Cephalosporin-3’-diazeniumdiolate NO-donor prodrug PYRRO-C3D enhances azithromycin susceptibility of non-typeable Haemophilus influenzae biofilms

Objectives: PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO)-donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against non-typeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. Methods: The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free LC/MS proteomic analyses were performed to identify differentially expressed proteins following NO treatment. Results: PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log-fold reduction in viability (p<0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log reduction was observed (p<0.01). Label-free proteomics showed that NO increased expression of sixteen proteins involved in metabolic and transcriptional/translational functions. Conclusions: NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm associated antibiotic tolerance.