Bacteria that have antibiotic resistance can appear naturally in the environment or they acquire it from other species(Bio2EE3 Lab 3 Manual, 2017). Horizontal gene transfer(HGT) also known as lateral gene transfer takes place between unrelated species and it is the movement of genetic information from one organism(donor) to another(unrelated recipient)(Clark D.P. et al., 2013). There are three mechanisms of HGT: transformation(recipients absorb the naked DNA from the environment), transduction(it involves transfer of DNA through phages) and conjugation(it includes the transfer of genetic material occurs through conjugal plasmids or conjugal transposons)( Charles T.C. et al., 2017) . Due to horizontal gene transfer of genetic material between organisms, bacteria with multiple genes coding for antibiotic resistance are emerging nowadays. This makes these organisms stronger as they are resistant to multiple antibiotics and require novel antibiotics to kill them(Bio2EE3 Lab 3 Manual, 2018). This is the reason why different antibiotics are required to cure distinctive diseases caused by the antibiotic resistant microorganisms.
The objectives of this lab are to “measure the process of conjugative plasmid transfer, monitor the differential susceptibility of microbes to antibiotics and to study the morphology of important antibiotic producers, the Streptomyces”(Bio2EE3 Lab 3 Manual, 2018). The hypothesis being tested in this lab is that Gram-negative bacteria are more resistant to antibiotics as compared to Gram-positive bacteria. The expectations form this lab are that the Gram-positive organisms(Saccharomyces cerevisiae and Micrococcus luteus) should have more zones of inhibitions depicting less resistance to antibiotics due to the lack of an outer cell membrane. The Gram-negative organisms(Escherichia coli and Pseudomonas fluorescens) should have less number of zones of inhibitions depicting more antibiotic resistance due to the presence of an outer cell membrane.
The methods used in the experiment performed have been followed as mentioned in parts 3.2 and 3.3 of experiment 3 in the BIO2EE3 Lab 3 Manual(2018). No changes were made to the method.
The hypothesis being tested in this experiment is that Gram- negative bacteria will be more resistant towards antibiotics as compared to Gram-positive bacteria. The results observed partially met our expectations as two of the species, Saccharomyces cerevisiae(Gram positive) and Escherichia coli(Gram negative) gave us the expected results while Micrococcus luteus(Gram positive) and Pseudomonas fluorenscens(Gram negative) did not give us the expected results. The unexpected results may be due to contamination of the microbial samples while plating them on the agar which then led us to results observed for M. luteus and P. fluorenscens as tabulated in Table 3.
In section 3.1 sample data for the morning labs was used to calculate the results as bacterial growth was not observed the experiment plates created by my lab group. This may have occurred due the incorrect isolation technique used to isolate the bacterial colonies at time t= 15 minutes, 30 minutes and 45 minutes. According to Table1 and Table 2 made using the sample data the number of colonies were observed to increase over time. The semi-log graph (Graph 2) is better than the linear graph(Graph 1) to present this data because it gives a better and clearer idea on how the colonies grow exponentially over time.
In section 3.4 the gram-negative organisms give a different result than Gram positive organisms as they have an outer membrane around their cells on top of the peptidoglycan layer. The outer membrane protects the Gram-negative organisms from antibiotic attack, making them more resistant as compared to the Gram-positive organisms which lack the outer layer(Charles T.C. et al., 2017). Different kinds of antibiotics attack the bacterial cell differently to kill the bacterial cell. Some antibiotics like the Beta-lactam antibiotics target the D-alanyl D- alanine part of the peptide chain which is normally bound to the penicillin binding proteins(PBPs). The lactam rings interact with the PBPs inhibiting the synthesis of new peptidoglycan, therefore lysing the bacterial cell due to the disturbances in the peptidoglycan layers. Other kinds of antibiotics like chloramphenicol attack the bacterial DNA and inhibit protein synthesis attacking the 30S or 50S subunits of the ribosomes and hence preventing the t-RNA from binding to the A site (Elongavan A. et al., 2017).
Ampicillin is a derivative of penicillin and it works on both gram-negative and gram-positive bacteria whereas, penicillin affects gram-positive bacteria and some gram-negative bacteria, but is required in higher concentrations for the gram-negative bacteria(Coppoc G.L., 1996). S. cerevisiae gave us zones of inhibition with seven antibiotics out of the eight. It did not give any results with chloramphenicol which means that it is only resistant to that antibiotic. Ampicillin, gentamicin, neomycin and streptomycin gave us zones of inhibition with diameters 2.4cm, 1.6cm, 1.3cm and 1.5cm respectively which indicates that these antibiotics were more effective in killing the S. cerevisiae cells(ampicillin being the most effective). Penicillin, kanamycin and tetracycline gave us zones of inhibitions with radiuses equal to 0.5 cm, 0.4cm and 0.4 cm respectively. This indicates that these antibiotics were less effective in killing the microbial cells as compared to the other antibiotics.
- Bio2EE3 Lab 3 Manual(2018). Production of antibiotics and horizontal gene transfer. McMaster University.
- Charles TC, Dupont C., Wessner DR. New Jersey: John Wiley & Sons; 2013.58-297p.
- Clark DP, Pazdernik NJ. 2013. Molecular biology. 2nd ed. [accessed 2019 November 9]. https://www.sciencedirect.com/topics/neuroscience/horizontal-gene-transfer
- Coppoc GL. 1996. Penicillin Derivatives. Purdue Research Foundation. [accessed 2019 November 10]. http://www.cyto.purdue.edu/cdroms/cyto2/17/chmrx/penems.htm
- Elongavan A, Kapoor G, Saigal S. 2017. Action and resistance mechanisms of antibiotics: A guide for clinicians. J Anaesthesiol Clin Pharmacol.[accessed 2019 November 9];33(3):300–305. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672523/doi:10.4103/joacp.JOACP_349_15.