Effect of ciprofloxacin concentration on the frequency and nature of resistant mutants selected from Pseudomonas aeruginosa mutS and mutT hypermutators

Abstract

The rapid emergence of drug resistance upon treatment of Pseudomonas aeruginosa infections with fluoroquinolones is a serious concern. In this study, we report the effect of hypermutability on the mutant selection window for ciprofloxacin (CIP) by comparing the hypermutator MPAO1 mutS and mutT strains with the wild-type strain. The mutant selection window was shifted to higher CIP concentrations for both hypermutators, presenting the mutS strain with a broader selection window in comparison to the wild-type strain. The mutation prevention concentrations (MPC) determined for mutT and mutS strains were increased 2- and 4-fold over the wild-type level, respectively. In addition, we analyzed the molecular bases for resistance in the bacterial subpopulations selected at different points in the window. At the top of the window, the resistant clones isolated were mainly mutated in GyrA and ParC topoisomerase subunits, while at the bottom of the window, resistance was associated with the overexpression of MexCD-OprJ and MexAB-OprM efflux pumps. Accordingly, a greater proportion of multidrug-resistant clones were found among the subpopulations isolated at the lower CIP concentrations. Furthermore, we found that the exposure to CIP subinhibitory concentrations favors the accumulation of cells overexpressing MexCD-OprJ (due to mutations in the transcriptional repressor NfxB) and MexAB-OprM efflux pumps. We discuss these results in the context of the possible participation of this antibiotic in a mutagenic process.

Fig. 1.

Fraction of cells recovered in LB plates containing ciprofloxacin (CIP) at different concentrations from the wild-type (WT; ●), mutT (○), and mutS (▾) MPAO1 strains (see Materials and Methods). The MICs required to inhibit the growth of 99% of the bacterial population (MIC₉₉) were determined from the plots (indicated by arrowheads). These values are 0.08, 0.45, and 0.52 μg ml⁻¹ for the wild-type, mutT, and mutS strains, respectively. Symbols marked with asterisks represent the CIP concentration at which no colony is recovered when 1 × 10¹⁰ cells are applied to the plates (mutant prevention concentration [MPC]). MPC values are 1.25, 2.25, and 5 μg ml⁻¹ for the wild-type, mutT, and mutS strains, respectively.

Fig. 2.

Effect of ciprofloxacin (CIP) at subinhibitory levels on nfxB mutagenesis. MPAO1 wild-type, mutT, and mutS reporter strains carrying a chromosomal transcriptional fusion of the mexCD-oprJ promoter to the lux operon were used to identify nfxB mutants. (a) Representative luminescence signal of an nfxB mutant (above) and the parental mutT strain (below) carrying the reporter construction. Approximately 300 CFU/plate were grown on LB media without (b) or with (c and d) CIP at subinhibitory concentrations (0.06 and 0.4 μg ml⁻¹ for the wild-type and mutator strains, respectively). Colonies with increased luminescence were observed from day 2 at the CIP plates and, after further isolation on LB medium, were shown to be composed of a mixed population (e). Sequence analysis of nfxB from mixed colonies also indicated the presence of both mutated and not mutated cells in the same colony. Insets in panels c and d present an expanded view of some mixed colonies. A representative chromatogram for one of the colonies grown on CIP plates is shown in panel f; here A→C transversion leading to Lys37Gln substitution does not affect the entire colony population (the residue C resulting from this mutation is underlined).

Fig. 3.

Percentage of nfxB mutants arising in solid media with or without the addition of ciprofloxacin (CIP) at subinhibitory levels (gray bars). These percentages are relative to the total bacterial populations, and their numerical values are also indicated inside the bars. The percentages of cells recovered in relation to the total number of cells applied to the plates are also indicated (white bars). In this assay, 20 independent cultures of wild-type, mutT, and mutS strains carrying a luminescent reporter of mexCD-oprJ expression were plated in solid media with or without the addition of CIP at subinhibitory levels (approximately 300 cells at each plate). After 48 h of growth, about 6 to 10 of the most luminescent clones derived from each strain and condition were further isolated in LB medium to determine its nfxB DNA sequence. The nfxB mutations detected in this assay are detailed in Table S3 in the supplemental material.