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

Abstract

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 for 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 sub-MIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at sub-MICs in 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 anitibiotics 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 antibiotic as well, suggesting that adaptation to the growth environment occurred in parallel with 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 sub-MIC exposure in real life settings.

IS1-related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli

“…Since nitrofurantoin MICs of both EC0026B and EC0880B were at the clinical breakpoint for nitrofurantoin resistance (>64 mg l −1 ) determined by the European Committee on Antimicrobial Susceptibility Testing [37], both isolates may grow under therapeutic or prophylactic dosing of nitrofurantoin [38,39] and gain adaptive mutations under sub-MIC exposure [40] in the urinary tract or gut. Moreover, the deletion of nfsB in these isolates, which already had inactivating mutations in nfsA, would cause an irreversible reduction in their nitrofurantoin susceptibility, paving the way to the emergence of nitrofurantoin-resistant cells if there were to be subsequent resistance mutations, such as those in ribE and the marA-marB intergenic region [40,41]. Importantly, however, because the loss of nfsB reduces the reproduction rate of E. coli [33], the establishment of ΔnfsB mutants may only occur under selective pressure of nitrofurantoin.…”

IS1-related large-scale deletion of chromosomal regions harbouring oxygen-insensitive nitroreductase genenfsBcauses nitrofurantoin heteroresistance inEscherichia coli

“…Nonetheless, neither the level of the reduction in nitrofurantoin susceptibility nor the nitrofurantoin MIC of each strain is as great as those of strains EC0026B and EC0880B. Because both EC0026B and EC0880B had an MIC of 64 mg/L, which is the EUCAST clinical breakpoint for nitrofurantoin resistance (>64 mg/L), these strains may grow under therapeutic or prophylactic dosing of nitrofurantoin [34,35] and gain adaptive mutations under sub-MIC exposure [36] in urinary tract or gut. Moreover, the deletion of nfsB in these strains would cause an irreversible reduction in their nitrofurantoin susceptibility, paving the way to the emergence of nitrofurantoin-resistant cells if there were to be subsequent resistance mutations, such as those in nfsA, ribE, and the marA-marB intergenic region [36,37].…”

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide

“…Common antibiotic concentrations in the water are usually low, on the order of the ng/L-μg/L, enough to select resistance against several antibiotics. [5,6] On the other hand, this range of concentrations will result in extremely minimal amounts of degradation products, significantly decreasing the risk of toxic effects on human and animal health. Overall, our results suggest that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic-contaminated environments.…”

“…[1,2] Moreover, the accumulation of antibiotics has the potential to generate reservoirs of antibioticresistant bacterial strains since the presence of these drugs in the environment strongly promotes the evolution of these strains, [3,4] even at very low concentrations. [5,6] Antibiotic resistance is a growing, widespread problem, with antibiotic-resistant infections causing in 2019 almost 1.3 million deaths worldwide and a huge economic burden. [7] Nevertheless, there are fewer and fewer antibiotics with novel modes of action available in the market and it is currently non-attractive for companies to spend resources on research to develop new antibiotics.…”

“…However, their continuous discharge into the environment, together with the low rates of degradation of some of them, is making antibiotics accumulate and become a new micro‐pollutant, which are currently poorly legislated [1,2] . Moreover, the accumulation of antibiotics has the potential to generate reservoirs of antibiotic‐resistant bacterial strains since the presence of these drugs in the environment strongly promotes the evolution of these strains, [3,4] even at very low concentrations [5,6] . Antibiotic resistance is a growing, widespread problem, with antibiotic‐resistant infections causing in 2019 almost 1.3 million deaths worldwide and a huge economic burden [7] .…”