Purification Of A11 Endophytic Antibacterial Substance And Its Biological Activities And Mode Of Action

Antibiotic industry was one of the most important businesses during the last few decades. While people enjoyed the fruition of this medical miracle, problems came about almost simultaneously. Antibiotic abuse and following bacterial resistance became serious issue which severely restricted the application of this once deemed'panacea'. Nowadays, almost each commonly used antibiotic has its corresponding resistant bacteria. Many bacteria such as MRSA can endure several different antibiotic through an actually not a very long selection and screening progress. All these hard situations impose on the requirement for searching the new antibiotics or antibacterial compounds. Recently endophytes have become a rich source for new products of various bioactivities. The lab has discovered a new endophytic bacterium named as A11 years ago with great ability to inhibit a board spectrum of microorganisms including several clinically and agriculturally important pathogens. The extracts from the bacterial culture remained this strong inhibitory property and primary study implied a potential novelty of the mixture. So it became a crucial object to isolate and define all the components with antibacterial activity and research their biological properties and ultimately mechanisms of action respectively. This study took on this track and did several key aspects of research focused on these antibacterial agents with methods and results as follows:1. Two main antibacterial agents were isolated and partially purified. Extracted from 48h of fermentation broth by adding to it triple volumes of ethanol, the dissolved antibacterial compounds were then dried and dissolved in methanol with minimal volume. An absorption chromatography method was used for crude isolation with silica gel (200-300 mesh) as polymeric sorbent. After gradient elution with proportion of mobile phases of ethyl acetate and methanol declined from 9:1 to 0:10, two inconsecutive gradients (ethyl acetate/methanol 3:7 and 5:5 respectively) named gradient 3 and 5 contained the antibacterial components. This process was repeated after previous method with only stationary phase replaced 300-400 mesh silica gel. The same results came out of this repetition. Each gradient was condensed and then applied to exclusion chromatography column stuffed with Sephadex LH-20 gel. The sample was washed with mobile phase methanol at 0.3mL/min and collected at interval of 10min. The target compounds were appeared in about No.20-26 collections. The condensed solution was then applied to reversed phase chromatography for final purification. Gradient elution with proportion of mobile phases of water and methanol declined from 9:1 to 0:10. The sample was washed at 1mL/min in temperature 25℃. The efflux representing each peak was gathered and tested for activity. The third peak showed bacterial activity and defined as target peak. The ultimate samples were then analyzed with mass spectrometry and the molecular weights of samples were 338.52 for gradient 3 and 339.41 for gradient 5. The following MNR tests failed to give some further details accurately due to insufficient amount and purity of two samples, but it can be deduced that the gradient 3 contained at least 7 carbon atoms.2. The biological properties of antibacterial extracts. An undefined but certain concentration of methanol extraction of antibacterial compounds was used for all the property tests and experiments. First the A11 extract and other antibiotics were tested for its inhibitory effect on Methicillin-resistant Staphylococcus aureus (MRSA) and normal nonpathogenic Staphylococcus aureus. The A11 extract showed an equal inhibitory ability for both bacteria whereas most of other antibiotics exhibited a different range of decreased inhibitory ability for MRSA except for Vancomycin, indicating a marvelous potential for further application. The MIC and MBC tests were conducted by classic serial dilution method. The resulting MIC was defined as between 1/1280 to 1/640 of the methanol extract and MBC between 1/40—1/20. The study also explored the effect of sub-inhibitory concentration of A11 extracts on the formation of Staphylococcus aureus biofilm. By 96-well culturing of Staphylococcus aureus with different concentrations of A11 extracts and assaying the cell mass of formed biofilms, the study discovered a decreased ability of biofilm formation when treated with A11 extract even in sub-MIC.3. The preliminary study of the mechanism of antibacterial action. The study mainly focused on two possible effects of A11 antibacterial agents:membrane change and protein synthesis. The SEM was applied to observe the morphological changes of bacterial cell. The results showed the cell massively shrunk, concave or even broke after exposed to MBC A11 extract while they showed no particular changes in MIC A11 extract. The membrane permeability of bacteria exposed to MIC was further studied based on the leakage of K+. The ICP scanning indicated a rapid decrease of K+ concentration after 1h exposure to A11 extract in inhibitory concentration and a relatively stable concentration K+ of 3h later. Lastly, the study researched the expression differentiation of total intracellular proteins by SDS-PAGE. The result showed a decrease of total protein after administration of MIC dosage of All extract. Although it should be taken into the consideration that the effects on those structures may not be initial--the blockage or deactivation of the real targets may lead to these consequential phenomena--the results are valuable for at least providing some useful clues for further study.