Green synthesis of zinc oxide nanoparticles using Murraya koenigii leaf extract: characterization and anti-infective efficacy against drug-resistant bacteria

A new challenge in treating infection-related diseases has arisen due to the widespread emergence and persistence of multidrug-resistant bacteria. Environmentally friendly methods for producing nanoparticles with potent therapeutic properties, such as antivirulence and antibiofilm effects, are being prioritised to combat the rise in multidrug resistance. An effective substitute for traditional chemical techniques in the formation of metal oxide nanoparticles is the use of extracts from medicinal plants. This work used an aqueous leaf extract of Murraya koenigii to synthesize zinc oxide nanoparticles in an environmentally friendly manner and subsequently characterized. The capacity of ZnO@MK-NPs to suppress biofilms and quorum-sensing-mediated virulence factors against newly isolated MDR strains, Pseudomonas aeruginosa PAO1, and Chromobacterium violaceum ATCC 12472 at sub-MIC levels was evaluated. ZnO@MK-NPs were shown to suppress the synthesis of violacein by 78.03% against C. violaceum 12472. Among the tested virulence factors of P. aeruginosa, 69.87%, 57.49%, 64.36%, 62.5%, and 65.15% reduction in pyocyanin, pyoverdin, protease, rhamnolipid, and swimming motility was observed in the presence of 64 μg/ml MK-ZnONPs. Furthermore, there was a 74.19%, 72.15%, and 84.65% decrease in Serratia marcescens virulence factors such as prodigiosin synthesis, exoprotease activity, and swarming motility at the highest tested sub-MIC. Additionally, ZnO@ MK-NPs showed broad-spectrum antibiofilm action, inhibiting biofilms produced by P. aeruginosa, Escherichia fergusonii, S. marcescens, and C. violaceum by 72.22%, 74.64%, 79.01%, and 80%, respectively, at the highest tested sub-MIC. SEM and CLSM examination revealed a considerable reduction in biofilm growth on glass coverslips. The findings highlight the effectiveness of ZnO@ MK-NPS in preventing newly isolated MDR bacteria from forming biofilms, suggesting that they may be used as biofilm inhibitors after further in vivo research, particularly for topical treatments and medical device coatings. 1. Introduction M. Koenigii (Family: Rutaceae), often called curry leaf or kari patta in various parts of India, has a range of medicinal properties. For instance, its leaves, roots, and bark can be used as tonics to promote digestion and possess anti-flatulent effects. The leaves, after decoction, become bitter and exhibit antipyretic properties. Both the leaves and roots show anti-inflammatory and anti-itching benefits. They can also be employed as analgesics, remedies for piles, agents to reduce heat, and suppressors of thirst. Additionally, they aid in treating blood disorders and leukoderma. Raw green leaves are used as a remedy for diarrhoea, while a paste made by boiling them in milk can be applied to treat poisonous bites and eruptions (Balakrishnan et al., 2020). The plant contains a significant amount of vital antioxidant phytochemicals, including alkaloids, flavonoids, terpenoids, and polyphenols, making it valuable for medicinal applications, food flavouring, and spice production. Its proximate composition is also notable, with 63.2% moisture, 8.8% protein, 39.4% carbohydrate, 1.15% total nitrogen, 6.15% fat, 18.92% total sugars, 14.6% starch, and 6.8% crude fibre. It is also rich in vitamins and minerals: vitamin A (β-carotene) at 6.04 ± 0.02 mg/100 g; vitamin B3 (niacin) at 2.73 ± 0.02 mg/100 g; vitamin B1 (thiamin) at 0.89 ± 0.01 mg/100 g; calcium at 19.73 ± 0.02 mg/100 g; magnesium at 49.06 ± 0.02 mg/100 g; and sodium at 16.50 ± 0.21 mg/100 g, all recognised as significant sources (Nandy & Das, 2023). During nanoparticle synthesis, these materials can act as reducing and stabilising agents. Nanoscience and nanotechnology are the most emerging fields in recent times and are moving forward sharply, along with physics, chemistry, biology, molecular engineering, and so on. A nanoparticle is a particle of matter with a diameter of 1–100 nm, possessing a large surface area. The NPs can be prepared by several physical, chemical, and biological methods, but physical and chemical methods are associated with high energy demand and sometimes generate poisoned and perilous chemicals, which may lead to related dangers (Akl M. Awwad et al., 2020). To minimize these problems, a safe, cost-effective and less hazardous synthesis procedure has already been developed by modern scientists, namely the biological or green method using plant extract with a low concentration of the chemicals. The ”green synthesis” approaches/methods are receiving much interest in the present research because they decrease waste generation, reduce pollution, and employ cleaner, renewable auxiliary materials. Hence, the green production of nanoparticles is likewise rated as an environmentally benign method (Qais et al., 2020). Nanoparticles may pass across the external exopolysaccharide membrane to release antimicrobials precisely to the targeted cells without degradation. Consequently, it serves as a potentially effective treatment method for the development of a new, efficient biofilm-targeting mechanism (Dolatabadi et al., 2018). Globally, infections brought on by multidrug-resistant bacteria are becoming more common causes of morbidity and mortality. There are few antibiotic choices for infections brought on by multidrug-resistant bacteria. These medical difficulties emphasize how urgently we need new, potent antimicrobial approaches to combat antimicrobial resistance (Lee et al., 2019). The formation of plant-based nanoparticles has played a significant role in the area of nanotechnology by establishing totally eco-friendly substances that can be used in nanomedicine with negligible or no toxicity (Ahmad et al., 2015). The use of nanotechnology in medicine is growing, particularly as a novel approach to infectious illnesses. Zinc oxide has been recognised as one of the safest metal oxides by the U.S. Food and Drug Administration (Sundrarajan et al., 2015). It has numerous applications in engineering, biological, and medical fields. ZnONPs possess several biological and medicinal uses, such as cytotoxic activity (Mishchenko et al., 2019), antimicrobial and fungicidal effects (Dadi et al., 2019), anti-inflammatory activity, wound healing, and antidiabetic properties (El-Gharbawy et al., 2016). Metal nanoparticles like ZnONPs, AgNPs, and CuONPs release metal ions that interact with DNA and generate reactive oxygen species, causing membrane damage and bacterial cell death through oxidative stress (Cherian et al., 2019, 2020; de Melo et al., 2022). Recently, green-synthesized nanoparticles have shown activity against QS-mediated virulence factors in pathogenic bacteria. However, data on broad-spectrum green-synthesized active nanoparticles with enhanced efficacy and stability remain limited. Consequently, we hypothesised that green synthesis of zinc oxide nanoparticles using bioactive plant extract could produce nanoparticles with improved efficacy and stability against bacterial pathogens. This study demonstrates the green synthesis of ZnO NPs employing leaf extract of M. koenigii for their anti-infective potential against QS-regulated virulence and biofilms of pathogenic bacteria and newly isolated MDR bacteria. 2. Materials and methods 2.1 Materials Triphenyl tetrazolium chloride (TTC) and azocasein were acquired from SRL Pvt. Ltd. and Sigma Aldrich, USA, respectively. We purchased orcinol and microbiological media (LB Agar) from Hi-Media, India. Every chemical, reagent, and organic solvent was of analytical grade. 2.2 Plant material collection and synthesis of zinc oxide nanoparticles M. koenigii leaves were collected locally from Aligarh, UP, India. The identity of the plant was confirmed by the Botany Department, AMU, Aligarh. After being cleaned of debris and dust, the leaves were dried in the shade for four days. The sample has been submitted to the departmental repository with the voucher specimen (MZK-MK-19/20). The M. koenigii aqueous extract was made by adding 5 g of powdered dry leaves in double-distilled water (100 ml), followed by heating at 80°C for 60 min while stirring using a magnetic stirrer at 800 rpm and then filter. 50 ml of extract was mixed with 50 ml of 0.45 M Zinc acetate dihydrate [Zn(CH 3 COO) 2 .2H 2 O], and then 50 ml of 0.45 M NaOH was added to the mixture to synthesize zinc oxide nanoparticles (ZnO@MK-NPs). After stirring continuously for two hours at 800 rpm, the three mixtures produced a yellow precipitate. The precipitates were then washed three times and dried in an oven at 100°C for 1 hour. The powders were mashed and calcined at 400°C in a muffle furnace (Demissie et al., 2020). The flow chart depicting the green synthesis of zinc oxide nanoparticles is shown in Fig. S1 . 2.3 Characterization of zinc oxide nanoparticles ZnO@MK-NPs were characterized at the preliminary stage using UV-visible spectroscopy. As previously mentioned, X-ray diffraction, FTIR, TEM, and SEM-EDX were performed after the UV-visible spectra of ZnO@MK-NPs were acquired in the wavelength range of 300–600 nm (Haris & Ahmad, 2024b; Qais et al., 2020). To evaluate the stability of ZnO@MK-NPs, the spectral measurements were carried out at regular intervals of 15 days for up to 6 months (Fig. S2) . 2.4 Bacterial cultures and growth conditions E. fergusonii (Previously isolated in the lab and characterized using 16S rRNA gene sequence analysis and showing multidrug resistance behaviour to 9 antibiotics; Accession no: PP157582), S. marcescens (Previously isolated in the lab and characterized using 16S rRNA gene sequence analysis and showing multidrug resistance behaviour to 10 antibiotics; Accession no: PP157584), P. aeruginosa PAO1 (gifted by Prof. R. J. C. McLean, Texas State, University, USA), and C. violaceum 12472 (ATCC, Manassas, VA, USA) were used in this study. All cultures were grown and maintained in Luria-Bertani medium (15.0 g tryptone, 0.5% yeast extract, and 0.5% NaCl) unless otherwise specified. 2.5 Assays for the determination of anti-QS activity of ZnO@MK-NPs 2.5.1 Growth curve analysis The effect of sub-MICs of ZnO@MK-NPs on cell growth kinetics was determined. Each bacterial strain was inoculated into 25 ml LB broth with or without different sub-MICs of ZnO@MK-NPs, and the OD at 600 nm was monitored at regular intervals of 2 h till 20 hr. 2.5.2 Extraction and quantification of violacein The previously described standard methodology (McLean et al., 2004) was used to qualitatively evaluate the violacein inhibitory action of ZnO@MK-NPs. After overlaying LB agar plates with five millilitres of LB soft agar (0.5% w/v agar) containing C. violaceum 12472, the plates were allowed to stand for twenty minutes. On the solid media, sterile discs (8 mm) impregnated with ZnO@MK-NPs in different concentrations were placed. Plates were incubated at 30°C for 24 hours, and the pigment inhibition was noted as a halo zone surrounding the discs. The diameter (mm) of pigment inhibition was used to express the findings. Quantitative evaluation of violacein inhibition in the presence of ZnO@MK-NPs was also carried out as previously described (Taganna et al., 2011). C. violaceum 12472 with and without different sub-MICs of ZnO@MK-NPs was cultured in liquid LB medium at 30°C for 24 h. Following the incubation, 1 ml of vortexed broth was centrifuged at 12,000 rpm for 10 min. In 1 ml of DMSO, the pellet was reconstituted and vortexed for 5 min to solubilize the cell-bound violacein. The solution was again centrifuged to remove the bacterial cells, and the OD 585 of the cell-free DMSO solution was acquired for violacein. 2.5.3 Inhibition of virulence factors of S. marcescens 2.5.3.1 Prodigiosin production In Luria-Bertani media, prodigiosin pigment was assessed using the established procedure described previously (Haris & Ahmad, 2024b; Slater et al., 2003). In brief, for 18 hours at 30ºC, S. marcescens was cultivated both in the absence and presence of sub-MICs of ZnO@MK-NPs. The bacterial cells were pelleted by centrifuging two millilitres of the growing culture at 10,000 rpm for five minutes. After a thorough 5-minute vortexing procedure, the pellet was dissolved in 1 millilitre of an acidified ethanol solution. After that, the sample was centrifuged again for five minutes at 13,000 rpm to remove debris. A UV-2600 spectrophotometer was used to detect the absorbance at 534 nm. 2.5.3.2 Exoprotease activity The exoprotease activity of S. marcescens was evaluated using the azocasein degradation assay described previously (Salini & Pandian, 2015). Briefly, S. marcescens was grown for 18 hours at 30ºC with and without sub-MICs of ZnO@MK-NPs. After centrifuging the culture, 100 microliters of the supernatant were mixed with one millilitre of 0.3% (w/v) azocasein. The reaction mixture was shaken and incubated at 37ºC for 15 minutes. After stopping the reaction with 0.5 ml of ice-cold TCA, the insoluble azocasein was removed by centrifugation. A UV-2600 spectrophotometer was used to measure the absorbance at 400 nm. 2.5.3.3 Motility assay To evaluate the swarming motility, 5 microliters of the overnight-cultivated bacterial culture were spotted on LB plates (0.5% agar) and left to dry at room temperature. The control group consisted of plates devoid of ZnO@MK-NPs. After 18 hours of incubation, the plates were examined, and the swarming motility was assessed by measuring the swarm’s zone diameter (Haris & Ahmad, 2024b). 2.5.4 Inhibition of virulence factors of P. aeruginosa 2.5.4.1 Pyocyanin production The pyocyanin test was conducted in a Pseudomonas broth medium to optimize the synthesis of pyocyanin using the described methodology (Essar et al., 1990; Haris & Ahmad, 2024a). The absorbance of the resulting deep red or pink aqueous phase was measured at 520 nm. 2.5.4.2 Pyoverdin production Pyoverdin levels were measured spectrophotometrically according to the prior standard approach (Ankenbauer et al., 1985). In brief, a cell-free supernatant was obtained by centrifuging P. aeruginosa cells that had been cultivated overnight, both with and without sub-MICs of ZnO@MK-NPs. 900 µl of 50 mM Tris-HCl (pH 7.4) was combined with 100 µl of supernatant. The RF-5301PC spectrofluorometer (Shimadzu, Japan) was used to measure the fluorescence emission signal from the sample at 460 nm following excitation at 400 nm. 2.5.4.3 Proteolytic activity The aforementioned azocasein degradation test was used to determine the proteolytic activity of the bacterial strains under the influence of various sub-MICs of the fractions (Husain et al., 2017). 2.5.4.4 Rhamnolipid production The rhamnolipid test was performed using the orcinol technique described previously (Husain et al., 2017). Briefly, the cell supernatant was obtained by centrifugation after P. aeruginosa was cultured for 18 hours at 37 °C with and without sub-MICs of ZnO@MK-NPs. A mixture of 300 microliters of cell-free culture supernatant and 600 microliters of diethyl ether was made. A minute was spent vortexing the mixture. Following separation and drying at 37°C, the organic phase was reconstituted using a 100 microliter solution of deionised water. To 900 µl of an orcinol solution, 100 µl of each sample was added. For thirty minutes, the mixture was heated to 80ºC. The sample was allowed to cool at room temperature for 15 minutes before the absorbance at 421 nm was measured. 2.5.4.5 Motility assay Soft agar plates were utilised in the previously described method to evaluate bacterial motility inhibition (Haris & Ahmad, 2024a). 2.6 Biofilm inhibition assay Crystal violet method. The quantitative assessment of biofilm inhibition was evaluated on a 96-well microtitre plate using the previously described crystal violet technique (O’Toole & Kolter, 1998). Following an overnight incubation, bacterial cultures with various sub-MICs of ZnO@MK-NPs were introduced into the wells containing LB media. After three rounds of cleaning with sterile phosphate buffer to get rid of extra broth and planktonic cells, the wells were left to air dry for twenty minutes. The biofilms were gently rinsed three times to get rid of the stain after being stained for 15 seconds with 200 µl of crystal violet. The crystal violet bound to the biofilm was extracted using 200 microlitres of 90% ethanol, and the absorbance at 620 nm was measured using a microplate reader (Thermo Scientific Multiskan EX, UK). Light microscopy of biofilm. Biofilms on glass coverslips were inhibited using the previously described method (Sybiya Vasantha Packiavathy et al., 2012). Briefly, 60 μl overnight-grown cultures of the bacterial pathogens were seeded into a 24-well culture plate containing 3 ml of culture media. Furthermore, sterile glass coverslips with the highest sub-MICs of ZnO@MK-NPs were placed in the wells. After a 24-hour incubation period, the loosely attached cells were rinsed three times with sterile phosphate buffer solution and allowed to air dry for 20 minutes. Slides were left to air dry for half an hour after being stained with crystal violet solution. A light microscope (Olympus BX60, Model BX60F5, Olympus Optical Co., Ltd., Japan) equipped with a colour VGA camera (Sony, Model no. SSC-DC-58AP, Japan) was used to view the biofilms. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) of biofilm. As previously indicated, biofilms developed on coverslips. Unbound bacterial cells were removed after being cleaned with sterile phosphate buffer and fixed with 2.5% glutaraldehyde. The adherent cells and biofilms were then dried for ten minutes using an ethanol gradient. The slides were air-dried and gold-coated before visualisation. A JEOL-JSM 6510 LV was utilised by the University Sophisticated Instrumentation Facility (USIF), AMU, Aligarh, to take the SEM micrographs. For CLSM, biofilms were grown on glass surfaces using the same procedure as previously mentioned. The biofilms were then stained for 20 minutes with acridine orange (0.1%). The images were taken at USIF, AMU, Aligarh, with a Zeiss LSM780. 2.7 Statistical analysis Three duplicates of each experiment were conducted. The study’s results are average values with plus minus standard deviation. The control and treatment groups were compared using the t-test. P values≤ 0.05 were considered significant. 3. Results 3.1 Characteristics of ZnO@MK-NPs The zinc oxide nanoparticles were synthesized using an aqueous extract of M. koenigii, initially characterized by UV-Vis spectroscopy. The absorption spectrum is shown in Fig.1 . ZnO@MK-NPs exhibited a sharp absorption at 360 nm, attributed to surface plasmon resonance (SPR). Fig. 1. UV–visible spectra of green synthesized zinc oxide nanoparticles (ZnO@MK-NPs). Fig. 2 shows the XRD pattern of ZnO@MK-NPs nanoparticles synthesized using zinc acetate dihydrate and M. koenigii leaf extract. The formation of biosynthesized ZnO@MK-NPs was also confirmed by X-ray diffraction measurements. The diffraction peaks appeared at a 2 θ value of ≈31.76°, 34.42°, 36.24°, 47.54°, 56.59°, 62.86°, 66.41°, 67.97, and 69.06° corresponding to (100), (002), (101), (102), (110), (103), (200), (112), and (201) crystal planes, respectively. All the diffraction peaks were properly assigned using the JCPDS file card No. 36-1451, and characteristic peaks for pure ZnO were observed in the XRD patterns, confirming the formation of ZnO NPs. Furthermore, using the Debye-Scherrer formula, the particle size averaged to be 10.72 nm. Fig. 2. X-ray diffraction pattern of ZnO@MK-NPs. Transmission electron microscopy was performed to decipher the size and shape of ZnO@MK-NPs. A TEM image of ZnO@MK-NPs at 50,000X magnification is shown in Fig. 3A . Most of the ZnO@MK-NPs were irregular in shape with variations in size. Moreover, the morphological analysis of ZnO@MK-NPs was performed by SEM analysis, as shown in Fig. 3B(i) . The nanoparticles were observed as spherical to oval or spheroidal in shape, further validating the TEM results. EDX revealed a high signal for Zinc and Oxygen, which confirms the presence of Zinc in the oxide form (Fig. 3 B(ii)) . The composition of each element contained in the analyte is obtained from the EDX, which gives strong peaks of 68.89% for Zinc and 31.11% for Oxygen (Fig. 3 B(iii)) . Fig. 3. (A) Transmission electron micrograph of synthesized ZnO@MK-NPs. (B) SEM and EDX analysis of ZnO@MK-NPs. Panel (B-i) shows the SEM images of ZnO@MK-NPs; Panel (B-ii) represents the energy dispersive X-ray spectrum of ZnO@MK-NPs; Panel (B-iii) represents the histogram of weight % of major elements in ZnO@MK-NPs. The chemical characterization of ZnO@MK-NPs was performed using FTIR spectroscopy. The FTIR spectrum of ZnO@MK-NPs is shown in Fig. 4 . Different bands were observed at 3416 cm −1, 2922 cm −1, 2854 cm −1, 2374 cm −1, 1714 cm −1, 1626 cm −1, 1454 cm −1, 1382 cm −1, 1162 cm −1, 1028 cm −1, 720 cm −1, 680 cm −1, and 542 cm −1 . A sharp absorption band at 542 cm -1 confirms the presence of ZnO nanoparticles. Fig. 4. FTIR spectrum of ZnO@MK-NPs 3.2 anti-QS efficacy of nanoparticles on certain QS-regulated bacterial traits At sub-inhibitory concentrations, ZnO@MK-NPs did not show any significant growth inhibitory effect against bacterial pathogens (Fig. S3) . All experiments on inhibition of QS and biofilms were performed at their respective sub-MICs. ZnO@MK-NPs were studied for their ability to inhibit the QS-regulated virulence factors against C. violaceum, P. aeruginosa, and S. marcescens . 3.2.1. Effect of ZnO@MK-NPs on violacein production ZnO@MK-NPs were primarily assessed against the production of violacein by C. violaceum 12472 by the disc diffusion method at 16 μg/ml/disc and 32 μg/ml/disc concentrations. ZnO@MK-NPs exhibited varying levels of pigment inhibition at tested concentrations (Fig.5A) . The quantitative estimation of violacein inhibition was done at varying levels of sub-MICs (2 – 32 μg/ml) of ZnO@MK-NPs. The ability of ZnO@MK-NPs to reduce violacein production was measured spectrophotometrically. At 2, 4, 8, 16, and 32 µg/ml, 23.36, 33.02, 48.59, 68.69, and 78.03% inhibition of violacein production was recorded as compared to the control (Fig. 5B) . Fig.5. (A) Effect of ZnO@MK-NPs on the violacein pigment production by C. violaceum 12472 by disc diffusion. A-i: ZnO@MK-NPs (16 µg/ml); A-ii: M. koenigii (Aqueous: 1 mg/ml); A-iii: ZnO@MK-NPs (32 µg/ml); A-iv: DMSO (control). (B) Quantitative analysis of violacein inhibition in C. violaceum in the absence and presence of ZnO@MK-NPs. Data are represented as mean values of triplicate readings, and the bar is the SD. The percent inhibition is shown on the secondary y-axis. * indicates P ≤0.05 with respect to control; ** indicates P ≤ 0.01 with respect to control; *** indicates P ≤ 0.005 with respect to control. With the increasing concentration of ZnO@MK-NPs, prodigiosin pigment synthesis was seen to decrease in a concentration-dependent manner. Prodigiosin production was reduced by 30.64, 37.09, 64.51 and 74.19% following treatment with 4, 8, 16, and 32 µg/ml of ZnO@MK-NPs, respectively (Table 1) . A concentration-dependent response to exoprotease activity was clearly shown by the findings of our study (Table 1) . Exoprotease activity was inhibited by 20.25, 34.17, 63.29, and 72.15%, at concentrations of 4, 8, 16, and 32 µg/ml of ZnO@MK-NPs, respectively. ZnO@MK-NPs were further evaluated for their ability to prevent S. marcescens from swarming over agar plates. Within 18 hours of incubation, the untreated control of S. marcescens swarmed through the whole plate with a 89 mm zone diameter . After being treated with 4, 8, and 16 µg/ml of ZnO@MK-NPs, the swarming motility was reduced by 25.84, 40.44, and 61.42% respectively. The swarming motility was decreased by 84.65% at the highest level of sub-MIC (32 µg/ml) used. | ZnO@MK-NPs (μg/ml) | Virulence factors production | || | Prodigiosin a | Protease activity b | Swarming motility c | | | Untreated control | 0.62 ± 0.02 | 0.79 ± 0.04 | 89.00 ± 1.00 | | 4 | 0.43 ± 0.04*** (30.64) | 0.63 ± 0.03** (20.25) | 66.00 ± 2.00*** (25.84) | | 8 | 0.39 ± 0.05*** (37.09) | 0.52 ± 0.04* (34.17) | 53.00 ± 2.64*** (40.44) | | 16 | 0.22 ± 0.03*** (64.51) | 0.29 ± 0.03*** (63.29) | 34.33 ± 2.30*** (61.42) | | 32 | 0.16 ± 0.05*** (74.19) | 0.22 ± 0.04*** (72.15) | 13.66 ± 1.15*** (84.65) | | a Prodigiosin were expressed as the absorbance at 534 nm. b Total protease activity is expressed as the absorbance at 400 nm. c Swarming motility is expressed as swarm diameter in mm. | ||| | The data represent the mean value of three independent experiments. *significance at P ≤ 0.05, ** significance at P ≤ 0.01, *** significance at P ≤ 0.005. The value in parentheses represents the percent reduction over control. | The data shown in Table 2 demonstrated the in vitro anti-quorum-sensing activity of ZnO@MK-NPs against QS-regulated virulence traits of P. aeruginosa PAO1. ZnO@MK-NPs significantly inhibited the virulence factors in a concentration-dependent manner. The production of pyocyanin was found to be significantly decreased in a concentration-dependent manner after the treatment with ZnO@MK-NPs. Treatment with 8, 16, 32, and 64 μg/ml ZnO@MK-NPs resulted in 14.06, 22.95, 51.79, and 69.87% inhibition of pyocyanin, respectively (Table 2) . Likewise, the pyoverdin fluorescence was maximally inhibited by 57.49% in the presence of 64 μg/ml ZnO@MK-NPs in a concentration-dependent manner. A similar pattern of decline in protease activity was observed. The protease activity was inhibited by 64.36% at the highest tested sub-MIC (64 μg/ml), compared to the untreated control. The rhamnolipid content was also decreased by 62.5% at the highest tested sub-MIC (64 μg/ml) in a concentration-dependent manner. There was a 65.15% reduction in swarming motility in the presence of 64 μg/ml ZnO@MK-NPs, as depicted in Table 2. Table 2: Effect of sub-MICs of ZnO@MK-NPs on inhibition of virulence factors in P. aeruginosa PAO1. & The suppression of biofilm production by ZnO@MK-NPs against test bacteria is shown in Table 3. In the presence of 4, 8, 16, and 32 µg/ml ZnO@MK-NPs, the biofilms of S. marcescens were inhibited by 16.04, 39.50, 60.49, and 79.01% respectively, indicating a concentration-dependent inhibition. Similarly, treatment with 4, 8, 16, and 32 µg/ml ZnO@MK-NPs resulted in biofilm suppression in C. violaceum 12472 by 32, 41.33, 61.33, and 80% respectively. The biofilms of E. fergusonii were inhibited by 18.30, 25.35, 42.25, and 74.64% on supplementation of 4, 8, 16, and 32 µg/ml ZnO@MK-NPs, respectively, in growth media. A similar trend of biofilm inhibition was also recorded against P. aeruginosa PAO1with maximum inhibition of 72.22%, at the highest tested concentration of 64 µg/ml ZnO@MK-NPs. | ZnO@MK-NPs (μg/ml) | OD 620nm | ||| | S. marcescens | E. fergusonii | C. violaceum 12472 | P.aeruginosa PAO1 | | | Untreated control | 0.81 ± 0.07 | 0.71 ± 0.04 | 0.75 ± 0.03 | 1.08 ± 0.06 | | 4 | 0.68 ± 0.04* (16.04) | 0.58 ± 0.04* (18.30) | 0.51 ± 0.05*** (32.00) | 0.96 ± 0.04* (11.11) | | 8 | 0.49 ± 0.03*** (39.5) | 0.53 ± 0.06*** (25.35) | 0.44 ± 0.05* (41.33) | 0.78 ± 0.05* (27.77) | | 16 | 0.32 ± 0.03*** (60.49) | 0.41 ± 0.05*** (42.25) | 0.29 ± 0.05*** (61.33) | 0.61 ± 0.04*** (43.51) | | 32 | 0.17±0.05*** (79.01) | 0.18 ± 0.03*** (74.64) | 0.15 ± 0.04*** (80.00) | 0.52 ± 0.07*** (51.85) | | 64 | NT | NT | NT | 0.3 ± 0.05*** (72.22) | | Biofilm formation is expressed as OD 620 after incubation with crystal violet. The data represent the mean value of three independent experiments. *significance at P ≤ 0.05, *** significance at P ≤ 0.005 with respect to control. NT represents Not Tested due to their respective MIC concentration. The value in parentheses represents the percent reduction over control. | Microscopic analysis on glass coverslips was used to further evaluate the quantitative results of the reduction in biofilm formation acquired by the microtitre plate assay. Untreated bacterial cells produced thick, mat-like materials on the surface of the glass, as shown by the light microscopic image (Fig. S4). Cell adhesion to the glass surface was decreased after treatment with the highest tested sub-MIC (MIC/2) of ZnO@MK-NPs. The treated cells are organized into small microcolonies with fewer biofilms. SEM images further showed the biofilm inhibition with scattered cells, in the presence of the highest tested sub-MIC (MIC/2) of ZnO@MK-NPs, as compared to the untreated control (Fig.S5). The above findings were also validated by the CLSM study. In the presence of the highest tested sub-MIC (MIC/2) of ZnO@MK-NPs, the group of cells that had grown on glass coverslips was extensively diminished (Fig.6). Fig.6. Confocal laser scanning microscopic images of E. fergusonii (EZP1), C. violaceum 12472 , S. marcescens (SZU1) and P. aeruginosa biofilm in the absence and presence of the highest tested sub-MIC (MIC/2) of ZnO@MK-NPs. 4. Discussion The plant-mediated synthesis of NPs is relatively underexplored; therefore, in this study, M. koenigii leaf extract was used for the synthesis of ZnO@MK-NPs. Aqueous extract of M. koenigii was used to synthesize the zinc oxide nanoparticles. Over the past decade, researchers have shown interest in bio-inspired green methods for the nanoscale synthesis of ZnONPs, which are increasingly used in various industries, pharmaceutics, and healthcare (Ahmed et al., 2017). Extracts of a huge fraction of plant varieties, such as leaf extract of Ailanthus altissima (Shabbir Awan et al., 2023) , Hibiscus subdariffa (Bala et al., 2015), Ochradenus baccatus (Al-Shabib et al., 2018), Pandanus odorifer (Hussain et al., 2019), Evolvulus alsinoides (Yadav et al., 2023), Cayratia pedata (Jayachandran et al., 2021) , Cocos nucifera (Rahman et al., 2022) , fruit extract of Ailanthus altissima (Akl M. Awwad et al., 2020) , Myristica fragrans (Faisal et al., 2021) , have been used in bio-inspired synthesis of stable and varied size and shape ZnONPs. The initial characterization of ZnO@MK-NPs was performed using UV–Vis spectroscopy. ZnO@MK-NPs exhibited a sharp absorption at 360 nm, which is due to the surface plasmon resonance (SPR), and it is assigned to the intrinsic band-gap absorption of ZnO owing to the electron transitions from the valence band to the conduction band (Elumalai & Velmurugan, 2015). Our results corroborate with a previous study that found ZnO-NPs produced using N. arbor-tristis flower extract displayed a sharp band at 365 nm (Jamdagni et al., 2018). XRD analysis also showed that all the diffraction peaks fit well with the hexagonal wurtzite structure of ZnO NPs (Jamdagni et al., 2018). Moreover, using the Debye-Scherrer formula, the particle size was averaged at 10.72 nm. The finding corroborates previous results in which the size of ZnO-NPs synthesized using M. koenigii leaf extract was 19.53 nm (Lakshmikandhan, 2020). Transmission electron microscopy was performed to decipher the size and shape of ZnO@MK-NPs. Most of the ZnO@MK-NPs were irregular in shape with variations in size. Moreover, the morphological analysis of ZnO@MK-NPs was performed by SEM analysis. The nanoparticles were observed as spherical to oval, or spheroidal in shape. Variations in the shape and size of green synthesized nanoparticles have been documented previously (Husain et al., 2022; Jamdagni et al., 2018). Such variation in the shape of green synthesized nanoparticles is due to the fact that some of the nanoparticles are capped and stabilized at smaller sizes, while other particles are stabilized at larger sizes. The phytocompounds present in the extract are responsible for the reduction of zinc and stabilization or capping of the particles. A similar report has been documented previously, where the size of ZnO-NPs ranged from 10 to 90 nm (Khatami et al., 2018). The chemical characterization of ZnO@MK-NPs was performed using FTIR spectroscopy. Different bands were observed at 3416 cm −1, 2922 cm −1, 2854 cm −1, 2374 cm −1, 1714 cm −1, 1626 cm −1, 1454 cm −1, 1382 cm −1, 1162 cm −1, 1028 cm −1, 720 cm −1, 680 cm −1, and 542 cm −1 . The broad absorption band observed in 3416 cm −1 can be assigned to O–H stretch and H–bonded in alcohol or phenol groups. The absorption peak observed at 2922 cm −1 corresponds to asymmetric C–H stretching in alkanes. A symmetric C-H stretching absorption peak at 2854 cm −1 was also found in the spectra. The absorption band at 2374 cm −1 is due to the presence of CO 2 molecules in the environment (Jamdagni et al., 2018). The peak at 1714 cm −1 referred to the presence of stretching vibrations of non-ionic carboxylic acids or acid esters. The absorption peak at 1626 cm −1 indicates the N–H bends in primary amines. A peak around 1454 cm −1 indicates the presence of methylene groups (CH 2 ). The presence of the C-O-H bending mode was confirmed by the peak at 1382 cm −1 . The peak observed at 1162 cm −1 was due to the C = C stretching in the aromatic ring in polyphenols and aliphatic amines. The peak at 1028 was due to the C–N stretch in aliphatic amines. The band ranging from 600 to 400 cm −1 is attributed to the M-O stretching (Zn-O) (Sangeetha et al., 2011). Previous studies reported a relatively small absorption peak at 575 cm −1, which confirms the existence of ZnO-NPs (Shabbir Awan et al., 2023). Serious concerns have been raised by the widespread prevalence and global expansion of antibiotic resistance in healthcare settings and the environment. Our findings demonstrated the significant inhibition of QS-regulated functions in C. violaceum, P. aeruginosa, and S. marcescens by green-synthesised ZnO@MK-NPs. Quorum sensing controls the production of prodigiosin, a red-pigmented secondary metabolite in S. marcescens (Sakuraoka et al., 2019). Our results clearly showed a concentration-dependent response in exoprotease activity. The primary source of proteolytic activity in S. marcescens is exoproteinases, which are considered one of its main pathogenic features (Qais et al., 2021). High motility is characteristic of virulent strains of S. marcescens and plays a role in certain hospital-acquired infections (Salini & Pandian, 2015). S. marcescens adheres to solid surfaces through motility mediated by flagella, promoting biofilm formation (Alagely et al., 2011). Additionally, ZnO@MK-NPs were tested for their antibiofilm activity against C. violaceum 12472, P. aeruginosa PAO1, S. marcescens, and E. fergusonii . Bacterial biofilms are communities that adhere to surfaces and are embedded in a self-secreted matrix of polymeric substances, including polysaccharides, proteins, and extracellular DNA. Biofilms are significant in medical science because of their role in bacterial pathogenesis and the challenges they pose to antibiotic treatment (Hall & Mah, 2017). Biofilms are believed to serve as reservoirs of drug-resistant genes. Moreover, antibiotic resistance can spread rapidly within biofilms through horizontal gene transfer (Abe et al., 2020). Bacteria within biofilms exhibit increased tolerance and resistance to antimicrobial agents due to restricted antibiotic penetration (Balcázar et al., 2015). Targeting bacterial biofilms is considered a novel approach in modern antimicrobial drug discovery, given their importance in antibiotic resistance and clinical infections. Nanoparticles can interact with biofilms and diffuse through water channels that act as nutrient pathways (Peulen & Wilkinson, 2011). Furthermore, they can attach to and penetrate the bacterial membrane, causing intracellular accumulation and bacterial cell death (Ma et al., 2014). As bacteria develop within biofilms, they coordinate gene expression, which enhances their resistance to both chemical and physical therapeutic agents (Pompilio et al., 2015). Our findings demonstrated a concentration-dependent antibiofilm activity against MDR bacteria. At a sub-MIC value of 32 µg/ml of ZnO@MK-NPs, maximum inhibition was observed against C. violaceum 12472 (80%), followed by S. marcescens (79.01%), E. fergusonii (74.64%), and P. aeruginosa PAO1 (51.85%), compared to untreated controls. The data align with previous research; for example, zinc oxide nanoparticles synthesised using leaf extract of Ochradenus baccatus inhibited biofilm formation of C. violaceum, P. aeruginosa, E. coli, K. pneumoniae, and S. marcescens, at sub-inhibitory concentrations (Al-Shabib et al., 2018). In another study, zinc oxide nanoparticles have been reported to generate reactive oxygen species within P. aeruginosa cells, inhibiting biofilm development as well (Dwivedi et al., 2014). 5. Conclusion The global increase in drug-resistant microbes has created a demand for new strategies to combat infections. Among these, developing or screening biofilm inhibitors is seen as a new target. In this study, ZnONPs were synthesized using the leaf extract of M. koenigii and subsequently characterized. They were spherical, oval, or spheroidal, with an average particle size of 10.72 nm. The therapeutic potential of ZnO@MK-NPs was demonstrated by their broad-spectrum anti-QS and anti-biofilm activities against bacterial pathogens. Disruptions in biofilm growth on abiotic surfaces were observed through confocal microscopy and SEM. The findings highlight the effectiveness of ZnO@ MK-NPs in preventing bacterial pathogens from forming biofilms, suggesting that they may be used as biofilm inhibitors after further in vivo research, particularly for topical treatments and medical device coatings.

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Authors Metrics & Citations Metrics Article Usage 366views 130downloads Citations Download citation Zarrin Haris, Iqbal Ahmad. Green synthesis of zinc oxide nanoparticles using Murraya koenigii leaf extract: characterization and anti-infective efficacy against drug-resistant bacteria. Authorea. 08 September 2025. DOI: https://doi.org/10.22541/au.175734016.63646601/v1 DOI: https://doi.org/10.22541/au.175734016.63646601/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.