Biology Assignment

Title

Identification of E. coli and S. aureus Susceptibility towards Chloramphenicol and Doxycyclin

a) Introduction

Microorganisms (Microbes) are the microscopic organisms that exist as a single cell or in the form of colonies. They can be classified in to various types including bacteria, viruses, fungi, protozoa, archaea, algae, and various multi-cellular animal parasites (Tortora et al., 2004). Microorganisms are causative agent of many diseases in human beings. Some of the common diseases caused by microbes are tuberculosis, plague, anthrax, malaria, dysentery, Influenza, ringworm, yellow fever, hepatitis, and many more (Wilson, 2005). Diseases caused by bacteria are responsible for a large number of deaths worldwide. Antibiotics are used to defend human body against bacterial infections. Antibiotics are the antimicrobial agents that are active against bacteria and are widely used to cure or prevent the bacterial infections (Walsh, 2003). They have ability to either kill of inhibit the growth of harmful bacteria. The antibiotics that are produced by one microorganism to fight against another are known as natural antibiotics such as penicillin, while the antibacterial agents that are produced artificially are known as synthetics antibiotics such as sulfonamides (Hutchings, 2019). Antibiotics are classified into five major groups on the basis of their mechanisms of actions:

1) Inhibit Cell Wall Synthesis:

?-lactams and glycopeptides are the main antibiotic classes that interfere with the biosynthesis of cell wall of bacterial. They inhibit the cell wall synthesis by inhibiting the synthesis of peptidoglycans, or preventing the cross-linking of peptidoglycans. Antibiotics that inhibit cell wall synthesis are: penicillin, cephalosporin, ?-lactamase inhibitors, vancomycin, polymycin, carbapenems, and more (Kohanski, 2010).

2) Inhibit Protein Synthesis:

Protein is translated from mRNA in the ribosome and the process of mRNA translation is initiated by the binding of tRNA with mRNA and binding of various initiation factors at 30S subunit of ribosome, which is constituted by two subunits of ribonucleoprotein i.e., 50S and 30S. Antimicrobial agents that inhibit synthesis of protein can be classified into 30S inhibitors and 50S inhibitors. The 30S inhibitors are aminoglycosides and tetracyclines. 50S inhibitors include chloramphenicol, macrolides (erethromycin), oxazolidinones (linezolid), and streptogramins (dalfopristin– quinupristin) (Reygaert, 2018).

3) Inhibit Nucleic Acid Synthesis:

Quinolones and Fluoroquinolones are used to inhibit the synthesis of DNA. Quniolones inhibit DNA synthesis by inhibiting DNA gyrase enzyme essential for the replication of DNA. Rifamycin antibiotics are used to inhibit the synthesis of mRNA (Tortora, 2004).

4) Depolarization of Cell Membranes:

One important class of antibiotics is Lipopeptides (such as daptomycin) that attck the bacterial cell and cause structural changes in the cell membrane.

5) Inhibit Essential metabolites Synthesis:

Sulfonamides and Trimethoprim antibiotics inhibits the synthesis of essential metabolites like folic acid.

b) Mechanism of Action of Doxyxyxline and Chloramphenicol:

In this experiment two antibiotics Doxycycline and Chloramphenicol were used. Doxycycline is a tetracycline antibiotic used in the treatment of bacterial infections. As mentioned above, tetracyclines like Doxyxyxline bind to the 16S RNA portion of ribosome and prevent the binding of tRNA with 30S subunit of ribosome, thus inhibiting the translation process and protein synthesis.

Chloramphenicol inhibits the protein synthesis by reversibly binding to the L16 protein of ribosome subunit 50S in order to prevent transfer of amino acids towards growing chains of peptide thereby incorporating the peptide bond formation and protein synthesis ultimately.

c) Cellular Differences between Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli):

E. coli is a bacterium (rod like structure and gram-negative). A thin layer of perptidoglycan covered by an outer layer (composed of lipopolysaccharides) constitute its cell wall. Some strains of E. coli also possess flagella and such strains are motile. The diameter and cell volume of E. coli cells are about 0.25–1.0 ?m and 0.6–0.7 ?m3 respectively (Tenaillon, 2010).

On the other hand, S. aureus is a bacterium having rod like structure and Gram-positive. A thick layer of peptidoglycan (murein) composes its cell wall. Outside the plasma membrane lies the peptidoglycan layer. S. aureus cells are about 1??m in diameter and 0.52??m3 in volume (Dmitriev et al., 2004).

d) Disc diffusion Method:

The antimicrobial effectiveness determination of antibiotics against specific bacteria is essential for choosing the accurate treatment. Among many other techniques, disc diffusion method (DDM) also known as agar diffusion test or Kibry-Bauer method is the most common technique to determine the effectiveness of antibiotics. DDM is a very simple method in which the microbes of interest are applied onto an agar surface to which filter papers (paper discs) impregnated with different antibiotics are applied. The resistance or susceptibility of microorganisms towards the antibiotic can be observed by assessing the zone diameter of inhibition around the discs (Wheat, 2001).

e) Aseptic Technique:

During the experiment aseptic technique was applied. BSL-2 procedures were followed to isolate the microbes. Cell culture was carried out in the laminar flow safety cabinet. The cabinet was wiped with the 70% ethanol before use. Gloves were also sprayed with the 70% ethanol. Bacterological loop was sterilized on Bunsen flame before isolation and sterile swab was used to inoculate the bacteria on agar plates. The aim of the experiment was to perform antimicrobial susceptibility test to determine the usefulness and efficacy of two different antibiotics on the above discussed E. coli and S. aureus.

Methods

Antimicrobial Susceptibility Test

The disc diffusion method was used in this experiment to determine the susceptibility of E. coli and S. aureus towards Deoxycycline and Chloramphenicol. The test involve following steps:

i) Preparation:

•Four sterile plates of standard antibiotic testing agar were taken and two were labeled for E. coli and two for S. aureus.

•The bacterial overnight cultures were utilized to create a bacteria lawn. The two labeled petri plates were injected with E. coli and other two with S. aureus with the aid of sterile swab.

ii) Setting up Plate:

•16 plain discs were added in an empty petri dish with the aid of Sterile Tweezers.

•Antibiotic stock solution (20??l) was poured in each plain disc and discs were dried for 15 minutes.

The amount of each antibiotic added to the discs was as follow:

a) Doxycycline (0.5 mg/ml): 10??g

b) Doxycycline (1.5 mg/ml): 30 ?g

c) Chloramphenicol (0.5 mg/ml): 10??g

d) Chloramphenicol (0.5 mg/ml): 30??g

•A marker was used to divide the plates into four quadrants.

•Discs were applied to the agar plates using sterile tweezers and incubated at 37C overnight.

•After incubation diameter of inhibition zone was measured.

Results

Results revealed clear zone of inhibitions on the agar plate for E. coli (Figure 1) and S. aureus (Figure 2). It was also observed from the results shown in table 1 that the zone of inhibition for E. coli duplicate 1 was 14mm and for duplicate 2 was 20mm when Chloramphenicol (10 ?g) was applied and it was 20 mm for both E. coli duplicate 1 and 2 when Chloramphenicol (30 ?g) was applied. Moreover, when Doxycycline (10 ?g) was applied on E. coli plates the zone of inhibition was 14 mm and 15 mm for duplicate 1 and 2 respectively when Doxycycline (30 ?g) was used zone of inhibition was 20 mm for both duplicates when. In case of S. aureus Chloramphenicol (10 ?g) produced 23 mm and 20 mm zone of inhibition for duplicate 1 and 2, while Chloramphenicol (30 ?g) produced 35 mm and 29 mm for both duplicates respectively. Furthermore, the diameter of inhibition zone for S. aureus (duplicate 1 and 2) was 34 mm and 33 mm when Doxycycline (10 ?g) was used while it was 37 mm for both duplicates when Doxycycline (30 ?g) was applied.

Discussion

It can be observed from the results that E. coli shows intermediate susceptibility towards chloramphenicol (10 ?g) which means that some strains of E. coli may have relative resistance towards the antibiotic at 10 ?g concentration. Moreover, at chloramphenicol (30 ?g) the E. coli was susceptible. So it can be concluded that E. coli is susceptible to chloramphenicol at higher concentrations. Researchers have demonstrated that the accumulation of RNA was slightly stimulated and the synthesis of proteins remained unaffected in E. coli at low concentration of chloramphenicol but at higher concentration of chloramphenicol the protein synthesis is inhibited in E. coli (Dennis, 1976). Furthermore, results also revealed that E. coli is susceptible to Doxycycline both at 10 ?g and 30 ?g.

Results also showed that S. aureus is intermediate susceptible chloramphenicol at 10 ?g concentration, while it is highly susceptible to chlormaphenicol at 30 ?g. Researchers have also demonstrated that chloramphenicol has bacteriostatic effect on S. aureus at low concentrations, while S. aureus is highly susceptible to chloramphenicol at higher concentrations (Nishimura et al., 2006; Fayyaz, 2013). In addition, S. aureus is also susceptible to Doxycycline but researchers have demonstrated that S. aures is resistant towards Doxycycline. The difference in results may be due to the limitations in disc diffusion method. Studies have suggested that this method may not be genuine and well-grounded for testing vulnerability and susceptibility because of the inaccuracy of the method (Lehtopolku et al., 2012). In contrast genetic testing like polymerase chain reaction (PCR), microarray, etc. can be used for accurate results.

References

•Dennis, P. P. (1976). Effects of chloramphenicol on the transcriptional activities of ribosomal RNA and ribosomal protein genes in Escherichia coli. Journal of molecular biology, 108(3), 535-546.

•Dmitriev, B. A., Toukach, F. V., Holst, O., Rietschel, E. T., & Ehlers, S. (2004). Tertiary structure of Staphylococcus aureus cell wall murein. Journal of bacteriology, 186(21), 7141-7148.

•Fayyaz, M., Mirza, I. A., Ahmed, Z., Abbasi, S. A., Hussain, A., & Ali, S. (2013). In vitro susceptibility of chloramphenicol against methicillin-resistant Staphylococcus aureus. J Coll Physicians Surg Pak, 23(9), 637-640.

•Hutchings, M. I., Truman, A. W., & Wilkinson, B. (2019). Antibiotics: past, present and future. Current opinion in microbiology, 51, 72-80.

•Kohanski, M. A., Dwyer, D. J., & Collins, J. J. (2010). How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology, 8(6), 423-435.

•Lehtopolku, M., Kotilainen, P., Puukka, P., Nakari, U. M., Siitonen, A., Eerola, E., ... & Hakanen, A. J. (2012). Inaccuracy of the disk diffusion method compared with the agar dilution method for susceptibility testing of Campylobacter spp. Journal of Clinical Microbiology, 50(1), 52-56.

•Nishimura, S., Tsurumoto, T., Yonekura, A., Adachi, K., & Shindo, H. (2006). Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus epidermidis biofilms isolated from infected total hip arthroplasty cases. Journal of Orthopaedic Science, 11(1), 46-50.

•Reygaert, W. C. (2018). An overview of the antimicrobial resistance mechanisms of bacteria. AIMS microbiology, 4(3), 482.

•Tenaillon, O., Skurnik, D., Picard, B., & Denamur, E. (2010). The population genetics of commensal Escherichia coli. Nature reviews microbiology, 8(3), 207-217.

•Tortora, G. J., Funke, B. R., Case, C. L., Weber, D., & Bair, W. (2004). Microbiology: an introduction (Vol. 9). San Francisco, CA: Benjamin Cummings.

•Walsh, C. (2003). Antibiotics: actions, origins, resistance. American Society for Microbiology (ASM).

•Wheat, P. F. (2001). History and development of antimicrobial susceptibility testing methodology. Journal of Antimicrobial Chemotherapy, 48(suppl_1), 1-4.

•Wilson, M. (2005). Microbial inhabitants of humans: their ecology and role in health and disease. Cambridge University Press.