Optimization of nitric oxide donors for investigating biofilm dispersal response in Pseudomonas aeruginosa clinical isolates

Yu-ming Cai,Jeremy S. Webb

doi:10.1007/s00253-020-10859-7

Yu-ming Cai, Jeremy S. Webb

Open Access

https://doi.org/10.1007/s00253-020-10859-7

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Abstract

Pseudomonas aeruginosa biofilms contribute heavily to chronic lung infection in cystic fibrosis patients, leading to morbidity and mortality. Nitric oxide (NO) has been shown to disperse P. aeruginosa biofilms in vitro, ex vivo and in clinical trials as a promising anti-biofilm agent. Traditional NO donors such as sodium nitroprusside (SNP) have been extensively employed in different studies. However, the dosage of SNP in different studies was not consistent, ranging from 500 nM to 500 μM. SNP is light sensitive and produces cyanide, which may lead to data misinterpretation and inaccurate predictions of dispersal responses in clinical settings. New NO donors and NO delivery methods have therefore been explored. Here we assessed 7 NO donors using P. aeruginosa PAO1 and determined that SNP and Spermine NONOate (S150) successfully reduced > 60% biomass within 24 and 2 h, respectively. While neither dosage posed toxicity towards bacterial cells, chemiluminescence assays showed that SNP only released NO upon light exposure in M9 media and S150 delivered much higher performance spontaneously. S150 was then tested on 13 different cystic fibrosis P. aeruginosa (CF-PA) isolates; most CF-PA biofilms were significantly dispersed by 250 μM S150. Our work therefore discovered a commercially available NO donor S150, which disperses CF-PA biofilms efficiently within a short period of time and without releasing cyanide, as an alternative of SNP in clinical trials in the future.Key points• S150 performs the best in dispersing P. aeruginosa biofilms among 7 NO donors.• SNP only releases NO in the presence of light, while S150 releases NO spontaneously.• S150 successfully disperses biofilms formed by P. aeruginosa cystic fibrosis clinical isolates.

Highlights

The major cause of morbidity and mortality in cystic fibrosis (CF) patients is the chronic bacterial colonization of patients’ lungs and airways leading to pulmonary dysfunction and infection (Gilligan 1991; Govan and Deretic 1996)
Our study showed that (1) S150 is superior to sodium nitroprusside (SNP), which can be applied in wider settings as it does not require light to release Nitric oxide (NO); (2) S150 can disperse biofilms formed by genetically different cystic fibrosis P. aeruginosa (CF-PA) strains, indicating its potential clinical applications
Due to the 1 ppm maximum detection limit of CLD 88Y, NO released from 5 μM S150 and SNP was quantified in M9 media at 37°C across ~ 1.5 h timeframe as shown in Fig. 2a and b

Summary

Introduction

The major cause of morbidity and mortality in cystic fibrosis (CF) patients is the chronic bacterial colonization of patients’ lungs and airways leading to pulmonary dysfunction and infection (Gilligan 1991; Govan and Deretic 1996). Bacteria invade healthy individuals, opportunistic pathogens that overcome mucociliary clearance can be targeted by phagocytic cells and specific opsonizing antibodies (Govan and Deretic 1996). In CF patients, the dehydrated surface liquid on respiratory epithelium results in defective mucociliary clearance and frustrated phagocytosis due to the impaired opsonisation process, contributing to the chronic colonization (Govan and Deretic 1996). P. aeruginosa tend to form aggregates/biofilms in vivo, leading to a much higher tolerance to treatments, driven by both the protective extracellular polymeric

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