Antibiotic Sensitivity

Introduction
Because of the recent anthrax threat, a news-hungry public has seen antibiotics moved to the frontline of the United Statesí war with terrorism. Whereas information about this tactical defense may be new to some, scientists have deployed antibiotics to fight microorganisms since the early 1900ís (Madigan, 2000), manipulating microorganismsí natural production of antibiotics to defend against attack. By considering the chemical structure that the antibiotic targets and the class of the targeted bacterium, one may predict whether the bacterium will be susceptible or resistant to a particular antibiotic (Madigan, 2000). This study of antibiotic diversity can help explain why the Center for Disease Control has recommended a particular antibiotic, Bayerís cipro, as the drug of choice against anthrax.

Most often antibiotics are classified either in terms of what species and/or what chemical structure of the microorganismósuch as the cell wall, cytoplasmic membrane, and players in protein and nucleic acid synthesis--they target (Madigan, 2000). A myriad of naturally occurring antibiotic classes exists that are produced by microorganisms through metabolic processes that either inhibit growth or kill other microorganisms. Organic chemists have developed successful methods of creating these natural products synthetically (Madigan, 2000). Treatment of diseases caused by gram-positive bacteria also has been advanced by biochemistsí alteration of genuine antibiotics to affect a larger variety of microorganisms and to lessen side effects to humans (Vilgalys, 2001). As the choice of antibiotic varies with the situation, scientists and doctors must choose an effective antibiotic by considering which classes are better fighting a specific microorganism and which are inactivated by the microorganismís resistance to attack (Madigan, 2000).

One method to determine proper treatment is the Paper-Disk-Plate technique (Vilgalys, 2001), which involves inoculating an agar plate with a microorganism-- in this study, a bacterium-- through the spread plate method. Filters permeated with a specific amount of antibiotic are dispensed onto the plate. After the plate has been incubated, the diameter of the clear zone around the disk is examined using the Kirby-Bauer antibiotic susceptibility chart (Vilgalys, 2001) to determine the extent that the drug inhibited the growth of the organism and, thereby, the most effective antibiotic for use in combating it.

This procedure studies the susceptibility of Staphylococcus aureus and Escherichia coli to eight different antibiotics, including both broad- and narrow-spectrum examples. The broad-spectrum antibiotics such as chloramphenicol and tetracycline should be effective for both of the bacteria even though S. aureus is gram positive and E. coli is gram negative. The rest of the antibiotics tested can be considered narrow-spectrum and will only affect one of the tested organisms.

Materials and Methods
The experimental setup used was taken from the BIO 103 Lab Manual (Vilgalys, 2001). No modifications were made.

Results
The data tables were elminated due to lack of space on the web site. Please contact me if you would like to look at the data. Contact information can be found on the main page.
The results show that E. coli was susceptible to all the antibiotics except bacitracin and penicillin. S. aureus was susceptible to all the antibiotics except bacitracin. The standard deviations were mostly between 2-3 mm. The highest deviations were for antibiotics that had trials where the disks were defective.

Discussion
The eight drugs studied fall into two main categories: antibiotics that target the cell wall and antibiotics that target protein synthesis. With a few exceptions, the results supported the hypothesis that broad-spectrum antibiotics affect both S. aureus and E. coli while the narrow-spectrum antibiotics affect only one. The greatest discrepancies were for the antibiotics that were thought to be specific to certain sites on the ribosome.

Bacitracin and penicillin, the antibiotics targeting the cell wall, both interfere with the cell wallís biosynthesis. In particular, bacitracin interferes with the dephosphorylation of C55-isoprenyl pyrophosphate (Toscano, 1982). This compound is essential in cell wall biosynthesis because isoprenyl phosphates are carriers during the synthesis of the repeat units of peptidoglycan (Pollock, 1984). Studies have shown that the outer membrane of gram negative bacteria protect it from bacitracin (Toscano, 1982). This experiment supported Toscanoís finding because E. coli was determined to be resistant to the drug. However, S. aureus was only intermediately susceptible to the drug. Previous antibiotic resistant studies have shown that even gram positive bacteria can become resistant to bacitracin either by developing a novel way of creating isoprenyl phosphates (Pollock, 1984) or by decreasing the cell wallís permeability (Toscano, 1982). Similarly, penicillin affects the biosynthesis of the cell wall, but its method is by preventing the crosslinking of peptides in the peptidoglycan layer (Madigan, 2000). Gram negative bacteria are also thought to protect themselves from this drug via their outer membranes. Indeed, E. coli was resistant to the drug while S. aureus was susceptible.

All the other antibiotics studied target the synthesis of proteins for the cell. Two of these, chloramphenicol and tetracycline, are known to be broad-spectrum antibiotics. Chloramphenicol inhibits the elongation process in protein synthesis by blocking the formation of peptide bonds and should be effective against most bacteria because all microorganisms need that ability (Gladwin, 1999). This experiment showed that both E. coli and S. aureus were susceptible. In a similar way, tetracycline targets the biosynthetic processes of protein synthesis, specifically those involving the 30S ribosomal unit (Madigan, 2000). It, too, was able to inhibit the growth of both bacteria.

The rest of the drugs studied were predicted to be specific to one class of bacteria or another. Erythromycin inhibits the 50S-ribosome unit of gram-positive bacteria (Pechere, 2001) during protein synthesis and was expected to only be active against S. aureus (Gladwin, 1999). The zone of inhibition around the erythromycin filter for S. aureus was greater than for E. coli, but both bacteria were determined experimentally to be susceptible. Erythromycin appeared to be more effective for gram positive bacteria. Similarly, the amine-glycoside antibiotics, which include kanamycin, neomycin, and streptomycin, inhibit the 30S-ribosome unit of gram-negative bacteria (Russell, 1969) during protein synthesis (Gladwin, 1999). The zone of inhibition around these antibiotic filters was larger for E. coli than S. aureus, but the standard deviations of the measurements overlap. Therefore, the differences between bacteria did not reach statistical significance. Perhaps the active sites of the ribosomes of E. coli and S. aureus are similar, thus explaining why they were equally susceptible to these narrow-spectrum antibiotics.

Although the standard deviations indicate the results were fairly repeatable, the method does have its flaws. For example, sometimes the zones of inhibition overlapped with other clear zones making the measurements of the diameter difficult. Additionally, the clear zones were not perfect circles; therefore, the choice of where to measure could have affected the results. Furthermore, there was no correction factor to account for the drugsí varying diffusion rates. These problems could be ameliorated by having fewer filter disks on the plates and by creating an objective way of measuring the irregular diameter. Additionally one study suggests that the irregularity of the circles can be diminished by inoculating an even amount of bacterium over the plate (Garrod, 1971). There were some cases where there were outliers and where the disk was defective, but by repeating the experiment, they were readily identified.

Conclusion
Overall, the hypothesis, which stated that broad-spectrum antibiotics would affect both S. aureus and E. coli while narrow-spectrum would affect only one, was fairly supported, and the discrepancies were easily explained. The Paper-Disk-Plate method is a fast, accurate way of determining a bacteriumís sensitivity to a variety of antibiotics and an effective, though expensive, tool (Vilgalys, 2001) for allowing doctors and hospitals to determine proper antibiotic treatment. The results can also provide information for such questions as what alternative drug to use when someone is allergic to the commonly chosen antibiotic (Madigan, 2000). However, this particular experiment did not give much information on how to determine what dosage is needed to inhibit all growth of the bacterium. In any case, the experiment demonstrated a tactical method of examining and appreciating the diversity of two combatants, bacteria and antibiotics.

References
Garrod, L.P.; Waterworth, P.M., DM Disease-a-Month, 1971, 7, 1-48.
Gladwin, M.; Trattler, B., Clinical Microbiology Made Ridiculously Simple, Medmaster, 1999.
Kotra, LP; Haddad, J.; Mobashery, S., Antimicrobial Agents and Chemotherapy, 2000, 44, 3249-3256.
Madigan, M.T.; Martinko, J.M.; Parker, J., Biology of Microorganisms, Prentice Hall, 2000.
Mingeot-Leclercq, M.P.; Glupczynsky; Tulkens, P.M., Antimicrobial Agents and Chemotheraphy, 1999, 43, 727-737.
Pechere, J.C., International Journal of Antimicrobial Agents, 2001, 18, S25-S28.
Pollock, T.J., Thorne, L.; Yamazaki, M., Journal of Bacteriology, 1984, 176, 6229-6237.
Russell, A.D., Progress in Medicinal Chemistry, 1969, 6, 135-199.
Toscano, W.A.; Storm, D.R., Pharmacology and Therapeutics, 1982, 16, 199-210.
Vilgalys, R., Laboratory Exercises for BIO 103 General Microbiology, Duke University, 2001.
 
 

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