Interplay in the selection of fluoroquinolone resistance and bacterial fitness

Abstract

Fluoroquinolones are antibacterial drugs that inhibit DNA Gyrase and Topoisomerase IV. These essential enzymes facilitate chromosome replication and RNA transcription by regulating chromosome supercoiling. High-level resistance to fluoroquinolones in E. coli requires the accumulation of multiple mutations, including those that alter target genes and genes regulating drug efflux. Previous studies have shown some drug-resistance mutations reduce bacterial fitness, leading to the selection of fitness-compensatory mutations. The impact of fluoroquinolone-resistance on bacterial fitness was analyzed in constructed isogenic strains carrying up to 5 resistance mutations. Some mutations significantly decreased bacterial fitness both in vitro and in vivo. We identified low-fitness triple-mutants where the acquisition of a fourth resistance mutation significantly increased fitness in vitro and in vivo while at the same time dramatically decreasing drug susceptibility. The largest effect occurred with the addition of a parC mutation (Topoisomerase IV) to a low-fitness strain carrying resistance mutations in gyrA (DNA Gyrase) and marR (drug efflux regulation). Increased fitness was accompanied by a significant change in the level of gyrA promoter activity as measured in an assay of DNA supercoiling. In selection and competition experiments made in the absence of drug, parC mutants that improved fitness and reduced susceptibility were selected. These data suggest that natural selection for improved growth in bacteria with low-level resistance to fluoroquinolones could in some cases select for further reductions in drug susceptibility. Thus, increased resistance to fluoroquinolones could be selected even in the absence of further exposure to the drug.

Figure 1. Resistance and fitness of constructed strains.

For the wild-type and each of the 28 constructed isogenic strains the MIC for ciprofloxacin (part A) and the relative fitness per generation in in vitro growth competition (part B) is shown as a function of the number of resistance mutations per strain. Each diamond symbol represents one strain. In some cases more than one symbol occupies the same space. Strain identification numbers are shown for four strains of particular interest (Table 2).

Figure 2. Fitness in vitro as a function of fitness in vivo in a mouse UTI model.

Samples from urine, kidney, and bladder are illustrated with black diamonds, triangles, and squares, respectively. The addition of one extra resistance mutation to either LM695, or LM882 created LM707, with increased fitness in vitro and in vivo and an increase in MIC for ciprofloxacin from 0.75 to 32 µg/ml.

Figure 3. A general model illustrating evolutionary paths for a lineage under antibiotic selection.

Mutual compensation, the acquisition of a mutation that simultaneously improves fitness and reduces susceptibility to the antibiotic, is the pathway presented in this manuscript.