ANTIBIOTIC INHIBITION OF BACTERIA

ANTIBIOTIC INHIBITION OF BACTERIA STANDARDS 3.2.10B, 3.2.12B Apply process knowledge and evaluate experimental information 3.3.10B, 3.3.12B Chemical and structural basis of living organisms Westminster College INTRODUCTION Single-celled organisms were the first life on the planet. Bacteria have adapted and evolved over millions of years, resulting in the numerous varieties of bacteria that exist on the planet today. Most bacteria can be divided into two classes, gram-negative and gram-positive, based on a differential staining process called the Gram stain. The Gram stain separates bacteria based on the ability of the cell wall to retain crystal violet stain when decolorized by an organic solvent like ethanol. The differences in the cell wall also play an important role in the types of antibiotics that will be effective against them. The ability to retain Gram stain is based on the structure of the bacterial cell membrane. Gram-positive bacteria, which retain the Gram stain, have a membrane which is composed of two parts, the cell wall and the cytoplasmic membrane (Fig. 1). The cell wall is composed primarily of peptidoglycan, a complex of linked polysaccharide chains which provide strength and rigidity. This peptidoglycan layer also is responsible for the ability of the cell to retain the Gram stain Figure 1. Cell Wall of a Gram-positive Bacterium Gram-negative bacteria have a cell wall which consists of an outer membrane, a periplasmic space and a cytoplasmic membrane. The peptidoglycan layer, found in the periplasmic space, is much smaller, and there is no teichoic acid present. The outer membrane and cytoplasmic membrane are comprised of phospholipids. The outer Westminster College SIM Page 1

membrane also contains lipopolysaccharides (LPS) and porins. The porins allow small molecules, like glucose, to diffuse through the outer membrane. A cell wall of this type does not retain the Gram stain. Figure 2. Cell Wall of a Gram - Bacterium Many microorganisms produce chemicals, or antibiotics, which protect them from bacteria. The first of these defensive chemicals to be isolated was penicillin. In 1928, Alexander Fleming noticed that a mold growing on his bacterial culture produced a clear zone around it, as though the bacterial growth was inhibited right near the mold. Upon further inspection, he determined that the mold Penicillium notatum produced a diffusible chemical, named penicillin, which was lethal to several bacterial species. Since that time, numerous other antibiotics have been discovered and isolated, as is evident by the wide selection of antibiotics available to the medical community. Antibiotics act against bacteria in two different ways. Some are bacteriocidal, and are capable of killing the bacteria. Other antibiotics are bacteriostatic and only inhibit the growth of the bacteria. The effect of bacteriostatic antibiotics is reversible. If an antibiotic is removed before the immune system can eliminate the bacteria, bacterial growth will begin again. Antibiotics are also classified as broad spectrum or narrow spectrum. A broad spectrum antibiotic is effective on many different bacteria, while a narrow spectrum drug only attacks a limited variety of pathogens (Table 1). It should be noted that some antibiotics are also used against protozoa, fungi, and viruses. The spectrum of an antibacterial drug is usually determined by its mode of action a gainst the bacteria. For example, penicillin is a bacteriocidal drug which inhibits the synthesis of the cell wall. Penicillin is a narrow spectrum antibiotic because it only affects gram-positive bacteria (Table 1). In contrast, tetracyclines inhibit protein Westminster College SIM Page 2

Table 1. Spectrum of Commonly Used Antibiotics Drug Primary Effect Drug Spectrum Chloramphenicol Static Broad (gram +/- ; rickettsia, chlamydia) Erythromycin Static Narrow (gram +, mycoplasma) Penicillin Cidal Narrow (gram +) Streptomycin Cidal Broad (gram +/- ; mycobacteria) Sulfonamides Static Broad (gram +/-) Tetracyclines Static Broad (gram +/- ; rickettsia, chlamydia) synthesis, a function common to all living organisms. Tetracyclines are considered broad spectrum, inhibiting the growth of both gram-positive and gram-negative bacteria. Other drugs interfere with nucleic acid synthesis (naladixic acid) or metabolite synthesis (sulfonamides). It is important to know which bacterium is causing an infection so that an antibiotic with the appropriate spectrum can be prescribed. An important point to remember is that many antibiotics are, in effect, poisons for living cells and are not specifically targeted to the bacteria causing an infection. Many of the side effects observed from antibiotic treatment are the result of the toxic effect of the drug on human cells as well as the bacterial cells. With the widespread use of antibiotics, particularly in the United States, an important problem has arisen. Many species of bacteria are acquiring resistance to antibiotics, rendering standard drug therapy for some infections useless. Two of the most common methods by which bacteria evade a drug are: a) the destruction or inactivation of the drug or b) the prevention of penetration to the target site. Alteration (mutation) of the drug target site and transfer of resistance between bacteria are also means by which bacteria are able to evade antibiotics. Some of this resistance has occurred naturally through spontaneous mutation in bacterial genomes. But there are multiple human practices which have led to an increase in resistance to antibiotics. Overuse of antibiotics, particularly in the case of colds and flu (which are not affected by these drugs) and in animal feed, gives subpopulations of bacteria a chance to acquire resistance. Likewise, not completing a prescribed treatment of antibiotics allows exposure to the drug without eradicating the entire population of bacteria. Also, many subpopulations of bacteria which are resistant to multiple antibiotics are spreading rapidly due to world travel. This laboratory uses a disc diffusion assay to examine the effectiveness of different antibiotics on gram-positive and gram-negative bacteria. A bacterial culture is spread on a nutrient agar plate and lines are drawn on the outside of the plate to create sectors. A sterile disc soaked with a particular antibiotic is placed in each sector, and the plates are allowed to grow overnight at 37ºC (human body temperature). After incubation, an even growth of bacteria, or lawn, should cover the plate. The only place where the bacteria do not grow is in a region around the antibiotic discs. This clear region is called the zone of inhibition. Measurement of this zone, in millimeters (mm), Westminster College SIM Page 3

gives an indication of the effectiveness of an antibiotic at a given dosage. The larger the zone, the more successful the antibiotic is at inhibiting bacterial growth. GUIDING QUESTIONS What is the major difference between Gram negative and Gram positive bacteria? Why are different antibiotics prescribed for different bacterial infections? Why is it necessary to include a sterile control disc on each bacterial plate? MATERIALS Tryptic soy agar plates (2) Bacillus cereus culture E. coli culture forceps 95% ethanol 1 ml pipets, sterile sterile antibiotic discs sterile control discs bacterial spreaders (2), sterile pipet bulb permanent marker 37ºC incubator ruler (metric) Kimwipes SAFETY This lab requires the use of live bacteria. The following precautions should be taken. a. Wash hands before and after handling the bacterial cultures b. Use some of the 95% ethanol to wipe down the lab bench after the lab is done. c. Students with long hair should always pull their hair back to avoid contamination. PROCEDURE 1. You will be given 2 tryptic soy agar plates. Use the permanent marker to label them as follows: a. Bacillis cereus = Gram-positive bacteria b. Escherichia coli = Gram-negative bacteria Remember to write your initials or group name on the plate for identification. 2. A culture of gram-negative (Escherichia coli) and gram-positive (Bacillus cereus) bacteria will be provided to each group. These bacteria must be spread on the appropriate agar plate using sterile technique. 3. Sterile Plating Technique. Sterile technique ensures that the only bacteria growing on the experimental plates are the ones from your culture. a. Open just one end of the plastic covering on the sterile 1 ml pipet. Be sure to open the end where the pipet bulb will attach, not the end at the tip of the pipet. As long as the lower portion of the pipet does not come in contact with anything Westminster College SIM Page 4

before pipetting the bacteria, it is considered sterile. Keeping the plastic wrapper around the pipet, fit the pipet bulb onto the top of the pipet (as shown in Fig. 3). b. Find the agar plate labeled E. coli. Uncap the top of the test tube labeled Gram -. Carefully remove the plastic wrapper from the pipet and place the pipet in the test tube without touching anything but the inside of the tube. Figure 3. Equipment for Sterile Plating Technique pipet bulb sterile pipet bacterial spreader plastic wrapper Sterile bottom edge antibiotic disc nutrient agar plate bacterial culture antibiotic disc dispenser c. Use the pipet bulb to withdraw approximately 0.4 ml of Gram - bacterial culture (between 0.30 and 0.50 ml is acceptable). Open the top of the petri dish and carefully dispense the culture onto the top of the agar plate. You do not want to splatter the culture all over your work area, because it increases the risk of contamination to your second plate. Replace the cover on the plate. d. Carefully unwrap the tinfoil from the end of the blue bacterial spreader. As long as nothing touches the triangular end of the spreader, everything under the tinfoil is considered sterile. Westminster College SIM Page 5

e. Using the bottom edge of the spreader, push the bacterial culture around the surface of the agar until there is a relatively even coating of bacteria. Do not press down hard on the agar plate! They are like very firm Jello, and will disintegrate with too much pressure. Set the plate aside and let the culture soak into the surface of the agar for 5 min. h. Obtain the Gram+ bacterial culture and the appropriate agar plate (labeled Bacillus cereus). Repeat Steps a. e. for these bacteria. Be sure to use a new sterile pipet and bacterial spreader or you will contaminate the second culture with the first. 4. Once the cultures have soaked into the agar plates, turn the plates over. Using a Sharpie or permanent marker, draw lines on the bottom of the petri dish, dividing it into 6 roughly equal sections. Label the sections: 1-6. Do this for both plates. 5. Sterile discs which contain no antibiotic are provided. These are a negative control which should show that placing a disc on the bacteria does not, in itself, inhibit bacterial growth. 6. Take the forceps provided and dip the tip of them into the ethanol. Let the excess ethanol drain off and wipe the remaining ethanol off with a Kimwipe. 7. Carefully remove a sterile control disk and place it in the center of the agar plate. It is easiest to do this for both plates at once. You do not need to re-sterilize the forceps between each control disc. 8. The antibiotics discs come in a cassette that will dispense them in a sterile fashion. There are a total of 6 different antibiotics, listed below. Choose one of the antibiotics, hold the disc dispenser over one of the sectors on the E. coli plate, and click the metal bar once to dispense a single antibiotic disc. Repeat this procedure with the same antibiotic for the B. cereus plate. Record the antibiotic name in the appropriate sector number on the Data Sheet. Antibiotic Disc Disc Designation kanamycin (30 µg) K 30 pencillin (10 IU) P 10 erythromycin (15 µg) E 15 tetracycline (30 µg) Te 30 chloramphenicol (30 µg) C 30 bacitracin (10 IU) B 10 9. Repeat step 8 for the five remaining antibiotics. Westminster College SIM Page 6

Note: If you forget to record which antibiotic is in which section, don t worry. Each disc is labeled with the abbreviations shown above. For example, the chloramphenicol disc will have C 30 printed on it. 10. Let the plates with the antibiotic discs rest on the bench for 5 minutes. During this time, the discs will absorb moisture from the agar and adhere to the plate. 11. Turn the plates upside down and place them in a 37ºC incubator for 24 hrs. Each group should label the plates in a way that they can identify them the next class period. The bacteria must grow for 18-24 hrs before the data is collected. DATA ANALYSIS 1. Remove the plates from the 37ºC incubator. After incubation, an even growth of bacteria, or lawn, should cover the plate. The only place where the bacteria do not grow is in a region around the antibiotic discs. This is the zone of inhibition. 2. Using a metric ruler, measure the zone of inhibition (in mm) around each antibiotic disc. Measure the zone for the control disc as well. Do this for the gram-positive and the gram-negative bacteria. 3. Record the data for the lab group in the Individual Data tables provided. Share this data with the rest of the class. Adapted from: Inhibition of Bacteria: Antibiotics and Antiseptics (1999) Juniata College Science in Motion. Lansing M. Prescott, John P. Harley and Donald Klein. Microbiology. Antimicrobial Chemotherapy Wm. C. Brown Publishers. 1996. 3 rd Edition. pp. 656-664. Gerard J. Tortora, Berdell R. Funke and Christine L. Case. Microbiology, An Introduction. Antimicrobial Drugs Addison Wesley Longman, Inc. (1998) 6 th Edition. pp. 531-538. CREDITS This lab was adapted and revised by Dr. Stephanie Corrette-Bennett. Westminster College SIM Page 7

DATA SHEET Name: Date: Individual Data Gram-negative (Escherichia coli) Agar Plate Section Antibiotic Zone of Inhibition (mm) C None (control) 1 2 3 4 5 6 Gram-positive (Bacillus cereus) Agar Plate Section Antibiotic Zone of Inhibition (mm) C None (control) 1 2 3 4 5 6 Group Data Antibiotic Average Zone of Inhibition (mm) Gram-negative / Gram-positive bacitracin (10 IU) / chloramphenicol (30 µg) / erythromycin (15 µg) / kanamycin (30 µg) / pencillin (10 IU) / tetracycline (30 µg) / Westminster College SIM Page 8

Name: Date: QUESTIONS AND ANALYSIS 1. What does the zone of inhibition indicate about each antibiotic? Which antibiotic is most effective against the E. coli (gram-negative)? the B. cereus (gram-positive)? Which is the least effective? 2. Each antibiotic disc has a certain low concentration based on doses commonly used to treat disease in humans. If low dose of an antibiotic is proven effective against a certain bacteria, why wouldn t a higher dose be better? 3. A common side effect of antibiotic use is intestinal distress and trouble digesting food. Hypothesize why this occurs. Westminster College SIM Page 9

4. The following data were obtained using a disc diffusion assay on a gram-negative bacterium. Can you deduce anything from the results of this particular experiment? Why or why not? Antibiotic Zone of Inhibition (mm) Chloramphenicol 8 Kanamycin 12 Penicillin 5 Tetracycline 7 5. Many parents can now request (and receive!) prescriptions from pediatricians over the phone, without the child ever being examined by the doctor. Describe why this is an unwise practice in terms of a) spectrum of antibiotic being used. b) bacterial resistance to antibiotics. Westminster College SIM Page 10