Evolution of Antibiotic Resistant Bacteria TEACHER BACKGROUND INFORMATION: Revised 9/05 Introduction: During normal bacterial growth in a nutrient rich culture, some cells develop an alteration (change) in their DNA. These changed cells are called mutants. It is likely that mutant cells develop when mistakes occur in the normal sequence of their nucleotides. If allowed to grow in a selective environment, mutant bacterial cells will reproduce successive generations of bacteria with this altered DNA. Consequently, these bacteria exhibit characteristics different from the parent strain. In normal populations, the incidence of mutants is estimated to be between 1 in 10,000 to 1 in 1,000,000 cells. Because bacterial populations in rich culture can reach one billion individuals in 12 hours, a certain number of mutants are predictable. The fate of mutant bacterial cells is determined by their ability to survive the environmental conditions surrounding them. Under normal conditions, mutants will have conditions less favorable than the parent strain and end up being crowded out. These mutants will not survive until the population is exposed to a natural selective agent. This supports Darwin s theory of natural selection and, in turn, can be compared to Lamarck s hypothesis of acquired traits. In this activity, the selective agent will be one that measures some degree of antibiotic (ampicillin) resistance. Ampicillin is a long established broad spectrum antibiotic with well-documented chemical and physical characteristics. E. coli strain MM 294 bacteria are grown in LB broth for 24 to 48 hours. When the bacteria are spread onto the antibiotic (ampicillin) gradient plate and incubated, the different concentrations of ampicillin in the media and the pattern of growth demonstrate several key molecular biology principles: 1) within a population of bacteria grown in a nutrient rich culture, random mutations occur in some bacteria, 2) bacteria containing the mutation for ampicillin resistance will survive on a media containing ampicillin, and 3) some bacteria show more resistance than others. The ability of bacteria that are normally susceptible to antibiotics to grow on a media plate with ampicillin is an indication that a genetic change or mutation has occurred in the bacteria. Antibiotic resistance in bacteria occurs mostly in a non-chromosome, circular piece of DNA called a plasmid. Plasmids replicate independently at a faster rate than individual bacteria cells. As the number of bacteria increases, each succeeding generation carries the original number of plasmids of the parent, and the new cells will produce greater numbers of plasmids, including those carrying mutations. Bacteria growing on media with a higher antibiotic concentration owe their resistance to the number of copies of the plasmid containing the mutation. The more copies of the mutation, the higher concentration of antibiotics the bacteria withstands. Bacteria also have a second ability to share their newly evolved resistant genes with other bacteria called conjugation. During conjugation a tube called the sex pilus will connect two bacteria. The bacteria exchange genetic material through the tube, which can include antibody resistance genes. This allows additional antibiotic resistant bacteria to develop and grow. Over time and in the continued presence of an antibiotic containing 1
environment, these bacteria will become the dominant strain. For this reason, antibiotics given to ill people must be carefully designed around concentration and duration in order to destroy all the disease causing bacteria, including those with mutations. If the prescribed regimen of antibiotics is interrupted or stopped too early, the resulting environment favors the surviving bacteria with the resistant mutation. As the surviving bacteria multiply, replicate plasmids and exchange genes, they develop more tolerance to the antibiotic. Bacteria s ability to develop resistance to antibiotics in this manner helps explain why it is necessary to take all prescribed antibiotics as directed by a doctor. In observing the activity s results, students will notice that the agar in the Petri dish containing no antibiotic has a lawn growth of bacteria. However, bacterial growth thins to just a few separate colonies and finally no colonies as you move away from the noantibiotic side toward higher antibiotic concentration in the agar. The separate individual colonies farthest away from the no-antibiotic side of the plate, represent bacteria that evolved a mutation to resist some ampicillin in the media. The media area with no growth represents the lethal concentration for all bacteria, including those that might contain a mutation for resistance. The correctly prescribed regimen of antibiotics by a doctor is intended to reach this 100% lethal environment. TEACHER PREPARATION: Procedure: A couple of days before the lab, start preparing the media for the activity. An alternative to the teacher preparing the media is to allow students to prepare their own. Equipment per group of two to three students: 3 - Empty sterile Petri dishes if students make their own plates, two for the gradient slant agar plates and one for the LB agar plate 1 - LB agar plate with no ampicillin * 4 - Large paper clips bent as spreaders (wrapped in aluminum foil and sterilized in a 350 o F oven for 30 minutes) 2 - Inoculating loops 2- sterile transfer pipettes 10 ml of sterile LB/nutrient broth for each student group * 1-50 ml culture tube MM294 strain of E.coli * 1 pencil Ampicillin solution- 0.05 grams ampicillin salt dissolved in 1 ml of sterile distilled water LB premix * Agar * Marking pen * Ready-to-pour media and bacterial strains can be purchased from most biological supply companies. 2
Media Preparation To be completed two to three days before the activity. LB broth solution: Calculate the amount of nutrient broth that is to be supplied to the students and add extra for spillage and other factors. Weigh out 2.5 grams of LB premix and dissolve it in 100 ml of distilled water. For a larger amount, use multiples of the ingredients listed previously. Cap the bottle tightly and place it in a boiling water bath for at least 30 minutes. Sterile Water: Place 50 ml of distilled water in a screw top bottle. Tighten the cap and place the bottle in a boiling water bath for 30 minutes. Remove the bottle and allow it to cool. Preparing Gradient Antibiotic LB Agar Plates: Mix 2.5 grams of Luria broth and 1.5 grams of agar in 100 ml of distilled water in a Kimax or Pyrex bottle with a screw-on cap. Microwave the solution until it is clear (free of suspension). Caution: Never microwave any solution in a bottle with a tight cap or lid. With the cap tightened down, place the bottle into a boiling water bath for at least 30 minutes. After 30 minutes, immediately pour enough of the agar into one sterile Petri dish to cover the bottom of each dish. Place the lid back on the plate and leave flat on the table top to cool and harden. Place the remaining agar in a 55 o C water bath until you are ready to pour the gradient plates. If students are preparing their own plates, they can let the agar cool a short time on the table top and proceed to the next step. For a step-by-step tutorial on the preparation of the LB broth, LB agar plates and sterile water visit: www.biotech.iastate.edu/publications/ppt_presentations/default.html and find the section on Transformation-Media Preparation. While the agar is cooling to 55 o C in a water bath or on your tabletop, prepare two ampicillin gradient agar plates for each group. To start, rest one edge of each sterile Petri dish on a pencil. 3
When the agar is cool enough, pour the agar containing no antibiotic into the Petri dish until it is two-thirds of the way across the bottom of the Petri dish and cover. Repeat this with the second dish. Allow the agar to cool and harden. After drying, continue to the next step. After the first layer has hardened, remove the pencil and lay the Petri dish flat on the table. If time allows, you can let the plates sit on the table top for several days to dry before proceeding to the next step. If you plan to complete the plates the same day, allow the remaining LB agar to cool until you can barely hold the warm flask in your hand and add 1 drop of ampicillin solution to the remaining LB agar. Pour the agar containing the antibiotic two-thirds of the way across the top of the first layer, leaving the thickest edge of the first layer uncovered. If you plan to complete the plates after several days of drying, prepare another bottle of LB agar exactly like the first day. Allow the LB agar to cool until you can barely hold the warm flask in your hand and add 2 drops of ampicillin solution per 100 ml of media. Pour the agar containing the antibiotic two-thirds of the way across the top of the first layer, leaving the thickest edge of the first layer uncovered. 4
Replace the lid of the Petri dish and allow the media to dry on the lab table until the condensation on the lid evaporates. The ampicillin will diffuse through the agars establishing a concentration gradient across the entire plate. The highest concentration of antibiotic is at the side with the thickest ampicillin agar and the lowest concentration is at the side of the thickest agar without ampicillin. After the agars have completely hardened, mark a small portion of the underside of the plate to indicate the position of the different concentrations. Place the name of your group on each plate with a marker. If after the drying time you do not plan to use the plates immediately, place them into a refrigerator for up to five days. Plates should be used within 2-5 days. The antibiotic will continue to diffuse into the media and eventually the concentration gradient will be lost. Results: No Antibiotic Lawn growth Antibiotic agar line Resistant bacteria High Antibiotic Conc. Safety and Clean Up: It is important to follow your school district s lab safety rules and use established safe lab practices especially when the handling microorganisms. All contaminated equipment can be made safe by soaking in a 10% Clorox solution for 15 minutes. Glassware can be washed with dish soap and water and plastic items disposed of in the regular trash. 5
STUDENT INSTRUCTIONS: Pre-Lab One day before the start of the lab, prepare a fresh culture of bacteria. Using a sterile inoculating loop, scrape a mass of cells from a slant culture of E. coli MM 294 and streak it on a LB agar plate. Incubate the plate at 37 o C. for approximately 24-48 hours or until a good growth of bacteria is established. Overnight Culture: Lab Day 1 Use a sterile pipet to transfer 10 ml of sterile Luria (LB) broth into a 50 ml culture tube. Take a sterile inoculating loop or toothpick and scrape a mass of E.coli MM294 from the LB agar plate that had been streaked and incubated for 24 hours. Transfer the loop of bacteria to the culture tube with the LB broth and loosely recap the tube. Place a piece of tape across the cap so will not come off during shaking. Mark the tube with the name of your group. Incubate for 24 hours at 37 o C. Occasional shaking of the tube will insure that the culture grows to its maximum potential. If an incubator is not available, let the culture sit at room temperature with occasional shaking for 3-5 days. You can judge whether the culture is ready by holding it up to the light. The solution should be cloudy enough so you cannot see through it. 6
Lab Day 2 Remove the culture from the incubator, water bath, or table top holder. Use a sterile transfer pipet to add three drops of the culture solution to each of your two gradient plates. Use a sterile large paperclip bent in the shape of a spreader and spread the culture evenly on the plate. Incubate with the agar down for two hours and then flip the plates. As an alternative to an incubator, you can let the plate grow at room temperature until the colonies are well formed. Observe the plates after 24 and 48 hours. Record Your Observation: Draw your results. Explain the significance of your results. 7