Real-Time Monitoring of nfxB Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin

Abstract

Biofilm infections caused by Pseudomonas aeruginosa are 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 in nfxB, 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 of nfxB mutants in the wild-type PAO1 flow cell biofilm model. For this purpose, we constructed fluorescent reporter strains (PAO1 background) with an mCherry tag for constitutive red fluorescence and chromosomal transcriptional fusion between the P _mexCD promoter and gfp leading to green fluorescence upon mutation of nfxB We observed a rapid development of nfxB mutants 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 that nfxB mutants emerged de novo in the biofilm during CIP treatment from filamentous cells, which might have arisen due to the stress responses induced by CIP. Identical nfxB mutations 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.

Keywords: Pseudomonas aeruginosa; biofilms; ciprofloxacin.

FIG 1

Characteristics of the P. aeruginosa fluorescent reporter strains. (A) Colonies of PAO1 and its isogenic ΔnfxB strain with the P_CD-gfp+ reporter grown on LB agar plates and examined under an epifluorescence microscope with blue excitation light (green fluorescence) and green excitation light (red fluorescence). (B) The green fluorescent signal together with the OD₆₀₀ was monitored during 24 h of planktonic growth of PAO1 and the ΔnfxB mutant with the P_CD-gfp+ reporter. Raw fluorescence values were divided by the corresponding OD₆₀₀ at each time point. The fluorescence of parent strains (resulting from the autofluorescence background) was subtracted from the fluorescence of reporter strains. The means and 95% confidence intervals based on 5 replicates are shown. (C) Growth curves of the reporter strains. The values represent the means and 95% confidence intervals of data for five replicate wells in a microtiter plate, where the cultures were grown and monitored.

FIG 2

Rapid development of nfxB mutants in a 24-h-old PAO1 flow cell biofilm treated with low-dose CIP. The biofilms of PAO1-mCherry-P_CD-gfp+ were grown in flow cell chambers with a continuous flow of minimal medium for 24 h at 37°C and then grown for an additional 24 h with 0.2 μg/ml CIP (24 h +CIP for 24 h) or without CIP (24 h −CIP for 24 h). Red shows wild-type cells (elongated or filamentous) due to the constitutive expression of mCherry, and green shows nfxB mutants due to the expression of GFP via the P_CD-gfp+ reporter. z-stacks were generated by using a Zeiss LSM 710 microscope and processed with Imaris 8.2 software (Bitplane). Top-row images show orthogonal 3D views, and middle and bottom (for treated biofilms only) rows show perspective 3D views of biofilms with an overlay of red and green fluorescence. The images shown are representative of results for two independent flow cell channels.

FIG 3

Development of nfxB mutants in a 72-h-old PAO1 flow cell biofilm during treatment with low-dose CIP. The biofilms of PAO1-mCherry-P_CD-gfp+ were grown in three independent channels of flow cell chambers with a continuous flow of minimal medium for 72 h and then treated with 0.2 μg/ml CIP for a total of 96 h. Imaging by CLSM was done every 24 h. At least 3 images were taken per channel at every time point. Red represents wild-type cells due to the constitutive expression of mCherry, and green shows nfxB mutants due to the expression of GFP+ via the P_CD-gfp+ reporter. z-stacks were generated by using a Zeiss LSM 710 microscope and processed with Imaris 8.2 software (Bitplane). The images show orthogonal 3D biofilm views (left) or a perspective view (right) with an overlay of red and green channel fluorescence.

FIG 4

Fraction of nfxB mutants in the 72-h-old PAO1 flow cell biofilm population at different time points of CIP treatment. Shown are the fractions of biomass expressing gfp in 72-h biofilms formed by PAO1-mCherry-P_CD-gfp+ and PAOΔnfxB-mCherry-P_CD-gfp+ at different time points of treatment with CIP. The positive control had significantly higher biomass with green fluorescence at time zero (*, P < 0.0001) and at 48 h (**, P = 0.01) than that of the wild type. After 72 h and 96 h of CIP treatment, the fraction of green fluorescent biomass in the PAO1-mCherry-P_CD-gfp+ (representing nfxB mutants) biofilm approaches that of the positive control PAOΔnfxB-mCherry-P_CD-gfp+.