In vitro and in vivo antimicrobial activity of combined therapy of silver nanoparticles and visible blue light against Pseudomonas aeruginosa

Abstract

Silver nanoparticles (AgNPs) have been used as potential antimicrobial agents against resistant pathogens. We investigated the possible therapeutic use of AgNPs in combination with visible blue light against a multidrug resistant clinical isolate of Pseudomonas aeruginosa in vitro and in vivo. The antibacterial activity of AgNPs against P. aeruginosa (1×10(5) colony forming unit/mL) was investigated at its minimal inhibitory concentration (MIC) and sub-MIC, alone and in combination with blue light at 460 nm and 250 mW for 2 hours. The effect of this combined therapy on the treated bacteria was then visualized using transmission electron microscope. The therapy was also assessed in the prevention of biofilm formation by P. aeruginosa on AgNP-impregnated gelatin biopolymer discs. Further, in vivo investigations were performed to evaluate the efficacy of the combined therapy to prevent burn-wound colonization and sepsis in mice and, finally, to treat a real infected horse with antibiotic-unresponsive chronic wound. The antimicrobial activity of AgNPs and visible blue light was significantly enhanced (P<0.001) when both agents were combined compared to each agent alone when AgNPs were tested at MIC, 1/2, or 1/4 MIC. Transmission electron microscope showed significant damage to the cells that were treated with the combined therapy compared to other cells that received either the AgNPs or blue light. In addition, the combined treatment significantly (P<0.001) inhibited biofilm formation by P. aeruginosa on gelatin discs compared to each agent individually. Finally, the combined therapy effectively treated a horse suffering from a chronic wound caused by mixed infection, where signs of improvement were observed after 1 week, and the wound completely healed after 4 weeks. To our knowledge, this combinatorial therapy has not been investigated before. It was proved efficient and promising in managing infections caused by multidrug resistant bacteria and could be used as an alternative to conventional antibiotic therapy.

Keywords: biofilm; bioplastic disc; invasive sepsis; murine model; nonconventional therapy; wound colonization.

Figure 1

Antimicrobial activity of silver nanoparticles (AgNPs) in combination with blue light against Pseudomonas aeruginosa. Notes: Cell suspensions were exposed to either the silver compound alone at minimal inhibitory concentration (MIC) and sub-MIC, blue light alone at 460 nm and 250 mW for 2 hours, or a combination of both agents. Viable colony count was recorded as the mean of three separate experiments. Note that the combination enhanced the antimicrobial activity of both agents when the AgNPs were tested at MIC (A), 1/2 MIC (B), and 1/4 MIC (C), but not at 1/8 MIC (D). Abbreviations: CFU, colony forming unit; SD, standard deviation.

Figure 2

Visualization of the effect of combination of silver nanoparticles (AgNPs) and blue light on Pseudomonas aeruginosa using transmission electron microscopy. Notes: The bacterial cells in Mueller Hinton Broth medium were treated as follows: (A) 8 mg/mL AgNPs alone at zero time. (B) 8 mg/mL AgNPs alone after 2 hours. (C) 8 mg/mL AgNPs in combination with blue light for 2 hours. (D) Blue light alone after 2 hours of exposure. Note that at zero time most of AgNPs adhered to the cell surfaces whereas the majority entered the cells after 2 hours. Prominent damage is clear in cells that received the combined therapy compared to other cells which received each treatment alone. Small arrows indicate the location of the AgNPs and long arrows show the cellular damage.

Figure 3

Effect of the combined antimicrobial therapy of silver nanoparticles (AgNPs) and blue light in preventing biofilm formation by Pseudomonas aeruginosa on gelatin-based bioplastic discs. Notes: AgNP-impregnated gelatin discs with or without combination with the blue light were inoculated with 1×10⁷ CFU/mL of P. aeruginosa. The formed biofilm was then dislodged by sonication and the viable adherent cells were counted. Viable colony count was recorded as the mean of three separate experiments. Note that the combined therapy significantly reduced the number of adherent cells in the biofilm compared to the control and the silver compound or blue light alone. Error bars represent SD. Abbreviations: CFU, colony forming unit; SD, standard deviation.

Figure 4

Effect of silver nanoparticles (AgNPs) in combination with blue light in preventing wound colonization and invasive sepsis by Pseudomonas aeruginosa in a murine model. Notes: The AgNPs at 8 mg/mL, alone and in combination with blue light, were tested to prevent burn wound infection and dissemination by P. aeruginosa. The combined therapy was more effective to reduce the bacterial load in the wound compared to other treatments; other treatments failed to prevent dissemination to internal organs except to the liver. Error bars represent SD. Abbreviation: SD, standard deviation.

Figure 5

Efficiency of the combined therapy of silver nanoparticles (AgNPs) with blue light in the treatment of antibiotic-unresponsive chronic wound in an infected horse. Notes: A 13-year-old female thoroughbred horse suffering antibiotic-unresponsive chronic skin wound on its back was treated with AgNPs in combination with blue light. The photos show the wound before treatment (A), and after 1 (B), 2 (C), and 4 weeks (D) of treatment. Note that the wound completely healed after 4 weeks of treatment.