Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence

Abstract

The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 2050(1). Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non-growing antibiotic-tolerant 'persister' state(2-6). Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment(7). Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20-100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models(8-10), we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of 'bet-hedging' theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.

In Vitro Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the ESKAPE Pathogens

“…Theoretically, such fluctuating stress conditions are predicted to promote the evolution of antibiotic tolerance through increased persister cell formation, rather than to select for resistant mutants (39). In agreement with our earlier observations in nonpathogenic E. coli (30), all ESKAPE pathogens showed a rapid evolution toward high persistence under cyclic aminoglycoside treatment (Fig. 3).…”

Should we develop screens for multi-drug antibiotic tolerance?

“…Second, we confirmed the longstanding prediction of a direct correlation between the evolved persister level and the frequency of antibiotic treatments in E. coli [24]. Daily aminoglycoside exposure selects for extreme persistence levels up to almost 100%, not only in E. coli [24] but also in all ESKAPE pathogens and any species we tested so far [25]. Moreover, all evolutionary changes take place in a short timeframe, less than 100 generations, and are caused by individual single nucleotide polymorphisms or small insertions or deletions.…”

“…Tolerance levels in E. coli and S. aureus increase tremendously upon repeated exposure to ampicillin and daptomycin [22,23]. Second, we confirmed the longstanding prediction of a direct correlation between the evolved persister level and the frequency of antibiotic treatments in E. coli [24]. Daily aminoglycoside exposure selects for extreme persistence levels up to almost 100%, not only in E. coli [24] but also in all ESKAPE pathogens and any species we tested so far [25].…”

“…Their prevalence in the genome is correlated with the level of persisters [31] and highpersistence mutations in HipA are abundant among clinical isolates of uropathogenic E. coli [32]. Mutations resulting in increased tolerance in experimental evolution studies represent other promising genetic markers [22][23][24].…”

The evolutionary advantage of heritable phenotypic heterogeneity

“…The dormant phenotypic state can be partially heritable upon cellular division, so that the offspring cell can "remember" and express the phenotypic state of its parent with a certain probability. In such bacterial populations, individuals typically acquire and relinquish the dormant phenotype at rates that exceed the rate of DNA mutation 14,15 , providing populations with the phenotypic plasticity required to persist through periodic environmental stresses. Even though persisters are a non-genetic form of inheritance, the capacity to generate persistent cells, and the propensity to retain the phenotype of a parent, are likely under genetic control 16 .…”