Nitric oxide-mediated dispersal in single- and multi-species biofilms of clinically and industrially relevant microorganisms

Abstract

Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilm formation and biofilm-related infections. Nitric oxide (NO), an important messenger molecule in biological systems, was previously identified as a signal for dispersal in biofilms of the model organism Pseudomonas aeruginosa. In the present study, the use of NO as an anti-biofilm agent more broadly was assessed. Various NO donors, at concentrations estimated to generate NO levels in the picomolar and low nanomolar range, were tested on single-species biofilms of relevant microorganisms and on multi-species biofilms from water distribution and treatment systems. Nitric oxide-induced dispersal was observed in all biofilms assessed, and the average reduction of total biofilm surface was 63%. Moreover, biofilms exposed to low doses of NO were more susceptible to antimicrobial treatments than untreated biofilms. For example, the efficacy of conventional chlorine treatments at removing multi-species biofilms from water systems was increased by 20-fold in biofilms treated with NO compared with untreated biofilms. These data suggest that combined treatments with NO may allow for novel and improved strategies to control biofilms and have widespread applications in many environmental, industrial and clinical settings.

Figure 1

Nitric oxide release profiles from the NO donor SNP. After the NO baseline signal was stabilized for at least 30 min in the PBS solution, SNP was added (arrow) at final concentrations of (a) 250 µM, (b) 500 µM and (c) 1 mM and the amount of NO released was quantified by using the NO electrode. The inset shows the linear relationship between SNP concentration (mM, x‐axis) and NO increase (µM, y‐axis); error bars indicate standard deviation (n = 3).

Figure 2

Nitric oxide effect on V. cholerae biofilm antimicrobial sensitivity. Pre‐established V. cholerae biofilms were treated for 24 h in the presence or absence of the NO donor SNP, and/or the antibiotic tetracycline (Tet) at 14 µM. Biofilm cells remaining on the slides were stained with SYTO 9 to allow analysis using fluorescence microscopy and quantification (per cent surface coverage) using digital image analysis. Data are mean values and error bars indicate standard error (n = 3).

Figure 3

Effect of NO on multi‐species biofilms established from water distribution systems. Three‐month‐old biofilms from recycled and potable water distribution systems were exposed to 0 (control), 100 nM and 500 nM SNP for 18 h and then (recycled water biofilms) treated for 10 min with free chlorine (HOCl) at 0.5 ppm and 1 ppm and no chlorine controls. (A) The images show microscopic pictures of recycled water biofilms after 1 ppm HOCl exposure (lower panels) or no chlorine controls (upper panels) and stained with the LIVE/DEAD reagents. Live cells appear green, dead cells appear red. Bar, 50 µm. Viability analyses of the biofilms were assessed by heterotrophic colony‐forming units (cfu) measurements of (B) recycled water biofilms and (C) potable water biofilms. Data are mean values and error bars indicate standard error (n = 3).

Figure 4

Multi‐species biofilms on a RO filtration membrane exposed to SNP or the fast NO donor PROLI in combination with chlorine.
A. Reverse osmosis membrane coupons harbouring multi‐species biofilms were treated: (i) in the presence or absence of 100 nM SNP and subsequently exposed to 5 ppm HOCl for 10 min; or (ii) simultaneously in the presence or absence of 20 nM or 500 nM PROLI and/or free chlorine at 5 ppm or 10 ppm for 2 h. Biofilms were analysed by performing cfu counts. Data are mean values and error bars indicate standard error (n ≥ 3).
B. Nitric oxide release profiles from PROLI in water. (a) Control, (b) 625 nM, (c) 1.25 µM and (d) 2.5 µM PROLI. Arrows indicate addition of NO scavenger PTIO (100 µM) to the solutions.