Lab Report: Antibiotic Evaluation by the Kirby-Bauer Method

Lab Report Antibiotic military rating by the Kirby-Bauer Method Introduction C stitchical antimicrobial agents atomic number 18 chemical substance compounds capable of either checking the growth of microorganisms or killing them outright. Those which are taken internally to alleviate the symptoms of or promote healing from disease are called chemotherapeutic drugs, and among these is a class of compounds called antibiotic drug drugs. In order for a chemotherapeutic drug to be classed as an antibiotic, it must be produced by a microorganism such as bacterium or fungus or at least derived from a chemical produced by hotshot.It must as well be capable of killing or inhibiting the growth of other microorganisms and of doing so when taken in very short quantities. To study whether a microbial output qualifies as an hard-hitting antibiotic, a amount mapping called the Kirby-Bauer method is employed. This method, which is the procedure recommended by the US Food and Drug Admini stration, was devised by William Kirby and A. W. Bauer in 1966. In the current protocols involved in the Kirby-Bauer method, Mueller-Hinton standard agar-agar is utilize as the medium for bacteriuml kitchen-gardening.The pH of the standard agar is 7. 2 to 7. 4 and it is poured exclusively to a depth of 4 mm. The medium is heavily inoculated with bacterium and paper disks containing enough of the antibiotic under study to create an optical density of 1 (the McFarland standard) are placed on top of the cultures. By examining the results of incubation in the form of a z ace of inhibition al close to to each one disk after incubation, it passel be determined how strong each antibiotic is against any given bacterium. A minimum inhibitory concentration can then be deduced for the given antibiotic vs. he specific bacterium tested so that appropriate dosage whitethorn be determined. Resistant bacteria cultures will show a small or no zone of inhibition if their growth is non suf ficiently inhibited for the antibiotic to be a viable candidate in treating transmittance by that organism. Sensitive cultures, on the other hand, will be appreciably inhibited in their growth or, ideally, eliminated entirely in a relatively large radius or so the McFarland standard disk. In this case, the antibiotic under study might be prescribed as a recitationful counter to illness brought on by that particularly bacterium.In the taste discussed here, we tested eight antibiotics against four common opportunistic pathogens, namely strep faecalis, staph aureus, Escherichia coli, and Pseudomonas aeruginosa. Of these, the first two are one thousand haughty and the latter pair are grand negative. The eight antibiotics tested were Ampicillin, a of import-lactam antibiotic that inhibits the final stage of bacterial cell wall deduction by binding to receptors within the cell wall. The result is a poriferous cell wall and subsequent lysis carried out by the bacterias own enzyme s.It is effective against many bacteria, both Gram cocksure and Gram negative, and is particularly use in treating infection by E. coli, Salmonella typhosa and Enterococcus faecalis, among others. (DrugBank) This antibiotic is a semi-synthetic derivative of penicillin, which is itself an antibiotic produced by the fungus Penicillium notatum. Bacitracin, a mixture of polypeptides obtained from Bacillus subtilis var Tracy. It inhibits tax write-off of the peptidoglycan layer in Gram positive bacteria by cloging the enjoyment of a molecule that transports components to synthesis sites.Bacitracin has a low threshold of perniciousness when taken orally or injected, but it has found application as a topical ointment in the prevention of wound infection by Staphylococci. (DrugBank) Chloramphenicol, a broad spectrum antibiotic that is produced synthetically but which was primarily discovered in a Streptomyces bacterium. It can be employed against some(prenominal) types of infection b ut most notably has found application in combating typhoid fever cholera. This antibiotic inhibits protein synthesis by suppressing the function of the 50S subunit in bacterial ribosomes.Chloramphenicol is bacteriostatic but does not kill bacteria. It to a fault has a low venomousity threshold when ingested, and so it is flat utilize almost exclusively to combat life-threatening illness or infection. (DrugBank) erythromycin, which is produced by a Streptomyces and functions as a protein synthesis inhibitor in much the alike way as Chloramphenicol. It is much little toxic than Chloramphenicol and is apply to combat such diseases as whooping cough, diptheria, and pelvic inflammation due to syphilis. (DrugBank) Novobiocin, one of the aminoglycoside antibiotics.This class of antibiotics whole works by binding to the bacterial 16S rRNA and causing the misread of tRNA. Because of this, the bacteria synthe size of its incomplete or toxic polypeptides, resulting in the death of the bacterial cell. Novobiocin can be use to treat infection by Gram negative bacteria and Mycobacteria, including Mycobacterium tuberculosis. It is not effective against anaerobic bacteria, however, and is not often employ against Gram positive infections because other antibiotics that are less toxic to the patient are available for this purpose. DrugBank) Moreover, Novobiocin is known to bind to and transfer the function of DNA gyrase, effectively stopping proper replication in the bacterial cell and thus bactericidal. Penicillin G, another antibiotic of the beta lactam class. It is employ primarily against Gram positive bacteria such as the strep but is also effective against some Gram negatives such as Neisseria gonorrhoeae and the spirochete Treponema pallidum, which is accountable for syphilis.Penicillin G inhibits synthesis if peptidoglycan by the alike weapon as in Ampicillin. (DrugBank) Polymyxin B, a mixture of polypeptides derived from Bacillus polymyxa. It can be used bactericidally against most Gram negative bacteria and is applied most often against urinary tract, blood, and meningal infections of Pseudomonas aeruginosa. It has no effect upon Gram positive bacteria. It kills bacterial cells by binding to a removing lipids in the cell membrane.Due to this mechanism, however, Polymyxin B also damages eukaryotic cells and thus sometimes proves to be a neuro- and nephrotoxic in humans. (DrugBank) Tetracycline, synthesized from chlortetracycline, a compound produced by a Streptomyces. It works by binding to the bacterial ribosome and interfering with protein synthesis and is effective against a wide range of Gram positive and negative bacteria, including the Mycoplasma and the bacteria responsible for Rocky Mountain Spotted Fever and nongonococcal urinary tract infections. DrugBank) physical and Methods CulturesStreptococcus faecalis Staphylococcus aureus Escherichia coli 1 Pseudomonas aeruginosa One person each in a team of four heavily inoculated two Mueller-Hinton agar plates with one of the cultures listed by aseptic transfer from a broth culture using a sterile cotton swab. Each plate was attach off into four segments, a total of eight sectors. One McFarland standard disk containing one of the eight antibiotics tested was placed, using alcohol-flame sterilized forceps, in the center of a sector.After incubating for 18 hours at 37C, the diameters of the clear zones (zones of inhibition) around each McFarland disk was measured with a standard ruler to the nighest millimeter. The measurements obtained were matched against a chart (Claus 407) to determine whether the bacterium was tolerant, handsome, or intermediate in susceptibility to the antibiotic used. The numbers against which these measurements are matched take into account the remnant in zone sizes caused by variations in diffusion roams through agar in the antibiotics tested. Results Bacterium Streptococcus faecalis Antibiotic used banning zone size (mm)Cul ture response Ampicillin 28 S Bacitracin 20 S Chloramphenicol 22 S Erythromycin 21 S Novobiocin 20 S Penicillin G 20 S Polymyxin B 0 R Tetracycline 24 S Bacterium Staphylococcus aureus Antibiotic used Inhibition zone size (mm)Culture response Ampicillin 48 S Bacitracin 22 S Chloramphenicol 24 S Erythromycin 25 S Novobiocin 39 S Penicillin G 43 S Polymyxin B 0 R Tetracycline 32 S Bacterium Escherichia coli Antibiotic used Inhibition zone size (mm)Culture response Ampicillin 20 S Bacitracin 11 R Chloramphenicol 0 R Erythromycin 25 S Novobiocin 21 S Penicillin G 8 R Polymyxin B 6 R Tetracycline 12 R Bacterium Streptococcus faecalis Antibiotic used Inhibition zone size (mm)Culture response Ampicillin 0 R Bacitracin 0 R Chloramphenicol 21 S Erythromycin 22 S Novobiocin 10 R Penicillin G 0 R Polymyxin B 18 S Tetracycline 25 S word S. faecalis was sensitive to all of the antibiotics tested except for Polymyxin B. Since that antibiotic is known to be effective only against Gram negative bacteria, this observation is in retentivity with expected results.As a Gram positive, one would expect, as we observed, that it would be sensitive to polypeptide inhibitors, such as Penicillin G and Ampicillin, as well as protein synthesis inhibitors, such as Tetracycline and Erythromycin. Novobiocin, while not often used against Gram positives due to concerns about its toxicity, is known to be effective against Gram positives as well, which is borne out by these observations. Like S. faecalis, S. aureus is a Gram positive, and so would be expected to be sensitive to the same antibiotics and resistant to Polymyxin B. Our observations moderate this as well. E. coli is Gram negative, and our observations show it to be sensitive only to Ampicillin, Erythromycin, Novobiocin.It was, however, resistant to Penicillin G, demonstrating that there is some chemical factor which allows one beta lactam antibiotic to inhibit this bacterium (Ampicillin) w hile another (Penicillin G) does not. This is likely due to the disparity in chemical structure of the two. Penicillin G lacks an amino base, which is present on Ampicillin. It may well be that the presence of a partially-charged amino group on Ampicillin allows it to be uptaken by the bacterium more readily than Penicillin G. (Deacon) More unexpectedly, E. coli was resistant to Chloramphenicol, even though this antibiotic is useful in inhibiting other Gram negative bacteria. It may be that the E. oli strain used in this experiment has developed a resistance to this particular antibiotic. P. aeruginosa also proved to be resistant to Ampicillin, Bacitracin, Novobiocin, and Penicillin G. This suggests that the chemical structure which allows Ampicillin to inhibit E. coli is not effective in the case of this bacterium, which is sensitive to neither of these beta lactams. Bacitracin is only useful against Gram positives as well, so it had no detectable affect upon P. aeruginosa. On the other hand, this culture was sensitive to Polymyxin B, Tetracycline, Chloramphenicol, and Erythromycin, as one would predict for a Gram negative. Its resistance to Novobiocin is due to its having a modify DNA gyrase (Miller 674).It is worth noting in this case that while the bacterium is resistant, Novobiocin does encounter some effect at a standard concentration, and higher concentrations can be used to kill the organism. Such concentrations, however, would also be toxic to the patient if taken as a chemotherapeutic dosage. The emergence of antibiotic-resistant strains of bacteria has been an ongoing phenomenon since shortly after the widespread use of penicillin, the first antibiotic, began. The rate at which this occurs has increased as the availability and employment has increased since then, and it has now become a significant medical problem. The major reason for this is that the use of antibiotics acts as a selective pressure. Those bacteria which carry a variance on t heir plasmids that make them able to survive treatment give hem access to a niche which non-resistant strains cannot exploit, leading to their proliferation both in infected individuals and ultimately in the environment in general. The use of antibiotics kills not only pathogens, but also normal microflora which might otherwise prevent virulent bacteria from establishing a foothold in the human body. give thanks to the phenomenon of transformation, in which living bacteria can hold naked heritable material left over when other cells are lysed for reasons other than antibiotic sensitivity, resistance genes can cross between genera. For example, the Staphylococci can incorporate resistance genes from Bacilli and Streptococci.Added to this are resistance genes that exist as transposons found in the main bacterial genome and which can be transmitted to other members of the same species by temperate phages via the process of transduction. These mechanisms have resulted in the arisal o f such things as Vancomycin-resistant strains of S. aureus, responsible for a usually-lethal nosocomial infection that, ironically, is carried by the very bacteria that Alexander Fleming first observed as being susceptible to penicillin, the headmaster antibiotic. (Deacon) Literature cited Bauer AW, Kirby WMM, Sherris JC, Turck M. 1966. Antibiotic susceptibility testing by a standardize single disk method. Am J Clin Pathol 45(4)493-6. Deacon, J. The Microbial World Penicillin and some other Antibiotics. http//helios. bto. ed. ac. k/bto/microbes/penicill. htm. Institute of Molecular and Cell Biology, The University of Edinburgh. August 2003. DrugBank, http//redpoll. pharmacy. ualberta. ca/drugbank/cgi-bin/getCard. cgi? bait=APRD00320. July 29 2006. DrugBank, http//redpoll. pharmacy. ualberta. ca/drugbank/cgi-bin/getCard. cgi? poster=APRD00816. txt. July 29, 2006. 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