Antioxidant, Antibacterial and Antibiofilm Potential of Green Synthesized Silver-Zinc Oxide Nanocomposites from Curcuma longa Extract against Multi-Drug-Resistant Enteroaggregative E. coli "2279

Enteroaggregative Escherichia coli (EAEC) has been widely recognized as one of the leading causes of infantile diarrhoea and nutrient malabsorption in developing as well as developed countries. In recent years, drug resistance, particularly multi-drug resistance (MDR), among the EAEC strains has been widely documented and could result in a therapeutic stumble. Antimicrobial alternatives are widely employed to curb this emerging public health crisis. In the present study, a facile one-pot synthesis of silver/zinc oxide nanocomposites (Ag/ZnO NCs) using a methanolic extract of stem and leaves of Curcuma longa was performed. The synthesis of Ag/ZnO NCs was confirmed using UV-vis spectroscopy and Fourier transform infrared spectroscopy, while the thermal stability was ascertained by thermogravimetric analysis with differential thermogravimetric analyses, and crystallinity was determined using powder X-ray diffraction. The shape and size of the green synthesized Ag/ZnO NCs, determined using field-emission–scanning-electron microscopy and transmission electron microscopy, revealed an irregular polycrystalline morphology with a size of 31.34 ± 1.27 nm. Later, the antibacterial potential of the green synthesized Ag/ZnO NCs evaluated against MDREAEC strains revealed a minimum inhibitory concentration of 31.25 μg/mL and a minimum bactericidal concentration ranging from 62.50 to 125 μg/mL. Moreover, the green synthesized Ag/ZnO NCs inhibited the biofilm-forming ability of the tested strains of MDR-EAEC. Furthermore, concentrationdependent antioxidant activity was exhibited by the green synthesized Ag/ZnO NCs, as evidenced by the ABTS assay and reducing power assay. Overall, this study demonstrated the antibacterial potential of Ag/ZnO NCs synthesized using C. longa extracts with antifouling as well as antioxidant properties, which could be used as an alternative therapeutic candidate.


Introduction
Antimicrobial resistance (AMR) is a significant global public health threat in recent times [1]. Enteroaggregative E. coli (EAEC) has recently been recognized as one of the major emerging enteric pathogens owing to its increased recovery from diarrhoeal episodes around the world [2]. Furthermore, the dissemination of multi-drug-resistant (MDR)-EAEC pathotypes has been closely linked to morbidity, case fatality and healthcare expenses [3]. 2 of 7 In this context, there is an urgent need to look into alternatives to traditional antibiotics, since they are becoming less effective in curing drug-resistant infections [4].
Recently, nanotechnology using metal or metal oxide nanoparticles (NPs) has been promoted as a viable alternative strategy for combating AMR [5]. The antibacterial properties of silver (Ag) NPs against a wide range of drug-resistant pathogens are well-known [6]. Similarly, zinc oxide (ZnO) NPs have received recent attention, since they have been designated as 'generally regarded as safe' (GRAS) by U.S. Food and Drug Administration (US-FDA) and are known to possess characteristic anti-inflammatory, antibacterial, antifungal, antidiabetic, and photocatalytic properties [7]. A combination of Ag-and ZnO-forming Ag/ZnO nanocomposite (NC) heterostructures with the synergistic generation of reactive oxygen species (ROS) has recently been recognized as a broad-spectrum antibacterial agent in tackling drug-resistant infections [8].
The green synthesis of NPs has recently received a lot of attention since these NPs are easier to produce, biodegradable, and more economical and eco-friendly when compared to the NPs synthesized by conventional physical and chemical methods [9]. Since time immemorial, C. longa, a perennial rhizomatous plant belonging to the Zingiberaceae family, has been reported to be an integral component of Asian food and has widely been used as a food preservative and colouring agent in culinary dishes [10]. In this regard, the present study aims to evaluate the antibacterial and anti-biofilm potential of green synthesized Ag/ZnO NCs using methanolic extract of stem and leaves of C. longa against MDR-EAEC pathogens and to explore its antioxidant properties.

Bacterial Strains
Antimicrobial susceptibility testing [11] and PCR assays [12] were used to revalidate the MDR field strains of EAEC (E1; E2; E3). E. coli ATCC 25922 served as the quality control strain.

Preparation of C. longa Methanolic Extract
The C. longa leaves and stems obtained from a turmeric plantation near the university campus (11 • 33 02.8" N; 76 • 01 46.1" E) were thoroughly washed and finely ground using a mortar and pestle. The ground material (100 g) was filtered using Whatman No. 1 filter paper after being soaked in methanol (Loba Chemie Pvt. Ltd., India) overnight. The extract thus obtained (100 mL) was kept at 4 • C and employed for the green synthesis of Ag/ZnO NCs.

In Vitro Antibacterial Activity of Ag/ZnO NCs
In order to assess the in vitro antibacterial efficacy of green synthesized Ag/ZnO NCs against the MDR field isolates of EAEC, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by micro-broth dilution technique [11].

In Vitro Antioxidant Activity of Ag/ZnO NCs
The in vitro antioxidant activity of green synthesized Ag/ZnO NCs were compared using the reducing power assay and an ABTS•+ (2,2 -azinobis (3-ethylbenzothiazoline-6sulfonic acid)-based free radical scavenging assay [13].

In Vitro Antibiofilm Efficacy
The in vitro antibiofilm efficacy of green synthesised Ag/ZnO NCs was determined by performing a crystal violet assay against the MDR-EAEC strains in 96-well microtiter plates at 24 and 48 h [14].

Green Synthesis of Ag/ZnO NCs
The methanolic extract of C. longa was used in the present study to successfully synthesize Ag/ZnO NCs under continual stirring at 25 • C for 12 h. A brown precipitate formed at the bottom of the conical flask indicated that Ag/ZnO NCs had been synthesized, and UV-vis spectroscopy confirmed the synthesis.

Characterization of Green Synthesised Ag/ZnO NCs
The results of characterizations were reported earlier [8] in our study. Briefly, Ag/ZnO NCs exhibited surface plasmon resonance (SPR) bands at 293 nm, 340 nm, and 450 nm [8]. These SPR peaks could be the result of interphase interactions between nearby colloidal NPs caused by electromagnetic coupling between Ag and ZnO [15]. When compared to the reference chart, the FTIR spectra of the greenly synthesised Ag/ZnO NCs showed distinctive peaks at 520 cm −1 , 730 cm −1 , 1020 cm −1 , 1380 cm −1 , 1620 cm −1 , 2920 cm −1 , and 3420 cm −1 [8]. An initial weight loss of around 4% from 40 • C to 100 • C was exhibited by the green synthesised Ag/ZnO NCs, which was further confirmed by the DTG graph [8]. This weight loss may have been caused by the loss of water molecules linked to the green synthesised Ag/ZnO NCs [4], with an exothermic peak observed at 97 • C. Furthermore, the progressive thermal degradation of green synthesised Ag/ZnO NCs between 200 • C and 450 • C was noted, with an exothermic peak at 320 • C. The weight of the green synthesised Ag/ZnO NCs steadily decreased as the annealing temperature has increased, leaving 65% of the sample at the endpoint temperature and undermining the thermal stability.
During FE-SEM evaluation, Ag/ZnO NCs exhibited an agglomerated poly-crystalline morphology [8]. In addition, the TEM images depicted the typical spherical shape of Ag/ZnO NCs [8], with a mean diameter of 31.34 ± 1.27 nm and a lattice fringe spacing of around 0.22 nm. Interestingly, the SAED pattern of Ag/ZnO NCs complemented our PXRD findings, which, in turn, proved the poly-crystalline structure [8].

In Vitro Antibacterial Activity of Green Synthesised Ag/ZnO NCs
The green synthesised Ag/ZnO NCs demonstrated MIC of 31.25 µg/mL and MBC values ranging from 62.50 µg/mL to 125 µg/mL against all the tested MDR-EAEC strains using the micro-broth dilution technique ( Table 1). The antibacterial activity of Ag/ZnO NCs might be due to the generation of ROS, such as hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), superoxide anion (O 2 •− ), hydroxyl radical (OH • ), and hypochlorous acid (HOCl), leading to oxidative stress and thereby inducing the desired bacterial cell toxicity [8]. Furthermore, the Ag NPs coated on the surface of the ZnO NPs contain electrons produced by the photocatalytic reactions of the ZnO NPs, resulting in a strong interaction between the semiconductor zinc oxide and the metallic silver, which ruptures the cell membrane and boosts the antibacterial activity [18]. Antioxidants are substances that shield cells from the harmful effects of reactive oxygen species (ROS) [19]. The in vitro antioxidant-scavenging capacity of green synthesised Ag/ZnO NCs was evaluated by employing ABTS and reducing power assays with ascorbic acid as the reference standard. In this study, a dose-dependent increase in their antioxidant capabilities, indicating an improved capacity to scavenge free radicals, was exhibited by the green synthesised Ag/ZnO NCs in ABTS (Figure 1a) and reducing power assays (Figure 1b). However, compared to the conventional antioxidant, ascorbic acid, the antioxidant activity of greenly synthesised Ag/ZnO NCs was lower. Furthermore, C. longa has been regarded as one of the most promising sources of natural antioxidants due to the high concentrations of polyphenols, flavonoids, tannins, and ascorbic acid it contains [20]. Hence, a combination of Ag-and ZnO-forming plant-based nanomaterials has been found to exhibit an increased antioxidant capacity and anti-proliferative action, which eliminates free radicals [21]. membrane and boosts the antibacterial activity [18].

In Vitro Antioxidant Activity of Green Synthesised Ag/ZnO NCs
Antioxidants are substances that shield cells from the harmful effects of reactive oxygen species (ROS) [19]. The in vitro antioxidant-scavenging capacity of green synthesised Ag/ZnO NCs was evaluated by employing ABTS and reducing power assays with ascorbic acid as the reference standard. In this study, a dose-dependent increase in their antioxidant capabilities, indicating an improved capacity to scavenge free radicals, was exhibited by the green synthesised Ag/ZnO NCs in ABTS (Figure 1a) and reducing power assays (Figure 1b). However, compared to the conventional antioxidant, ascorbic acid, the antioxidant activity of greenly synthesised Ag/ZnO NCs was lower. Furthermore, C. longa has been regarded as one of the most promising sources of natural antioxidants due to the high concentrations of polyphenols, flavonoids, tannins, and ascorbic acid it contains [20]. Hence, a combination of Ag-and ZnO-forming plant-based nanomaterials has been found to exhibit an increased antioxidant capacity and antiproliferative action, which eliminates free radicals [21].

In Vitro Antibiofilm Efficacy of Green Synthesised Ag/ZnO NCs
The present scenario necessitates the research on an antibacterial agent that can work against biofilms since these are known to be closely linked to numerous microbial diseases [22]. Ag/ZnO NCs might be regarded as a potential therapeutic alternative against bacterial biofilms owing to their intrinsic antibacterial activity. In this study, the in vitro anti-biofilm efficacy of green synthesised Ag/ZnO NCs against the MDR-EAEC

In Vitro Antibiofilm Efficacy of Green Synthesised Ag/ZnO NCs
The present scenario necessitates the research on an antibacterial agent that can work against biofilms since these are known to be closely linked to numerous microbial diseases [22]. Ag/ZnO NCs might be regarded as a potential therapeutic alternative against bacterial biofilms owing to their intrinsic antibacterial activity. In this study, the in vitro anti-biofilm efficacy of green synthesised Ag/ZnO NCs against the MDR-EAEC pathotypes was evaluated by employing a crystal violet staining assay at 24 and 48 h. The green synthesised Ag/ZnO NCs (1X MIC) demonstrated a highly significant (p < 0.001) biofilm inhibition after 24 h and 48 h, since all the tested MDR-EAEC isolates exhibited a drop in biomass compared to their respective controls (untreated bacterial cultures) (Figure 2). The antibiofilm activity of green synthesised Ag/ZnO NCs might be due to the increased generation of ROS along with the suppression of exopolysaccharides of MDR-EAEC, which are a crucial component of bacterial biofilms [4,23]. Hence, the green synthesised Ag/ZnO NCs can be thought of as an effective choice to prevent and inhibit the formation of bacterial biofilms since the MDR-EAEC cultures are highly susceptible to these.
( Figure 2). The antibiofilm activity of green synthesised Ag/ZnO NCs might be due to th increased generation of ROS along with the suppression of exopolysaccharides of MDR EAEC, which are a crucial component of bacterial biofilms [4,23]. Hence, the gree synthesised Ag/ZnO NCs can be thought of as an effective choice to prevent and inhibi the formation of bacterial biofilms since the MDR-EAEC cultures are highly susceptible t these.

Conclusions
Using the methanolic extract of C. longa leaves and stem, we successfully synthesised Ag/ZnO NCs in this study. While the green synthesised Ag/ZnO NCs exhibited absorbance peaks (340 nm and 450 nm) that corresponded to ZnO NPs and Ag NPs in UV Vis spectroscopy, a progressive thermal deterioration was seen between 200 °C and 45 °C that reflected a 21% weight loss in TGA/DTA plots. The poly-crystalline morpholog of Ag/ZnO NCs with homogeneous Ag distribution on the surface of ZnO was furthe supported by the PXRD, SEM, and TEM. In addition, the promising antibacterial efficac of green synthesised Ag/ZnO NCs against MDR pathogens was also discovered using th micro-broth dilution technique. Furthermore, the green synthesised Ag/ZnO NC exhibited significant anti-biofilm and concentration-dependent antioxidant activit against the tested MDR-EAEC strains. Overall, the study demonstrated a environmentally benign way to make Ag/ZnO NCs from C. longa extract, which might b used as a promising antibacterial candidate with potential antioxidant and antibiofilm action. However, the effectiveness of Ag/ZnO NCs must be further validated b conducting in vivo clinical trials in appropriate laboratory models.

Conclusions
Using the methanolic extract of C. longa leaves and stem, we successfully synthesised Ag/ZnO NCs in this study. While the green synthesised Ag/ZnO NCs exhibited absorbance peaks (340 nm and 450 nm) that corresponded to ZnO NPs and Ag NPs in UV-Vis spectroscopy, a progressive thermal deterioration was seen between 200 • C and 450 • C that reflected a 21% weight loss in TGA/DTA plots. The poly-crystalline morphology of Ag/ZnO NCs with homogeneous Ag distribution on the surface of ZnO was further supported by the PXRD, SEM, and TEM. In addition, the promising antibacterial efficacy of green synthesised Ag/ZnO NCs against MDR pathogens was also discovered using the micro-broth dilution technique. Furthermore, the green synthesised Ag/ZnO NCs exhibited significant anti-biofilm and concentration-dependent antioxidant activity against the tested MDR-EAEC strains. Overall, the study demonstrated an environmentally benign way to make Ag/ZnO NCs from C. longa extract, which might be used as a promising antibacterial candidate with potential antioxidant and antibiofilm action. However, the effectiveness of Ag/ZnO NCs must be further validated by conducting in vivo clinical trials in appropriate laboratory models.