Focus: Students investigate the growth of bacteria in the presence of antibiotics and use the results to explain a case of antibiotic-resistant tuberculosis, presented in an Internet-based interview.
Major Concepts: The re-emergence of some diseases can be explained by evolution of the infectious agent (for example, mutations in bacterial genes that confer resistance to antibiotics used to treat the diseases).
Objectives: After completing this activity, students will
Prerequisite Knowledge: Students should be familiar with bacterial growth and with evolution by natural selection.
Basic Science-Public Health Connection: In this activity, students learn that the evolution of antibiotic resistance among bacteria observed in laboratory experiments occurs in the natural environment as well, and that such evolution has serious consequences for the effectiveness of treatments for some diseases.
In 1943, penicillin was introduced as the "magic bullet" for curing many infectious diseases. By 1946, however, approximately 14 percent of Staphylococcus aureus strains isolated at a London hospital were resistant to penicillin. Today, scientists estimate that more than 95 percent of all S. aureus strains are penicillin-resistant.
After the introduction of penicillin, additional antibiotics were rapidly isolated and developed, including streptomycin and the tetracylines. Today, there are more than 100 antibiotics available. Nevertheless, some strains of at least three bacterial species (Enterococcus faecium, Mycobacterium tuberculosis, Pseudomonas aeruginosa) are resistant to all of these antibiotics, and health care workers fear the time is rapidly approaching when more deadly organisms escape the effects of all known antibiotics.
The primary reason for the increase in antibiotic resistance is evolution. When mutant genes arise that make a bacterium less sensitive to an antibiotic, that bacterium survives and produces descendants in an environment rich in antibiotics. That is, the process of natural selection operates. Multiple mutations may be required to result in fully resistant bacteria. However, once resistant genes appear, bacteria have a variety of mechanisms for exchanging those (and other) genes both within and across species. These mechanisms include conjugation, transformation, transduction, and transposon-mediated exchange. This exchange allows for "accelerated evolution" of bacterial species (accelerated in the sense that random mutations that result in antibiotic resistance need not occur in every individual bacterium, nor even in every species of pathogen, but can simply be acquired from another organism).
This activity invites students to explore one reason for the re-emergence of some infectious diseases: the evolution of antibiotic resistance among pathogens. In Activity 4, Protecting the Herd, students explore another reason for the re-emergence of infectious diseases.
You will need to prepare the following materials before conducting this activity:
Students complete this activity across a five-to-seven-day period. You will need to prepare the materials for the laboratory exercise. Ordering information and preparation directions immediately follow the activity.
Information about the safe use of microorganisms in classrooms, including lists of organisms considered safe for students at various levels of school, can be found at: http://www.science-projects.com/safemicrobes.htm. A number of leaders in infectious diseases, including scientists from NIH, contributed to the Web site. Pseudomonas fluorescens, the organism used in the laboratory exercise in this activity, is included on the list of microorganisms considered appropriate for students in grades 9 or higher. Nevertheless, experts acknowledge that people who are immunocompromised may be at risk for infection by organisms that do not affect healthy individuals. We recommend that you read a statement such as the following to your classes before beginning the activity:
|Pseudomonas fluorescens, the bacterium used in the laboratory exercise you will begin soon, does not cause disease in healthy people. However, people who have weakened immune systems should not have contact with most microorganisms or with people who handle those organisms. Your immune system may be weakened if you are undergoing antibiotic therapy, if you are taking immunosuppressive drugs or drugs for cancer treatment, or if you have AIDS or are HIV-positive. If you have a weakened immune system for these or any other reasons, let me know and I will provide you with an alternative experience that is safer for you.|
Students who should not participate in the laboratory exercise can view a video demonstration of it on the Web site as described in the following paragraphs.
They can rejoin the class in Day 3 of the activity, after the other students have recorded their results and discarded their bacterial cultures.
If you do not have the time or facilities for conducting the laboratory exercise, you will need only one day to complete this activity. Complete Steps 1 to 3, Day 1, and then have students view a video demonstration of the laboratory exercise, Bacterial Growth Experiment on the Emerging and Re-emerging Infectious Diseases Web site. Students will need copies of Master 3.1 to help them follow the steps in the demonstration. Then move to Day 3 of the activity.
Note to teachers: If you do not have enough computers equipped with Internet access to conduct this activity, you can use the print-based alternative.
DAY 1 (5 to 7 days before Day 3 of the activity)
1. Remind students of the theory of evolution by natural selection and tell them that a powerful feature of theories is that they lead to hypotheses that can be experimentally tested.
Students should be able to state the basic elements of the theory of evolution: (1) there is variation among the individuals in a population; (2) some of these differences can be inherited; (3) some individuals will be better adapted to their environment than others; (4) the better adapted individuals will reproduce more successfully; and (5) thus, the heritable characteristics that make individuals better adapted will increase in frequency in the population.
2. Organize students into teams of three and challenge the teams to use their understanding of evolution by natural selection to write a hypothesis about what will happen in a population of bacteria after growing for several generations in the presence of an antibiotic.
If students have difficulty with this, stimulate their thinking by asking questions such as, "What effect does an antibiotic usually have on a bacteria? Do you know of cases in which that effect did not occur? What does that suggest about variations that exist in the bacteria population? Which bacteria survived? What trait did they pass on to other progeny?"
3. Convene a class discussion in which you ask several teams to share the hypotheses they developed. Challenge the class to work together to refine them into one hypothesis similar to the following:
If a bacterial culture is grown in a medium containing an antibiotic, then after several generations, all of the bacteria in the culture will be resistant to the antibiotic.
4. Tell students that they will conduct an experiment to test this hypothesis and explain that they will also consider the implications of their results for controlling infectious diseases in an activity the following week. Then distribute Master 3.1, Bacterial Growth Experiment, and instruct students to complete Steps 1 through 4 with their team members.
Emphasize that for safety reasons as well as the success of their experiments, students must use aseptic techniques. If students are not familiar with aseptic techniques for handling bacterial cultures, you will need to demonstrate them. Alternatively, you can have your students view the "Day 1" video segment of Bacterial Growth Experiment, which shows students using aseptic techniques as they prepare the initial cultures in the experiment.
DAY 2 (2 to 3 days before Day 3 of the activity)
1. Direct teams to complete the remaining steps on Bacterial Growth Experiment.
1. Tell students that today they will analyze the results of the bacterial growth experiment they have been running and will use those results to help explain what happened to a high school student who had tuberculosis.
2. Organize students into teams and instruct them to collect their bacterial growth plates. While they do this, distribute a copy of Master 3.2, Discussion Questions for the Bacterial Growth Experiment, to each student. Tell the teams to draw (or describe) their results on the flow chart on Bacterial Growth Experiment first, then refer to those results as they discuss and write answers to the discussion questions.
Depending on students' microbiology background, you may need to explain that when a single, microscopic bacterium is placed on an agar plate, it will grow and divide into two progeny cells. Each progeny cell will grow and divide, and so on, until thousands and thousands of individual bacteria are growing right in that spot. At this point, the growth becomes visible to us as a colony of bacteria. Each colony came from a single original bacterium on the plate. When approximately 10,000 or more bacteria are plated, each individual bacterium is close enough to a neighboring bacterium that the colonies they produce merge together, and we observe confluent growth, or a "lawn," of bacteria across the plate.
Move among the teams as they discuss each question and help lead students to the following understandings.
Question 1 Compare the bacterial growth on the two plates from the parental culture (Plates 1 and 2). Which has more growth? Explain why. How do you explain the presence of bacteria on the plate containing kanamycin?
The nutrient agar plate (Plate 1) should show a lawn of bacteria, or confluent growth, whereas the plate containing kanamycin should show only 50 to 100 colonies. Students should explain that the antibiotic prevented the growth of most of the bacteria on Plate 2. A simple, straightforward answer is all students need to provide for the last question: The bacteria that grew on Plate 2 were resistant to the antibiotic.
Question 2 Compare the growth on Plates 3 and 4, which you prepared from culture A (without kanamycin). How does the growth on the plates with and without kanamycin appear? What does this tell you about the bacteria grown in culture A?
The plate without kanamycin (Plate 3) should show a lawn of bacterial growth, whereas the plate with kanamycin (Plate 4) should show 50 to 100 colonies. The results on Plate 3 indicate that a lot of bacteria were growing in the sample plated from culture A. Comparing the results on that plate with the results on Plate 4 indicates that some of the bacteria in the culture (for example, 50 out of 10,000 or more) were resistant to the antibiotic, but most were not.
Question 3 Compare the growth on Plates 5 and 6, which you prepared from culture B (with kanamycin). How does the growth on the plates with and without kanamycin appear? What does this tell you about the bacteria grown in culture B?
Both plates should show a lawn of bacterial growth. This indicates that most or all of the bacteria growing in this culture were resistant to kanamycin.
Question 4 Compare the growth of cultures A and B on Plates 4 and 6 (with kanamycin). Explain how culture B could have so many more resistant bacteria than culture A, even though they both came from the same parental culture.
If, after a minute or two of discussion, students cannot offer an explanation, suggest that they use their understanding of natural selection to explain the difference in the results on the plates for the two cultures. They should be able to explain that the environment in culture B (which contained kanamycin) selected for the growth of those bacteria that were resistant to kanamycin. By the time students plated a sample from that culture, all of the bacteria in the sample were resistant, so they all grew on the plate with kanamycin, resulting in a lawn of bacterial growth (Plate 6). Culture A did not contain kanamycin, so there was no selection for kanamycin resistance, and most of the bacteria they plated from that culture were not resistant. Thus, most did not grow on the plate with kanamycin (Plate 4).
Question 5 How do you explain the presence of some resistant bacteria in the parental culture and culture A?
To answer this question, students must recognize that bacteria become resistant (for example, through mutation) before natural selection operates. In other words, the bacteria in the parental strain did not "know" that some of them would be placed in growth medium with kanamycin and "respond" by becoming resistant. Instead, in the parental strain, a few bacteria were already present that were resistant to kanamycin, even though there was no kanamycin present. Similarly, a few bacteria in culture A were resistant to kanamycin even though no antibiotic was present. When the resistant and nonresistant bacteria from the parental culture were placed in medium containing kanamycin (culture B), only the resistant bacteria survived and reproduced, passing their kanamycin resistance trait on to their progeny. Soon, virtually all of the bacteria in the culture—the progeny of the original resistant bacteria—were resistant to kanamycin, as observed on the students' plates.
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