Table 1. The zone of inhibition and antimicrobial susceptibility for the unknown bacteria on a Kirby-Bauer Test. A Kirby-Bauer disk diffusion test was conducted on a Mueller-Hinton plate containing the unknown bacteria to examine its susceptibility for three antimicrobial agents. These antimicrobial disks contained the same amount of antibiotic concentration, 30 μg. Observed are the zones of inhibition for each antibiotic disk, control disk (0mm), Chloramphenicol (20mm), Tetracycline (26mm), and Neomycin (27mm). Although the unknown bacteria was susceptible to all antimicrobial agents, the unknown bacterium was most susceptible to Neomycin because it contained the large zone of inhibition compared to other treatment disks.
Determination for the treatment of the antimicrobial was based on the level of susceptibility of the unknown bacteria to different antibiotics. A Kirby-Bauer test was conducted using the standard Mueller-Hinton agar. The antimicrobial disks were placed on an evenly plated lawn of unknown bacteria. In addition, to control for inhibition levels all three antimicrobial disks contained the same amount of antibiotic concentration. These disks absorbed the moisture from the surrounding media causing the antibiotic to disperse into the media, which leads to inhibition of bacterial growth. The media closer to the antimicrobial disk contain a higher concentration of bacterial inhibition compared to media located farther away from the disk. Thus this inhibition of bacterial growth will produce no lawns leading to clear media and creating a zone of inhibition. The larger the zone of inhibition is for the antimicrobial disk, the more susceptible the bacterium is to the antibiotic. Three different antimicrobials agents were tested in the Kirby-Bauer Test. Chloramphenicol and Tetracycline are both bacteriostatic antibiotics, which inhibit protein synthesis of gram-negative and gram-positive bacteria. Chloramphenicol’s inhibition of the bacteria’s protein synthesis is caused by the interruption of amino acids transferred to ribosomes. Tetracycline’s inhibition of the bacteria’s protein synthesis is caused by the interference of tRNA and mRNA binding. Neomycin is a bactericidal antibiotic, which inhibits protein synthesis for gram-negative rods and occasionally gram-positive bacteria. Neomycin’s inhibition of the bacterial protein synthesis occurs during translation of mRNA. In addition, the Chloramphenicol, Tetracycline, and Neomycin disks contained an antibiotic concentration of 30 μg. A blank disk was also placed on the Mueller-Hinton agar to act as a no treatment control and show correct growth of the unknown bacteria by not having a zone of inhibition (CMMB Department, 2017). In Table 1, the antibiotic disks used were Chloramphenicol, Tetracycline, and Neomycin. The measured zones of inhibition were 0mm for the blank disk, 20mm for Chloramphenicol, 26 mm for Tetracycline, and 27mm for Neomycin. The unknown bacteria was susceptible to all the antimicrobial agents. However, the bacteria was most susceptible to the Neomycin treatment based on the largest zone of inhibition compared to the other antibiotics.
Discussion
Determination of the unknown bacteria was first analyzed with Gram staining, which allowed for differentiate of bacterial species based on their cell wall composition. By retaining the secondary stain this signifies the bacteria had a thin peptidoglycan layer, which indicated the bacteria was gram negative. In addition, the cellular morphology of the stained bacteria showed bacillus shaped cell walls. In addition, adding 3% KOH to the bacteria resulted a product with thick viscosity. Meaning the thin cell wall was disrupted with the KOH and the cell released its DNA, indicating the bacteria is gram-negative. Specialized purpose media plates were then used to further differentiate the gram-negative bacteria. EMB agar differentiates the gram-negative bacteria by indicating lactose fermentation. Bacteria that can ferment lactose are coliforms and bacteria that are unable to ferment lactose are paracolon. The inoculated EMB agar resulted in purple colonies indicating the bacteria can ferment lactose and is coliform. These purple colonies occurred because of the production of acidic products that lowered the pH of the media. Citrate agar was another special purpose media plate used to examine whether the bacterial production of citrase enzyme when citrate is the only carbon source to ferment and no other fermentable carbohydrates are available in the media. The inoculated citrate agar resulted in a green media after incubation. Indicating the unknown bacteria did not produce the enzyme citrase to ferment the sodium citrate causing no production of the alkaline product, CO2. Thus the citrate agar pH remained the same (CMMB Department, 2017). Analysis of the results from the Gram stain, 3% KOH, EMB agar, and citrate agar, indicate the causative agent as Escherichia coli (E. coli).
E. coli is a bacterium and foodborne pathogen known to cause secretory diarrhea within the intestines of humans after consumption of contaminated water or food. Once E. coli is consumed it is able to survive in acidic environment, such as the human intestines, and allow for colonization (Thorpe, 2004). However, E. coli cannot invade the intestinal epithelium to directly induce secretory diarrhea because the intestinal lining contains a layer of epithelial cells held together by tight intercellular junctions that prevent microbial invasion (Paton and Paton, 1998). Thus to cause secretory diarrhea, E. coli will produce and secrete shiga toxin (ST) within the small intestine to translocate into the cells. ST will translocate into the cell by apical membrane attachment, endocytosis, and retrograde vesicle trafficking to invade the intestinal epithelial cell’s cytosol (Paton and Paton, 1998).
The ST structure consists of one A-subunit and a pentameric B-subunit (Thorpe, 2004). After ST’s secretion, initiation of ST binds to the ganglioside GM3 receptor on the apical membrane of the intestinal epithelial cell (Gallegos et al., 2012 and Paton and Paton, 1998). During endocytosis of the ST and GM3, a COPI-coated vesicle assembles around the complex and generates retrograde transport movement. Therefore the folded ST and GM3 are transported from the apical membrane to the Golgi cisternae, then into the endoplasmic reticulum (ER), and finally the cytosol. During translocation of ST into the cytosol the A-subunit is cleaved from the AB-subunit by the protease furin. Cleavage of the ST causes activation of the A-subunits N-glycosidase activity. The A-subunit will then cleave a specific adenine nucleotide on the 28s rRNA, located in the cells 60s ribosomal subunit (Paton and Paton, 1998). Thus preventing the tRNA to bind to the ribosome, which inhibits the cells elongation process during protein synthesis and eventually causing the intestinal epithelial cells death (Gallegos et al., 2012 and Thorpe 2004).
Furthermore, the Kirby-Bauer test was conducted for the E. coli to find the most susceptible antimicrobial agent in inhibiting the growth of bacteria. Finding the correct antimicrobial agent is based on finding the largest zone of inhibition for the antimicrobial disk, which indicates the bacteria is more susceptible to the specific antibiotic. Chloramphenicol, Tetracycline, and Neomycin antimicrobials agents were tested in the Kirby-Bauer Test because these antimicrobial agents targeted gram-negative bacteria (CMMB Department, 2017). The measured zones of inhibition were 0mm for the blank disk, 20mm for Chloramphenicol, 26 mm for Tetracycline, and 27mm for Neomycin. These results indicate E. coli was most susceptible to the Neomycin treatment because it contained the largest zone of inhibition compared to the other antibiotics. Thus the antibiotic suggested for the patient treatment is Neomycin. Neomycin targets E. coli by inhibiting its protein synthesis during translation (CMMB Department, 2017). Thus E. coli will be unable to replicate, which eventually leads to its death and inability to further colonize the intestine (CMMB Department, 2017 and Paton and Paton, 1998).
There are a few suggestions to further improve the identification of the unknown bacteria. One suggestion is to improve the streak plate technique when inoculating the bacteria onto the media. Inadequate streaking caused a low amount of bacteria to be inoculated onto the EMB agar. Resulting in very few bacterial colonies to appear on the EMB agar after incubation. Thus repeating the EMB agar test with adequate streaking may help further support the identification of E. coli. Another suggested improvement is to repeat the Kirby-Bauer Test because the zone of inhibition measurements for all three antimicrobials agents were similar in size. By repeating the Kirby-Bauer Test and averaging the multiple results for each antimicrobial agent, this would further strengthen the reasoning behind treatment recommendation.