Bio lab Report - Bacteria

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Dec 6, 2023

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Joseph Kuo Lab Section: BISC-120 13157 The Effect of Tetracycline, Nalidixic Acid and Vancomycin on Growth Sensitivity of Staphylococcus aureus and Salmonella through Antibiotic Test Introduction: Bacteria can be both harmful and essential for healthy body function. Lactobacillus is a species of beneficial microbial that prevent and treat cancer. But for other types of bacteria, it could be harmful for human health. Therefore, antibiotics are developed as chemical substances that fight bacterial infections. Antibiotics work by killing the bacteria or stopping it from spreading in the body’s immune system. There are various types of antimicrobial drugs that are used for the prevention of bacterial infections, but all have only one of two functions. They either kill the bacteria or inhibit their growth. A bactericidal antibiotic interferes with the formation of the bacterium’s cell wall, which is the purpose of the antibiotic test for this experiment. Staphylococcus aureus is a gram-positive bacterial human pathogen that causes invasive infections or toxin-mediated diseases. It’s evasion of the immune system is by either producing an antiphagocytic capsule, isolating the host antibodies or antigen masking (Taylor and Unakal, 2023). Since neutrophils are the most prevalent kind of leukocyte in the blood, S aureus is extremely harmful because it may eradicate neutrophils with cytolytic toxins and cause apoptosis in the phagocyte. This is particularly efficient in impeding a normal immune response to infection (Taylor and Unakal, 2023). Salmonella is a rod-shaped and gram-negative pathogen. It’s the most common type of bacteria that is exploited for developing gene manipulation techniques such as crispr gene recombinant technology. Salmonella is a common inhabitant of the human and animal gut. 1

Joseph Kuo Lab Section: BISC-120 13157 Human infections of the urinary tract, ears, wounds, and other tissues are primarily caused by Salmonella. Just like S. aureus, antimicrobial resistance in Salmonella has an overall susceptibility pattern of high resistance to many antibiotics (Kibret, 2011). The antimicrobials that worked best against Salmonella isolates were determined to be norfloxacin, ciprofloxacin, gentamicin, and chloramphenicol (Kibret, 2011). Salmonella has a fast reproduction rate, so is S. aureus. In a short span of time, random mutation can build up in many generations. Therefore, the observation can be clearly retrieved and evaluated that which antibiotics of the three mentioned in the title are most effective to treat pathogen infections. The one antibiotic treatment has the most effect is supposed to be Tetracycline because of its broad spectrum. Tetracyclines are a wide range of antibiotic class used in the treatment of a variety of infectious diseases. They are extremely effective on both gram-positive and negative. It prevents the infections by inhibiting the bacterial protein synthesis (Shutter and Akhondi, 2023). Semi synthetic tetracyclines are lymecycline, methacycline, minocycline, rolitetracycline, and doxycycline. These drugs can treat rickettsial infections, ehrlichiosis, anaplasmosis, leptospirosis, amebiasis, actinomycosis, nocardiosis, brucellosis, melioidosis, tularemia, chlamydial infections, pelvic inflammatory disease, syphilis, traveler's diarrhea, early Lyme disease, acne, legionnaire's disease, and Whipple disease (Shutter and Akhondi, 2023). Another antibiotic, Vancomycin, is a type of glycopeptide antibiotic that works by inhibiting cell wall formation (Scholar, 2007). It is mainly used up against infections caused by gram-positive bacteria, since it is ineffective against Gram-negative bacteria. Vancomycin has 2

Joseph Kuo Lab Section: BISC-120 13157 been considered the primary drug in treating methicillin resistant Staphylococcus aureus (MRSA). It targets the synthesis of the bacterial cell walls. Vancomycin inhibits the synthesis if peptidoglycan. The transglycosylation and transpeptidation steps the bacteria must perform during cell wall assembly (Scholar, 2007). Although it has posed some serious concerns for the kidney, it is still an effective treatment for the Chloridoids difficile-associated diarrhea (Scholar, 2007). The chance of vancomycin nephrotoxicity increases with the bacterial resistance when the clinical use has also increased. The bacterial resistance will be seen in strains as Vancomycin-resistant S. aureus (VRSA) (Cong et al., 2019). In order to determine the effectiveness of tetracycline, vancomycin and nalidixic acid in the prevention of both S. aureus and Salmonella, the experiment carries out the most capable antibiotic for each bacterium through the observation of the relative size zone of inhibitions. The objective is to find out which of the three antibiotics has the best inhibitory ability. Therefore, the goal of this experiment is to compare the antibiotic treatment that functions the most against S. aureus and Salmonella. Our hypothesis is that Vancomycin will be most effective prohibiting against S. aureus, which has already been tested in clinical practices (Nwankwo and EOK, 211). The design of the experiment separates the growth environment of both bacteria, and dividing the whole petri dish into four sections that include the control with no antibiotic on it, and three with each antibiotic in its particular sections. After the bacterial growth, we perform a gram-stain process as a part of the antibiotic test to examine our hypothesis that Vancomycin is the most effective antibiotic for S. aureus. We can also hypothesize that nalidixic acid is the most effective antibiotic for Salmonella, and then test our hypothesis by comparing the zone of inhibition around their disc, which will be the largest. 3

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Joseph Kuo Lab Section: BISC-120 13157 Materials and Methods: To test the three antibiotics, we have obtained agar plates that contain only bacteria, either S. aureus or Salmonella. To determine which antibiotic treatment is the most effective, we perform antibiotic sensitivity test that is related to zones of inhibition, and a gram staining process to verify that the bacteria have their corresponding cell wall structure. Each petri plate was labeled with 4 quadrants, which A: tetracycline, B: vancomycin, C: control and D: nalidixic acid. Half of the class was assigned to grow S. aureus from VWR (catalog #470179-208), and the other half Salmonella (catalog #470179-144). Samples from both bacteria were then collected from their host environment and transferred to two tryptic agar plates using a sterile swab from Hardy Diagnostics (catalog #G60BX). After the bacteria is spread evenly over the surface of the agar, a blank disc was put into the control quadrant, and each antibiotic disc was then placed in the center of their quadrants. To prevent cross contamination, all discs were transferred onto the medium with tweezers, cleaned with 70% ethanol after each use. The antibiotic discs were retrieved from BBL Sensi-Disc Antimicrobial Susceptibility Test Discs from Becton Dickson (BD). The plates were then sealed for a week for the bacteria to reproduce. After a week of growth, the zone of inhibition for each disc in both bacteria culture was then observed. The diameters of the zones of inhibition were measured in millimeters. The diameters were then organized into a table for each type of bacteria. A larger diameter signifies that a higher ability of the specific antibiotic to inhibit the growth of the particular bacteria on the plate. The diameters then calculated into averages for each treatment group. The statistics were then presented as bar graphs to describe the effectiveness of each antibiotic on each bacterial environment. 4

Joseph Kuo Lab Section: BISC-120 13157 As of gram-staining, a sample of S. aureus and Salmonella were collected from the petri dish with a sterile inoculation loop and transferred onto a microscope slide. A drop of DI water was placed on top of the bacteria and smeared to spread the bacteria. After the water has semi- dried up with air, a Bunsen burner was used to evaporate the left-over water and to form a thin layer of bacteria sample on each slide. The heat fixed slides were squirted with crystal violet stain for a minute and then washed with DI water. And we repeated the same procedure with Gram’s iodine for one minute, and rinse with DI water afterward. 70% ethanol was then dropped for around 5-10 and immediately rinsed with DI water again. And in order to counter stain the samples, the slides were flooded with safranin and left for one minute, and then rinsed with DI water as again. All the Gram-stains were obtained form Carolina Biological. Finally, we blotted dry the gram-stained slide with bibulous paper to remove any excess stains. A coverslip was placed on the smear, and we placed the slide in a light compound microscope to determine if the samples contained bacteria that were gram-positive or negative. The gram staining process was repeated for a total of four trials. The results were photographed through the lens of the light compound microscope in 100x with oil immersion. Results: The gram staining process indicated that in the Salmonella cultures, all three antibiotics inhibit the bacterial growth, but Tetracycline was the most effective one preventing Salmonella growth around its antibiotic disc with a zone of inhibition average 19.4 mm (fig. 1). Although nalidixic acid did not have the best inhibiting ability, it came up to second best with an average 16.2mm zone of inhibition (fig. 1). Vancomycin turned out to be the least effective one, having a 5 mm zone of inhibition in Salmonella (fig. 1). 5

Joseph Kuo Lab Section: BISC-120 13157 For S. aureus, Tetracycline was still the most effective antibiotic preventing the growth around the disc with a zone of inhibition average 20.4 mm (fig. 2). Although it did not prove that our hypothesis was correct, it showed that Tetracycline has the best inhibiting ability among both gram-positive and negative bacteria, and where in both gram-positive, Vancomycin has second best inhibition for bacterial growth. In gram-positive bacteria environments, Vancomycin has the least effect of the three antibiotics. The control discs in both bacteria cultures showed no ability in inhibiting bacterial growth (fig. 1 and fig. 2). Overall, the table showed a similar trend of effectiveness of Tetracycline in the average values between Salmonella and S. aureus bacteria (Table 1). Tetracycline produced an average 19.4 mm of zone of inhibition on Salmonella (fig. 1) and 20.4 mm on S. aureus (fig. 2). Tetracycline has the best ability in inhibiting the growth of bacteria, although the standard deviation may indicate that Nalidixic acid might be more effective. Nevertheless, through Gram- staining, we figured out that Salmonella is gram-negative because it turned red-ish (fig. 2), and S. aureus is gram-positive because it turned out to be purple, which we observed under the light compound microscope (fig. 2). 6

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Joseph Kuo Lab Section: BISC-120 13157 Table 1: diameters of zones of inhibition in millimeters for each treatment group for both Salmonella and S. aureus. There are nine trials of average and standard dieviations for each treatment group. 1 0 5 10 15 20 25 16.2 19.4 5 Zones of Inhibition on Salmonella Zone of Inhibition (mm) Nalidixic Acid Zone of Inhibition (mm) Tetracycline Zone of Inhibition (mm) Vancomycin Zone of Inhibition (mm) Control Figure 1: average diameter of zones of inhibitions for Salmonella in response to Nalidixic acid, Tetracycline and Vancomycin and a blank disc (the control group). The diameters are measured in millimeters and include the diameter of the antibacterial disc with them. 7

Joseph Kuo Lab Section: BISC-120 13157 1 0 5 10 15 20 25 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 6.28 20.44 11.61 Zone of Inhibitions on S. aureus Zone of Inhibition (mm) Nalidixic Acid Zone of Inhibition (mm) Tetracycline Zone of Inhibition (mm) Vancomycin Zone of Inhibition (mm) Control Figure 2: Average diameter of zones of inhibitions for S. aureus in response to Nalidixic acid, Tetracycline and Vancomycin and a blank disc (the control group). The diameters are measured in millimeters and include the diameter of the antibacterial disc with them. Figure 3: Photo of gram-stained S. aureus (left) and gram-stained Salmonella (right). Photos were taken using an iPhone through a light microscope. Discussion: 8

Joseph Kuo Lab Section: BISC-120 13157 The gram-staining process helped us identify that Salmonella is gram-negative because of its red-ish color (fig. 2), and S. aureus is gram-positive because it is purple (fig. 2). Bacteria that stained purple is gram-positive because the crystal violet dye can stay on the thick layer of peptidoglycan in the cell wall. On the flip side, the bacteria that is stained pink is gram-negative with thinner outermembrane. Therefore, crystal violet dye is way easier to penetrate through the membrane, instead of remaining on and in between the surface, when the water runs through the bacteria with the dye. This is the exact reason why gram-negative bacteria can be dyed pink by safranin. In the previous research in 2016 by Cold Spring Harb Perspect Med., the optimization of tetracyclines in the process of semisynthetic derivatives are discovered to treat the multidrug- resistant (MDR) Gram-negative and positive pathogens. In recent years, two new tetracyclines have entered clinical development, such as a semisynthetic aminomethylcycline called Omadacycline and Eravacycline, a fully synthetic flouorocycline (Cold Spring Harb Perspect Med., 2016). And in recent studies, the prevalence of tetracycline resistance in some European countries was found to be 66.9% (Cold Spring Harb Perspect Med., 2016). There altogether showed the importance of tetracycline for combating both gram-negative and positive bacteria. And in relativity, the more tetracycline is applied in clinical and environmental setting, the more healthy individuals are because it can treat infections caused by bacteria. And furthermore, by inhibiting the growing of bacteria, it’s rather more effective to prevent multiplying harmful microorganism than killing infectious agent (Cold Spring Harb Perspect Med., 2016). The result of the experiment was not as accurate as we imagined. There might be errors such as malfunctioning of the antibiotic in the disc, or even human error when measuring the 9

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Joseph Kuo Lab Section: BISC-120 13157 zone of inhibitions. And it’s possible that the bacteria were not smeared evenly across the agar plate, and thus affect the growth of the bacteria. Other factors can also contribute to growth limitation, such as unequal distribution of the light, regardless of the presence of antibiotic. Also, not all the petri dishes were completed at the same time, some team may work faster than the other, which can cause more bacteria to grow in the medium, and thus influence the diameters of the zone of inhibition. Therefore, the next time we perform the same experiment, we should reduce the confounding variables introduced either by outward factor like differing light amount, unequal heat distribution when incubating, or even to avoid human error by verifying measurement with the team individually. In future experiment, we could test other antibiotics, or sub-class of it. Or we can develop an alternative method to determine if Tetracycline has the best inhibiting ability on bacteria growth. There might be a possibility that we can also develop an experiment with combined antibiotics and its effectiveness. Bacteria are famous in its capability of developing resistance, so sensitivity test must be continuous for finding the most effective antibiotics for treatment, especially in health care and health related field. We should be aware of bacterial resistance and be reminded that the treatment option we have accessed to before new antibacterial is discovered. References: Cong, Y., Yang, S., & Rao, X. (2019, October 12). Vancomycin resistant Staphylococcus aureus infections: A review of case updating and clinical features . NCBI. Retrieved October 26, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015472/ 10

Joseph Kuo Lab Section: BISC-120 13157 Kibret, M. (2011). Antimicrobial susceptibility patterns of E. coli from clinical sources in northeast Ethiopia . NCBI. Retrieved October 26, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220125/ Nwankwo, E. O., & EOK, N. (2011, January 26). Antibiotic sensitivity pattern of Staphylococcus aureus from clinical isolates in a tertiary health institution in Kano, Northwestern Nigeria . NCBI. Retrieved October 27, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201603/ Scholar, E. (2007). Vancomycin . ScienceDirect. Retrieved 10 26, 2023, from https://www.sciencedirect.com/topics/chemistry/vancomycin Shutter, M. C., & Akhondi, H. (2023, June 5). Tetracycline - StatPearls . NCBI. Retrieved October 26, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK549905/ Taylor, T. A., & Unakal, C. G. (2023, July 17). Staphylococcus aureus Infection - StatPearls . NCBI. Retrieved October 26, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK441868/ 11

Bio lab Report - Bacteria

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