The spread of pathogenic bacteria that are resistant to certain antibiotics constitutes a growing threat to public health. One strategy to counter this problem is to minimize the probability that antibiotic resistance arises. To achieve this we need to understand the factors that drive and constrain its evolution . The fitness costs associated with resistance have received considerable attention since they determine the long-term success of resistant bacteria. Such costs may arise from modifications of cellular components with vital functions, such as ribosomes or the cell wall, or from metabolic costs due to the expression of enzymes that break down the antibiotic. These pleiotropic costs would allow resistance to be selected only when bacteria are confronted with antibiotics. Surprisingly, a study published in BMC Evolutionary Biology  now reports the fixation of mutations causing rifampicin resistance in Escherichia coli populations that evolved at high temperature in medium without any antibiotics. By gauging the fitness consequences of these mutations in other environments and in other bacterial strains, the authors show that the beneficial effects of these mutations are highly specific and highlight two factors that are crucial for the evolution of antibiotic resistance: pleiotropy and epistasis.
The study by Rodríguez-Verdugo et al.  analyses E. coli lines from a previously reported large-scale evolution experiment with 114 populations that have evolved for 2,000 generations at an increased temperature of 42.2°C . Genome sequencing of clones from all evolved populations showed that 74 clones contained at least one of 46 unique non-synonymous mutations in the rpoB gene , which encodes the β-subunit of RNA polymerase and is a known target of rifampicin resistance . Thirteen of the 114 lines did acquire intermediate to very high levels of rifampicin resistance. Interestingly, all 13 resistant lines had a non-synonymous mutation in rpoB, and 12 showed one of three mutations at codon position 572, thus showing signatures of parallel evolution - a hallmark of their involvement in adaptation. By looking at the temporal dynamics the researchers showed that these mutations appeared early in the selection, again consistent with their involvement in adaptation. The authors obtained definitive proof that the three mutations at position 572 increase fitness in the evolutionary environment by testing their effect in the ancestral strain, revealing fitness increases of about 20%.
How surprising are these results? Resistance mechanisms are generally thought to be costly since antibiotics target fundamental cellular processes, including the synthesis of mRNA, proteins and cell structures . Fitness costs associated with resistance may arise from modifications in the cellular targets of antibiotics, which prevent binding of the drug but also compromise their cellular role. This is, for instance, the case for resistance to rifampicin and streptomycin, which target the β-subunit of RNA polymerase and ribosomal protein S12, respectively. Costs may also be due to the synthesis of enzymes that break down antibiotics, as is the case for β-lactams, or when porin mutations that interfere with the antibiotic entering the cell also limit the uptake of nutrients, as for penicillin and tetracycline. Many studies have quantified such costs in the absence of the antibiotic under various in vitro and in vivo conditions . But a 'cost of resistance' was not always observed, and neutral or even beneficial effects in the absence of the drug have previously been observed for resistance to a number of other antibiotics (reviewed in ). Complementary to the results of Rodríguez-Verdugo et al., a recent study showed that E. coli selected in the presence of rifampicin acquired mutations in the rpoB gene that were beneficial in minimal medium at increased temperature . However, other than the study by Rodríguez-Verdugo et al., these studies lacked genomic information and could not rule out the possibility that secondary mutations have occurred that compensated for the negative pleiotropic effects of the resistance mutations.