanocomposite to the microbial cell membrane (Fig. 2a). Changes in the cell morphology, as well as cellular raptures, could easily be observed in the sample treated with nanocomposite (Fig. 2a), while no such changes were observed within control samples (Fig. 2b). Compositional analysis indicates the abundance of nanocomposite elements in treated cells as well as a lower percentage of electrolytes (Supplementary Fig. S4). Nanocomposite-induced cellular changes could be easily monitored by observing the cellular metabolomic profile [32]. It would provide a holistic view of molecular changes induced by external agents [32]. Metabolomic profiling of the nanocomposite treated microbial cell was identified to have 1274 and 407 statistically significant features (P < 0.01) after analysis of the metabolic profile captured with positive and negative ESI mode (Fig. 3a and b).

Fig. 2.

Mechanism of antimicrobial action of TiO₂/CdS/ZnS nanocomposite. Nanocomposite induced changes in the cell morphology (a) in reference to the untreated microbial cells (b)

Fig. 3.

Metabolomic analysis identifies statistically significant features (P < 0.01) after analysis of the metabolic profile captured with positive (a) and negative ESI mode (b)

These differentially abundant metabolic features were mapped with metabolic pathways associated with nucleotide metabolism, amino acid metabolism, and membrane precursor synthesis (Tables 1, 2). The metabolic precursors for nucleotide biosynthesis (P ≤ 0.002), cell wall synthesis (P ≤ 0.002), protein synthesis (P ≤ 0.0019), lipid biosynthesis (P = 0.0019), pH resistance (P = 0.00039), and siderophore formation (P = 5.9e-7) were significantly reduced (Table 1). These results indicate that the introduction of TiO₂/CdS/ZnS nanocomposite arrested the bacterial cell growth by inhibiting electron transport system (by reducing the precursors that mediate the electron transfer by 9.7 folds), vital pathways for protein, lipid, and nucleotide biosynthesis (by reducing the precursor molecules such as Uridine triphosphate (UTP), Cytidine monophosphate (CMP), deoxythymidine triphosphate (dTTP), uracil, deoxyguanosine triphosphate (dGDP); tryptophan, L-methionine, and histidine biosynthesis), cell membrane disruption as well as suppression of cell wall synthesis (by inhibition of precursor molecules required for cell wall synthesis such as teichoic acid, Meso-diaminopimelate, CMP, UTP), destruction of nucleotides and intermediates such as folates that facilitate the methionine, purine, and pyrimidine biosynthesis. Nanocomposite treatment also significantly affected the osmoprotection (P = 0.00029) and pH-resistance (P = 0.00039) properties of the microbial cell. Even a significant reduction in the overall energy status of the cell due to a decrease in the energy molecules (P = 0.00039), lipid (P = 0.0019), and protein (P ≤ 0.0019) biosynthesis has been observed. Additionally, the iron-quenching capacity is also reduced due to suppression of siderophores that lead to iron deficiency within the bacterial cell that altogether reduces the energy status of the bacterial cell. These results indicate that nanocomposite induces a holistic effect on the cellular functions (Fig. 4), not only cellular damages as reported by previous studies [30].

Table 1.

Metabolic pathways and their associated metabolic features down regulated in the microbial cell after the exposure of the nanocomposite

ESI-Mode	Metabolites	Pathway(s) involved	Fold change	p-value	m/z	Retention time	Adduct
Positive	UTP	UDP-N-acetyl-D glucosamine biosynthesis I Anhydromuropeptides recycling	204.8	2.0e-3	506.9561	4.05	M + Na[1 +]
Negative	CDP-glycerol	Teichoic acid (poly-glycerol) biosynthesis	162.8	2.6e-4	460.0502	3.59	M-H2O + H[1 +]
Negative	dTTP	Pyrimidine deoxyribonucleotide phosphorylation	125.5	1.1e-3	504.9785	3.73	M + Na[1 +]
Positive	dGDP	Guanosinedeoxyribonucleotides de novo biosynthesis I	16.0	9.6e-4	410.0251	5.96	M-H2O + H[1 +
Negative	(2S,3S)-2,3-dihydroxy-2,3- dihydrobenzoate	2,3-dihydroxybenzoate biosynthesis	15.6	5.9e-7	140.0233	5.28	M-NH3 + H[1 +]
Positive	Succinate	4-aminobutanoate degradation II	9.7	3.9e-4	136.0610	5.41	M + NH3 + H[1
Positive	CMP	Teichoic acid (poly-glycerol) biosynthesis	7.6	1.1e-4	324.0582	5.46	M + H[1 +]
Negative	Indole	L-tryptophan biosynthesis	4.2	7.1e-4	135.0918	4.59	M + NH3 + H[1 +
Positive	Meso-diaminopimelate	UDP-N-acetylmuramoylpentapeptide biosynthesis I	3.9	1.9e-3	175.1069	5.29	M-H2O + H[1 +
Positive	Histidinal	L-histidine biosynthesis	3.2	7.7e-4	82.0391	2.01	M + H + Na[2 +]
Positive	Uracil	Pyrimidine deoxyribonucleosides degradation	2.0	1.4e-4	113.0343	1.68	M + H[1 +]
Positive	N-acetyl-L-glutamate 5- semialdehyde	L-arginine biosynthesis II (acetyl cycle) L-ornithine biosynthesis I	1.7	1.9e-3	191.1022	5.89	M + NH3 + H[1

Table 2.

Metabolic pathways and their associated metabolic features upregulated in the microbial cell after the exposure of the nanocomposite

ESI-Mode	Metabolites	Pathway(s) involved	Fold change	p-value	m/z	Retention time	Adduct
Positive	(1R,6R)-6-hydroxy-2- succinylcyclohexa-2,4- diene-1-carboxylate	1,4-dihydroxy-2-naphthoate biosynthesis	13.7	2.9e-4	263.0524	4.79	M + Na[1 +]
Positive	D-mannitol 1- phosphate	Mannitol degradation I	13.7	2.9e-4	263.0524	4.79	M + H[1 +]
Positive	Palmitate	Palmitate biosynthesis II	12.1	1.4e-5	239.2366	1.73	M-H2O + H[1 +
Positive	Guanosine	Guanine and guanosine salvage	5.9	7.5e-5	306.0810	2.01	M + Na[1 +]
Negative	L-valine	L-alanine biosynthesis I	4.4	5.5e-3	119.0942	4.41	M + H[1 +]
Negative	5-aminopentanoate	L-ornithine degradation II (Stickland reaction)	4.4	5.5e-3	119.0942	4.41	M + H[1 +]
Positive	Adenosine	Adenine and adenosine salvage I	3.2	9.6e-5	290.0854	2.15	M + Na[1 +]
Positive	2'-deoxyguanosine	Purine deoxyribonucleosides degradation I	3.2	9.6e-5	290.0854	2.15	M + Na[1 +]
Positive	4-amino-4- deoxychorismate	4-aminobenzoate biosynthesis	3.1	3.6e-3	249.0606	2.50	M + Na[1 +]
Negative	3'-phosphoadenylylsulfate	Sulfate activation for sulfonation	2.8	7.7e-3	525.0204	3.27	M + NH3 + H[1 +
			2.8	9.0e-3	525.0205	2.96	M + NH3 + H[1 +
Positive	5-phospho-β-Dribosylamine	5- aminoimidazoleribonucleotide biosynthesis	2.5	1.6e-3	248.0770	1.64	M + NH3 + H[1
Positive	Protoporphyrinogen IX	Heme biosynthesis	2.2	9.3e-4	569.3127	2.68	M + H[1 +]
Positive	Pyridoxine	Pyridoxal 5'-phosphate salvage I	2.2	3.9e-3	187.1070	4.33	M + NH3 + H[1
Positive	Cytidine	UTP and CTP dephosphorylation I	2.2	1.2e-3	244.0933	4.82	M + H[1 +]
Positive	Guanine	Guanine and guanosinesalvage	2.1	3.4e-3	174.0381	2.07	M + Na[1 +]
Positive	4-aminobenzoate	4- aminobenzoatebiosynthesistetrahydrofolate biosynthesis	2.0	9.0e-4	155.0808	4.31	M + NH3 + H[1
Positive	Anthranilate	L-tryptophan biosynthesis	2.0	9.0e-4	155.0808	4.31	M + NH3 + H[1
Positive	Protoporphyrin IX	Heme biosynthesis	2.0	4.9e-3	293.1278	1.59	M + H + Na[2 +]
Negative	L-arginine	L-arginine biosynthesis II	1.5	8.8e-3	160.1088	4.34	M-NH3 + H[1 +]

Fig. 4.

Metabolomics and structural datasets described the holistic effects of the nanocomposite on the bacterial cell

Discussion

The nanomaterials can be produced through physical, chemical and biological routs with unique features such as their physical (morphology, size and particles distribution, and surface area), and chemical (composition, surface functional groups and photocatalytic activity) properties that are suitable for biotechnological application either directly or with few modifications [33–40]. TiO₂ nanocomposites are used in various therapeutic and industrial applications [25]. However, the small bandgap and limited photocatalytic activity in the visible range of the electromagnetic spectrum limit its photocatalytic activity. Efforts were made to introduce bandgap in the visible region to improve the photocatalytic activity of TiO₂ nanocomposites [26]. TiO₂ coupling with a narrow band semiconductor like CdS, Fe2O3, Cu2O, BiVO4, GaP with a bandgap of 2.4, 2.1, 2.1, 2.4, 2.3 eV respectively makes the composite absorb within the visible region, and the formation of heterojunctions reduces the recombination of charge carriers [27]. Further, CdS–ZnS, CdS–NiS, CdS–Cu₂O, CdS–SnS₂ binary nano-composites also absorb in the visible region and show good photocatalytic activity due to the formation of heterojunctions [28]. However, photo corrosion of these nanocomposites limits their use at a broad level. Ternary TiO₂ composites with metal oxides or sulfides in different coupling combinations not only exhibit visible absorbance and formation of heterojunctions but also reduce the photo corrosion of the catalyst [41]. In this regard, the composites of TiO₂ with other semiconductor materials are the potential candidates because they not only enhance the life span of charge carriers but also improve the absorption in the visible region [41]. In the present study, a ternary nanocomposite of TiO₂ with CdS and ZnS were synthesized. Characterization of TiO₂/CdS/ZnS nanocomposite revealed that the sample was free from impurities and highly crystalline. Even, presence of the CdS and ZnS did not affect the basic structure of the lattice. The ternary composites contain spherical nanoparticles that are well-dispersed and uniform. These results are in line with the previous studies [28].

Photo-induced antimicrobial activity of these nanocomposites needs to be assessed as the second objective of the study. The antimicrobial activity of TiO₂ nanoparticles has been reported [28–30]. Nanoparticles in general, affect the microbial electron transport, protein synthesis, nucleic acids, cell membrane, and cell wall [29, 30, 32]. Likewise, TiO₂ nanoparticles were found to attack the bacterial cell with a deteriorating effect on the cell wall, cell membrane, genetic material, transportation, communication, and respiratory processes [42]. TiO₂ induced reactive oxygen species (ROS) generation was correlated with antimicrobial activity [30]. However, the process behind TiO₂-induced ROS-based microbial death was unveiled. Additionally, microbes were characterized for the presence of cellular repair and stress tolerance machinery [43]. So, how do the nanoparticles elude these defense systems to introduce antimicrobial effects? These are a few preliminary questions that need to be addressed before describing the mechanism of antimicrobial activity. Efforts are being made to decode antimicrobial mechanisms [42], however, a holistic approach is still lacking.

Photo-induced antimicrobial activity TiO₂/CdS/ZnS nanocomposite was confirmed in the current study using disc diffusion assay, time-kill kinetics, and microbroth dilution assay. Even the TiO₂/CdS/ZnS nanocomposite induced ROS generation, lipid peroxidation, and membrane disintegration was observed. These results are in line with previous studies [30] indicating ROS-based bioactivity. However, to unveil the holistic effect of the nanocomposite, additional efforts were performed to map the complete cellular metabolome using the whole-cell metabolomics approach. The cellular metabolite profile gives a precise outlook of the cellular environment [32] and is commonly employed to define the holistic influence of any external agent on cellular functioning [23, 24, 42]. Accordingly, a metabolomics approach was employed to unveil the antimicrobial mechanism of action of the nanocomposite. A total of 1681 metabolic features were differentially (P < 0.001) abundant among nanocomposite treated cells. These metabolites were mapped onto metabolic pathways associated with cell survivability and functioning. TiO₂/CdS/ZnS nanocomposite affects the bacterial cells similarly by influencing the vital pathways for protein, lipid, and nucleotide biosynthesis, cell membrane disruption as well as suppression of cell wall synthesis, and destruction of nucleotides and intermediates such as folates that facilitate the methionine, purine, and pyrimidine biosynthesis. Thus, the nanocomposite not only suppresses the vital microbial processes but also inhibits the repair processes. Nanocomposite treatment also significantly affects the osmoprotectant ability of the cell that in turn affects cell survivability [44]. Similarly, the osmoprotectant and pH-resistance properties of the microbial cell were affected due to the presence of TiO₂/CdS/ZnS nanocomposite. The effects on the life-sustaining processes influence microbial cell survival [45]. In the current study, the introduction of nanoparticles arrested the bacterial cell growth through inhibition of the electron transport system (by reducing the precursors that mediate the electron transfer by 9.7 folds). Disruption of electron transfer generally leads to microbial cell death [45]. A normal energy status is required for microbial cell survival [45]. A significant reduction in the overall energy status of the cell due to a decrease in the energy molecules, lipid, and protein biosynthesis has been observed similar to the observations in the previous studies [41, 42, 45]. Additionally, the iron-quenching capacity is also reduced due to suppression of siderophores that lead to iron-deficiency within the bacterial cell that altogether reduces the energy status of the bacterial cell [41, 42, 45]. These results indicate that nanocomposite induces a holistic effect on the cellular functions, not only cellular damages as reported by previous studies [30].

In this study, we prepared the ternary TiO₂ nanocomposite with a lower band gap with higher light absorption in the visible spectrum of the light. The current study also elucidated the detailed mechanism of antibacterial action of TiO₂ nanocomposite on Bacillus subtilis. Exposure to nanocomposite influenced microbial physiology, metabolism, and structure (Fig. 4). These changes cumulatively affect microbial growth, repair, and maintenance that in turn lead to microbial death. Cumulatively, TiO₂/CdS/ZnS nanocomposite is a photocatalyst that embarks its potential application for the fabrication of self-sterilizing materials in various therapeutic and environment management systems.

Conflicts of interest

The authors report no conflict of interest.s

Ethics Approval

This article does not contain any studies with animals and human subjects.

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Supplementary Information

Below is the link to the electronic supplementary material.

Authors' Contributions

NSC and NK designed the study. AM carried out nanocomposite synthesis and characterization, MY and TK assessed the antimicrobial properties. RC and SG performed metabolomics analysis. NSC, NK, AM, and MY performed data analysis and integration. All authors contributed to the manuscript preparation.

Funding

The work was supported by grant from Principle Scientific Advisor, Government of India for the project entitled “Delhi Research Implementation and Innovation (DRIIV)”.