Resistance on a background of tolerance
Bacteria survive antibiotic exposure either because they are quiescent when antibiotics are around in the highest concentrations (i.e., tolerance) or because they acquire active biochemical resistance mechanisms (i.e., resistance). Both tolerance and resistance involve the acquisition of mutations from the wild type. Levin-Reisman et al. used in vitro evolution experiments to show that populations of bacteria that become genetically resistant to the antibiotic ampicillin most quickly do so on a background of tolerance mutations (see the Perspective by Lewis and Shan). Because the probability of a tolerant organism surviving is higher, it has a greater chance of subsequently acquiring resistance mutations. Tolerance is often overlooked in the clinic but should in future be screened for and targeted more precisely to reduce the rates of acquired resistance.
Controlled experimental evolution during antibiotic treatment can help to explain the processes leading to antibiotic resistance in bacteria. Recently, intermittent antibiotic exposures have been shown to lead rapidly to the evolution of tolerance—that is, the ability to survive under treatment without developing resistance. However, whether tolerance delays or promotes the eventual emergence of resistance is unclear. Here we used in vitro evolution experiments to explore this question. We found that in all cases, tolerance preceded resistance. A mathematical population-genetics model showed how tolerance boosts the chances for resistance mutations to spread in the population. Thus, tolerance mutations pave the way for the rapid subsequent evolution of resistance. Preventing the evolution of tolerance may offer a new strategy for delaying the emergence of resistance.
Antibiotic-treatment failure is typically attributed to resistance. Many resistance mechanisms have been identified, including mutations that decrease the binding of the drug to its target and increased expression of efflux pumps (1). Resistance mutations result in a decrease in the effective concentration of the drug. The effect of such mutations is measured by the minimum inhibitory concentration (MIC), i.e., the lowest drug concentration needed to prevent visible growth of the microorganism. However, it has long been realized that other mechanisms can help bacteria survive antibiotic exposure (2). Nongrowing or slow-growing bacteria can survive bactericidal antibiotics that require active growth for killing. This property is known as “tolerance” (3). When the nongrowing phenotype occurs in only part of a clonal population, such as in biofilms, this subpopulation of “persisters” underlies treatment failure (4–6).
Recent studies (7–9) have shown that tolerance and persistence evolve rapidly under intermittent antibiotic exposure. When Escherichia coli populations were subjected to daily intermittent exposures to ampicillin, separated by intervals in fresh medium, the cultures became tolerant to ampicillin by acquiring mutations that extended their lag phase, i.e., the period before exponential growth is resumed after stationary phase, without any change in the MIC. The evolved mutants did not become resistant; they survived antibiotic treatment as long as they remained in the lag phase but were efficiently killed by ampicillin once growth resumed (7).
Whether tolerant strains that can evolve rapidly, impede or accelerate the evolution of antibiotic resistance is the subject of debate (10–13). To understand the interplay between resistance and tolerance, we evolved bacterial cultures using a slightly modified treatment protocol from (7) (fig. S1). We now used a lower dose of ampicillin (50 μg/ml), but which was still comparable to therapeutic doses and a fixed residual level during growth (14, 15). We continued daily intermittent exposures until resistance was established as defined by clinical standards (16). Starting with three different E. coli strains, including an enteropathogenic (EPEC) strain (table S1), we found that 11 of the 14 cultures reached an MIC at least sevenfold greater than the MIC of the ancestral strains (Fig. 1A). Further analysis of the resistant cultures with whole-genome sequencing revealed that they all harbored mutations in the promoter of ampC, which codes for a beta-lactamase known to confer resistance to ampicillin when overexpressed (17–19) (Fig. 1B).