Nitric oxide donor sodium nitroprusside serves as a source of iron supporting Pseudomonas aeruginosa growth and biofilm formation.

Biofilm dispersal agents, like nitric oxide (NO), restore antimicrobial effectiveness against biofilm infections by inducing bacteria to shift from a biofilm to a planktonic state, thereby overcoming the antimicrobial tolerance typically associated with biofilms. Sodium nitroprusside (SNP) is a widely used NO donor for investigating the molecular mechanisms underlying NO-mediated biofilm dispersal in the nosocomial pathogen Pseudomonas aeruginosa. However, the biofilm effects of SNP are variable depending on the in vitro experimental conditions, with some studies reporting enhanced growth in both planktonic and biofilm forms instead of dispersal. These discrepancies suggest that SNP affects P. aeruginosa biofilm-residing cells beyond the release of NO. In this study, we compared SNP with another NO donor, Spermine NONOate, to systematically contrast their effects on biofilm and planktonic cultures of P. aeruginosa. We found that SNP, but not Spermine NONOate, increased the biomass of P. aeruginosa biofilms in microplate cultures. This effect was also observed when biofilm cultures were supplemented with iron. Additionally, supplementation with SNP rescued the planktonic growth of P. aeruginosa in iron-depleted media similarly to FeSO₄, suggesting that SNP may serve as an iron source. Our findings indicate that the use of SNP as an NO donor in biofilm dispersal may be compromised by its role in promoting both biofilm and planktonic growth through its iron center. Our study cautions investigators using SNP for studying NO-mediated biofilm dispersal.

Importance: Research into biofilm dispersal agent nitric oxide (NO) holds promise for treating biofilm-associated infections. Sodium nitroprusside (SNP), an NO donor widely used in antibiofilm research, has been shown in this study to enhance cell growth and biofilm formation in Pseudomonas aeruginosa by acting as a source of iron. Our results suggest that SNP functions both as an NO and an iron donor, with its iron-releasing properties playing a more dominant role in promoting biofilm growth in closed culture systems. This study underscores the dual but conflicting roles of SNP in biofilm growth, which caution its future development as an NO-based therapeutic strategy for biofilm-associated infections.