Microwave Accelerated Green Synthesis of Stable Silver Nanoparticles with Eucalyptus globulus Leaf Extract and Their Antibacterial and Antibiofilm Activity on Clinical Isolates

Abstract

A simple and rapid microwave assisted method of green synthesis of silver nanoparticles (AgNPs) was developed using aqueous leaf extract of Eucalyptus globulus(ELE), and their antibacterial and antibiofilm potential investigated. With this aim, the aqueous solutions of ELE and AgNO3(1 mM) were mixed (1:4 v/v), and microwave irradiated at 2450 Mhz, for 30 sec. The instant color change of the ELE-AgNO3 mixture from pale yellow to dark brown indicated ELE-AgNPs synthesis. The intensity of peak at 428 nm in UV-Vis spectra, due to the surface plasmon resonance of AgNPs, varied with the amount of ELE, AgNO3 concentration, pH and time of incubation. The biosynthesized ELE-AgNPs were characterized by UV-visible spectroscopy, XRD, TEM, SEM-EDX, FTIR and TGA analyses. The size of ELE-AgNPs was determined to be in range of 1.9-4.3 nm and 5-25 nm, with and without microwave treatment, respectively. SEM exhibited the capping of AgNPs with the ELE constituents, and validated by FTIR analysis. The FTIR data revealed the presence of plant organic constituents and metabolites bound to ELE-AgNPs, which contributes for their stability. The antimicrobial activity of ELE-AgNPs was assessed by growth and biofilm inhibition of extended spectrum β-lactamase (ESBL) producing Pseudomonas aeruginosa, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) clinical bacterial isolates. The results demonstrated that S. aureus were more sensitive to ELE-AgNPs than E. coli and P. aeruginosa. MRSA exhibited higher sensitive than MSSA, whereas P. aeruginosa were more sensitive than E. coli to ELE-AgNPs treatment. Also, significant (83 ± 3% and 84 ± 5%) biofilm inhibition was observed in case of S. aureus and P. aeruginosa, respectively. The results elucidated environmentally friendly, economical and quick method for production of colloidal bio-functionalized ELE-AgNPs, for effectual clinical applications, as broad spectrum antibacterial agents and biofilm inhibitors.

Fig 1. Graphical representation of ELE-AgNPs synthesis depicting scheme-I and-II.

Fig 2. UV-vis absorption spectra of ELE-AgNPs.

Panels show absorbance measurement during ELE-AgNPs synthesis as a function of time, as: (A) without microwave irradiation; a—c represents incubation time as 1, 2 and 3 h at 37°C, and (B) with microwave irradiation for 30 sec, followed by incubation in dark; a-g represents 0, 5, 10, 15, 20, 25 and 30 min at ambient temperature.

Fig 3. UV-vis absorption spectra of ELE-AgNPs synthesized by microwave assisted method.

Spectra under different reaction conditions during synthesis were recorded as a function of: (A) AgNO₃ concentration (a-e as 0.2, 0.4, 0.6, 0.8 and 1.0 mM) in presence of 1 ml ELE; (B) time (a-g as 0, 5, 10, 15, 20, 25 and 30 min in presence of 1:4 v/v ELE-AgNO₃); (C) ELE (a-d as 1, 2, 3 and 4 ml) with 1 mM AgNO₃; and (D) pH (a-d as 2.0, 4.0, 6.0 and 8.0) in 1:4 v/v ELE-AgNO₃ reaction mixture.

Fig 4. X-ray pattern of ELE-AgNPs synthesized following the scheme-I.

Fig 5. TEM micrograph of synthesized ELE-AgNPs.

Panel (A) depicts the TEM images of ELE-AgNPs synthesized by microwave assisted approach, as specified in Fig 1, scheme-II; Panel (B) shows the images of ELE-AgNPs synthesized at 37°C.

Fig 6. SEM and EDX analyses of ELE-AgNPs.

Panel (a) shows the SEM images of ELE-AgNPs; Panel (b) represents the energy dispersive X-ray spectrum of ELE-AgNPs.

Fig 7. FTIR spectra of synthesized ELE-AgNPs.

Panels (a) and (b) depict the spectra of ELE- AgNPs and ELE alone, respectively.

Fig 8. Antibacterial activity of ELE-AgNPs.

Histogram showing change in absorbance of growth culture of clinical bacterial isolates at ELE-AgNPs concentrations of 3 and 30 μg/ml after 2, 6 and 10 h treatment at 37°C. Panel (a) Gram-negative (ESBL positive) E.coli (b) Gram-negative (ESBL positive) P. aeruginosa; (c) Gram-positive (methicillin-sensitive) S. aureus and (d) Gram-positive (methicillin-sensitive) S. aureus. The data represent the mean ± S.D of two independent experiments done in triplicate.

Fig 9. Interaction of ELE-AgNPs with bacterial cells.

Representative SEM images showing the cellular damage and surface binding of ELE-AgNPs with (b) methicillin resistant S. aureus and (d) E.coli cells. The images in panels (a) and (c) show the untreated controls of S. aureus and E.coli cells, respectively.

Fig 10. ELE-AgNPs concentration dependent inhibition of bacterial biofilm formation.

Panels (a) and (b) show the inhibition of biofilm formation by P. aeruginosa (ESBL positive), and methicillin resistant S. aureus (MRSA), respectively. The data represent the mean ± S.D of two independent experiments done in triplicate. *p< 0.01; **p<0.001.