Biocidal mechanism of green synthesized thyme loaded silver nanoparticles (GTAgNPs) against immune evading tricky methicillin-resistant Staphylococcus aureus 090 (MRSA090) at a homeostatic environment

2.1 Preparation of Thymus vulgaris leaf extract

The dried 100 g of thyme (T. vulgaris L.) leaves were purchased from local market Mysuru, Karnataka, India. Leaves were ground to a fine powder and refluxed at 70 °C with 500 mL of distilled water for 5 h. The mixture was allowed to cool to room temperature. Then aqueous extract was centrifuged at 6000 rpm and the supernatant was collected then filter stored for further study.

2.2 Green synthesis of thyme-loaded silver nanoparticles (GTAgNPs)

The thyme (2 mL extract) was transferred to a 1 mM AgNO₃ solution and heated at 60 °C for 15 min. The liquid color changed indicates the formation of green thyme-loaded silver nanoparticles (GTAgNPs), centrifuged, the pellet was retained and after drying sample was stored for further study (Qiu et al., 2015; Jafari et al., 2015).

2.3 Characterization of GTAgNPs

Prepared GTAgNPs were vacuum dried to obtain fine powder to study size, shape, crystallinity and elemental analysis. GTAgNPs were subjected to scanning electron microscope (SEM) for size, X-ray diffraction (XRD) for determination of phase purity, Fourier transform infrared (FTIR) spectroscopy to study bonding pattern of NPs acquired between 600 and 4000 cm⁻¹. The size, distribution and surface charge of GTAgNPs were studied through dynamic light scattering (DLS) and zeta potential analysis (Wani et al., 2014; Das et al., 2015).

2.4 Physicochemical interaction at nano-bio interface

Physicochemical interaction at nano-bio interface was elucidated by ionic strength, pH, and temperature for GTAgNPs suspension. The ionic strength of NPs suspension was studied from 0.1 M to 1.5 M by direct addition of calculated amount of NaCl at constant pH of 7.0 and suspension was analyzed by UV–Vis spectroscopy. The stability of NPs was studied between temperatures of 30–100 °C for 1 h. The pH of the stability was measured by altering the pH of the NPs suspension by the addition of 1 M HCl or NaOH until pH values were reached and pH of the NPs were studied at 2–12 according to Das et al. (2012).

2.5 Catalytic activity

The GTAgNPs catalyst property was deduced according to the protocol described by Venkatesham et al. (2014). The 3 mL of reaction mixture contain freshly prepared 0.2 M NaBH₄ solution (1 mL), 0.2 mM of 4-nitrophenol (1.9 mL) solution was mixed and UV-absorbance was recorded. After GTAgNPs (0.1 mL, 0.1%) solution added, mixed vigorously then O.D was recorded.

2.6 DPPH radical scavenging activity

The radical scavenging property was measured for synthesized GTAgNPs using stable DPPH radical assay described by Li et al. (2012) with slight modifications. The synthesized GTAgNPs antioxidant capacity was expressed in% of DPPH radical scavenging activity. Briefly, activity was assessed by mixing different concentration of GTAgNPs to the 2 mL of methanolic DPPH solution (40 mg/L). At the same time control was prepared by adding 1 mL of water to the DPPH solution, then keep in the dark for 30 min the remaining DPPH was measured by reading at 517 nm. The radical scavenging activity (RSA) was calculated using following equation: $$\%\text{RSA}=100\times 1-\text{A sample}/\text{A control}$$ where A-sample: represents the absorbance of the test sample solution and A-control: represents the absorbance of the DPPH solution without test sample. All the experiments were carried out in triplicates and twice.

2.7 Antimicrobial activity

2.8 Release of cellular material

Effect of TE and GTAgNPs were analyzed by measuring cellular material (DNA) from MRSA090 according to the protocol of Chauhan and Kang (2014). The experiment was carried out by inoculating log phase culture into 0.1% sterile peptone water and without samples as a control. After incubation at 37 °C (for 0, 30, 60, and 120 min), 1 mL of broth was transferred to an Eppendorf tube, centrifuged at 3500 rpm and the supernatant was measured at 260 nm using a spectrophotometer. Results were expressed in the form of optical density for the sample collected from different time interval incubated samples. Assay performed in triplicates and repeated thrice.

2.9 Evaluation of potassium efflux

The membrane damage by samples was measured by the alternation in the potassium efflux as described by Wang et al. (2015). Briefly, as described above MRSA090 cells were grown and centrifuged to pellet the cells, then pellet was re-suspended in 100 mM sodium phosphate buffer. Cell density was adjusted to an A₆₀₀ of 1.0. 70 mL of cell suspension was transferred to 100 mL beaker and stirred magnetically at 30 °C. 1 mM of test samples were added to cell suspension and then, 9 mL of cell suspension was withdrawn at regular intervals using 10 mL pipettes, centrifuged and collected supernatant were measured for potassium concentration using flame photometer read at absorbance at 766.5 nm with a unit width of 0.1 mm. standard calibration curve prepared using KCl in HCl (1:10, final concentration 5% (v/v)) in milli-Q water. All values recorded from triplicate measurements comparing control to ensure, none of the elements interfered with the assay. Assay performed in triplicates and repeated thrice.

2.10 Action of GTAgNPs on production of MRSA090 biofilm

2.11 Molecular action of GTAgNPs on S. aureus

2.12 Bacterial cell microscopy

The scanning electron microscopy (SEM) and atomic force microscopy (AFM) carried out to study MRSA090 membrane damage by treating 1 mg/mL concentration of TE and GTAgNPs for 2 h, then cells were pelleted by centrifugation (10,000 rpm for 5 min) at 4 °C. Cells were fixed by using glutaraldehyde (2.5%) in PBS, pelleted and deposited on glass slide followed by stepwise treatment of 30–100% ethanol drying. After, 2 days drying under room temperature used for SEM and AFM analysis as per the protocol of Wang et al. (2015) and Di Pasqua et al. (2007).

2.13 Membrane damaging effect

2.14 Nano-toxicity study

2.15 RBCs collection and handing

Citrate-stabilized fresh sheep blood (1:9) was collected at a slaughterhouse in Mysore, Karnataka, India. Briefly, collected whole sheep blood was added to 20 mL of phosphate-buffered saline (PBS) and centrifuged at 1000 rpm for 10 min to isolate RBCs from serum. The collected RBCs were further washed twice with PBS solution. Following the last wash, the isolated RBCs were diluted to 10 mL with PBS solution for further use.

2.16 Inhibition of coagulase enzyme and gene regulation

3.1 Synthesis and characterization of GTAgNPs

The characterized GTAgNPs had narrow surface plasmon resonance (SPR) band indicates the formation of well-controlled particle sizes (spherical shape) and narrow distribution size of 75 nm was achieved by reducing and capping agent of T. vulgaris leaf extract (TE). The intensity of color increased as a function of time shows the reduction of Ag⁺. The change in color associated with the reduction of silver ions was visibly evident because bio-mass extract may act as reducing and capping agent. The formation of colloidal GTAgNPs was controlled by UV–visible spectroscopy and observed narrow band with a maximum at 430 nm after 5 min of reaction completed. The FTIR analysis was carried out for GTAgNPs to identify the interactions of possible molecules present in T. vulgaris L. extract playing responsible functions in reducing and stabilizing AgNPs. The results of FT-IR study showed for TE at 3335, 2975, 1650, 1380, 1087, 1046 and 880 cm-1 represents free OH in molecule and OH group involved in the formation of hydrogen bonds, aliphatic compounds, primary amines, aliphatic compounds, aliphatic ethers, primary alcohols and vinylidenes observed respectively. Because of these functional groups present in the plant extracts could be probably responsible for the reduction of noble metal ions and helps in the formation of GTAgNPs. This can be confirmed by —OH group stretching broad band in 3352 cm⁻¹, band around 2928 generally attributed to loss of O or N from the compounds, deformation of amines in 1605 cm⁻¹ indicated by broadening of band, at 1351 and 1038 cm⁻¹ broadening confirms the CH₃ deformation and C—O stretch once TE reduced the AgNO₃ to form GTAgNPs.

The size, shape, and morphology of GTAgNPs were characterized further by SEM analysis. The SEM image shows morphology had spherical GTAgNPs. The observed size in SEM and TEM was larger than hydrodynamic diameter obtained from DLS analysis. The XRD pattern of GTAgNPs confirmed the crystalline nature of the nanoparticles. All the Brag reflections of pure silver metal of 2θ peak values at 37.95, 43.93, 64.39 and 77.15 corresponds to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) respectively of lattice planes. This deviation from perfect crystallinity is the reason for broadening of the diffraction peaks of materials. Crystallite size is a measure of the size of a coherently diffracting domain, and the crystallite size of the particles is not generally the same as the particle size due to the presence of polycrystalline aggregates. XRD pattern indicates the formation of lattice plane of GTAgNPs which is in agreement with the electron diffraction results. The peak broadening in the XRD pattern clearly indicates that small nanocrystals are present in the samples and it expresses no evidence of bulk remnant materials and impurity. According to the Scherrer equation, the calculated theoretical value of GTAgNPs size was 74.21 nm not exactly matching to the DLS, which is experimental value. The peaks appear at different 2θ values as shown and sharp diffraction peaks indicate the good crystallinity of the prepared particles. The actual signal of silver (Ag) comes from the Ag nanoparticles and obtained the 56.73% atomic silver content. Apart from the Ag, also some other peaks such as Carbon (C), Oxygen (O) 24.26 and 19.01% respectively. These signals represent the adsorbed carboxylic component present in T. vulgaris L. The signal of C and O is also due to adsorption of plant element over GTAgNPs.

This GTAgNPs showed well nano-bio interface and strong catalytic activity by relaying electron from donor BH₄⁻ to acceptor p-nitrophenolate for conquering the kinetic barrier (Supporting Information, Figs. S1–S9). Particle size reported to be the one of a determinant of action and nanoparticle was synthesized by loading green thyme to AgNPs (GTAgNPs) eco-friendly procedure as described by Nasrollahzadeh et al. (2016). It is well-known that the plant extract containing phenolic compounds, proteins, lipid molecules and there NPs had tremendous biological properties also, still mechanism of green stabilized NPs was not understood clearly. Researchers debated mechanism by which NPs exert toxicity to bacteria and another organism independent of hyperosmotic shock. The doses of NPs selectively participate and disturb the process need for the cell growth influenced by both metal atoms and accessible donors within intracellular bio-molecules by abolishing enzyme activities, membrane disruption then interfering with cell respiration (Lemire et al., 2013). It is well-known that the plant extract containing phenolic compounds, proteins, lipid molecules and there NPs had tremendous biological properties also, still mechanism of green stabilized NPs was not understood clearly.

3.2 GTAgNPs action on methicillin-resistant Staphylococcus aureus 090 (MRSA090) can be solely explained by the dose dependent manner

The antioxidant activity of synthesized GTAgNPs increased in a dose-dependent manner comparable to TE (Supporting Information, Fig. S10). This pattern was correlated with minimum inhibitory concentration (MIC) for GTAgNPs was 20 mg/mL compared to standard streptomycin (10 µg). The plants are rich source of antioxidants containing different phytochemicial involved in antioxidant activity by neutralizing reactive oxygen species or free radicals, as antimicrobials, as anthelmintic, as anti-inflammatory, anti-diabetic, and as anti-thrombus etc, (Manukumar et al., 2013a, 2013b, 2013c, 2014, 2016; Manukumar and Madhu, 2013; Manukumar and Thribhuvan, 2014; Manukumar and Vanitha, 2014; Madhu et al., 2014a, 2014b; Manukumar and Shruthi, 2014; Manukumar and Umesha, 2015; Umesha et al., 2016; Manukumar and Umesha, 2017b). The antimicrobial activity was found to be higher in GTAgNPs compared to TE and growth curve analysis (0.0625–20 mg/mL concentration) showed promising action against S .aureus 090 at the minimum inhibitory concentration (MIC) of 1 mg/mL (Supporting Information, Fig. S11, Fig. 1A and B) and it is higher with respect to TE to kill MRSA 090 effectively. Researchers debated mechanism by which NPs exert toxicity to bacteria and another organism independent of hyperosmotic shock. The doses of NPs selectively participate and disturb the process need for the cell growth influenced by both metal atoms and accessible donors within intracellular bio-molecules by abolishing enzyme activities, membrane disruption then interfering with cell respiration (Lemire et al., 2013). These results highlight the identification of actively involved molecule on NPs surface could be an important molecule for the development of novel anti-S. aureus drugs (Smith-Palmer et al., 2004). However MIC of GTAgNPs significantly decreased the growth rate after 60 min of treatment and support, the GTAgNPs would gain a viable alternative treatment to synthetic drugs (Duncan et al., 2015; Chauhan and Kang, 2014).

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Fig. 1 Growth curve of methicillin-resistant S. aureus 090 (MRSA090). Different concentrations of TE (A) and GTAgNPs (B) were treated with MRSA090 and measured the efficient sub-inhibitory concentration. TE and GTAgNPs showed a sub-inhibitory concentration of 5 mg/mL and 1 mg/mL respectively towards MRSA090 at different intervals of time. The effect of GTAgNPs at 1 mg/mL on MRSA090 shows cellular leakage (a) and potassium ion efflux (b) effectively compared to TE. The results presented as mean values of triplicate biological determinations. The scanning electron microscopic analysis of MRSA 090 shows control (C—A) cells (12,000×) showing regular and intact morphology. Cells (12,000×) after treatment with GTAgNPs (C—B) and TE (C—C) at 1 mg/mL and 5 mg/mL concentration respectively, showed altered MRSA 090 cell morphology due to the disruption of a cell membrane. The GTAgNPs showed anti-biofilm property (D) excellent at 1 and 5 mg/mL concentration compared to TE was observed. SEM morphology of patchy biofilm formed by MRSA 090 (E) on the untreated slide and restricted colonization of MRSA090 (F) on treated slide.

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The GTAgNPs showed time-dependent enhancement of optical density for cell filtrates due to the release of cellular materials compared to a stable release of cellular materials in untreated cells. And also showed dose-dependent efflux of potassium ions from MRSA090 occurred immediately after the treatments of TE and GTAgNPs (Fig. 1a and b). The investigation on MRSA090 morphology was done by SEM and AFM, revealed a clear alteration in the membrane after treatment of GTAgNPs compared to untreated MRSA090, by retaining a normal morphological feature (Fig. 1A–C). Bacterial cells are highly metabolically active and cytoplasmic membranes are very delicate in nature. Thus, any active molecule having potential interaction with the cytoplasmic membrane leads to damage in membrane anatomical structure and release of potassium ions, DNA, and other cellular materials (Kim et al., 2007). The present study suggests an effect of NPs had a great effect on cytoplasmic membrane of MRSA090 and stimulated to release the cellular materials. Our results showed significant for GTAgNPs activity which is in accordance with reports of Chauhan and Kang (2014) and Kim et al. (2007).

3.3 Anti-biofilm properties

The microorganisms grow on inert or living surfaces forming microbial biofilms, by dense communities of microbial cells surrounded by a self-secreted matrix. We evaluated the anti-biofilm efficacy of GTAgNPs against MRSA090 using crystal violet method and showed prominent to eradicate the biofilm forming MRSA090 at two concentrations (1 and 5 mg/mL) compared to TE (Fig. 1D and E). Even, higher dose shows major changes in the biofilm formation by MRSA090. The biofilm formed by MRSA090 was very much patchy (Fig. 1F) and increased cell surfaces of MRSA090 suggest that it has been damaged by GTAgNPs (Fig. 1G). The failure in the prevention and eradication of microbial biofilms might create a number of serious problems such as bio-deterioration, food contamination and infectious diseases such as endocarditis, periodontitis, bacteremia and chronic lung infections in cystic fibrosis patients being the prominent ailments. The microscopic evacuation clearly shows that the GTAgNPs treated well restraining the MRSA090 to colonize effectively on the surface compared to untreated (dos Santos Rodrigues et al., 2016; Ansari et al., 2015).

3.4 Bioelectrical changes at the site of electron transport chain and membrane integrity

Metabolic pathway activation: The inactive cell does not transfer electrons to DCPIP and a redox mediator interaction with MRSA 090 is attributed to activation of cellular metabolism. Even though electron transfer reaction dependent on metabolically active cells, the energy carbon source plays a very important role in the formation of reduced electron shuttle (DIPIPH₂). Therefore, effects of carbon sources on MRSA 090 electrochemical behavior depends on and significantly, glucose showed increased oxidation current compared to other carbon sources (Fig. 2a and b).

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Fig. 2 The cyclic voltammogram (CV) analysis. The CV of (a) MRSA090 response to different carbon sources, (b) cell suspension of MRSA 090 in FCN/DCPIP in the absence (−) and presence (+) of glucose (10 g/L), (c) studied at 1 mM FCN, 40 µM DCPIP and combination in PB at pH 7.0 and (d) the effect of GTAgNPs and TE at 1 mg/mL concentration in presence of FCN affects the MRSA 090 CV was represented. All CV measurements were conducted after incubation of MRSA090 suspension (0.5 OD at A₆₀₀).

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Bioelectrochemistry of MRSA090 According to cyclic voltammetric (CV) signals with different intervals of time, FCN alone showed no significant electrochemical signal response and failed to show bacterial respiration connections at electrode system in the first case. In the second case, DCPIP alone leads to generating low oxidation peak indicates the weak wiring of MRSA 090 with electrode takes place. But in a case of DCPIP-FCN double mediator assay showed significant oxidation peak (Fig. 2c). This evidence claims, due to hydrophilic nature of FCN struggled to cross the cell peptidoglycan layer and no significant oxidation was determined in this system. Even DCPIP had lipophilic property, failed to transfer extracellular electron efficiently to an electrode. With this epitome, we used GTAgNPs as a paragon for its embodiment against MRSA090 in presence of FCN alone and showed the significant increase in current by transferring electrons from intracellular to extracellular followed by exchanging electrons to FCN to reach electrode efficiently. This way GTAgNPs impel the bioelectrical changes attributed by transporting electrons efficiently from intramembrane space to outside the cell by forming pores to MRSA090. This process plays a very important role in transferring electrons from intracellular redox center to FCN to reduce ferrocyanide and access the electrode system. The hydrophilic FCN used as a probe to assay the permeability created on MRSA090 in presence GTAgNPs mediated electrochemical method. Increase in the current in presence GTAgNPs and prominent increase action on MRSA 090 membrane indicate mobility of GTAgNPs (as like DCPIP) across the cell membrane (Fig. 2d).

The alterations in the membrane morphology of MRSA090 after subjecting into 1 mg of GTAgNP for 2 h accelerate the reaction of GTAgNPs with cell wall instead of air drying compared to MRSA 090 in absence of GTAgNPs (Supporting Information Fig. S12). As an increased action of GTAgNPs, the appearance of grooves indicates expel membrane components from MRSA 090 which facilitate the interaction of FCN for a mediated electrochemical process (Fig. 2c). Also, membrane toxicity of GTAgNPs showed significant action on MRSA 090 by decreasing fatty acids contents from treated cells compared to untreated cells (Supporting Information Table S2). Interestingly in the present study, molecular evidence explored for GTAgNPs by deducing insight physiological state of the MRSA090 by studying a change in the intracellular redox activities. Bioelectrochemistry evidence of MRSA090 defined as the unaltered peak height was observed in presence of metabolically inactive cell suspensions and consequently, electrochemical response peak observed when treating metabolically inactive cells to 10 g/L of glucose (Hassan and Bilitewski, 2011; Rawson et al., 2012). The absence of porins in S. aureus is very important to use artificial electron shuttle to study the bioelectrochemistry of MRSA 090 (Zhao et al., 2008). With respect to the formation of porin-like structures by GTAgNPs was also supported by AFM. The native MRSA090 showed relatively smooth surface without any grooves/ruptures and this result was similar to report of Jevon et al. (1999). The AFM and fatty acid analysis demonstrates the interaction of GTAgNPs on lipid and membrane-embedded proteins, resulting in destabilization of membrane integrity waee established (Veldhuizen et al., 2006; Fitzgerald et al., 2004). Hence, consequently, FCN mediated electrochemical process, cellular leakage, potassium efflux, SEM, and AFM showed a consistent effect and agreeing to make use of GTAgNPs for the establishment of anti-Staphylococcal drugs in future (Diao et al., 2013).

3.5 Cytotoxicity and growth kinetics of breast and lung carcinoma cells

Interestingly, the bio-reduced GTAgNPs do not show toxic to the non-cancerous peripheral blood mononuclear cells (PMBC) cells up to 500 µg/mL (Fig. 3B). When TE (Fig. 3A) treatment to PBMC cells in a dose dependent manner (25–1000 µg/mL) induced to kill PMBC cells at 300 µg/mL concentration itself and started to prevent growth of lung cancer (A549) and breast cancer (MCF-7) cells at concentrations of 400 and 500 µg/mL respectively. Thus, a free active component of TE was toxic to both cancerous and non-cancerous cells at higher doses. On the other hand, GTAgNPs showed toxicity at 500, 150 and 200 µg/mL against PBMC, A549, and MCF-7 cells respectively, indicating silver NPs with TE active synergistic components increased the bioavailability at lower doses apart from increasing its therapeutic efficacy. Based on evidence of cytotoxicity data, we further analyzed non-killing doses of GTAgNPs (50–300 µg/mL for A549 and MCF-7) for assessing further biological performance. The analyzed concentrations on growth of cancer cells for 24–72 h, Fig. 3C and D showed a dose-dependent reduction in the growth kinetics of A549 and MCF-7 significantly.

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Fig. 3 Cytotoxicity analysis and growth kinetics of GTAgNPs. The non-cancerous cell PBMC, cancerous cells (A549 and MCF-7) were treated different concentrations (0–1000 µg/mL) of TE (A) and GTAgNPs (B) for 24 h and analyzed for cell viability using MTT assay. The TE showed less toxicity to PBMC up to 300 µg/mL and showed anti-cancer activity to A549 (400 µg/mL) and MCF-7 (at 500 µg/mL) concentration onwards. GTAgNPs showed toxicity above 500 µg/mL concentrations onwards to PBMC and significantly respond to cancerous A549 (at 150 µg/mL) and MCF-7 (at 200 µg/mL). All the experiment represented after triplicate independent assays and showed a significant difference compared to control group. The GTAgNPs growth curve analysis showed a effectively decreased the number of A549 and MCF-7 cancerous cells. The GTAgNPs showed significant decrease in the cell number counted from the four quadrants and average of each has been plotted in C (A549) and D (MCF-7).

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The biokinetis of NPs was measured as the rate of nanoparticle uptake, distribution, exocytosis tremendously, dependent on NPs size, shape, surface area and stability which are the major factors that influence biokinetics and toxicity. The cell viability assays are vital steps in toxicology research that explain the cellular response to a toxicant and give information of cell death, survival, and metabolic activities (AshaRani et al., 2008). Many anticancer drugs and natural bioactives such as genistein, gallic acid, curcumin, silibinin and wogonin studied for anticancer and its bioavailability. The literature reveals that the tagged bioactive showed enhanced efficacy compared to free drug in terms of MIC and IC₅₀. The AgNPs studied majorly for drug delivery of anticancer agents apart from negligible toxicity to non-cancerous cells due to specificity and sustained release of properties (Jain et al., 2005). Thus, tagging orientation of GTAgNPs reduced the toxicity induced by TE and it comparable to chemotherapeutic drugs (Berry and Curtis, 2003).

3.6 Pristine nanoparticles at circulatory environment

The present study examined the agglomeration of NPs in fluids such as PBS, DW, FBS, and DMEM (with or without FBS) by monitoring their turbidity at 540 nm. GTAgNPs showed excellent hydrophilicity in all tested solutions (Fig. 4A). The impact of GTAgNPs on erythrocytes performance found to be biocompatible with no detectable hemolysis (<5%) at a concentration ranging from 0.06 to 5 mg/mL compared to TE (Fig. 4B). At the concentration of above, 10 mg/mL showed a significant effect on RBCs and studied the concentration of 20 mg/mL showed hemolysis (<10%) comparing to TE (Fig. 4B and C). The morphology of RBCs was observed after treatment with GTAgNPs, which showed no significant changes in membrane indicates the absence of aggregation index at the concentration even at 5 mg/mL of GTAgNPs (Fig. 4D, Supporting Information Fig. S13). The anticoagulant and thrombolytic activities of GTAgNPs indicate the maintaining healthy state of blood coagulation system by preventing aggregation of blood cells helps in the formation of thrombus. On another hand, GTAgNPs also exhibited thrombolytic property by dissolving preformed blood clot compared to negative controls (Supporting Information Fig. S14).

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Fig. 4 GTAgNPs stability and hemolytic activity. The stability of GTAgNPs was studied in various media at different time intervals of time. The dispersion of GTAgNPs well maintained and showed excellent stability in water (A). The percentage of hemolysis induced by TE and GTAgNPs presented in figure B in a dose-dependent manner. Compared to TE, GTAgNPs showed excellent biocompatibility after 1 h exposure. And visually inspect the difference of hemolysis before and after centrifugation. Saline and distilled water (DW) were used as a negative and positive control respectively (C). The exposure of GTAgNPs (1 mg/mL) with RBCs for 4 h at room temperature. Figure D clearly indicates, there is no alteration in the cell membrane of RBCs after exposure of GTAgNPs (b, 1 mg) compared to TE (c, 5 mg/mL). After treatment nanoparticles attached to the cell surface are distinguished by particle shape.

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The GTAgNPs does not involve in the normal blood coagulation mechanism and showed delaying of re-calcification time of citrated plasma in presence of EDTA and EGTA are nonspecific divalent metal ion chelator and specific calcium ion chelator respectively (Fig. 5A). Also, GTAgNPs showed significant hydrolysis of a fibrin clot in a dose-dependent manner showing plasmin-like activity (Fig. 5B). GTAgNPs do not turbulence the membrane integrity of RBCs up to a concentration of 1 mg/mL (Fig. 4D). As a concentration of GTAgNPs increases induces the turbulence in RBCs membrane to induce the reactive oxygen species (ROS) and this may be one of the reasons for the production of inflammation agents in the body was confirmed. When NPs enter to blood get in contact with RBCs, in such a condition stability and biocompatibility of NPs are important prerequisites for their application in a biological domain (Kim and Shin, 2014; Huang et al., 2016). Normally, microbial infections and cancer cells induce blood coagulation through triggering induction of procoagulant molecules and proinflammatory cytokines on their surfaces lead to the formation of a blood clot causing injuries organ failure, autoimmune disorders and cardiovascular disorders (Jurasz et al., 2004; ten Cate and Falanga, 2007). Even though blood clotting is necessary to hold back excessive bleeding, maintaining homeostasis is an equally important key factor to achieve a desirable outcome in patient with ischemia (Ilinskaya and Dobrovolskaia, 2013).

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Fig. 5 The effect of GTAgNPs on homeostatic condition. Dose-dependent action of GTAgNPs showed a potential effect on citrated plasma clotting time and fibrin clot lysis. The GTAgNPs does not show any effect up to 1 mg/mL concentration on plasma clotting (A) and dose-dependent lysis of fibrin thread (B) showed effectively by GTAgNPs compared to TE at 1 mg/mL concentration exhibiting strong effect at circulatory system was plotted. Anti-coagulation property of GTAgNPs (C). The test tube assay performed at the different concentration at 35 °C. Even though TE and GTAgNPs showed anti-coagulation property against MRSA 090, TE showed clotting initiation up 1 mg/mL and at 5 mg/mL, showed delaying the clotting of rabbit plasma. But GTAgNPs showed excellent anti-coagulation property against MRSA 090 by delaying the coagulation of rabbit plasma in a given time of recording clotting percentage. Nc-negative control (only rabbit plasma) and Pc-positive control (rabbit plasma with MRSA 090). Regulation of clotting responsible coagulase (Coa) and surface Ig binding protein (SpA) genes (D). In the figure, TE and GTAgNPs treatment showing action against gene regulation in MRSA 090 at 1 h and 24 h of treatment at 1 mg/mL concentration. The GTAgNPs exhibited the profound action against MRSA 090 gene regulation was seen by relative expression of genes (Coa and SpA) compared to housekeeping 16S rRNA gene.

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3.7 Action of GTAgNPs on transcription of Coa and SpA in MRSA090

The rabbit plasma coagulation test was performed at different concentrations of GTAgNPs, showed a significant delay in coagulation of MRSA 090 with respect to positive control (Fig. 5C). Since, the GTAgNPs showed the antimicrobial, thrombolytic, fibrinolytic, and anti-coagulase properties in the present investigation. This remarkable evidence promoted us to deal GTAgNPs on the regulation of Coa and SpA gene expression, we hypothesized that the GTAgNPs effectively affected the transcription of Coa and SpA genes. Attended semi-quantitative PCR results confirmed the down-regulation of Coa and SpA exoprotein genes transcription at 1 mg/mL concentration after 24 h treatment (Fig. 5D). This indicates the significant inhibition of one of the most important virulence factors in MRSA during food poisoning and serious infections such as bacteremia. We presume that the reduction of the staphylococcal coagulation cascade and Ig binding surface virulence factors production was observed due to GTAgNPs induced regulation. The molecules having broad antimicrobial spectrum shows greater efficacy towards some bacteria.

The AgNPs used in many food industries, and biomedical applications than other nanomaterials. Even though the mechanism of toxicity studied in various cell lines and animal model, a rare study has been conducted to provide a clear understanding toxicity and effect on blood system after exposing of nanoparticles (Kim and Shin, 2014; Huang et al., 2016). Microbial clearance from the body is one of the greatest jobs of the immune system which is facilitated by opsonization of target microbe by a component of the complement system and immunoglobulins (Ig). Interestingly, MRSA interference or diminish or delay the opsonization by targeting immune universal step of complement activation (Jongerius et al., 2007; Berends et al., 2014). At hemostasis level, even in presence of coagulation inhibitors such as hirudin, warfarin, heparin and calcium chelators MRSA clots the human or animal blood (Much, 1908). MRSA evolved with a good strategy by exhibiting Ig-binding factors, such as staphylococcal protein A (SpA) and staphylococcal superantigen-like 10 (SSL 10) to block Fcγ effector domain of opsonizing Ig (Forsgren and Quie, 1974; Smith et al., 2011). Another interesting nature of MRSA is, it bypasses the host hemostasis via secreting coagulase (Coa) enzyme usurps the zymogen one of clotting factor (prothrombin) to alter the coagulation cascade of human blood. Which causes an exuberant formation of the network like fibrin shield on staphylococci leads to effective protection from uptake by phagocytes (Friedrich et al., 2003; Cheng et al., 2010; Adams and Bird, 2009). Hence, clearance of MRSA from homeostasis depends on the opening of fibrin shield, regulation of Coa and SpA genes are focused in part of the present study to give molecular approach based evidence to eradicate from the homeostasis (Supporting Information Table S1). The shape of the GTAgNPs in SEM and TEM, and antibacterial activity against MRSA090 was represented (Fig. 6).

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Fig. 6 Synthesis route and mechanism of GTAgNPs against MRSA090 at homeostasis environment. A. The shape of the GTAgNPs: The synthesized GTAgNPs were analyzed by SEM (a) and TEM (b) to confirm the shape and it was depicted. B. The antibacterial activity of GTAgNPs: The different concentration of raw thyme extract (TE) (b) and GTAgNPs (c) was treated against MRSA090 compared to Streptomycin as a positive control and water as negative control (a).

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Despite the global use of nanomaterials in food and medicine applications, it is a prerequisite to evaluate the detailed mechanism of action against the target and their biological effects prior to use are still required. This was elaborated clearly in the present research and showed the overall probable mechanism of GTAgNPs (Fig. 7). The remarkable increase in the emergence of MRSA accelerated and broadened the interest for searching for potent molecule for the development of new therapeutics to treat life-threatening diseases caused by MRSA.

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Fig. 7 Overall mechanism of GTAgNPs on MRSA090: Once GTAgNPs entering to blood environment during MRSA infection, and the tackling effect of GTAgNPs against MRSA090 pathogen at different steps of defending routes described in the present research was depicted. This highlights the exact mechanism of GTAgNPs and in general was drawn.

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