1.2.3 Attack Of The Superbugs - 2023

Streptococcus pneumoniae, commonly referred to as pneumococcus, is a bacterium commonly found in the back of the nose in healthy people. Occasionally, pneumococcal bacteria spread to other parts of the body and cause diseases such as ear infections, sinus infections, pneumonia, and meningitis. Before the introduction of penicillin in the early 1940s, a person might die from a pneumococcal infection. After the introduction of penicillin, pneumococcal infections were effectively treated. However, strains of pneumococcal bacteria resistant to penicillin were discovered in the 1960s. Since then, new strains of pneumococcal bacteria resistant to multiple antibiotics have been emerging. HISTORICAL HIGHLIGHT Breaking the Mold In 1945 the Nobel Prize in Physiology or Medicine was given to Sir Alexander Fleming, Howard Florey, and Sir Ernst Boris Chain for their role in the “discovery of penicillin and its curative effect in various infectious diseases.” Although Fleming discovered penicillin in 1928, Florey, Chain, and their colleagues at the Sir William Dunn School of Pathology at Oxford University turned penicillin from a laboratory curiosity into a life-saving drug. Another vital figure in the lab was a biochemist, Dr. Norman Heatley, who used every available container, bottle, and bedpan in the lab to grow vats of the penicillin mold. After growth appeared, he suctioned off the fluid, purified the antibiotic, and finally measured the strength of the penicillin. Before introducing penicillin, there was no effective treatment for diseases such as pneumonia, gonorrhea, rheumatic fever, 1 or even everyday infections. Hospitals were full of people with blood poisoning contracted from a cut or a scratch, and doctors could do little for them but wait and hope. As the world’s first antibiotic, Penicillin marked a turning point in human history. But World War II halted the mass production needed for clinical trials. In 1941, Florey and Heatley went to the United States, where they worked with American scientists to develop a means of mass-producing penicillin. However, the fungus, Penicillium notatum that Fleming discovered, did not yield enough penicillin to treat people reliably. Laboratory assistant Mary Hunt purchased a cantaloupe from a market with “pretty, golden mold” that provided the final key. The mold turned out to be the fungus Penicillium chrysogeum that yielded 200 times the amount of penicillin! American pharmaceutical companies began producing 650 billion units a month throughout the war, drastically cutting the death rate from bacterial pneumonia to less than one percent. Over the last decade, almost every type of bacteria known to cause disease has become stronger and less responsive to antibiotic treatment. Antibiotic resistant bacteria are threatening communities with strains of infectious diseases. Tuberculosis, malaria, skin infections, pneumonia, and ear infections are just a few of the diseases that have become harder to treat. For this reason, antibiotic resistance has been called one of the world’s most pressing public health problems and is one of the Centers for Disease Control's top concerns. In this activity, you will investigate the mechanisms by which DNA from one bacterial cell is transferred to another bacterial cell. When the DNA that is transferred carries an antibiotic-resistant gene, the intercellular transfer enables the new cell to become antibiotic resistant. Over time, one bacterial cell containing an antibiotic-resistant gene could lead to an army of superbugs. 2 ● Computer with internet access ● Carolina Introductory Bacterial Conjugation Kit ● Aseptic Technique Procedure Read the first section of Daniel Zwerdling’s Antibiotics on the Farm article titled A New Public Health Threat on the Farm. Brainstorm why you believe Ciprofloxacin is not working on Culbreath. Record your ideas in the space below and discuss the validity of each idea. Read your assigned patient story and their incorporated case notes. Working with your group, use prior knowledge and clues in the case report to brainstorm why you believe antibiotics did not work on the individual in your patient story. Share your group’s ideas with the class. As a class, once each group has presented, discuss the validity of each idea and eliminate any ideas that the class believes are inaccurate. Complete the table as each group presents their information. 3 Case Files Why Antibiotics Failed Paolo Mohammed Lill-Karin You learned in the previous activity that bacterial cells can carry genetic information in two ways. The genes necessary for growth and reproduction are carried in the bacterial chromosomes, but bacteria also contain genes carried on rings of DNA outside of the nucleoid region, called plasmids. In the following experiment, you will work with two strains of E. coli. The first strain, E. coli I, contains a gene found on the chromosomal DNA coding for streptomycin resistance. The second strain, E. coli II, contains a gene found on the plasmid DNA coding for ampicillin resistance. Predict what will happen when the E. coli I strain is mixed with the E. coli II strain. Record your prediction in the space below. Review the Aseptic Technique resource document. Why is it important to use aseptic technique when working with bacteria in the lab? Place your answer in the space below. 4 Read Part I of the experiment to see how to create isolated colonies for the two strains of E. coli bacteria. Put on safety gloves and goggles. Tie back any loose hair. Wipe down your laboratory table using a 75% ethyl alcohol in water solution or other effective disinfectant solution. Working teacher: ○ ○ ○ ○ ○ ○ ○ ○ with your partners, obtain the following materials from your 1 LB agar plate 1 LB agar plate with streptomycin 1 LB agar plate with ampicillin 1 LB agar plate with streptomycin and ampicillin 8 sterile loops Alcohol lamp Lighter Permanent marker (fine point) Write your initials or first names on the outside edge of each Petri dish. Do not write too large or your writing will interfere with your observation of the results. On the bottom surface of each agar plate, draw a line down the middle. Write “I” on one side of the line, and “II” on the other side (Figure 1). 5 Obtain the E. coli I and E. coli II broth cultures from your teacher. You will have to share these cultures with another group. Remove a loop from its wrapper. Do this by opening the package at the end farthest from the inoculating loop, being careful not to touch anything. Watch as your teacher demonstrates the sterile technique. Use the sterile technique to remove and hold the cap from the E. coli I broth culture bottle. Dip the sterile loop into the culture while holding the cap so that it does not touch anything. Withdraw the loop and replace the cap immediately. Be careful not to touch the loop to anything before placing it into the bottle or after withdrawing it. Note that you will not need the culture tube again. Touch the loop lightly to the agar at the top edge of the plate in the section marked “I” on one of the four plates. Note that in the following steps you will be instructed on how to streak the plate. The purpose of streaking a plate in this manner is to dilute a concentrated sample and grow isolated bacterial colonies. Gently move the loop back and forth across the agar surface, creating a series of parallel lines at the top of the plate. Turn the plate 90 degrees. Repeat the streaking procedure beginning at the top edge of the plate (but do not dip the loop into the culture tube). Gently move the loop back and forth across the agar surface, making sure to 6 cross the previous material two to three times to pick up cells from the first section you streaked. Turn the plate another 90 degrees in the same direction. Repeat the streaking procedure beginning at the top edge of the plate. Gently move the loop back and forth across the agar surface, making sure to cross the previous material one to two times to pick up cells from the second section you streaked. Follow the same procedure to inoculate and streak the E. coli I culture onto all of the sections marked “I” on the remaining three plates. Make sure to use a new loop for each plate. 7 Using a new loop, follow the same procedure to inoculate and streak the E. coli II culture onto the sections marked “II” on each agar plate. Follow your teacher's directions to properly dispose of the contaminated loops. 8 Allow the liquid to soak into the agar. Invert the plates and tape the four plates together for incubation. It is important to incubate the plates upside down to prevent condensation from dripping onto the culture surface. Incubate the plates at 37°C for 24 hours. Spray disinfectant on the work area and wipe it clean. Remove your gloves and dispose of them as directed by your teacher. Wash your hands and forearms thoroughly with soap and warm water. Predict what growth you will see on both sections for each of the four plates. Record your predictions in the space below. Give your reasons for your predictions. Retrieve your plates from the incubator and observe the bacterial growth. Record the observed results in the table below. Use + for bacterial growth and – for lack of growth. Plate Strain I Results Strain II Results LB Agar LB Agar + Streptomycin LB Agar + Ampicillin LB Agar + Streptomycin & Ampicillin Working with your partners, obtain the following materials from your teacher: ○ Agar plates from previous class 9 ○ 1 LB agar plate ○ 3 sterile loops ○ Permanent marker (fine point) Write your initials or first names on the outside edge of the Petri dish. Label this dish Mix Plate. Note that the streaking procedures completed in the previous steps should have resulted in the growth of individual bacterial colonies. Bacterial colonies can be seen as separated clusters on the agar plates. Use the sterile technique to transfer one E. coli I colony from the LB agar + streptomycin plate to the Mix Plate. Make sure to use a new sterile loop. Open the lid of the plate just enough to slip the loop under, being careful not to touch anything. Do not take the lid completely off the plate. Gently touch the end of the inoculating loop to one of the isolated colonies. Withdraw the inoculating loop from the plate, being careful not to touch the loop to anything. The bacterial colony is now on the end of the inoculating loop. Open the lid of the Mix Plate just enough to slip the loop under and touch the loop lightly to the agar in the middle of the plate. Follow your teacher’s directions on how to dispose of the contaminated loop. Use a permanent marker to mark the spot where the colony is on the plate. 10 Follow the procedure in the previous step and use a new sterile loop to transfer one E. coli II colony from the LB agar + ampicillin plate to the Mix Plate approximately ½ inch apart from the strain I colony. Use a permanent marker to mark the spot where the colony is on the plate. Use a new sterile loop to mix strains I and II. Streak the plate by gently sweeping the loop across the top of the agar to mix the two bacterial cultures. 11 Seal the Petri dish using transparent tape. Invert the plate and incubate at 37°C for 24 hours. Properly dispose of the contaminated loop. Spray disinfectant on the work area and wipe it clean. Remove your gloves and dispose of them as directed by your teacher. Wash your hands and forearms thoroughly with soap and warm water. Answer Conclusion questions 1–4. Working teacher: ● ● ● ● ● ● with your partners, obtain the following materials from your 1 LB agar plate with streptomycin 1 LB agar plate with ampicillin 1 LB agar plate with streptomycin and ampicillin Plate with both E. coli strains from previous class 3 sterile loops Permanent marker (fine point) Write your initials or first names on the outside edge of each Petri dish. Remove one sterile loop from the package using the handle end. Do not touch the loop to anything. Slightly lift the lid of the agar plate labeled Mix Plate and use the sterile loop to pick up some bacteria. Close the lid of the plate. Streak some of these bacteria onto the LB agar plate with streptomycin. Note that for this step, you are not streaking for isolation but instead streaking in a random pattern to test for growth. Streak the plate as shown. 12 Repeat this procedure to streak the LB agar plate with ampicillin and the LB agar plate with streptomycin and ampicillin. Make sure to use a new sterile loop for each plate. Properly dispose of each loop. Invert the plates and tape the four plates together for incubation. Incubate the plates at 37°C for 24 hours. Spray disinfectant on the work area and wipe it clean. Remove your gloves and dispose of them as directed by your teacher. Wash your hands and forearms thoroughly with soap and warm water. Retrieve your plates from the incubator and observe the bacterial growth. Record the observed results in the table below. Use + for bacterial growth and – for lack of growth. Plate Left Side LB Agar +Streptomycin LB Agar+ Ampicillin LB Agar +Streptomycin & Ampicillin 13 Right Side Follow your teacher's directions on how to properly dispose of Petri dishes. Spray disinfectant on the work area and wipe it clean. Wash your hands and forearms thoroughly with soap and warm water. Answer Conclusion question 5. Given what you know about bacterial cells and antibiotics, brainstorm answers to the following questions and place your responses in the space below: ● How would a genetic mutation in the bacteria’s DNA cause the cell to become resistant to a particular antibiotic? ● How would a bacterial cell “share” this mutation with other bacterial cells? Given what I know about bacterial cells and antibiotics, a genetic mutation in the bacteria’s DNA would cause the cell to become resistant to a particular antibiotic because. A bacterial cell would “share” this mutation with other bacterial cells by Access the Animation of Antimicrobial Resistance produced by The Food and Drug Administration’s Center for Veterinary Medicine. 14 View the following sections that provide information about the mechanisms of antibiotic resistance. Note: Antibiotics are referred to as “antimicrobials” throughout the animation. ○ Mutation ○ Destruction or Inactivation ○ Efflux View the following sections that provide information about gene transfer between bacterial cells: ● Conjugation ● Transformation ● Transduction ● Naked DNA Work with your group to create a poster that illustrates one of the gene transfer or resistance mechanisms assigned by your teacher that bacteria use to transfer beneficial genes throughout a population. You must include a title, relevant illustrations, explanations of how it works, limitations and what types of bacteria use that method. Feel free to add any additional information gained from your research. In a well-crafted paragraph, summarize the information about all of the mechanisms of antibiotic resistance in the space below. Your summary should include the terms listed below. Highlight each term in your paragraph. Place your paragraph in the space provided. Target protein Genes Antibiotic Conjugation Resistance gene Plasmid Pilus Transformation “Naked” DNA Transduction Bacteriophage 15 Answer the remaining Conclusion questions. 1. What was the purpose of culturing bacteria on these four plates? The purpose of culturing bacteria on these four plates 2. How did your predicted results compare to your observed results? My predicted results are compared to my observed results 3. Why did the E. coli I strain grow on both the LB agar plate and the LB agar plate with streptomycin but not on the LB agar plate with ampicillin? The E. coli I Strain grew on both the LB agar plate and the Lb agar plate with streptomycin but not on the LB agar plate with ampicillin because 4. Why did neither strain grow on the plate containing both streptomycin and ampicillin? Neither strain grew on the plate containing both streptomycin and ampicillin because 5. Explain what the results indicate about the new strain of bacteria produced when both strains of E. coli were mixed together. The results indicate that the new strain of bacteria 16 6. Based on the results of the experiment, as well as what you learned about the mechanism of gene transfer between bacterial cells, was the streptomycin resistant gene transferred from Strain I to Strain II, or was the ampicillin resistant gene transferred from Strain II to Strain I? How do you know? The streptomycin resistant gene was transferred from 7. What mechanism of gene transfer do you think was responsible for transferring the antibiotic resistant gene between these two strains of bacteria? Explain your answer. The mechanism of gene transfer I think was responsible for transferring the antibiotic resistant gene between these two strains of bacteria was 17