Hydrogel Containing Biogenic Silver Nanoparticles and Origanum vulgare Essential Oil for Burn Wounds: Antimicrobial Efficacy Using Ex Vivo and In Vivo Methods Against Multidrug-Resistant Microorganisms

Abstract

Background/Objectives: Wounds from burns are susceptible to infections, allowing multidrug-resistant microorganisms to complicate treatments and patient recovery. This highlights the development of new strategies to control these microorganisms. This work evaluated the antibacterial activity of hydrogels containing biogenic silver nanoparticles (bio-AgNP) and Origanum vulgare essential oil (OEO) against multidrug-resistant bacteria. Methods: The formulations were subjected to organoleptic, pharmacotechnical, and stability characterization and antimicrobial activity assessment by time-kill tests and alternative methods, an ex vivo model using porcine skin, and an in vivo model using Galleria mellonella. Results: All hydrogels maintained their stability after the thermal stress. The hydrogel containing bio-AgNP + OEO 1% (HAgNP + OEO1) presented bactericidal effectiveness, within 2 h, against both Gram-positive and Gram-negative multidrug-resistant bacteria in the time-kill test. For alternative testing, HAgNP + OEO1 was compared with 1% silver sulfadiazine (SS) and the base formulation. In the ex vivo test, both HAgNP + OEO1 and SS treatments showed a similar reduction in superficial washing of the burn for S. aureus 999, while for P. aeruginosa, the reduction was more expressive for SS treatment. In the burn tissue, HAgNP + OEO1 treatment was more effective against S. aureus 999, while for P. aeruginosa 1461, both formulations were similarly effective. In the Galleria mellonella test, survival rates after 48 h were 84% for the control group (base) and 50% for both HAgNP + OEO1 and SS treatment groups. Conclusions: This study demonstrates that the hydrogel combining antimicrobials is effective against multidrug-resistant microorganisms, offering a promising alternative for the treatment of infected burns.

Keywords: Galleria mellonella; Klebsiella pneumoniae; Pseudomonas aeruginosa; Staphylococcus aureus; green nanotechnology; oregano essential oil; porcine skin.

Figure 1

Spreadability of hydrogels at the preliminary stability test. Ei: spreadability of the sample for a given weight I; mm²: square millimeters; T0: zero time; T15: after 15 days of thermal stress; Base: formulation without actives; HAgNP: hydrogel containing bio-AgNP 500 µM; HO1: hydrogel containing OEO 1%; HO0.5: hydrogel containing OEO 0.5%; HAgNP + OEO1: hydrogel containing bio-AgNP 500 µM + OEO 1%; HAgNP + OEO0.5: hydrogel containing bio-AgNP 500 µM + OEO 0.5%.

Figure 2

Time–kill curve of clinical isolates: (A) P. aeruginosa 1461; (B) P. aeruginosa 1634; (C) P. aeruginosa 117; (D) K. pneumoniae 11091; (E) S. aureus 999. Each isolate presented the p-value at different times: (A) * Base: 0–24 h; 2–8 h; 2–24 h; 8–24 h: p < 0.0001; ** HOEO0.5: 0–2 h; 0–8 h; 0–24 h; 2 -24 h; 8–24 h: p < 0.0001; *** HAgNP; HOEO1; HAgNP + OEO0.5; HAgNP + OEO1; silver sulfadiazine 1%: 0–2 h; 0–8 h; 0–24 h: p < 0.0001; (B) * Base; HAgNP; HOEO1; HAgNP + OEO0.5; HAgNP + OEO1; silver sulfadiazine 1%: 0–2 h; 0–8 h; 0–24 h: p < 0.0001; ** HOEO0.5: 0–2 h; 0–8 h; 0–24 h; 2–8 h; 2–24 h: p < 0.0001; (C) * Base: 0–24 h; 2–8 h; 2–24 h; 8–24 h: p < 0.0001; ** HOEO0.5: 0–24 h; 2–24 h; 8–24 h: p < 0.0001; *** HAgNP; HOEO1; HAgNP + OEO0.5; HAgNP + OEO1; silver sulfadiazine 1%: 0–2 h; 0–8 h; 0–24 h: p < 0.0001; (D) * Base: 0–2 h; 0–8 h; 0–24 h; 2–8 h; 2–24 h: p < 0.0001; ** HOEO0.5: 0–2 h; 0–8 h; 0–24 h; 2–8 h; 8–24 h: p < 0.0001; *** HAgNP; HOEO1; HAgNP + OEO0.5; HAgNP + OEO1; silver sulfadiazine 1%: 0–2 h; 0–8 h; 0–24 h: p < 0.0001; (E) * Base: 0–8 h; 0–24 h; 2–8 h; 2–24 h: p < 0.0001; ** HOEO0.5: 0–2 h; 0–8 h; 0–24 h; 2–24 h; 8–24 h: p < 0.0001; *** HAgNP; HAgNP + OEO0.5; silver sulfadiazine 1%: 0–2 h; 0–8 h; 0–24 h; 2–8 h; 2–24 h: p < 0.0001; **** HOEO1: 0–2 h; 0–8 h; 0–24 h; 2–8 h; 2–24 h; 8–24 h: p < 0.0001; ***** HAgNP + OEO1: 0–2 h; 0–8 h; 0–24 h: p < 0.0001.

Figure 3

Evaluation of antimicrobial treatments in the ex vivo model. (A) P. aeruginosa 1461 and (B) S. aureus 999. Treatments that presented a value of p ≤ 0.005 are indicated by the symbol (*). Base: hydrogel without the actives; HAgNP + OEO1: hydrogel containing bio-AgNP 500 µM + OEO 1%.

Figure 4

Survival curve in the in vivo model. Galleria mellonella was infected with P. aeruginosa 1461 and treated with the antimicrobial hydrogel containing bio-AgNP 500 µM + OEO 1% (HAgNP + OEO1). Log-rank p-value 0.1386. Base: hydrogel without the actives; Bacterial control: bacteria with saline (PBS 0.1 M, pH 7.2).

Figure 5

Larvae during the healing process. Figures (A–D) are related to time zero, that is, burning, infection, and treatment at the first time point, while Figures (E–G) represent the larvae after 48 h (end of the experiment). (A) Larvae before burning; (B) Larvae after burning and infection; (C) Larvae infected and after application of HAgNP + OEO1 formulation; (D) Larvae infected and after application of SS; (E) Larvae infected and without treatment; (F) Larvae infected and after treatment with HAgNP + OEO1, during the healing process; (G) Larvae infected and after treatment with SS, during the healing process.