Introduction:
Antimicrobial chemotherapy uses chemicals to inhibit or kill microbes, and is based on selective toxicity. The goal is to inhibit or kill the organisms targeted without seriously harming the host. This is accomplished by interacting with a microbial structure or function that is not present or is significantly different than what occurs in the host. In prokaryotic bacterial infections, this is frequently done by targeting the peptidoglycans or bacterial ribosomes which are not present in the human host. Eukaryotic organisms such as parasites or fungi, have structures and functions more similar to humans, and therefore the number of agents effective at inhibiting or killing those organisms without significantly harming the host is relatively limited. Viruses are not cells and do not have the structures to be targeted by antibiotics, and therefore are not treated with antibiotics.
Antibiotics are substances which are produced as a metabolic product of one microorganisms which inhibit or kill another microorganism. Antimicrobial chemotherapeutics chemicals are synthesized in the lab to be used therapeutically on microorganisms. May antibiotics today are semisynthetic and modified or even completely synthesized in the lab.. The three major groups that are used to produce useful antibiotics are the actinomycetes (filamentous soil bacteria), the Bacillus genus, and the saprophytics molds penicillium & Cephalosprium.
Cidal agents kill microorganisms such as the penicillins, cephalosporins, streptomycin, and neomycin, while other are static in action and only inhibit microbial growth. Those that inhibit microbial growth allow for the body’s own defenses to attach the organisms, such as the tetracyclines, erythromycin, and sulfonamides. Drugs that target a variety of both gram positive and gram negative organisms are said to be broad-spectrum (tetracycline, streptomycin, cephalosporins, ampicillin, sulfonamides), while those that only target gram positive or gram negative are narrow spectrum (penicllin G, erythromycin, clindamycin, gentamicin). Whenever possible, it is preferred to use the more narrow spectrum antibiotic to preserve the body’s natural flora , reduce opportunistic infections, and decreases selection for resistant strains of microorganisms.
1. Antimicrobial agents that inhibit peptidoglycan synthesis. This results in lysis of actively dividing bacteria.
a. penicillins
i. Natural penicillins – active against gram positive, but are inactivated by penicillinase (Penicillin G, F, X, K, O, V)
ii. Semisynthetic penicillins – active against gram positive but not inactivated by penicillinase (methicillin, dicloxacillin, nafcillin)
iii. Semisynthetic braod-spectrum penicillin – both gram positive & gram negative but inactivated by penicillinase (ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, piperacillin)
iv. Semisynthetic broad-spectrum penicillins with beta lactamase inhibitors – the beta lactamase inhibitors inhibit the penicillinase enzyme
b. cephalosporins – active agains a variety of gram positive and negative organisms and resistant to penicillinase (cephalothin, cephapirin, and cephalexin, cefamandole, cefaclor, cefazolin, cefuroxime, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, deftazidine, moxalactam)
c. carbapenems – broad spectrum beta lactam to inhibit peptidoglycan synthesis (imipenem)
d. monobacterms – broad spectrum beta lactam resistant to beta lactamase (aztreonam)
e. carbacephem – synthetic cephalosporin
f. glycopeptides – target gram positive bacteria (vancomysin, teichoplanin)
g. bacitracin – targets gram positive, used topically
h. fosfomycin
2. Altering the cytoplasmic membrane
a. Polymyxin B – used in severe Pseudomonas infections
b. Amphoterocin B – systemic fungal infections
c. Nystatin – Candida infections
d. Imidazoles – antifungals (clotrimazole, miconazole, ketoconazole, itraconazole, fluconazole)
3. Agents that inhibit protein synthesis
a. Agents that block transcription (rifampins) inhibit RNA polymerase, some gram positive and negative, M. tuberculosis
b. Agents that block translation – (streptomycin, kanamycin, tobramycin, amikacin, tetracycline, minocycline, doxycycline, erythromycin, roxithromycin, clarithromycin, azithromycin) – target gram positive and gram negative, (lincomycin & clindamycin) – usually used against gram positive, (oxazolidinones, streptogramins)
4. agents that interfere with DNA synthesis
a. fluroquinolones – inhibits enzymes called topoisomerases needed for bacterial nucleic acid synthesis (norfloxacin, ciprofloxacin, enoxacin, levofloxacin, trovacloxacin)
b. sulfonamides & trimethoprim – block enzymes needed for synthesis of tetrahydrofolate to make bases thymine, guanine, uracil, & adenine
c. metronidzaole – nicks microbial DNA strands
Methods of microbial resistance:
1. enzymes that detoxify or inactivate the antibiotic
2. altering the target site or reduce / block the binding site of antibiotic
3. prevent transport of agent into the bacterium
4. develop alternate metabolic pathway to by-pass step blocked by the agent
5. increased production of certain enzymes
These changes occur naturally that allow a bacterium to resist the antimicrobial agent as the result of mutation and genetic recombination. With exposure, the resistant organisms are selected for because the others die out, leaving only the resistant organisms to multiply. These bacteria may also transfer resistance to other bacteria, both of similar and different genus & species by the use of R plasmids. R (resistance) plasmids have genes coding for multiple antibiotic resistance, and are transferred between bacteria through the use of a sex pilus. The normal flora may acquire the R plasmids from pathogenic bacteria. Resistant normal flora may be transmitted person to person as well. Antibiotic susceptibility testing can be done in the clinical laboratory to determine which antimicrobial agents will most likely be effective at targeting that particular strain of organism.
In some cases the susceptibility to a chemotherapeutic agent is predictable, but in many cases there is no reliable way of predicting which antimicrobial agent will be effective, especially with the emergence of so many antibiotic resistant strains. This may be tested using tube dilution tests, the agar diffusion test (Bauer-Kirby test), and automated tests. The automated tests are able to give results within a few hours, but equipment is very expensive.
Tube dilution tests are performed using a series of cultures with different concentrations of the chemotherapeutic agent. The tubes are inoculated with the test organism and incubated 16-20 hours and examined for growth. The MIC (minimum inhibitory concentration) is the lowest concentration of the chemotherapeutic agent that prevents growth of the test organism. Additional tests to determine MBC (minimum bactericidal concentration) to determine the lowest concentration of a chemotherapeutic agent that inhibits growth of the cultures, are time consuming and expensive.
Procedure:
In this lab we will be using the Bauer-Kirby disc diffusion method. The standardized antibiotic-containing disc has been correlated with the clinical response of patients given that drug. Zones of growth inhibition are correlated with the MIC for each agent to establish categories of “resistant”, “intermediate”, and “sensitive”.
1. Prepare a standard Mueller-Hinton agar plate with a standardized inoculum covering the entire agar surface with bacteria. Also prepare 1 plate with a throat culture collected using a sterile swab. Cover the entire plate.
2. Place standardized antibiotic-containing discs on the plate.
3. Incubate the plate for 18-20 hours at 35 degrees Celsius.
4. Measure the diameter of any resulting zones of inhibition in millimeters.
5. Determine if the bacterium is susceptible, moderately susceptible, intermediate, or resistant to each antimicrobial agent using a standardized table. (Zone Size Interpretive Chart for Bauer-Kirby Test)
Prepare a table of your results:
| Organism | Chemotherapeutic agent | Zone size (mm) | Sensitivity |
| Zone of Inhibition Example: |
http://www.life.umd.edu/classroom/bsci424/Images/PathogenImages/AntibioticZonesofInhibition.jpg | ||
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