Subinhibitory Concentrations of Biogenic Silver Nanoparticles Affect Motility and Biofilm Formation in Pseudomonas aeruginosa

Abstract

Biogenic silver nanoparticles (bio-AgNPs) are increasingly recognized as an antibiofilm and antivirulence strategy against P. aeruginosa, a bacterium that causes chronic infections in immunocompromised and cystic fibrosis patients. This study aimed to investigate the effects of subinhibitory concentrations of bio-AgNPs on motility and biofilm formation in P. aeruginosa. Bio-AgNPs were synthesized via reduction of ionic silver catalyzed by cell-free culture filtrate from Fusarium oxysporum. A total of 17 P. aeruginosa isolates and strains were evaluated for swarming, swimming, and twitching motility in the presence and absence (control) of bio-AgNPs, including 10 clinical isolates from patients with and without cystic fibrosis, 5 environmental isolates obtained from the public water supply system, and 2 reference strains (PAO1 and PA14). Isolates were identified by biochemical and molecular methods. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Swarming, swimming, and twitching motility assays were performed in Petri dishes. Biofilm formation capacity was assessed quantitatively by the crystal violet method. MIC values ranged from 15.62 to 62.50 µM. The results showed that subinhibitory concentrations of bio-AgNPs (½ MIC, 7.81-31.25 µM) significantly increased (p < 0.05) swarming, swimming, and twitching motility in 40.0, 40.0, and 46.7% of isolates, respectively. Subinhibitory bio-AgNP treatment enhanced (p < 0.05) biofilm formation capacity in PA14 and a cystic fibrosis isolate (P11). It is concluded that subinhibitory concentrations of bio-AgNPs increased biofilm formation and swarming, swimming, and twitching motility in PA14 and some P. aeruginosa isolates. These virulence factors are directly involved with quorum-sensing systems. Further research should investigate the effects of AgNPs on P. aeruginosa quorum sensing to help elucidate their mechanism of action at subinhibitory concentrations.

Keywords: antibiofilm; antivirulence; metal nanoparticle; quorum sensing; virulence factor.

Figure 1

Agarose gel electrophoresis (1.0%) of Pseudomonas aeruginosa isolates showing amplified PCR products for the oprL gene (209 bp). Lane 1 (M), molecular weight marker; P1–P5, environmental isolates; P6–P10, clinical isolates obtained from non-cystic fibrosis patients; P11–P15, clinical isolates obtained from cystic fibrosis patients.

Figure 2

Characterization by transmission electron microscopy (TEM) of bio-AgNP synthesized by Fusarium oxysporum (300,000×).

Figure 3

Pseudomonas aeruginosa motility in the absence (control) and presence of biogenic silver nanoparticles (bio-AgNPs). (A) Swarming motility of P2 isolate, (B) mean swarming motility, (C) swimming motility of P2 isolate, (D) mean swimming motility, (E) twitching motility of P6 isolate, and (F) mean twitching motility.

Figure 4

Biofilm formation capacity of reference strains, environmental isolates, and clinical isolates of Pseudomonas aeruginosa in the absence (control) and presence of biogenic silver nanoparticles (bio-AgNPs). Results are the mean ± standard deviation of three independent experiments. *p < 0.05 compared with the control.