A combination of silver nanoparticles and visible blue light enhances the antibacterial efficacy of ineffective antibiotics against methicillin-resistant Staphylococcus aureus (MRSA)

Abstract

Background: Silver nanoparticles (AgNPs) are potential antimicrobials agents, which can be considered as an alternative to antibiotics for the treatment of infections caused by multi-drug resistant bacteria. The antimicrobial effects of double and triple combinations of AgNPs, visible blue light, and the conventional antibiotics amoxicillin, azithromycin, clarithromycin, linezolid, and vancomycin, against ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were investigated.

Methods: The antimicrobial activity of AgNPs, applied in combination with blue light, against selected isolates of MRSA was investigated at 1/2-1/128 of its minimal inhibitory concentration (MIC) in 24-well plates. The wells were exposed to blue light source at 460 nm and 250 mW for 1 h using a photon emitting diode. Samples were taken at different time intervals, and viable bacterial counts were determined. The double combinations of AgNPs and each of the antibiotics were assessed by the checkerboard method. The killing assay was used to test possible synergistic effects when blue light was further combined to AgNPs and each antibiotic at a time against selected isolates of MRSA.

Results: The bactericidal activity of AgNPs, at sub-MIC, and blue light was significantly (p < 0.001) enhanced when both agents were applied in combination compared to each agent alone. Similarly, synergistic interactions were observed when AgNPs were combined with amoxicillin, azithromycin, clarithromycin or linezolid in 30-40 % of the double combinations with no observed antagonistic interaction against the tested isolates. Combination of the AgNPs with vancomycin did not result in enhanced killing against all isolates tested. The antimicrobial activity against MRSA isolates was significantly enhanced in triple combinations of AgNPs, blue light and antibiotic, compared to treatments involving one or two agents. The bactericidal activities were highest when azithromycin or clarithromycin was included in the triple therapy compared to the other antibiotics tested.

Conclusions: A new strategy can be used to combat serious infections caused by MRSA by combining AgNPs, blue light, and antibiotics. This triple therapy may include antibiotics, which have been proven to be ineffective against MRSA. The suggested approach would be useful to face the fast-growing drug-resistance with the slow development of new antimicrobial agents, and to preserve last resort antibiotics such as vancomycin.

Keywords: Azithromycin; Checkerboard assay; Clarithromycin; Double and triple combinations; Linezolid; Multidrug-resistance; Nonconventional antimicrobials; Vancomycin.

Fig. 1

Antimicrobial activity of the AgNPs at different concentrations in combination with blue light against MRSA isolates. Cell suspensions were exposed to either the silver compound alone at sub-MICs (a 1/2, b 1/4, c 1/8, and d 1/16 MIC), or blue light alone at 460 nm and 250 mW for 1 h, or combination of both agents. Viable colony count was recorded as mean ± SD of three independent experiments. AgNPs silver nanoparticles, CFU colony forming unit, MIC minimum inhibitory concentration, SD standard deviation

Fig. 2

Double combination of AgNPs with the amoxicillin, vancomycin, linezolid, azithromycin or clarithromycin against ten MRSA isolates. The combination was assessed by the checkerboard method and the response was evaluated by calculation of the fraction inhibitory index (FIC) as follow: synergistic if the FIC index is 0.5 or less, indifference if the FIC index more than 0.5 and less than four, and antagonistic if the FIC index more than four. AgNPs silver nanoparticles, AMX amoxicillin, AZM azithromycin, CLR clarithromycin, LNZ linezolid, VAN vancomycin

Fig. 3

Combination of AgNPs, blue light, and azithromycin against two isolates of MRSA. The triple combination of AgNPs with the blue light and azithromycin against two isolates of MRSA was assessed. The isolates were selected on the basis of synergistic response in checkerboard assay. Based on the best result of the combination in the checkerboard assay, the concentrations of the two agents were used as follow: a Isolate C41: AgNPs at 1/16 of the MIC, and azithromycin at 2 µg/mL. b Isolate N8: AgNPs at 1/16 of the MIC, and azithromycin at 0.25 µg/mL. AgNPs silver nanoparticles, CFU colony forming unit, AZM azithromycin, SD standard deviation

Fig. 4

Combination of AgNPs, blue light, and clarithromycin against two isolates of MRSA. The triple combination of AgNPs with the blue light and clarithromycin against two isolates of MRSA was assessed. The isolates were selected on the basis of synergistic response in checkerboard assay. Based on the best result of the combination in the checkerboard assay, the concentrations of the two agents were used as follow: a Isolate C51: AgNPs at 1/8 of the MIC, and azithromycin at 0.25 µg/mL. b Isolate C41: AgNPs at 1/512 of the MIC, and azithromycin at 0.25 µg/mL. AgNPs silver nanoparticles, CFU colony forming unit, CLR clarithromycin, SD standard deviation

Fig. 5

Combination of AgNPs, blue light, and linezolid against two isolates of MRSA. The triple combination of AgNPs with the blue light and linezolid against two isolates of MRSA was assessed. The isolates were selected on the basis of synergistic response in checkerboard assay. Based on the best result of the combination in the checkerboard assay, the concentrations of the two agents were used as follow: a Isolate C19: AgNPs at 1/2 of the MIC, and azithromycin at 0.25 µg/mL. b Isolate N5: AgNPs at 1/2 of the MIC, and azithromycin at 8 µg/mL. AgNPs silver nanoparticles, CFU colony forming unit, LNZ linezolid, SD standard deviation

Fig. 6

Visualization of the effect of combination of AgNPs, blue light, and azithromycin on MRSA isolate N8 using transmission electron microscope (TEM). The antimicrobial efficacy of the AgNPs at 1/16 MIC, blue light and azithromycin at 0.25 µg/mL alone and in triple combination against isolate N8 was visualized by TEM at ×80,000. The photos show the response of the bacteria to the following treatments: a Drug-free and light-free (control). b AgNPs alone at 1/16 of its MIC. c Blue light exposure at 460 nm and 250 mW for 1 h. d Azithromycin alone at 0.25 µg/mL. e Triple combination of AgNPs, blue light and the azithromycin. Signs of membrane damage and cell lysis were more pronounced in cells treated with a combination of three agents compared to cells treated with each agent alone. Small arrows indicate the location of the AgNPs and the sites of the damage