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Pre-Lab Preparation: Place saved culture of S. epidermidis (from previous lab) in incubator 12-24 hours prior to the start of the experiment.
Discussion and Review: Antimicrobial therapy is the use of chemicals to inhibit or kill microorganisms in or on the host. Drug therapy is based on selective toxicity. This means that the agent used must inhibit or kill the microorganism in question without seriously harming the host.
In order to be selectively toxic, a therapeutic agent must interact with some microbial function or microbial structure that is either not present or is substantially different from that of the host. For example, in treating infections caused by prokaryotic bacteria, the agent may inhibit peptidoglycan synthesis or alter bacterial (prokaryotic) ribosomes. Human cells do not contain peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows little if any effect on the host (selective toxicity). Eukaryotic microorganisms, on the other hand, have structures and functions more closely related to those of the host. As a result, the variety of agents selectively effective against eukaryotic microorganisms such as fungi and protozoans is small when compared to the number available against prokaryotes. Also keep in mind that viruses are not cells Hands-On Labs, Inc.
and, therefore, lack the structures and functions altered by antibiotics so antibiotics are not effective against viruses.
Based on their origin, there are 2 general classes of antimicrobial agents:
Antibiotics: substances produced as metabolic products microorganism which inhibit or kill other microorganisms.
Antimicrobial chemicals: chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.
Today the distinction between the 2 classes is not as clear, since many antibiotics are extensively modified in the laboratory (semisynthetic) or even synthesized without the help of microorganisms.
Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since then have come about by modifying the older forms. In fact, only 3 major groups of microorganisms have yielded useful antibiotics: the actinomycetes (filamentous, branching soil bacteria such as Streptomyces), bacteria of the genus Bacillus, and the saprophytic molds Penicillium and Cephalosporium. To produce antibiotics, manufacturers inoculate large quantities of medium with carefully selected strains of the appropriate species of antibiotic-producing microorganism. After incubation, the drug is extracted from the medium and purified. Its activity is standardized and it is put into a form suitable for administration. Some antimicrobial agents are cidal in action: they kill microorganisms (e.g., penicillins, cephalosporins, streptomycin, neomycin). Others are static in action: they inhibit microbial growth long enough for the body’s own defenses to remove the organisms (e.g., tetracyclines, gentamicin, sulfonamides).
Antimicrobial agents also vary in their spectrum. Drugs that are effective against a variety of both gram-positive and gram-negative bacteria are said to be broad spectrum (e.g., tetracycline, streptomycin, cephalosporins, ampicillin, sulfonamides). Those effective against just gram-positive bacteria, just gram-negative bacteria, or only a few species are termed narrow spectrum (e.g. penicillin G, clindamycin, gentamicin). If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body’s normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to superinfection by opportunistic microorganisms, such as Candida (yeast infections) and Clostridium difficile (antibiotic-associated ulcerative colitis), when the body’s normal flora is destroyed. Other dangers from indiscriminate use of antimicrobial chemotherapeutic agents include drug toxicity, allergic reactions to the drug, and selection for resistant strains of microorganisms.
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Below are examples of commonly used antimicrobial agents arranged according to their modes of action:
Antimicrobial agents that inhibit peptidoglycan synthesis: Inhibition of peptidoglycan synthesis in actively-dividing bacteria results in osmotic lysis. These include: Penicillins, Cephalosporins, Carbapenems, Monobactems, Carbacephem, Vancomycin, and Bacitracin.
Antimicrobial agents that alter the cytoplasmic membrane: Alteration of the cytoplasmic membrane of microorganisms results in leakage of cellular materials. These include: Polymyxin B, Amphotericin B, Nystatin, and Imidazoles.
Antimicrobial agents that inhibit protein synthesis: These agents prevent bacteria from synthesizing structural proteins and enzymes. These include Rifampins, streptomycin, kanamycin, tetracycline, minocycline, doxycycline, and gentamicin.
Antimicrobial agents that interfere with DNA synthesis: These agents inhibit one or more enzymes in the DNA synthesis pathway and include: norfloxacin, ciprofloxacin, Sulfonamides and Metronidazole.
A common problem in antimicrobial therapy is the development of resistant strains of bacteria. Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms:
Producing enzymes which detoxify or inactivate the antibiotic: penicillinase and other beta-lactamases.
Altering the target site in the bacterium to reduce or block binding of the antibiotic: producing a slightly altered ribosomal subunit that still functions but to which the drug can’t bind.
Preventing transport of the antimicrobial agent into the bacterium: producing an altered cytoplasmic membrane or outer membrane.
Developing an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent: overcoming drugs that resemble substrates and tie up bacterial enzymes.
Increasing the production of a certain bacterial enzyme: overcoming drugs that resemble substrates and tie up bacterial selection of antibiotic resistant pathogens at the site of infection. Indirect selection enzymes.
These changes in the bacterium that enable it to resist the antimicrobial agent occur naturally as a result of mutation or genetic recombination of the DNA in the nucleoid, or as a result of obtaining plasmids from other bacteria. Exposure to the antimicrobial agent then selects for these resistant strains of organism.
The spread of antibiotic resistance in pathogenic bacteria is due to both direct selection and indirect selection. Direct selection refers to the selection of antibiotic-resistant normal floras within an individual anytime an antibiotic is given. At a later date, these resistant normal floras may transfer resistance genes to pathogens that enter the body. In addition, these resistant normal flora may be transmitted from person to person through such means as the fecal-oral route or through respiratory secretions. The direct selection process can be significantly accelerated by both the improper use and overuse of antibiotics.
For some microorganisms, susceptibility to antimicrobial agents is predictable. However, for many microorganisms there is no reliable way of predicting which antimicrobial agent will be effective in a given case. This is especially true with the emergence of many antibiotic-resistant strains of bacteria. Because of this, antibiotic susceptibility testing is often essential in order to determine which antimicrobial agent to use against a specific strain of bacterium.
Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen:
1. Tube dilution tests: In this test, a series of culture tubes are prepared, each containing a liquid medium and a different concentration of an antimicrobial agent. The tubes are then inoculated with the test organism and incubated. After incubation, the tubes are examined for turbidity (growth). The lowest concentration of antimicrobial agent capable of preventing growth of the test organism is the minimum inhibitory concentration (MIC).
Subculturing of tubes showing no turbidity into tubes containing medium but no antimicrobial agent can determine the minimum bactericidal concentration (MBC). MBC is the lowest concentration of the antimicrobial agent that results in no growth (turbidity) of the subcultures. These tests,
however, are rather time-consuming and expensive to perform.
2. The agar diffusion test (Kirby-Bauer test): A procedure commonly used in clinical labs to determine antimicrobial susceptibility is the
Kirby-Bauer disc diffusion method. In this test, the in vitro response of bacteria to a standardized antibiotic-containing disc has been correlated with the clinical response of patients given that drug.
In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test). The MIC for each agent was then compared to the usually-attained blood level in the patient with adequate dosage. Categories of “Resistant,” “Intermediate,” and “Sensitive” were then established.
PROCEDURES: The Kirby-Bauer Test
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes may have been incorporated into the sample. Any culture that may contain an unknown organism should be treated as potentially pathogenic. Therefore be certain to wear the gloves and mask provided when handling the cultures to protect yourself from unintended exposure. When you have completed the experiment dispose of the mask and gloves you have been using. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, as with all cultures and materials in your LabPaq, use and store them out of the reach of children, other individuals and pets.
Preparation of Bacterial Cultures:
Place your stock culture of S. epidermidis in the incubator 12 – 24 hours prior to starting the experiment.
1. Disinfect your work area.
2. Using the nutrient agar plate that was made previously, and a sterile swab, coat the surface of the agar thoroughly with liquid S. epidermidis – do not leave any unswabbed areas on the agar dish.
3. After completely swabbing the dish, turn it 90 degrees and repeat the swabbing process. (It is not necessary to re-moisten the swab.)
4. Run the swab around the circumference of the dish before discarding it in the 10% bleach. Allow the plate to dry upright for 5 minutes to allow the S. epidermidis culture to absorb completely.
5. Using your marker on the outside bottom surface divide the dish into three sections triangular segments, similar to dividing a pie.
6. Label the first section novobiacin, the second penicillin, and the third gentamicin. Hands-On Labs, Inc.