The validity of antibiotics as a category of antimicrobial drugs was determined by a method that was developed by Kirby and Bauer, and standardized by the World Health Organization in 1961 to ensure reliable results. The Bauer-Kirby method was highly suggested by the U.S. Food and Drug Administration to determine the antimicrobial disk susceptibility. (Liberman et al., 1966) Therefore, extreme care is required to meet to the standards. To begin meeting the standards, a Mueller-Hinton agar is used for the bacteria culture. The standard Mueller-Hinton agar usually requires a pH between 7.2 and 7.4, which is then poured to a depth of 4mm in either 150 mm or 100mm Petri dishes. The petri dish is then heavily inoculated with a specific bacteria and different paper disks that contain different antibiotics to examine.
With the disks on the inoculated petri dish, the petri dish must be sealed and placed into an incubator at an exact temperature of 37°C for 24 hours and allow the bacteria to grow. Within 24 hours, the bacteria must be put into a refrigerator with an estimated temperature of 4.4°C to prevent overproduction of bacteria. By examining the results of the incubation of bacteria, the zone of inhibition, which is the area without bacterial growth that surrounds the antimicrobial disk, can determine how effective the antibiotics were against the bacteria. Depending on the length of the radius of the clear region around the paper disk, an estimated measure from the edge of the paper disk of 1-2 cm can determine how powerful the antibiotics were. Some of the bacteria may show small or no zones of inhibition if their growth was completely resistant to the antibiotic. Though this may be the case, the more sensitive cultures will be utilized efficiently to counter the different diseases that are brought on by specific bacterium.
With the experiment explained and discussed, a group of two students including I tested one antibiotic against three different pathogens. Erythromycin, a compound produced by a strain of Saccharopolyspora erythraea that is categorized as a macrolide group of antibiotics. This antibiotic is an extremely useful antibiotic often prescribed to patients who are allergic to penicillin. Erythromycin inhibits the synthesis of specific proteins that is essential to bacteria reproduction. (Weisblum, 1995) With the inhibition of protein synthesis, erythromycin does not necessarily kill the bacteria, but leaves them unable to replicate. The remaining bacteria are incapable of surviving and are destroyed by the immune system.
The three different pathogens, _Escherichia coli, Shigella flexneri, and Staphylococcus aureus_ were exposed by Erythromycin and the effects were examined.
_Escherichia coli (E. coli),_ a prokaryote with one circular chromosome composed of monocistronic (single genes) and polycistronic genes is a Gram-negative, rod shaped, bacterium that essentially lives in the intestine of warm-blooded organisms. The cell structure of an _E. coli_ possesses a cell wall that is constructed by lipopolysaccharides, a peptidoglycan layer, and a cytoplasmic membrane. _E. coli_ can cause severe infections to animals by ascending infections of the urethra and kidneys, which lead to bloody diarrhea or urinal tract infections. Though not a very dangerous bacterium, it can be prevented with amoxicillin, penicillin, and many other antibiotics. (Chan et al., 2006) _Shigella flexneri (S. flexneri),_ a physiologically similar prokaryote to _Escherichia coli,_ may very well have a common evolutionary basis.
This gram-negative rod shaped bacteria, is notoriously known to cause shigellosis, an acute bloody diarrhea. _S. flexneri_ can be treated with Beta-lactams antibiotics such as ampicillin and amoxicillin. (Chan et al., 2006) The last bacteria being tested, _Staphylococcus aureus (S. aureus),_ a Gram-positive, cocci-shaped bacterium, is the most common species among the entire Staphylococcus bacteria. Because its Gram-positive, _S. aureus_ is formed from a thick layer of peptidoglycan.
Out of the entire microbial species, _S. aureus_ is the most complete genome sequence. This bacterium causes mild skin infections, invasive diseases, and toxic mediated diseases by colonization. Though _S. aureus_ causes painful infections, they are becoming increasingly resistant to common antibiotics such as penicillin, erythromycin, tetracycline, and aminoglycosides. (Chan et al., 2006) Since Erythromycin is more effective towards Gram-positive bacteria, the radius of the zone of inhibition will most likely be longer for _Staphylococcus aureus_ than _Escherichia coli_ and _Shigella flexneri._
MATERIAL AND METHODS:
In our lab, with a team of three, each person distinguished the different areas of pathogens that were divided in fourths with a black permanent marker on either a 150 mm or 100 mm petri dish that is coated with a 4 mm deep Mueller-Hinton agar. In this case we used _Escherichia coli, Shigella flexneri, and Staphylococcus aureus_ as our pathogens. It is very important to label the pathogens where it was swabbed because all of the bacteria contained a transparent color, which were difficult to distinguish. After applying the bacteria onto the Mueller-Hinton agar petri dish, one McFarland standard disk that contained 15 Î¼g of Erythromycin dosage was placed in the center of each sector.
It is important to take note that the stainless precision tweezers must be sterilized by flame or alcohol before placing the disk onto the center of the sector because there may be different bacteria on the tweezers before. When the McFarland disks were placed, the Mueller-Hinton agar petri dish were sealed and placed in an incubator that was set at a 37°C temperature for 24 hours. After 24 hours, the Mueller-Hinton agar petri dish was moved out of the incubator and placed into a refrigerator with an approximate temperature of 4.4°C to inhibit continuous bacterial growth. After incubation, the radiuses from the edge of the McFarland disks to the end of the clear zones were measured with a standard ruler to the nearest millimeter.
DISCUSSION AND CONCLUSION:
As a result from the experiment, _Escherichia coli_ was not very sensitive to Erythromycin. With a zone of inhibition of 2.6 mm, Erythromycin had a small effect on _Escherichia coli_. Erythromycin is not very effective against Gram-negative bacteria because they contain hydrophobic molecules and macrolides do not have the ability to penetrate both inner and outer membranes of Gram-negative bacteria. As a Gram-negative bacterium, the peptidoglycan layer is thinner, but contains a much more complex cell wall that contains polysaccharides, proteins, and lipids. Referencing back to the results, according to Figure 5 under oil-immersion objective at 1000x magnification, _Escherichia coli_ had a stained color of pink/red, which categorizes itself as a Gram-negative bacterium.
Similar to _Escherichia coli, Shigella flexneri_ is Gram-negative as well. Although these two bacteria are Gram-negative, _Shigella flexneri_ is less sensitive than _Escherichia coli._ The radius of the zone of inhibition resulted to be 0 mm when exposed to the Erythromycin antibiotic, which proved that _Shigella flexneri_ was completely resistant to Erythromycin. Since Erythromycin is categorized as macrolides, it is most likely that _Shigella flexneri_ is resistant to all the macrolides. In Figure 4, _Shigella flexneri_ had a stain color of red/orange, which is also a characteristic of a Gram-negative bacterium.
As for _Staphylococcus aureus,_ the radius of the zone of inhibition was 6.8 mm. This proved _Staphylococcus aureus_ to be Gram-positive when exposed by Erythromycin and would be expected to be very sensitive to the rest of the macrolide antibiotics. As a Gram-positive bacterium, _Staphylococcus aureus_ results with a blue/purple colored stain. (Figure 3)
The radiuses of the zone of inhibition could have been a lot more accurate because as I placed the McFarland disk with Erythromycin, it didnt completely stick onto the bacteria. As I flipped the Mueller-Hinton agar petri dish over, the disk fell onto the cover of the petri dish. Without intelligently thinking, I quickly flipped the petri dish over back to its original position and the disks landed back onto the bacterial colonies. Luckily, my results still supported my hypothesis, which meant that as I flipped the petri dish over, all four of the Erythromycin disks spontaneously landed onto the correct sectors. Although the disks landed on the sector, they were not centered, which was difficult to determine the clear area radius since the disks were close to the constant. If they were centered, the results would have provided me a much more accurate measurement.
Ever since the discovery of penicillin in 1928, people have been utilizing this antibiotic to the utmost of their abilities. The rate of antibiotic discoveries has been phenomenal, but at the same time, different bacteria that undergo different mutations rapidly eclipse the rate of discovery for antibiotics. This puts pressure to find solutions to prevent the spread of renovating bacteria and continuing to ensure safety around the world with the utmost of our abilities.
Boyle, V.D., Fancher, E.M., Jr. Ross, W.R. (1973). Rapid, Modified Kirby-Bauer Susceptibility Test with Single, High-Concentration Antimicrobial Disks. _Antimicrobial Agents and Chemotherapy,_ 3(3): 418-424
Liberman, F.D., Robertson, G.R. (1975). Evaluation of a Rapid Bauer-Kirby Antibiotic Susceptibility Determination, _Antimicrobial Agents and Chemotherapy,_ 7(3): 250-255
Bauer, A.W., Kirby W.M., Sherris, J.C., Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method, _Am J Clin Pathol,_ 45(4): 493-496
Boyle, V.J., Fancher M.E., Ross R.W., Jr Rapid. (1973) Modified Kirby-Bauer Susceptibility Test With Single, High-Concentration Antimicrobial Disks. _Antimicrobial Agents and Chemotherapy_, 3(3): 418-424.
Weisblum, Bernard (1995). Erythromycin Resistance by Ribosome Modification.
_Antimicrobial Agents and Chemotherapy_, Mar. 1995, 577-585.
Chan, V.L., Sherman M.P., Bourke, Billy (2006) Bacterial genomes and infectious diseases, _Humana Press._