Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli

Abstract

ObjectivesTo determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC.MethodsIsogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR.ResultsThe spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin.ConclusionsMutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

The SNAP hypothesis: Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation

“…This process inevitably leads to the accumulation of inactivating mutations in the genes of the duplicated region that are not under positive selection. Gene-inactivating mutations (for example, frameshift, nonsense, deletion) occur with spontaneous rates of 10 −5 to 10 −6 per gene [46,47]. Recombination between repeat sequences that lie within the duplicated region (IS elements for example, or other repeat sequences) could lead to a loss of parts of a duplication, including a copy of an essential gene, at much higher rates.…”

“…A minimal mathematical model of SNAP is presented in Fig 2. The spontaneous rates of duplication and mutational gene inactivation used in modelling are taken from published literature [30,46,47]. The only variable parameter in the model is the range of potential effects on relative fitness of duplications and mutations within duplicated regions.…”

“…Alternatively, if the duplication segregates (for example, because selection is relieved) the original gene order will be maintained. The minimal model is illustrated here with rates for each step that are conservative estimates based on experimentally determined values [30,46,47].…”

“…The model allows the appearance of cells with a small duplication (Dup) that includes two essential genes, and cells with the duplication and a single (Δ1) or double (Δ2) inactivation of essential genes. Rates of duplication formation and mutational gene inactivation were estimated based on previous experimental data [30,46,47]. All possible directions and rates of evolution are displayed in Fig 2 and the inactivation of two essential genes is assumed to stabilize the duplication.…”

Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition

“…Notably, AcrAB+, which overexpresses efflux pumps, was less evolvable and never achieved as high levels of resistance as the WT strain, suggesting that both having pumps and the ability to precisely control their expression together are important in the evolution of resistance. This reduced evolvability of AcrAB+ may be a result of the fitness burden of constitutive pump overexpression (25,46). In contrast, we found that under certain conditions the acrB strain evolved to survive on 40x MIC 0 .…”