Pervasive Selection for Clinically Relevant Resistance and Media Adaptive Mutations at Very Low Antibiotic Concentrations

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e., when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched? Third, are the mutations selected under laboratory settings at subMIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at subMICs in the presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline, and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the antibiotics tested, and selection for fosfomycin- and nitrofurantoin-resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotics as well, suggesting that adaptation to the growth environment occurred in parallel with the selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during subMIC exposure in real-life settings.

Automated summary

Experimental evolution studies have shown that weak antibiotic selective pressures can select resistant mutants even at concentrations below the minimum inhibitory concentration (MIC). This study addressed several important questions, including the lowest antibiotic concentrations at which de novo resistance mutations can occur, the types of adaptive mutations that are enriched under weak antibiotic selections, and whether the mutations selected in laboratory settings at subMIC are also observed in clinical isolates. The results showed rapid evolution for all antibiotics tested, with selection for resistance observed at concentrations as low as 1/2000th of the MICsusceptible. The evolved resistant mutants showed increased growth yield and outcompeted the susceptible ancestral strain even in the absence of antibiotics, indicating adaptation to the growth environment. Genomic analysis revealed that some of the mutations selected under these conditions are also found in clinical isolates, highlighting the importance of experimental evolution at very low antibiotic levels in identifying novel mutations contributing to bacterial adaptation during subMIC exposure.