Many drugs with antimicrobial effects do not directly kill microorganisms but block the expression of essential proteins, which affects pathogen dysfunction. The antimicrobial drug that was discovered in this assignment should have a similar effect on Methicillin-resistant Staphylococcus aureus (MRSA). However, hypothetical claims require empirical examination, and it is for this reason that this paper critically evaluates methods and techniques that can be used to assess the effect of an antimicrobial drug on global gene expression.
One of the basic yet extremely accurate tools of expression analysis is DNA microarray, which allows one to determine whether a particular sample of genetic material — DNA or cDNA — can encode a particular gene. The methodology of this method is based on the hybridization of complementary single-stranded molecules previously labeled with a fluorescent pigment. If nucleotide chains that can hybridize with a gene encoding a specific protein were used as a matrix, the use of the DNA microarray technique and subsequent UV-lighting allows us to determine which samples can express that gene (NIH, 2020). With MRSA, this method proves to be highly successful because the genomic profile of MRSA is documented and recorded in digital data libraries. Accordingly, comparing the ability to encode specific proteins with the same ability in the microorganism after antimicrobial treatment helps to answer the question about the effect of this drug on global gene expression in MRSA.
The DNA microarray method as part of the expression assay is an excellent biotechnological strategy, which, however, is not without drawbacks. This method can be used comprehensively so that the ability of a sample to express multiple genes at once can be tested. In addition, the microarray device is straightforward and technically uncomplicated. Microarray also helps to identify mutations in individual genes, rather than complete shutdowns, which can give a complete picture of the nature of the active drug. Nevertheless, this is a very expensive test that requires a lot of probes and nucleotide fragments. Moreover, there is no control in the design of this test, which leads to varied results even when working on the same sample. Although the disadvantages are apparent, the DNA microarray method deserves to be used for this case.
Western blotting can also be used to test the ability of a particular cell to express a specific protein, and thus this technique is applicable to determine the effect of an antimicrobial agent on MRSA indirectly. This method uses the principle of complementary binding of antibodies to antigens, which means that if protein samples on a nitrocellulose membrane, previously separated by electrophoresis by polypeptide chain length, are treated with prepared antibodies, only the proteins existing in the initial mixture will bind to the antibodies. Hence, by the absence of an analytical signal on the part of some proteins, one can judge that these proteins were not present in the cell suspension, which means that MRSA cannot produce them. If it is known for certain that the bacteria could have produced these proteins before, the absence of a signal indicates the antimicrobial effect of the drug through suppression of the expression of the responsible genes.
A critical evaluation of the effectiveness of the Western blotting method provides information on both its advantages and serious disadvantages. Firstly, this method is extremely sensitive because it can detect even trace amounts of protein. Second, it is specific because it allows indirect expression analysis on specific proteins (genes). However, it is still an expensive process, requiring considerable stocks of ready-specific antibodies. In addition, the proteins cannot be quantified using this method, which means that it is not possible to say for sure whether the expression has been entirely suppressed or not. There may be additional proteins in the MRSA cell that were not known beforehand, but for some reason were able to react with the antibody. In this case, scientific errors and false positives occur.
A third technique can be real-time PCR, which allows us to refine the presence of the target nucleotide sequence in the sample, allowing us to judge the activity of specific genes. This technique is based on the classical PCR method with the difference that after each cycle of the procedure, the number of amplified genes modified by the fluorescent signal is recorded to construct an amplification schedule. When compared to a reference graph — for MRSA without treatment — this method is very accurate in indicating the number of specific genes. This method provides a quantitative assessment and gives an indication of the dynamics of the number of genes over time. However, among its disadvantages are the reduced sensitivity and the limited amount of DNA that can be introduced at the sample preparation step. Thus, a moderate combination of this method with microarray analysis may be the right strategy for expression analysis in MRSA.
Reference
NIH. (2020). DNA microarray technology fact sheet. Genome. Web.