Comparison of methods to detect the in vitro activity of silver nanoparticles (AgNP) against multidrug resistant bacteria

Abstract

Background: Multidrug resistant microorganisms are a growing challenge and new substances that can be useful to treat infections due to these microorganisms are needed. Silver nanoparticle may be a future option for treatment of these infections, however, the methods described in vitro to evaluate the inhibitory effect are controversial.

Results: This study evaluated the in vitro activity of silver nanoparticles against 36 susceptible and 54 multidrug resistant Gram-positive and Gram-negative bacteria from clinical sources. The multidrug resistant bacteria were oxacilin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus spp., carbapenem- and polymyxin B-resistant A. baumannii, carbapenem-resistant P. aeruginosa and carbapenem-resistant Enterobacteriaceae. We analyzed silver nanoparticles stabilized with citrate, chitosan and polyvinyl alcohol and commercial silver nanoparticle. Silver sulfadiazine and silver nitrate were used as control. Different methods were used: agar diffusion, minimum inhibitory concentration, minimum bactericidal concentration and time-kill. The activity of AgNPs using diffusion in solid media and the MIC methods showed similar effect against MDR and antimicrobial-susceptible isolates, with a higher effect against Gram-negative isolates. The better results were achieved with citrate and chitosan silver nanoparticle, both with MIC90 of 6.75 μg mL(-1), which can be due the lower stability of these particles and, consequently, release of Ag(+) ions as revealed by X-ray diffraction (XRD). The bactericidal effect was higher against antimicrobial-susceptible bacteria.

Conclusion: It seems that agar diffusion method can be used as screening test, minimum inhibitory concentration/minimum bactericidal concentration and time kill showed to be useful methods. The activity of commercial silver nanoparticle and silver controls did not exceed the activity of the citrate and chitosan silver nanoparticles. The in vitro inhibitory effect was stronger against Gram-negative than Gram-positive, and similar against multidrug resistant and susceptible bacteria, with best result achieved using citrate and chitosan silver nanoparticles. The bactericidal effect of silver nanoparticle may, in the future, be translated into important therapeutic and clinical options, especially considering the shortage of new antimicrobials against the emerging antimicrobial resistant microorganisms, in particular against Gram-negative bacteria.

Fig. 1

FEG-SEM micrographs of AgNPs stabilized by a PVA, b chitosan and c citrate

Fig. 2

UV-vis spectroscopy spectra of PVA, chitosan and citrate AgNPs

Fig. 3

X-ray diffraction patterns for AgNPs stabilized by a PVA, b chitosan and c citrate

Fig. 4

Inhibition by diffusion in depth with AgNPs particles synthesized by IFSC-USP (citrate, chitosan and PVA AgNPs), for P. aeruginosa INCQS 230 in agar MHA (left) and the same microorganism in MHA blood 5 % (right)

Fig. 5

Distribution of size of inhibition zones (mm) obtained by diffusion in depth with AgNPs (citrate, chitosan and PVA) and controls against multidrug-resistant (MDR) (n = 54) and antimicrobial-susceptible bacteria (n = 36). Asterisk Only inhibition zones >6 mm were presented. All silver compounds showed absence of inhibition zones (<6 mm) when tested in MHA 5 % blood against the same microorganisms

Fig. 6

MIC₉₀ for AgNPs (citrate, chitosan and PVA) and controls comparing the subgroups MDR Gram-negative GN (n = 31) versus MDR Gram-positive GP (n = 23) (totalizing n = 54) and antimicrobial-susceptible GN (n = 22) versus GP (n = 14) (totalizing n = 36)

Fig. 7

Results of minimum bactericidal concentration/minimum inhibitory concentration (MBC/MIC) ratio of AgNPs particles (citrate, chitosan and PVA) and controls against multidrug-resistant (MDR) (n = 54) and susceptible (n = 36) bacteria

Fig. 8

Comparison of time-kill curves for one oxacillin-resistant S. aureus (MRSA) and one oxacillin-suscpetible S. aureus (MSSA) isolate, using AgNPs particles (citrate, chitosan and PVA) and controls (silver sulfadiazine, silver nitrate and commercial AgNPs). For MRSA was made the comparison using MHB II broth and MHB II blood 1.25 %. MHB II- Mueller–Hinton Broth cation adjusted and microorganism; MHB II CTL-control without microorganisms. MHB II SGE-microorganism and broth enriched with blood; MHB II CTL SGE-only broth and blood. For oxacilin-susceptible S. aureus (MSSA3), silver sulfadiazine and silver nitrate curves were not done due to high MICs (Ag Sulfad and Ag Nitrate: MIC ≥27 μg mL⁻¹; MBC ≥27 μg mL⁻¹)

Fig. 9

Comparison of time-kill curves for a carbapenem-resistant K. pneumoniae (KPC) isolate and an isolate of carbapenem-susceptible E. aerogenes, using AgNPs particles (citrate, chitosan and PVA) and controls (silver sulfadiazine and silver nitrate). MHB II- Mueller Hinton Broth cation adjusted and microorganism; MHB II CTL- control without microorganisms. For commercial AgNPs control the curve was not done due to high MICs (MIC ≥ 10 μg mL⁻¹; MBC ≥ 10 μg mL⁻¹)