01 Jul Lab assignment 2
BACTERIA AND ANTIBIOTIC RESISTANCE LAB INSTRUCTIONS Please read the following lab case study. You will need a ruler for one part of the lab. Please write your answers in the boxes that are provided. The sections for each of this lab are intended to be completed in order. PART 1: DOES JIMMY HAVE MRSA? Of course Jimmy always had cuts and scrapes—he was a five-year-old boy! He’d been playing in the neighborhood playground and cut his lip when he fell off the jungle gym. This time though, his lip swelled up and he developed a fever. When his mother took him to the doctor, the pediatrician said the cut was infected and had prescribed cephalothin, an antibiotic related to penicillin, and recommended flushing the cut regularly to help clear up the infection. Two days later, Jimmy was in the hospital with a fever of 103°F, coughing up blood and having trouble breathing. The emergency room doctors told the family that Jimmy had developed pneumonia. They started him on IV antibiotics, including ceftriaxone and nafcillin, both also relatives of penicillin. Figure 1. Example of a Kirby-Bauer plate. Note the lawn of bacteria covering the plate and the antibiotic disk at the center. A clear zone of inhibition has formed, which indicates that the bacteria have not grown in the area in which the antibiotic has diffused out of the antibiotic disk. To determine the extent of antibiotic susceptibility or resistance, measure the diameter of the zone of inhibition Jimmy’s doctors decided to check for MRSA (methicillin-resistant Staphylococcus aureus). He hasn’t gotten better, and MRSA is resistant to most of the penicillin derivatives. To test for MRSA, the doctors took a swab from the site of Jimmy’s wound. They then isolated bacteria from the swab and tested it biochemically. A Kirby-Bauer disk diffusion test was performed next. To do this test, paper disks containing different antibiotics are placed on agar petri plates. Agar is a gelatinous material that has nutrients required for the growth of bacteria. The suspected bacteria are spread in a thin layer on the agar plate. The bacteria is grown until it has “lawned,” or completely coated with microbes to make a thick hazy layer over the agar surface. The area around each disk is examined for a clear zone where the microbe’s growth has been inhibited (Fig. 1). Jimmy’s Kirby- Bauer test results are shown in figure 2. Figure 2. Jimmy’s test results (bottom). The control plate (top) was isolated from a patient not infected with MRSA. PE: penicillin, ME: methicillin, CE: cephalothin, VA: vancomycin, CTL: control disk that contains no antibiotic. The Kirby-Bauer method is standardized so that no zone of inhibition is scored as a zero, and all others include the disk as part of the zone. Measure the zones of inhibition in centimeters (in science we use the metric system) for each plate in Figure 2. Record your results in the table below. Disk Zone Size – Control Zone Size – Jimmy PE VA ME CE CTL 1. Why is a control disk included on each of the Kirby-Bauer plates? 2. Why is a control sample included in this laboratory test? 3. Does Jimmy have MRSA? How did you come to this conclusion? 4. What antibiotic should Jimmy’s doctors prescribe? Explain your choice. PART 2: DID JIMMY INFECT HIS FAMILY? Jimmy’s doctors have prescribed a new antibiotic based on his Kirby-Bauer results, and he’s quickly recovering. However, his now doctors have a new question. Did anyone catch Jimmy’s MRSA? MRSA and other forms of Staphylococcus aureus can be spread through direct skin-to-skin contact. Jimmy’s doctors are also epidemiologists, scientists that study the incidence, spread, and cause of disease. It’s not surprising that Jimmy’s doctors would be concerned about his family. The first MRSA cases arose in the 1970s. However, MRSA isn’t the first variety of Staphylococcus aureus (commonly shortened to S. aureus or staph) to become resistant to antibiotics. Penicillin-resistant strains first arose in the mid-1940s, shortly after penicillin was widely introduced to treat staph infections. Initially, these resistant varieties arise in hospitals and other health care settings where antibiotic use is common. However, as time elapses, infection with resistant strains can be acquired in the community, independent of health care environments. After penicillin resistance in S. aureus became common, doctors began to treat infections with methicillin. Similarly, methiciliin resistance was first observed in health care settings and then rapidly spread to the community. Complicating this issue of resistance, individuals can asymptomatically carry and shed these bacteria for years, making identification of sources of outbreaks difficult. And, as can be seen in Jimmy’s case, these antibiotic-resistant infections can be very severe, even causing death. To answer their question about Jimmy’s family, his doctors took nasal swabs from each of his asymptomatic family members and their isolates were subjected to KirbyBauer tests. The results can be seen in Figure 3. Figure 3. Test results from Jimmy’s family. The control plate was isolated from a patient not infected with MRSA. PE: penicillin, ME: methicillin, CE: cephalothin, VA: vancomycin. The Kirby-Bauer method is standardized so that no zone of inhibition is scored as a zero, and all others include the disk as part of the zone Measure the zones of inhibition in centimeters for each plate in Figure 3. Record your results in the table below. Disk Zone Size – Mother Zone Size – Sister Zone Size – Father Zone Size – Control PE VA ME CE CTL 5. Do any of Jimmy’s family members carry MRSA? Explain your answer. PART 3: VRSA, THE FUTURE With the introduction of vancomycin to treat MRSA, vancomycin-resistant Staphylococcus aureus (VRSA) strains have also arisen. 6. In what setting is VRSA most likely to first arise? 7. Predict what a Kirby-Bauer VRSA plate would look like. 8. What might the emergence of VRSA mean for future treatment of S. aureus? Adapted from: Leonard, M. (2012, December 3). Antibiotic Resistance: Can we ever Win? National Center for Case Study Teaching in Science. Retrieved from http://sciencecases.lib.buffalo.edu/cs/ References: Chambers, H.F. & DeLeo, F. R. (2009). Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews: Microbiology, 7, 629-641. doi: 10.1038/nrmicro2200 …
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