Abstract
The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.
Highlights
Antibiotics represent one of mankind’s most important medical inventions but during the last decades the continuing rapid development of antibiotic resistance has emerged as one of the most serious health care problems, both in community and hospital settings [1,2,3]
At a concentration 1/30 of the MICsusc, the susceptible strain grew about 15% slower than without antibiotic whereas the resistant mutant seemed unaffected, suggesting that resistant strains are strongly selected at these low concentrations
The strains were genetically tagged with variants of the green fluorescent protein gene to allow counting of large populations of competing cells by fluorescence activated cell sorting (FACS), thereby significantly reducing any experimental errors associated with counting of small populations
Summary
Antibiotics represent one of mankind’s most important medical inventions but during the last decades the continuing rapid development of antibiotic resistance has emerged as one of the most serious health care problems, both in community and hospital settings [1,2,3]. While it is evident that the high concentrations of antibiotics used therapeutically can select for resistant mutants, it still remains unclear how important the low antibiotic concentrations that due to anthropogenic input pollute natural (e.g. aquatic or soil) environments [8,9,10], that are produced naturally by antibiotic-producing micro-organisms or that are present in certain human/animal body compartments during therapeutic or growth promotion use, are for the selection and enrichment of resistant mutants. Earlier studies on selection with small differences in bacterial susceptibility to antibiotics show that selection can efficiently act on minute differences to select for resistance [13,14,15]. We further explore the mutant selective window assumption and as outlined schematically, we examine for two bacterial species and three antibiotics how far below the MICsusc pre-existing and de novo generated resistant mutants can be selectively enriched because of minute reductions in the growth rate of their susceptible counterparts We further explore the mutant selective window assumption and as outlined schematically in Fig. 1A, we examine for two bacterial species and three antibiotics how far below the MICsusc pre-existing and de novo generated resistant mutants can be selectively enriched because of minute reductions in the growth rate of their susceptible counterparts
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