Cephalosporin-3′-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms

Author:

Collins Samuel A.12,Kelso Michael J.3,Rineh Ardeshir3,Yepuri Nageshwar R.3,Coles Janice1,Jackson Claire L.1,Halladay Georgia D.1,Walker Woolf T.12,Webb Jeremy S.24,Hall-Stoodley Luanne5,Connett Gary J.12,Feelisch Martin12,Faust Saul N.126,Lucas Jane S. A.12,Allan Raymond N.16

Affiliation:

1. Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

2. Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom

3. Illawarra Health and Medical Research Institute, School of Chemistry, University of Wollongong, Wollongong, NSW, Australia

4. Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom

5. Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, College of Medicine, The Ohio State University, Columbus, Ohio, USA

6. Southampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom

Abstract

ABSTRACT 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 nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. 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 liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. 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 (10-fold reduction or 1-log-unit 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-unit reduction was observed ( P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. 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.

Funder

Australian Cystic Fibrosis Research Trust

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Pharmacology (medical),Pharmacology

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