title: Real-Time Monitoring ofnfxBMutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin.
abstract: Biofilm infections caused byPseudomonas aeruginosaare frequently treated with ciprofloxacin (CIP); however, resistance rapidly develops. One of the primary resistance mechanisms is the overexpression of the MexCD-OprJ pump due to a mutation innfxB, encoding the transcriptional repressor of this pump. The aim of this study was to investigate the effect of subinhibitory concentrations of CIP on the occurrence ofnfxBmutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with anmCherrytag for constitutive red fluorescence and chromosomal transcriptional fusion between the PmexCDpromoter andgfpleading to green fluorescence upon mutation ofnfxBWe observed a rapid development ofnfxBmutants by live confocal laser scanning microscopy (CLSM) imaging of the flow cell biofilm (reaching 80 to 90% of the whole population) when treated with 1/10 minimal biofilm inhibitory concentration of CIP for 24 h and 96 h. Based on the observed developmental stages, we propose thatnfxBmutants emergedde novoin the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. IdenticalnfxBmutations were found in fluorescent colonies from the same flow cell biofilm, especially in 24-h biofilms, suggesting selection and clonal expansion of the mutants during biofilm growth. Our findings point at the significant role of high-enough antibiotic dosages or appropriate combination therapy to avoid the emergence of resistant mutants in biofilms.
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Greta
authors.initials: G
authors.lastname: Zaborskyte
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jens Bo
authors.initials: JB
authors.lastname: Andersen
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kasper Nørskov
authors.initials: KN
authors.lastname: Kragh
authors.affiliation: Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark ociofu@sund.ku.dk.
authors.forename: Oana
authors.initials: O
authors.lastname: Ciofu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects / Q000254:growth & development; D002939:Ciprofloxacin / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012212:Rheology; D012641:Selection, Genetic; D059008:Time-Lapse Imaging; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002939:Ciprofloxacin; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein; C470189:MexC protein, Pseudomonas aeruginosa; C121980:MexD protein, Pseudomonas aeruginosa; C077901:NfxB protein, Pseudomonas aeruginosa; C470190:OprJ protein, Pseudomonas aeruginosa
keywords: Pseudomonas aeruginosa; biofilms; ciprofloxacin