title: Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms.
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.
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Collins
authors.affiliation: Illawarra Health and Medical Research Institute, School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kelso
authors.affiliation: Illawarra Health and Medical Research Institute, School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Ardeshir
authors.initials: A
authors.lastname: Rineh
authors.affiliation: Illawarra Health and Medical Research Institute, School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Nageshwar R
authors.initials: NR
authors.lastname: Yepuri
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.
authors.forename: Janice
authors.initials: J
authors.lastname: Coles
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.
authors.forename: Claire L
authors.initials: CL
authors.lastname: Jackson
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.
authors.forename: Georgia D
authors.initials: GD
authors.lastname: Halladay
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Woolf T
authors.initials: WT
authors.lastname: Walker
authors.affiliation: Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.|Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom.
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Webb
authors.affiliation: Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, College of Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Luanne
authors.initials: L
authors.lastname: Hall-Stoodley
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Connett
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Feelisch
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.|Southampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Saul N
authors.initials: SN
authors.lastname: Faust
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom jlucas1@soton.ac.uk.|Southampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Jane S A
authors.initials: JS
authors.lastname: Lucas
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine and Institute for Life Sciences, University of Southampton, Southampton, United Kingdom.|Southampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom.
authors.forename: Raymond N
authors.initials: RN
authors.lastname: Allan
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017963:Azithromycin / Q000494:pharmacology; D001391:Azo Compounds / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D002511:Cephalosporins / Q000494:pharmacology*; D002853:Chromatography, Liquid; D024881:Drug Resistance, Bacterial; D006193:Haemophilus influenzae / Q000187:drug effects*; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009589:Nitrogen Oxides / Q000378:metabolism; D011355:Prodrugs / Q000494:pharmacology*; D040901:Proteomics; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001391:Azo Compounds; D002511:Cephalosporins; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D011355:Prodrugs; C452539:diazeniumdiolate; D017963:Azithromycin; D001618:beta-Lactamases
keywords: Haemophilus influenzae; antibiotic resistance; biofilms; nitric oxide; proteomics