Experimental Evolution of Antibiotic Resistance in Bacteria

Abstract

Evolution is typically measured as a change in allele or genotype frequencies over one or more generations. Consequently, evolution is difficult to show experimentally in a semester-long lab course because most organisms have longer generation times than fifteen weeks. Therefore, we designed an experiment to demonstrate and study evolution using bacteria as a model system. Bacteria are ideal organisms for studying evolution because they reproduce quickly and asexually, they permit the study of large sample sizes, they are easy to propagate, and it is easy to manipulate their environment (Elena & Lenski, 2003). In this simple experiment, the common soil bacterium, Bacillus thuringiensis, evolves resistance to the antibiotic streptomycin. Evolution of antibiotic resistance can be observed over a few weeks due to the short generation time of B. thuringiensis, which can divide every 45 minutes; the natural occurrence of random spontaneous mutations that occur during cell division; and experimental conditions that allow natural selection to occur. Thus, mutation and natural selection, the higher rate of reproduction or survival of organisms possessing favorable heritable traits, are the mechanisms of evolutionary change in this experiment. The antibiotic streptomycin prevents bacterial growth and division by binding to the bacterial ribosome and inhibiting protein synthesis. Two different spontaneous mutations may cause B. thuringiensis to become resistant to streptomycin. One mutation alters the shape of the ribosome so that the mutated ribosome is still capable of synthesizing protein but streptomycin is unable to bind to and inactivate the altered ribosome. Another mutation alters the shape of a protein in the cell membrane that is involved in transporting the antibiotic into the cell. This mutation results in lower permeability of the cell membrane to streptomycin. Bacteria that possess either of these mutations are able to grow and divide in the presence of streptomycin. There is a high probability that these mutations will occur in the experiment because of a combination of the frequency of spontaneous mutations, the number of cell divisions that occur in a short period of time, and the sheer number of bacteria present on each experimental plate. In bacteria, spontaneous mutations in a gene occur at an average frequency of one in every [10.sup.-9] generations. When this mutation rate is applied to the millions of cells on each Petri plate that will each experience over 16 million cell divisions, this means that most students will observe mutations. Hence, due to the intrinsic properties of bacteria and some simple manipulations, the students are able to observe a bacterial strain that is initially susceptible to the antibiotic evolve into one that is resistant. This experiment allows students to observe evolution in action and it illustrates how easily pathogenic bacteria can evolve into resistant forms that are much more difficult to treat. Since we are currently experiencing a major crisis in public health with the emergence of antibiotic resistant strains of bacteria (reviewed in Cohen, 2000) the subject matter of this laboratory experiment is highly relevant and, in our experience, very interesting to freshman college students. In addition, the topic provides the class an opportunity to explore the crisis of antibiotic resistance in greater depth (e.g., through articles, film, discussion). The major goals of this experiment are to observe evolution in real time, to understand mutation and natural selection and how they cause the bacteria to evolve, and to provide opportunities for the students to use evolutionary terminology. In the following sections, we will address the specific methods for setting up and performing the lab. In addition, we will address the results that are expected, the meaning of the results, how we assess students' understanding of the lab, and how we meet the goals of the experiment. …