title: Increased bactericidal activity of colistin on Pseudomonas aeruginosa biofilms in anaerobic conditions.
abstract: Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions.
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark Department of Immunology and Microbiology, UC-CARE, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark mettekolpen@gmail.com.
authors.forename: Mette
authors.initials: M
authors.lastname: Kolpen
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Cecilie F
authors.initials: CF
authors.lastname: Appeldorff
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Sarah
authors.initials: S
authors.lastname: Brandt
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Nabi
authors.initials: N
authors.lastname: Mousavi
authors.affiliation: Department of Immunology and Microbiology, UC-CARE, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Kasper N
authors.initials: KN
authors.lastname: Kragh
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Sevtap
authors.initials: S
authors.lastname: Aydogan
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Haleema A
authors.initials: HA
authors.lastname: Uppal
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark Department of Immunology and Microbiology, UC-CARE, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bjarnsholt
authors.affiliation: Department of Immunology and Microbiology, UC-CARE, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Oana
authors.initials: O
authors.lastname: Ciofu
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark Department of Immunology and Microbiology, UC-CARE, Faculty of Health Sciences University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Høiby
authors.affiliation: Department of Clinical Microbiology, Rigshospitalet, 2100 Copenhagen, Denmark.
authors.forename: Peter Ø
authors.initials: PØ
authors.lastname: Jensen
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D003091:Colistin / Q000494:pharmacology*; D015169:Colony Count, Microbial; D003550:Cystic Fibrosis / Q000150:complications; D005452:Fluoresceins / Q000032:analysis; D005470:Fluorometry; D006801:Humans; D017665:Hydroxyl Radical / Q000032:analysis; D050296:Microbial Viability / Q000187:drug effects*; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D005452:Fluoresceins; C543635:hydroxyphenyl fluoroscein; D017665:Hydroxyl Radical; D003091:Colistin
keywords: Pseudomonas aeruginosa; anaerobic conditions; biofilm; colistin; hydroxyl radicals