Sub-inhibitory concentrations of antibiotics and bacterial evolution

Antibiotic resistance is one of the greatest threats to human health in the 21st century. It is predicted that by 2050 antibiotic resistant infections will account for 10 million deaths annually. The mechanisms by which therapeutic concentrations of antibiotics select for resistance mutations and the lateral transfer of resistance genes is well known. However, the effect of lower concentrations of antibiotics, particularly when these are environmental contaminants, is relatively unknown. Antibiotics can enter the environment through human waste streams, agricultural run-off and pharmaceutical effluent. They can then persist in the environment at low concentrations. These low levels of antibiotics can induce the SOS response, a general response to DNA damage. Amongst the various effects of the SOS response are an increase in mutation rates driven by expression of error prone DNA polymerases, and a general increase in rates of recombination, transposition, conjugation and transformation. All these effects increase the likelihood of cells becoming antibiotic resistant. In this thesis, I collated data on the concentrations of clinically relevant antibiotics that have been reported from diverse environmental compartments. I then used these findings to design experiments that simulated the likely concentrations experienced by environmental bacteria. Once the environmental concentrations of antibiotics had been established, I carried out experimental evolution experiments, exposing bacteria to appropriate concentrations, by performing serial plating across multiple generations. Concentrations of antibiotics equivalent to 1/10 the minimum inhibitory concentrations promoted resistance after as little as 15 single colony passages on media. To identify the mechanisms of resistance, whole genome sequencing was performed. Point mutations were identified in relevant genes from all the lines with increased resistance. In ciprofloxacin treated lines, the relevant mutation occurred in gyrA, and was identical to a resistance mutation described in clinical pathogens. In kanamycin treated lines, a point mutation in fusA was detected. Again, this mutation and gene have previously been implicated in kanamycin resistance. To determine the role of the SOS response in the fixation of these mutations I performed similar experiments using RecA knockout mutants. Most environmental bacteria are not planktonic cells, but grow in biofilms. Consequently, to better mimic the likely effects of antibiotic pollution on environmental bacteria, I investigated the effects of environmental concentrations of antibiotics on biofilm bacteria. To study biofilms, I first had to modify protocols used for liquid and plate experiments, since biofilms display significantly higher resistance to antibiotics compared to their planktonic counterparts. This resulted in a novel method to determine the minimal inhibitory concentration of antibiotics in biofilms. I then exposed bacterial biofilms to environmentally relevant concentrations of antibiotics and using whole genome sequencing identified point mutations known to be associated with antibiotic resistance. The results of this work have clear implications. Antibiotics persist in the environment at low, but biologically relevant concentrations where they can have significant impacts on normal microbial processes. These low concentrations up-regulate mutation rates and generate increasing bacterial resistance to antibiotics amongst all bacteria, not just those of clinical concern. We expect that the phenomena I describe under experimental conditions are mirrored in the general environment. Acquisition of resistance is essentially stochastic, relying on rare events at a single point in time, coincident with relevant selection pressures that allow newly resistant lineages to compete and increase in abundance. Widespread pollution with antibiotics enhances the rates at which key mutational events are likely to occur, while simultaneously providing the selection regime to promote survival of newly resistant cells. The potential is clear for environmental organisms to acquire resistance, which could then be disseminated globally through horizontal gene transfer or become significant pathogens in their own right. Antibiotic pollution joins overuse and misuse as a significant threat to human health and the preservation of the efficacy of antibiotics.