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Antimicrob Agents Chemother. 2017 Jan 24;61(2). pii: e02086-16. doi: 10.1128/AAC.02086-16. Print 2017 Feb.

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

Author information

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.
7
Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom jlucas1@soton.ac.uk.

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.

KEYWORDS:

Haemophilus influenzae; antibiotic resistance; biofilms; nitric oxide; proteomics

PMID:
27919896
PMCID:
PMC5278716
DOI:
10.1128/AAC.02086-16
[Indexed for MEDLINE]
Free PMC Article

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