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. Antibiotic resistance has emerged as a very significant health care problem due to the extensive use and misuse of antibiotics in human and veterinary medicine and in agriculture. It remains unclear where most of the resistant bacteria have been selected, and in particular if the low antibiotic concentrations that are present in natural environments or in human/animal body compartments during therapeutic or growth promotion use, are important for the selection and enrichment of resistant mutants. The presented data shows that for several clinically used antibiotics extremely low concentrations, similar to the concentrations found in natural environments, can select for resistant bacteria. These results suggest that antibiotic release into the environment might be a significant contributor to the emergence and maintenance of resistance and emphasize the importance of introducing measures to reduce antibiotic pollution.

Cationic antimicrobial peptides (AMPs) are an intrinsic part of the human innate immune system. Over 100 different human AMPs are known to exhibit broad-spectrum antibacterial activity. Because of the increased frequency of resistance to conventional antibiotics there is an interest in developing AMPs as an alternative antibacterial therapy. Several cationic peptides that are derivatives of AMPs from the human innate immune system are currently in clinical development. There are also ongoing clinical studies aimed at modulating the expression of AMPs to boost the human innate immune response. In this review we discuss the potential problems associated with these therapeutic approaches. There is considerable experimental data describing mechanisms by which bacteria can develop resistance to AMPs. As for any type of drug resistance, the rate by which AMP resistance would emerge and spread in a population of bacteria in a natural setting will be determined by a complex interplay of several different factors, including the mutation supply rate, the fitness of the resistant mutant at different AMP concentrations, and the strength of the selective pressure. Several studies have already shown that AMP-resistant bacterial mutants display broad cross-resistance to a variety of AMPs with different structures and modes of action. Therefore, routine clinical administration of AMPs to treat bacterial infections may select for resistant bacterial pathogens capable of better evading the innate immune system. The ramifications of therapeutic levels of exposure on the development of AMP resistance and bacterial pathogenesis are not yet understood. This is something that needs to be carefully studied and monitored if AMPs are used in clinical settings.

Most types of antibiotic resistance impose a biological cost on bacterial fitness. These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium. Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium. This difference in mutation spectra was caused by either a growth condition–specific formation or selection of the compensated mutants. These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

Gene duplication-amplification (GDA) processes are highly relevant biologically because they generate extensive and reversible genetic variation on which adaptive evolution can act. Whenever cellular growth is restricted, escape from these growth restrictions often occurs by GDA events that resolve the selective problem. In addition, GDA may facilitate subsequent genetic change by allowing a population to grow and increase in number, thereby increasing the probability for subsequent adaptive mutations to occur in the amplified genes or in unrelated genes. Mathematical modeling of the effect of GDA on the rate of adaptive evolution shows that GDA will facilitate adaptation, especially when the supply of mutations in the population is rate-limiting. GDA can form via several mechanisms, both RecA-dependent and RecA-independent, including rolling-circle amplification and nonequal crossing over between sister chromatids. Due to the high intrinsic instability and fitness costs associated with GDAs, they are generally transient in nature, and consequently their evolutionary and medical importance is often underestimated.