MazEF homologous Modules System and A Post-segregational killing Mechanism (Bacteriostatic & Bactericidal Mechanism) of Novel Compound Isolated from Spondias monbinon Escherichia Coli and Bacillus Subtilis

The basic objective of this research work is to evaluate the mechanism of action of compound Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), Aspidofractinine-3-methanol) and Terephthalic acid, dodecyl 2-ethylhexyl ester with Selected clinical isolates by using molecular biomarker MazEF9 TA system. Toxin-Antitoxin (TA) systems are highly conserved in members of the Gram positive +ve and Gram negative –ve bacteria which has been proposed to play an important role in physiology and virulence. Clinical microorganisms were cultured and Sub-culturing in Department of Microbiology and Centre for Biocomputing and Drug Development (CBDD), Adekunle Ajasin University, Akungba Akoko, Ondo-state, Nigeria. A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000 μg mL in a total volume of 500μl for 0, 15, 30, 45, 60, and 180 minutes. The cells were pelleted by centrifugation at 5000 revolution for 5 minutes, to isolate the DNA and total RNA was quantified using spectrophotometric absorbance at 260nm, the DNA was removed with Turbo DNA-free (Ambion, Inc.). The Removal of DNA from the RNA samples was performed using DNA-freeTM DNA Removal Kit Reverse Transcription–PCR reaction in a 15.0μL final volume. 1μl template cDNA (~40ng) were combined with 1.0μL of forward primer (5nM), 1.0μL of reverse primer (5nM), 4.5mL nuclease-free water and 7.5μL of Taq 2X Master Mix. Assessment of Polymerase Chain Reaction products (amplicons) were electrophoresed in 0.5% of agarose gel using 0.5 × TBE buffer (2.6g of Tris base, 5g of Tris boric acid and 2mL of 0.5M EDTA and adjusted to pH 8.3 with the sodium hydroxide pellet) with 0.5μLethidum bromide. The bacteriostatic mechanism of action of isolated compounds A1, A3 and F3 from the Stem bark of Spondiasmombin. The bacteriostatic mechanism of action was demonstrated with the selected biomarker MazF against E. coli and B. subtilis. It was observed that at 30 minutes, the mechanism of action is bacteriostatic action on the test organism (B. subtilis). By overexpressing putative MazF homologues in clinical isolates (MazF9) induce bacteriostasis. MazFribo nucleases contribute synergistically to the ability of clinical isolates to adapt to conditions such as nutrient depletion and rapid progress to the death thereby measuring the reversible bacteriostatic mechanism of action of isolated novel compound from Spondiasmombin the novel compound is A1 (Epigallocatechin, Epicatechin and Stigmasterol phytosterol (synergy), A3(Aspidofractinine-3-methanol) and F3(terephthalic dodecyl 2-ethylhexyl ester) isolated from Spondiasmombin. The use of medicinal plant like Spondiasmombin should be encourage because of its therapeutic properties in the prophylaxis cure of infectious disease.