Synergy of silver nanoparticles and aztreonam against Pseudomonas aeruginosa PAO1 biofilms - PubMed (original) (raw)
Synergy of silver nanoparticles and aztreonam against Pseudomonas aeruginosa PAO1 biofilms
Marc B Habash et al. Antimicrob Agents Chemother. 2014 Oct.
Abstract
Pathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibit Pseudomonas aeruginosa PAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery of P. aeruginosa biofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope of P. aeruginosa within the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam against P. aeruginosa in vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Figures
FIG 1
High-throughput MBEC biofilm antimicrobial susceptibility assay. (A) Transmission electron micrographs of AgNPs (10 nm to 100 nm) used in this study. Bars, 100 nm. (B) Schematic illustration of the workflow used (i) to culture P. aeruginosa PAO1 planktonic and biofilm cultures (growth phase), (ii) to incubate biofilms in the presence of AgNP-aztreonam treatments and to determine MIC values for the planktonic cultures (challenge phase), and (iii) to determine biofilm biomass, viability, and MBEC values following incubation in fresh medium without antimicrobials (recovery phase) (see Materials and Methods for more details). (C) Reproducibility of biofilm formation, assessed in the growth phase of the experiment by plotting planktonic growth (based on planktonic cell density in the wells of the growth phase) versus biofilm growth (based on accumulated bacterial cell density, stained with crystal violet [CV], in biofilms in the growth phase) (n = 288). _A_590, absorbance at 590 nm.
FIG 2
Effects of AgNPs or aztreonam alone on P. aeruginosa PAO1 biofilm biomass and viability. Values were normalized to those obtained from untreated control samples after biofilms were incubated in fresh medium in the recovery phase. (A) Biofilm biomass and viability following challenge with aztreonam (0.0 μg/ml to 512 μg/ml). (B and C) Biofilm biomass (B) and viability (C) following challenge with different sizes (10 nm to 100 nm) and concentrations (0.0 μg/ml to 10.0 μg/ml) of AgNPs.
FIG 3
Effects of different AgNP-aztreonam combinations on P. aeruginosa PAO1 biofilm biomass in the recovery phase. Normalized values of percent recovery of biofilm biomass following challenge with different combinations of aztreonam (0.0 μg/ml to 512 μg/ml) and AgNP sizes (10 nm to 100 nm) at the specific AgNP concentration (0.0 μg/ml to 10.0 μg/ml) indicated in the top left corner of each panel are shown (panel A represents replicate samples treated with aztreonam only). Biofilm biomass values for treated samples were normalized to values for untreated cells (i.e., 0.0 μg/ml AgNPs and 0.0 μg/ml aztreonam). Dashed lines, 100% recovery (i.e., levels observed for untreated cells).
FIG 4
Effects of different AgNP-aztreonam combinations on P. aeruginosa PAO1 biofilm viability in the recovery phase. Normalized values of percent viability of biofilms following challenge with different combinations of aztreonam (0.0 μg/ml to 512 μg/ml) and AgNP sizes (10 nm to 100 nm) at the specific AgNP concentration (0.0 μg/ml to 10.0 μg/ml) indicated in the top left corner of each panel are shown (panel A represents replicate samples treated with aztreonam only). Biofilm viability values were normalized to values for untreated control samples (i.e., 0.0 μg/ml AgNPs and 0.0 μg/ml aztreonam). Dashed lines, 100% recovery (i.e., levels observed for untreated cells).
FIG 5
Comparison of effects of different AgNP-aztreonam combinations on biofilm biomass (left) and biofilm viability (right) in the recovery phase, according to AgNP size (indicated on the right). Results are presented as heat plots illustrating the normalized values of percent recovery of biofilm biomass and viability after treatment with AgNPs and aztreonam. One hundred percent recovery equals levels observed for untreated control samples. Darker blue boxes, greater percent recoveries (i.e., greater biofilm biomass or viability); lighter blue boxes, poorer biofilm recovery (i.e., greater antimicrobial effect).
FIG 6
Bliss independence model of synergy, confirming synergistic antimicrobial effects of AgNP-aztreonam combinations against P. aeruginosa biofilms. The degree of synergy was quantified using the Bliss model of synergy for recovery based on the normalized values of percent biofilm biomass after 20 h of incubation in fresh medium. For each size of AgNPs (indicated on the right), each point on the graphs represents a specific combination of AgNPs (0.0 μg/ml to 10.0 μg/ml) and aztreonam (512 μg/ml, 64.0 μg/ml, or 4.00 μg/ml). Dashed lines, no combined effects of the antimicrobials. Positive Bliss coefficient values reflect synergy and negative values represent antagonism between the combined antimicrobials.
FIG 7
Effects of AgNP-aztreonam combinations on biofilm architecture. (A) Optical coherence tomographic slice of an untreated P. aeruginosa biofilm cultured for 72 h in silicone tubing. White arrows, biofilm; black arrowheads, areas where the biofilm detached from the inside of the silicone tubing. Black dashed lines, areas where measurements of biofilm thickness were derived. Bar, 400 μm. (B) Mean thickness (with standard deviation) of biofilms treated with various antimicrobials for 18 h (n = 60 measurements per condition). Antimicrobial treatments included 10-nm or 100-nm AgNPs at 0.625 μg/ml alone, aztreonam (Azt) at 64.0 μg/ml alone, and combinations of these treatments. *, P < 0.05 versus untreated control; **, P < 0.05 versus 10-nm AgNPs alone; ***, P < 0.05 versus 100-nm AgNPs alone.
FIG 8
Penetration of smaller AgNPs into P. aeruginosa biofilms. (A to F) Negatively stained transmission electron micrographs of P. aeruginosa PAO1 biofilms cultured in silicone tubing for 72 h and either left untreated (A) or treated for 18 h with aztreonam (64.0 μg/ml) (B), 10-nm AgNPs (0.625 μg/ml) (C), 100-nm AgNPs (0.625 μg/ml) (D), 10-nm AgNPs (0.625 μg/ml) plus aztreonam (64.0 μg/ml) (E), or 100-nm AgNPs (0.625 μg/ml) plus aztreonam (64.0 μg/ml) (F). Bars, 500 nm. (G) Inset images from panel E, highlighting the presence of 10-nm AgNPs (arrowheads). Bars, 50 nm.
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