title: Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome.
abstract: Pseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM+). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies.
authors.affiliation: Department of Infectious Disease and Immunology, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia william.klare@sydney.edu.au.
authors.forename: William
authors.identifier: 0000-0002-1490-9704
authors.initials: W
authors.lastname: Klare
authors.affiliation: Department of Infectious Disease and Immunology, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW, Australia.
authors.forename: Theerthankar
authors.initials: T
authors.lastname: Das
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW, Australia.
authors.forename: Amaye
authors.initials: A
authors.lastname: Ibugo
authors.affiliation: Department of Infectious Disease and Immunology, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia.
authors.forename: Edwina
authors.initials: E
authors.lastname: Buckle
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW, Australia.
authors.forename: Mike
authors.initials: M
authors.lastname: Manefield
authors.affiliation: Department of Infectious Disease and Immunology, Charles Perkins Centre, University of Sydney, Sydney, NSW, Australia.
authors.forename: Jim
authors.initials: J
authors.lastname: Manos
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000187:drug effects*; D005978:Glutathione / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D059467:Transcriptome / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005978:Glutathione