Using biologically synthesized TiO2 nanoparticles as potential remedy against multiple drug resistant Staphylococcus aureus of bovine mastitis

Presently, there is considerable emphasis on biological synthesis of nanoparticles containing bioactive reducing compounds with an aim to mitigate the harmful effects of pollutants. The approach under study is simple and ideal for the production of durable antimicrobial nanomaterials by novel single-step green synthesis of TiO2 metal oxide nanostructures using ginger and garlic crude aqueous extracts with bactericidal and catalytic activity. A variety of experimental techniques were used to characterize the synthesized nanomaterials. As demonstrated using x-ray diffraction and ultra-violet visible spectroscopy, the produced nanoparticles exhibited high absorption at 318 nm with size varying between 23.38 nm for ginger and 58.64 nm for garlic in biologically-reduced TiO2. At increasing concentrations (500, 1000 µg/50 µl), nanoparticles reduced with garlic exhibited enhanced bactericidal efficacy against multiple drug-resistant S. aureus and effectively decomposed toxic methylene blue (MB) dye. In conclusion, biologically-reduced TiO2 nanoparticles may prove an effective tool in the fight against microbial illnesses and drug resistance.

www.nature.com/scientificreports/Nanoparticles (NPs) are often classified as entities between 1 and 100 nm in size.These particles are intriguing because their physicochemical characteristics vary significantly from those of their macroscale counterparts 25 .Metal oxide nanoparticles are most frequently exploited nanostructures, based on the number of NPs generated annually by the industrial sector.TiO 2 , ZnO, and SiO 2 are widely produced NPs.Particularly, global TiO 2 production has surpassed 10,000 tons per annum 26 .TiO 2 nanoparticles have several uses in numerous domains, including photocatalysis 27 , sensors 28 , and antibacterial agents 29 .It is a useful semiconducting transitioning metal oxide material that exhibits particular characteristics such as simple control, low cost, non-toxicity, and strong resistance to chemical erosion; thus, it is used in solar cells, chemical sensors, and environmental desalination 30,31 .These nanoparticles exhibit unique electrical, magnetic, and optical capabilities compared to their bulk counterparts.TiO 2 may exist in amorphous and crystalline forms, with anatase, rutile, and brookite being the most common crystalline polymorphs 32 .
Ginger, also known as the bulb of Zingiber officinale, is native to tropical Asia.The plant's rhizomes are widely used as a spice, flavoring, or aroma in foods, drinks, soaps, cosmetics, and most importantly in medicine.It contains antioxidant and bactericidal characteristics and is used to treat stomach pain, cough, and also lowers the intensity of transient chemotherapy-induced nausea in young cancer patients 33,34 .
Garlic (Allium sativum), extensively used in medicine as an antibiotic, serves as a renowned natural ingredient used in green synthesis.It is composed of allicin (76%) (diallyl thiosulphinate), methyl allyl thiosulphinate (5-7%), allyl methyl thiosulphinate (14.5%), and dimethyl thiosulphinates (2.6%) 35 .Garlic's bactericidal effect is often linked to the constituent allicin, which contains sulfhydryl-modifying action and may thus block sulfhydryl-containing enzymes 36 .Allicin has also been shown to hinder RNA synthesis in microorganisms 37 and lipid production, resulting in cell wall disruption 38 .In addition, it has broad antibacterial action against both gram-negative and positive microbes 39 .Garlic antibacterial action has been linked to the radical scavenging ability of its organo-sulfur compounds 40 .Its tendency to produce free radicals is also a contributing factor towards its antibacterial activity 41 .
Nanoparticle production can be divided into two broad categories: bottom-up and top-down approaches.The atom, cluster, and nanoparticle are progressively built up in bottom-up or constructive procedure.Spinning (involves a chemical solution serving as precursor to a combined system of distinct fragments) and sol-gel (involves chemical mixture serving as precursor to an interconnected network of distinct particles) are two examples of wet-chemical processes that allow for the regulation of physical characteristics.Nitrogen or additional inert gases are often used to purge the accelerator of oxygen and prevent chemical processes 42 .The prelude and water are injected into the disc as it rotates at a distinct speed.Fusion of atoms or molecules form precipitates, which are lumped together and dried due to the act of spinning 43 , in chemical vapor deposition (CVD) chemical response takes place where an exploded material comes into contact with merged gas 44 ), whereas pyrolysis includes burning a precursor using flame.The precursor could be a liquid or vapor, and is introduced inside the furnace via a tiny hole under high pressure before being burned 45 .In order to retrieve nanoparticles from combustibles or by-product gases, bottom-up approaches such as biosynthesis (which employs bacteria, herbal extracts, fungi, etc. together with precursors to generate the nanoparticle rather than conventional chemicals for bio-reduction and capping) and air classification are widely used.The top-down approach involves breaking a substance down to its atomic building blocks such as milling and subsequent annealing of nanoparticles in an inert environment 46 , nanolithography (constitutes printing an essential construct on a light-sensitive matter by deliberately eliminating an area of material to generate the intended shape), laser ablation (the illumination of metal immersed in water solution due to laser beam compressing a plasma plume that protrudes from the exterior of metal), and thermal degradation (an endothermic chemical breakdown induced by heat which destroys chemical connections in the substance 47 ) and sputtering (a formation of thin film of nanoparticles accompanied by annealing are common steps in the sputtering process).Among the most popular approaches to create nanoparticles is application of thermal breakdown (when an element is heated to its own unique disintegration temperature).Various diverse methods such as plasma enhanced chemical vapor deposition (PE-CVD), in situ fabrication, sol-gel procedure, solid state techniques [48][49][50] and biosynthesis, are used to produce TiO 2 nanoparticles.Among these approaches, biosynthesis of TiO 2 nanoparticles has attracted the most interest owing to its simplicity, nontoxicity, and cost-effectiveness, etc. Various plant components (roots, stem, leaf, flower, peel) act as oxidizing, reducing, and capping substances and are employed to modulate NPs' formation and agglomeration 51 .The comparative analysis with published literature is depicted in Table S1.
BM has emerged as a threat to the farming sector.The prevalence and resilience of S. aureus across microbes is on the rise, suggesting that microbes are a significant source of infection.The recent development of antibiotic resistance is an important concern for public health.Curing illnesses and maximizing safe milk yield by the use of antibiotics will no longer be acceptable in the future.Due to these issues, antibiotic overuse must be reduced, and substitute therapeutic methods must be implemented.New antibacterial substances are being developed due to breakthroughs in nanobiotechnology, particularly the ability to produce metal oxide nanomaterials of specified form and dimension.The current investigation is aimed at evaluating the antibacterial properties and catalytic effectiveness of single-step green-produced low-cost, non-toxic and unique TiO 2 nanostructures using ginger and garlic CAE to combat MDR S. aureus isolates of bovine mastitis, that results in enormous production decline in dairy sector and adverse effects on public health and economy due to resistant subclinical or persistent infections in humans and animals.This investigation is unique from veterinary standpoint since it uses costeffective and eco-friendly synthesis approach and evidential molecular docking analysis.

Materials
Titanium ethoxide Ti 4 (OCH 2 CH 3 ) 16 and sodium hydroxide (NaOH) and DPPH (2,2-diphenyl-1-picryl-hydrazylhydrate) of scientific grade were obtained from Sigma Aldrich for green fabrication of TiO 2 nanostructures.Ginger and garlic roots were procured from a local market.All additional chemicals and growth medium used were of laboratory grade.Collection of ginger and garlic roots material complied with our institutional, national, and international guidelines and legislation.

Crude aqueous roots extract (CAE) preparation
Ginger and garlic root flour was produced using an electric mill and stored in acrylic flasks after the recovery of fine dust.The resulting root powder was mixed with a regulated amount of DI water (1:10) and agitated at 70 ℃ for thirty (30) minutes using a magnetic stirrer.After filtering, the generated extracts were filtered using Whatman No. 1 paper.The resulting filtrate was refrigerated at 4 ℃ until subsequent processing 52 .

Green synthesis of TiO 2 nanostructures
Numerous proportions of ginger and garlic CAE (1.2, 1.8, 2.4, 3.0, 3.6 and 4.2 ml) were applied to 0.1 M (50 mL) titanium ethoxide Ti 4 (OCH 2 CH 3 ) 16 with stirring.After achieving the appropriate pH (12) with the inclusion of 2.0 M sodium hydroxide, the mixture was stirred for two hours at 90 °C till precipitates formed.The pellet was produced after 10 min of centrifugation at 10,000 rpm and washed with deionized water (DIW) followed by drying overnight in a hot air oven at 90 °C.Different ratios of CAE in salt solution (1.2 ml:1, 1.8 ml:1, 2.4 ml:1, 3.0 ml:1, 3.6 ml:1 and 4.2 ml:1) were used to optimize green nanostructures as depicted in Fig. 1 53 .

Characterization
In order to measure absorbance (ƛ max ), the synthesized NPs were evaluated with UV-visible spectrophotometer across the wavelength ranges of 250-800 nm.Employing a BRUKER D2 Phaser (XRD) with a 2θ range (10°-80°) and Cu K α radiation of = 1.540Å, the composition and structure of loaded nanostructures were examined.Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate the functionality of finished product.The elemental content of generated metal oxide nanostructures was examined using energy-dispersive X-ray spectroscopy (EDS).JEOL FE-SEM and JEM-2100F TEM instruments were deployed to validate the size and shape of produced nanostructures.The sample frame and concomitant band gap analysis were explored with X-ray photoelectron spectroscopy (XPS).

Acquisition and characterization of multiple drug resistant S. aureus
Specimen collection Specimens of mastitic fluid were acquired from clinically positive cattle at a number of local and state veterinary facilities and dairies in Punjab, Pakistan.The samples were purified by growing in order to check for microorganisms.All experimental protocols were approved by Muhammad Nawaz Shareef University of Agriculture (MNSUA) Multan, Pakistan.All methods were carried out in accordance with relevant guidelines and regulations and reported in accordance with ARRIVE guidelines.www.nature.com/scientificreports/

Isolation of multiple drug resistant S. aureus
The cattle milk specimens were maintained on 5% sheep blood agar (SBA) and stored at 37 °C for 24-48 h 54 .The acquired benchmark colonies were striped in triplicate on Mannitol Salt Agar to purify S. aureus.Employing the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) for separation of multidrugresistant S. aureus, the susceptibility of characteristic isolates to specified antibiotics was evaluated through disc diffusion.Microbicidal discs containing Oxytetracycline (30 μg), Tylosine (30 μg), Gentamicin (10 μg), Ciprofloxacin (5 μg), and Trimethoprim + Sulfamethoxazole (1.25 μg + 23.75 μg) procured from Bioanalyse® (Turkey) were coated to Mueller-Hinton agar (MHA).Bacteria exhibiting resistance to at least three antibiotics were declared as multiple drug resistant (MDR) 55 after overnight incubation at 37 °C.Microbial colonies were classified based on visual characteristics using Gram's stain and biochemically with coagulase and catalase tests in accordance with Burgey's Manual of Determinative Bacteriology 56 .

In-vitro bactericidal evaluation
In-vitro antibacterial assessment of green metallic oxide nanostructures was conducted upon prevalent bovine mastitogen, MDR S. aureus isolates.A total of 10 MDR S. aureus field isolates were collected.Using well diffusion technique, the in-vitro microbicidal activity was measured.The petri plates were swabbed with 0.5 Mc-Farland standard culture of S. aureus on MSA 55 .A sterilized cork borer was used to form 6-mm-diameter bores.Various proportions of CAE of ginger and garlic were infused as (10 mg and 50 mg/100 µl) and TiO 2 at values of 0.5 and 1.0 mg/50 µl.Positive reference was Ciprofloxacin (0.005 mg/50 µl) while neutral control was DIW (50 µl).Following an overnight incubation at 37 °C, the microbicidal effectiveness was determined by measuring inhibition areas using a Vernier caliper.

Statistical analysis
The inhibition regions were evaluated using SPSS 20.0 with one-way variance test containing significance threshold of 5%,

Radical scavenging assay (DPPH)
The anti-oxidant efficacy and free radical scavenging capacity of synthesized nanostructures were studied using a modified version of DPPH scavenging assay.For both ginger and garlic, a 0.1 mM DPPH solution was infused with TiO 2 nanoparticles at concentrations ranging from 50 to 200 µg/mL.This was then vortexed and allowed to incubate in the dark for 30 min at ambient temperature.Ascorbic acid reference sample solutions were used.Each sample's scavenging rate (%) was determined using degradation of DPPH solution at 517 nm via corresponding Eq. (1).

Molecular docking analysis
TiO 2 nanostructures were submitted to molecular docking analysis for determination of their binding interactions with potential substrates that are critical for bacterial cell proliferation.The enzymes engaged in DNA formation and folic acid production are well-known, desirable, and realistic targets for identification of antibiotics.Several protein targets, including dihydrofolate reductase (DHFR), Thymidylate kinase (TMK), and DNA Gyrase, were chosen for molecular interaction study.The 3D-structures of dihydrofolate reductase (PDB ID: 3FY8), resolution:2.20Å 57 , thymidylate kinase (PDB ID: 4HEJ), resolution:2.00Å 58 , and DNA gyrase (PDB ID: 5CTU), resolution:1.45Å 59 , were obtained from protein database bank Fig. 2a-c.The program SYBYL-X 2.0 was used for docking analysis 60 Sybyl-X2.0/SKETCHmodule was used to generate 3D structures of selected compounds as illustrated in Fig. 3 61 , accompanied by energy reduction pursuant to the Tripos force field with Gasteigere Hückel atomic charge 62 .Flexible molecular docking simulations were conducted using Surflex-Dock module of molecular modelling software program SYBYL-X 2.0 62 .To study the binding affinities of nanoparticles with the active region residues of chosen proteins, required hydrogens were added.Atom kinds and atomic charges were determined based on the AMBER 7 FF99 force field.To avoid steric conflicts, the energy was reduced using Powell method with a convergence rate of 0.5 kcal/(mol・A) over 1000 cycles.At least 20 optimal docking positions were preserved for every ligand-receptor complex system.Using the Hammerhead scoring function, top potential positions of ligands were ranked.Surflex dock module utilizes an empirically determined consensus scoring (cScore) 63 function which combines Hammerhead's empirical scoring function 59 , that is, D-score (dock score), G-score (gold score), Chem-score, potential mean force (PMF) score, and the total score, with a molecular resemblance technique (morphological identity) to create and evaluate hypothetical poses of ligand fragments 64 .

Catalytic activity
For catalytic evaluation of produced TiO 2 nanostructures, 3 mL of methylene blue (0.03 × 10 −3 M) and 300 µL of newly produced sodium boro-hydride hydro solution were mixed.The ideal concentration (3600 µL:1) of the material at a concentration of 6.0 mg/300 µL was then administered to the solutions.As a consequence, the methylene blue (MB) dye pigment disappeared, as seen in Fig. 4, indicating a degradation of the dye to leuco methylene blue (LMB).The UV-Vis spectrophotometer was employed to determine absorption within the range of 200 to 800 nm.

Results
Visual attributes of ginger and garlic-reduced TiO 2 between 200 and 500 nm are shown in Fig. 5a,b.Following a blueshift, the total absorbance (λ max ) of TiO 2 -NPs measured at 318 nm (1:3.6 ml) increased with higher extract concentration.Ginger and garlic CAE absorption peaks were exhibited at 275 and 280 nm, correspondingly.The breadth of peak showed aggregation of particles, and the passage of electrons to conduction bands was indicated by the significant absorption.Consequently, Fig. 5a,b depicts a reduction in NP absorption with increasing or decreasing extract volumes beyond the optimal range (1:3.6 ml).
The FTIR spectrum of TiO 2 produced using CAE of roots is shown in Fig. 7a,b as a broad absorption at 3640 cm −1 corresponding to OH and a carbonyl group with (N-H) amine stretching.Sharp absorptions at 3430 and 1625 cm −1 show the existence of water and hydroxyl groups.Absorption bands below 1200 cm −1 are indicative of Ti-O-Ti vibrations, whereas a high absorption spike at 2335 cm −1 confirms C≡N stretching.The absorption peak between 500 and 700 cm −1 corresponds to Ti-O stretch and Ti-O-Ti bridge stretching modes.The signal at 653 cm −1 suggested that phytochemically biologically-synthesized TiO 2 anatase phase contribution was present.The significant peaks at 978 and 687 cm −1 correspond to the O-Ti-O bond and metal oxygen stretching frequency, correspondingly.After TiO 2 reduction, changes in the peaks at 2335-2535 and 1625-1834 cm −1 were observed.
As illustrated in Fig. 8a,d, the morphology and size of biologically-reduced TiO 2 -NPs were determined using scanning (FE-SEM) and transmission (TEM) electron microscopes.FE-SEM analysis revealed spherical topography with dense aggregation.The TiO 2 -NPs, TEM images displayed more spherical (< 50 nm) morphology with aggregation and indicated that increasing extract concentration may enhance aggregation.
As illustrated in Fig. 9a,b, energy dispersive spectroscopy (EDS) was used to quantify the elemental composition of produced TiO 2 -NPs.The EDS spectrum of samples evaluated between 1 and 10 keV exhibited three    Well diffusion technique by estimating inhibition areas (mm) as seen in the Fig. 11a-d and Table 1, tested the microbicidal potential of ginger and garlic roots CAE and TiO 2 -NPs.The comparative analysis with published literature is depicted in Table S1.The results revealed a strong relationship between concentration and inhibitory regions (mm).Statistically significant inhibition regions (mm) (p < 0.05) were exhibited for samples 1 (1.2 ml:1), 2 (1.8 ml:1), 3 (2.4 ml:1), 4 (3 ml:1), 5 (3.6 ml:1) and 6 (4.2 ml:1) with a range of (0.95-1.55 mm) and (1.05-2.65 mm) at the minimum and maximum concentrations for ginger CAE reduced TiO 2 -NPs as seen in  The oxidative destruction induced by NPs is dependent upon their morphology, concentration, and scale, which are inversely attributed to the doped substance's characteristics 65 .The reactive oxygen species (ROS) generated by NPs persist effectively inside the pathogen cell membrane, causing cytoplasmic expulsion and pathogen violence as presented in Fig. 12 66 .Strong cationic interaction of Ti 4+ at greater concentrations with the negative virulent organism causes cellular degeneration and destruction of bacteria 67 .To gain insight into the mechanism of interactions between TiO 2 nanoparticles and target enzymes, a molecular docking study was conducted.Folate biosynthetic process results in the production of tetra-hydrofolate, which is required for biosynthesis of several bioactive elements, including thymidylate enzyme, pan-tothenic acid, nitrogenous bases as purine, ribonucleic acid, and amino acids.Dihydrofolate reductase and thymidylate kinase, enzymes related to such pathway, have been suggested as interesting candidates for antibiotic development 68,69 .Keeping in mind the significance and need of folate biosynthesis pathway for the development and survival of bacteria, the binding interface pattern of such nanoparticles was examined against S. aureus DHFR, TMK, and DNA gyrase enzymes.The best-docked shape of TiO 2 nanoparticle into active pocket of DHFR has a binding energy of 4.57.As demonstrated in Fig. 13C, TiO 2 is linked with THR121, Thr46, Ser49, Asn18, and Gln19 through H-bonding.Additionally, in the case of TMK, the highest binding score of 4.73 is attributed to H-bonding interactions with Ser97 and Gln101, as shown in Fig. 13A-C.www.nature.com/scientificreports/As illustrated in Fig. 13B, TiO 2 nanoparticles exhibited H-bonding interactions with Gly0, Asp81, and Ser55 in case of S. aureus DNA gyrase through binding score of 4.40.Table 2 provides a summary of docking values and critical residues implicated in H-bonding for all proteins.The considerable binding score and interaction of TiO 2 nanoparticles revealed that they are a potential blocker of dihydrofolate reductase, TMK, and DNA gyrase, whose inhibitory potential can be further investigated.
Figure 14a-e illustrates the substantial decrease in catalytic MB at room temperature with roots CAE and biologically reduced TiO 2 -NPs.Figure 14() demonstrates the catalytic activity of standard TiO 2 -NPs purchased from Sigma Aldrich. Figure 14b,d indicates the catalytic potential of garlic CAE and reduced TiO 2 -NPs with garlic CAE. Figure 14c,e depicts the catalytic activity of CAE of ginger and doped TiO 2 -NPs with ginger CAE.Generally, TiO 2 and CAE of garlic and ginger roots diminished MB in 62, 38, and 43 min, respectively (see Fig. 14a,b, and e) compared to a time of 10 min and 100% reduction of dye in 3 min for garlic and ginger CAE doped TiO 2 NPs, as presented in Fig. 14d,e.
The DPPH scavenging experiment in Fig. 15 is used to evaluate and quantify anti-oxidant capabilities of scavenging DPPH radicals, which are active radical species.Anti-oxidative properties of compounds are linked to their capacity to donate hydrogen or electrons to DPPH free radicals, leading to diamagnetic compounds with high stability 70 .All compounds exhibited anti-oxidant potential that scaled with increasing doses.TiO 2 -NPs with maximum ginger and garlic concentrations inhibited DPPH radicals by donating hydrogen atoms at concentrations of 200 g/mL (50.3% for ginger and 61.4% for garlic) in 3.6 ml: 1 sample whereas, turbidity of sample may be responsible for the modest drop in 4.    103), ( 200), ( 105), ( 211), ( 204), ( 116), ( 220) and ( 215) planes (JCPDS card no: 00-021-1272) 73 .The large peaks demonstrate oxygen presence 74 .The FTIR analysis of TiO 2 synthesized with roots extracts, as seen in Fig. 7a,b, showed wide absorption at 3640 cm −1 , which corresponds to the presence of OH while the peak width validated the presence of carbonyl group with (N-H) amine stretching 75 .Absorption peaks below 1200 cm −1 depicted Ti-O-Ti vibrations while, intense peak at 2335 cm −1 confirmed C≡N stretching 73 .The absorption peak at 500-700 cm −1 corresponded to Ti-O stretching and bridging-stretching modes of Ti-O-Ti 76,77 .The peak present at 653 cm −1 indicated contribution from phytochemically synthesized TiO 2 anatase phase 78 .Peaks at 978 and 687 cm −1 prominently corresponded to O-Ti-O bond and metal oxygen stretching frequency 79,80 .After TiO 2 reduction, peak changes were observed at 2335-2535 and 1625-1834 cm −1 indicating presence of phytochemicals, flavonoids, proteins containing ketones, carboxylic acid and amines that are considered significant for reduction 81 .The TiO 2 -NPs showed pleomorphism during FE-SEM examination with exhibition of cubical and spherical morphology (< 50 nm) accompanied by increased agglomeration 82 .Agglomeration of NPs suggests polymer conformity and the presence of magnetic forces between particles 83 .In XPS analysis, the O1s contribution located at 530.3 eV is ascribed to Ti 2 O 3 and the peak at 531.3 eV is attributed to non-lattice oxygen 84,85 .Peaks appearing at 286.5 and 288.5 eV corresponded with C-O and C=O bonds, respectively 86,87 .The Ti 2p spectrum containing Ti 2p 3/2 and Ti 2p 1/2 peaks at B.E 458.7 and 464.5 eV, respectively depict typical characteristic of Ti 4+ -O bond of TiO 2 88,89 .The production of highly reactive • OH and • O 2 radical species can interact with DPPH free radicals, triggering the breakdown of DPPH, which is intrinsically linked to standard ascorbic acid 90 .
The primary challenge in producing NPs sustainably has been in regulating their final dimensions and forms.Various phytochemical constitutions in plants have specific molecular parameters, and their forms and dimensions are reflected accordingly.Furthermore, the chemical makeup of similar plants cultivated in diverse regions or harvested at distinct times of year might also lead to differences in active ingredients.As a result, the diameter and form of the precipitated NPs would be affected.These might also reduce their worth in the market, since commercialized nanoparticles are often well-suited to their intended application.As a result, it might be even more challenging to identify suitable uses and marketplaces for phyto-based NPs.When contrasted with preparing solutions employed in chemical procedures, botanical extracts have a far higher concentration of active compounds.Researchers are encouraged to further refine their methods for producing phyto-based NPs since the benefits are seen to outweigh the drawbacks.To get a deeper comprehension of the processes involved in the manufacturing and use of TiO 2 NPs, the following future studies are recommended: • Substantial tuning is needed to produce TiO 2 NPs with appropriate morphology and size using a green production approach.• The metabolites in botanical extracts ought to be analyzed further to identify their effectiveness regarding NPs production.• The mechanical characteristic of TiO 2 NPs generated using a green technique needs further research.
• Further research is required to determine the durability of TiO 2 NPs generated using a green synthesis tech- nique.

Conclusion
This is the first research to quantify the bactericidal susceptibility of doped TiO 2 nanostructures against MDR S. aureus from bovine mastitis.Incorporating ginger and garlic roots CAE in varying proportions had significant impact on the development and optimization of metal oxide nanostructures.Analysis by means of X-ray aureus.This research indicated that the emergence of antibiotic resistance might be considerably mitigated by using green fabricated metal oxide nanostructures as alternatives for antibiotic treatments.Molecular documentation, toxicological investigations and in-vivo efficacy tests that target infectious and resistant Gram-negative pathogenic microbes ought to be the primary concern of future investigations that address uses of green synthesized metal oxide NPs produced with economical and non-toxic green methods.

Figure 4 .
Figure 4. Diagrammatic representation for catalytic reduction of MB to LMB by green-synthesized NPs.

Figure 8 .
Figure 8. FE-SEM analysis of biologically-reduced TiO 2 with ginger (a) garlic (b) TEM analysis of reduced TiO 2 with ginger (c) and garlic (d).

Figure 11 .Table 1 .
Figure 11.In-vitro bactericidal potential of biologically-reduced TiO 2 -NPs with ginger CAE at the minimum and maximum concentrations (a, b) garlic (c, d).

Figure 12 .
Figure 12.Mechanistic illustration of antibacterial activity of garlic and ginger doped TiO 2 NPs.

Figure 13 .
Figure 13.Binding interaction pattern of TiO 2 nanoparticles with active site residues of (A) Thymidylate kinase, (B) DNA gyrase and (C) Dihydrofolate reductase from S. aureus.

Table 2 .
2 ml: 1 sample 71 .Surflex score of docked ligand TiO 2 .a CScore is a consensus scoring system that ranks the attraction of ligands based on different scoring functions.b Crash-score indicating improper binding site piercing.c Polar domain of ligand.d G-score representing hydrogen bonding, complex (ligand-protein), and internal (ligandligand) energies.e PMF-score showing Helmholtz free energies for protein-ligand atom pairings (Potential of Mean Force, PMF).f D-score for charge and van der Waals interactions among protein and ligand.g Chemscore in addition to an intercept term points are awarded for hydrogen bonding, lipophilic contact, and rotational flexibility.