title: Azithromycin in Pseudomonas aeruginosa biofilms: bactericidal activity and selection of nfxB mutants.
abstract: Azithromycin (AZM) has shown promising results in the treatment of Pseudomonas aeruginosa chronic lung infections such as those occurring in cystic fibrosis (CF) patients. We evaluated the effect of hypermutation and alginate hyperproduction on the bactericidal activity and resistance development to AZM in P. aeruginosa biofilms. Strains PAO1, its microcA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were used. Biofilms were incubated with several AZM concentrations for 1, 2, 4, or 7 days, and the numbers of viable cells were determined. During the first 2 days, AZM showed bactericidal activity for all the strains, but in extended AZM incubation for strain PAOMS and especially strain PAOMSA, a marked increased in the number of viable cells was observed, particularly at 4 microg/ml. Biofilms formed by the lineages recovered from the 7-day experiments showed enhanced AZM resistance. Furthermore, most of the independent lineages studied, including those obtained from biofilms treated with AZM concentrations as low as 0.5 microg/ml, showed MexCD-OprJ hyperexpression and mutations in nfxB. The role of nfxB mutation in AZM resistance was further confirmed through the characterization of nfxB and mexD knockout mutants. Results from this work show that, although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected and that, furthermore, they frequently show cross-resistance to other unrelated antipseudomonal agents such as ciprofloxacin or cefepime but hypersusceptibility to others such as imipenem or tobramycin. Therefore, these results should help guide the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, Palma de Mallorca, Spain. antonio.oliver@ssib.es
authors.forename: Xavier
authors.initials: X
authors.lastname: Mulet
authors.forename: María D
authors.initials: MD
authors.lastname: Maciá
authors.forename: Ana
authors.initials: A
authors.lastname: Mena
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.forename: José L
authors.initials: JL
authors.lastname: Pérez
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017963:Azithromycin / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D018441:Biofilms / Q000187:drug effects*; D000077723:Cefepime; D002511:Cephalosporins / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000188:drug therapy; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins / Q000502:physiology; D008826:Microbial Sensitivity Tests; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002511:Cephalosporins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C121980:MexD protein, Pseudomonas aeruginosa; C107895:MucA protein, Pseudomonas; C077901:NfxB protein, Pseudomonas aeruginosa; C470190:OprJ protein, Pseudomonas aeruginosa; D014157:Transcription Factors; D002939:Ciprofloxacin; D000077723:Cefepime; D017963:Azithromycin; D051719:MutS DNA Mismatch-Binding Protein