The effect of sub inhibitory concentrations of antibiotics on bacterial evolution

Antibiotics and the genetic elements that confer resistance to them are both disseminated into aquatic environments via human waste streams and agricultural run-off. Antibiotics are not readily broken down or degraded and therefore persist in the environment at low concentrations. Such pollution with antibiotics establishes a selection gradient, but may also raise the frequency of events that can generate resistance : point mutations; recombination; and lateral gene transfer. The effect of clinical levels of antibiotics on the evolution of antibiotic resistance has been extensively studied. This study examined the response of bacteria to sub inhibitory levels of antibiotics, such as those found in aquatic environments subject to human pollution. Two species, Pseudomonas aeruginosa and Pseudomonas protogens were exposed to subclinical levels of the antibiotics kanamycin, tetracycline and ciprofloxacin. The minimum inhibitory concentration (MIC) of the bacteria to these antibiotics was determined, and then bacteria were subjected to 1/10 the MIC in a serial streaking experiment over 40 generations. Repetitive Element polymerase chain reactions were carried out to monitor changes in genomic DNA every five generations. Significant changes in the banding patterns of both species were discovered, even at five generations, suggesting that 1/10 the MIC induces mutation and/or recombination events. Lines exposed to sub-clinical levels of antibiotics also exhibited phenotypic effects. There were significant changes to colony morphology in exposed lines. The final MICs at generation 40 were significantly higher in some lineages of Ps. protogens, showing that even subclinical levels of antibiotics can select for resistance. In conclusion, these experiments demonstrate that exposure to sub-clinical levels of antibiotics, such as those found in waste streams, can induce significant genotypic and phenotypic changes.