Plasmids are important in the inner workings of most organisms. Over the years, these molecules have proved significant to microbiologists and geneticists in forming and transferring new genetic sequences.
Plasmids incorporate varying properties, including antibiotic and metal resistance, hydrocarbon degradation, and antibiotic and bacteriocin synthesis.
Notably, plasmids resistant to antibiotics are transferred seamlessly from one bacterium variation to the other. This transfer is made possible by conjugation, transformation, or mobilization.
Due to the antibody-resisting attributes of several plasmids, clinical researchers have encountered several bumps in their quest to find effective treatment modes for most infections. Besides this resistance, several bacterial organisms transmit pathogenicity to the cells embedded in their hosts.
Scientists employ a procedure called plasmid curing to understand the negative attributes of plasmids and how to deal with them effectively.
Plasmid Curing Explained
Plasmid curing denotes the loss of plasmids. This process involves sieving out the attributes existing in a plasmid, such as bacteria-themed aromatic compound degradation, antibiotic resistance, and virulence.
Although AAV science has introduced several plasmid curing agents, none of them have successfully eliminated bacteria from their host organisms.
Thus, the clinical world needs to obtain a valid plasmid curing agent to reverse the negative attributes embedded in plasmids.
Notable plasmid curing agents that have found their way into medical literature include:
- Ethidium Bromide
- Lauryl Sulfate
- Acridine Orange
However, research suggests that some plasmid curing agents (ethidium bromide and acridine dyes) can’t be engaged in-vivo due to their teratogenicity, mutagenicity, and carcinogenicity — resulting from detergent activity.
These characteristics have made these plasmid agents useless in checkmating the spread of bacteria antibody resistance in a healthcare facility. These agents are handy for studying the AAV plasmid transfection and phenotypes in various laboratories
So, although most plasmid curing agents are essential in studying the activity of plasmids, none have been able to dispel plasmids completely from a bacteria class. However, multiple variations of non-toxic and highly potent plasmid removing agents are created to continue effective treatment.
With the inefficient nature of most plasmid curing agents in view, most scientists are focusing on plants. For example, several extracts of Plumbago zeylanica were seen to be effective in the removal of plasmid antibiotic resistance in a bacterium form tagged Escherichia coli. However, note that this medium wasn’t scientifically proven or purified.
Therefore, researchers studied alternatives such as P. Zeylanica to see if they contained potent plasmid curing agents that would prove helpful in plasmid antibiotic resistance in Salmonella Typhi, Escherichia coli, and Acinetobacter baumannii.
Notable Material and Method Used to Facilitate the Curation of Novel Plasmid Curing Agents
As seen in the AAV production addgene phase, several materials and methods have been integrated to foster the seamless creation of effective plasmid curing agents. However, one material and method stood out due to its comprehensiveness:
For this process, plant samples of P. Zeylanica were obtained from India (Western Ghat Region). This plant had its roots harvested, dried, and powered to foster future extractions.
This plant material received the “seal of approval” from the subsection of India’s Ministry of Environments and Forests — the Botanical Survey of India. A specimen, also referred to as the RBPUP1, was transferred and deposited at the Herbarium of Botanical Survey of India (the Joint Director’s office).
Extracting Plant Curing Agents from P. Zeylanica
Here, 2.5 KG of powdered and dried Plumbago zeylanica were extracted successfully using a Soxhlet Apparatus. Scientists used solvents like petroleum ether, ethanol, benzene, methanol, acetone, diethyl ether, and chloroform at boiling temperature to make this possible.
Afterward, each extract underwent filtration and concentration. Once this process was completed, extracts were dissolved in 10 milliliters of dimethyl sulfoxide (DMSO).
Upon dissolution, column chromatography was executed on P. Zeylanica roots with a solvent called silica gel (measured at 100 to 200 mesh).
The ethanol extract showing signs of plasmid curing was fractionated with silica gel via a hexane-containing gradient solvent structure.
Upon TLC analysis and chromatographic fractions using several solvents, the compound detected by the former — hexane-ethyl acetate — underwent further purification with preparative TLC.
As we’ve established, plastic curing is a procedure that’s vital in determining antibiotic resistance levels in various bacteria classes. However, the lack of effective agents has made the development of treatment modes difficult. Thus, novel methods like the one we gave credence to in the latter parts of this article are vital.
Despite showcasing complexities highlighted in AAV vector production, plasmid curing techniques continue to evolve and break milestones in clinical research.