Biosynthesis of silver nano-drug using Juniperus excelsa and its synergistic antibacterial activity against multidrug-resistant bacteria for wound dressing applications

Abstract

We report here the synthesis of silver nanoparticles (AgNPs) from an aqueous extract of Juniperus excelsa and their use as an antimicrobial agent on their own or in combination with antibiotics in inhibiting multidrug-resistant bacteria (MDR). One strategy of bacterial infection control in wound healing is AgNP biosynthesis. We collected bacterial strains of patient skin infections from Al-Adwani Hospital. Phenotyping, biotyping, and molecular characterizations were applied using 16S rRNA gene analysis of bacterial isolates. Our results identified tested MDR bacteria Staphylococcus aureus strains (methicillin-resistant and methicillin-susceptible) and Proteus mirabilis. Gas chromatography/mass spectrometry (GC/MS) analysis was used to identify the Juniperus excelsa biomolecules in the leaf extract acting as both reducing and capping agents in the biosynthesis of AgNPs. The AgNPs appeared hexagonal and spherical in shape upon transmission electron microscope (TEM) analysis. The AgNP sizes ranged from 16.08 to 24.42 nm. X-ray diffraction (XRD) analysis confirmed the crystalline nature of the particles. The minimum inhibitory concentrations (MICs) of the AgNPs against the tested MDR bacteria ranged from 48 to 56 µg/ml, while the minimum bactericidal concentrations (MBCs) of the AgNPs against the tested strains ranged from 72 to 96 µg/ml. The AgNPs showed a good synergistic efficacy with Cefaclor, Cefoxitin, and Erythromycin. Their efficiency showed a threefold increase in the inhibition of tested strains when used in wound dressing, due to the AgNPs potentially activating the antibiotics. Consequently, we can use AgNPs with Cefaclor, Cefoxitin, and Erythromycin antibiotics as alternative antimicrobial agents, and they could be utilized in wound dressing to prevent microbial infections.

Keywords: Antibiotic resistance; Silver nanoparticles; Synergistic effect; Wound dressing.

Fig. 1

Scheme of silver biosynthesis explained change of the solution colour to brown by adding Juniperus excelsa leaf extract to silver nitrate at optimum conditions

Fig. 2

Effect of AgNO₃ concentration (a), Juniperus excelsa leaf extract concentration (b), pH (c), temperature (d), and incubation time (e) on the formation of AgNPs as recorded by the UV–Vis spectroscopy

Fig. 3

TEM images of AgNPs (a) and XRD analysis of phytosynthesized AgNPs using Juniperus excelsa leaf extract (b)

Fig. 4

FTIR analysis of Juniperus excelsa leaf extract (a) and AgNPs (b)

Fig. 5

A phylogenetic tree of multidrug-resistant bacterial isolates from skin relied on the nucleotide sequences of 16S rRNA genes, constructed by neighbour-joining method. The scale bar displays the genetic distance. The number presented next to each node displays the percentage bootstrap value of 1000 replicates. The Pseudomonas aeruginosa SP12 was treated as the out-group. The GenBank accession numbers of the bacteria are presented in parentheses

Fig. 6

Inhibition zone of Juniperus excelsa leaf extract (1) and AgNPs (2) against (a) S. aureus STA6, b S. aureus STA7 and Proteus mirabilis PRO3 (c).

Fig. 7

Inhibition zone of Juniperus excelsa leaf extract dressing (1) antibiotic-dressing (2) and AgNPs + antibiotic-dressing (3) against (a) S. aureus STA6, b S. aureus STA7 and Proteus mirabilis PRO3 (c)

Fig. 8

Transmission electron micrographs of Proteus mirabilis incubated with 56 μg mL⁻¹ AgNPs + antibiotic. a Arrows point to binding sites of NPs with cells; b Arrows denote lysed cells