Resistance/fitness trade-off is a barrier to the evolution of MarR inactivation mutants in Escherichia coli.

BACKGROUND: Mutations that inactivate MarR reduce susceptibility to ciprofloxacin and competitive growth fitness in Escherichia coli. Both phenotypes are caused by overexpression of the MarA regulon, which includes the AcrAB-TolC drug efflux pump. OBJECTIVES: We asked whether compensatory evolution could reduce the fitness cost of MarR-inactivating mutations without affecting resistance to ciprofloxacin. METHODS: The cost of overexpressing the AcrAB-TolC efflux pump was measured independently of MarA overexpression. Experimental evolution of MarR-inactive strains was used to select mutants with increased fitness. The acquired mutations were identified and their effects on drug susceptibility were measured. RESULTS: Overexpression of the AcrAB-TolC efflux pump was found not to contribute to the fitness cost of MarA regulon overexpression. Fitness-compensatory mutations were selected in marA and lon. The mutations reduced the level of MarA protein thus reducing expression of the MarA regulon. They restored growth fitness but also reduced resistance to ciprofloxacin. CONCLUSIONS: The fitness cost caused by overexpression of the MarA regulon has multiple contributing factors. Experimental evolution did not identify any single pump-independent cost factor. Instead, efficient fitness compensation occurred only by mechanisms that reduce MarA concentration, which simultaneously reduce the drug resistance phenotype. This resistance/fitness trade-off is a barrier to the successful spread of MarR inactivation mutations in clinical isolates where growth fitness is essential.

Antibiotic resistance by high-level intrinsic suppression of a frameshift mutation in an essential gene.

A fundamental feature of life is that ribosomes read the genetic code in messenger RNA (mRNA) as triplets of nucleotides in a single reading frame. Mutations that shift the reading frame generally cause gene inactivation and in essential genes cause loss of viability. Here we report and characterize a +1-nt frameshift mutation, centrally located in rpoB, an essential gene encoding the beta-subunit of RNA polymerase. Mutant Escherichia coli carrying this mutation are viable and highly resistant to rifampicin. Genetic and proteomic experiments reveal a very high rate (5%) of spontaneous frameshift suppression occurring on a heptanucleotide sequence downstream of the mutation. Production of active protein is stimulated to 61-71% of wild-type level by a feedback mechanism increasing translation initiation. The phenomenon described here could have broad significance for predictions of phenotype from genotype. Several frameshift mutations have been reported in rpoB in rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb). These mutations have never been experimentally validated, and no mechanisms of action have been proposed. This work shows that frameshift mutations in rpoB can be a mutational mechanism generating antibiotic resistance. Our analysis further suggests that genetic elements supporting productive frameshifting could rapidly evolve de novo, even in essential genes.

Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli.

Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump. Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin. Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance. Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to reduce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhIJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics (chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim). Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to resistance development by up-regulation of an array of efflux pumps.

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

OBJECTIVES: To 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. METHODS: Isogenic 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. RESULTS: The 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. CONCLUSIONS: Mutations 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.