Protocol to Experiment Antibacterial Agents Originated from Natural Products.

These days, I am studying the antimicrobial activity of phytochemicals. In prior of this, I should know an adequate protocol to investigate natural product originated antimicrobial agents. Therefore, I found a paper (Anti-infective potential of natural products: How to develop a stronger in vitro ‘proof-of-concept’) giving detail about it. It is basically about how to choose plants, extraction, and how to conduct the appropriate bioassays. Things that I focused on are the extraction part and the bioassay part and I'm going to summarize it.

 

First, about extraction, it starts with general information. ‘How to extract’ is the point. When we extract, we can macerate, which means putting plants (no matter whether it's fresh or dried powder) into warm water or organic solvent and making them flabby. Through this process, we can extract phytochemicals. The solvents that we use are methanol, ethanol, ethyl acetate, etc. According to the paper, dichloromethane or a mixture of 1:1 dichloromethane and methanol are used to extract more lipophilic compounds. To remove chlorophyll, hexane is used. After extracting like this, we need to remove the solvents that we have used. By evaporation in vacuo, we can only remove the solvent, leaving extracted phytochemicals in the flask. This process should be conducted at a low temperature as the chemicals we need could be destructed at high temperatures. However, this process might cause precipitation or co-precipitation, disturbing proper experiments. Therefore, we have to introduce different pH solutions to enhance the separation of acid, neutral and basic constituents.

 

Plus, there is another standard way of extracting phytochemicals; using solvents from high to low polarity. Pros of this method are logical, low cost, feasible, and highly performing starting approach. It can better discriminate by the polarity of chemicals and well maybe this could help us find their interesting ability. However, there is a thing that we have to be careful of. As acidic polysaccharides and tannins are already proven to have the antimicrobial ability, we need to remove them unless we are not studying about it. This can be done by adding 50% ethanol and/or chromatography on a polyamide 6 or 8 column, using water and methanol as eluents after extracting and precipitation.

After the extraction, we have to store them as stable as possible, so many researchers use DMSO (dimethyl sulfoxide), methanol, and ethanol as storage solvents. However, methanol and ethanol are easily evaporated, therefore, extracted compounds are stored in 100% DMSO and tightly sealed as it is hygroscopic. There are other reasons why we have to store them in 100% DMSO at -20℃ (to prevent chemical modification by freeze-thaw cycles and humidity). First, it eliminates microbial contamination, so we don’t have to sterilize (by autoclave or other strenuous methods) before using them. Next, its solubility is compatibly better than other solvents.  But, as I said, DMSO itself can kill microorganisms; therefore before we use them at the test, its concentration should not exceed 1%.

 

When we experiment with microorganisms, we should choose an appropriate bioassay. When conducting in vitro experiments, sometimes we design the experiment wrong due to too focusing on ethnomedical data. We have to search about it for the projects we have, but it could be wrong and this can lead to false positives. Also maybe we can suffer from the lack of discrimination from aspecific cell toxicity. Therefore, we need to conduct experiments to check cytotoxicity using human cell lines. Therefore, before using the chemicals on the patient, we must experiment in order. First, we need to check with the computer whether it has the effect or not. After that, through in vitro test, we can check if it is effective to the bacteria we are targeting or not. At this point, there are 2 types of in vitro tests. One is done at a subcellular level. Through this experiment, we need to identify which molecule the chemical we study is targeting. It could be microbial enzymes, receptors, and processes at the molecular level. However, usually, most extracts are a mixture, therefore it is hard to find which one is effective against which target. And the other one is targeting the whole organism. This test is conducted on the dishes that we commonly use. By introducing the compounds or the extracts that we want to know, we can see how effective they are. At this point, it is important to discriminate from aspecific cell toxicity as it can kill human cells as they terminate infectious microorganisms. To prevent this, it is important to keep the highest concentration below 100-150μM or μg/ml. If it’s over this concentration, it could be led to false positives. Usually, this test is conducted on the standard 96-well microplate format.

 

Before talking about this 96-well microplate format, we need to know several ways to test their antimicrobial ability. As they are not equally sensitive or not based upon the same principle, results could be different depending on the method we use. ‘Agar-diffusion method’ is a basic way to check its antimicrobial ability. To check its ability, we need to make a hole at the inoculated agar and drop the extract or solution with the specific compound in it. Another way to do this is; instead of making a hole, we can just place a filter paper disc, stainless steel cylinders with the extract, or the solution with the specific compound. After this, we have to incubate the plate and check the inhibition diameter. There are a few things that we need to be careful of. To enhance the detection limit, we have to keep the inoculated plate at a lower temperature for several hours. Plus, this method should be done with aqueous extracts or compounds as the agar is polar material. If we need to test with non-polar compounds, well then, we have to use other materials as a medium. The next method is the ‘Dilution method’. This method is a way to find MIC (minimal inhibitory concentration). By dividing the concentration in half each time, we can test it at various concentrations. Plus, we need to apply fluorescein-diacetate, as living cells will convert them into fluorescein which produces a yellowish-green fluorescence under UV light. The last method that we can use is the ‘Bio-autographic method’. This method is directly done with the TLC plate. After all the compounds are separated, we inoculate bacteria on the plate directly and incubate them. Then, we can find at which part bacterial growth was hindered. In the end, we can compare them with the plate without inoculation and scratch out the silica with antimicrobial activity, and extract the compounds. These methods could be conducted separately or combined. IC50 values should be lower than 100μg/ml for extracts and lower than 25μM for a pure compound. The reason why we need to keep the concentration below them is to prevent false-positive by aspecific toxic action of the compound itself which could be led to killing human cells, too.

Now, I’m going to explain the usage of 96-well plate.

As you see in this picture, there are 2 ways to use 96-well microplate format. One row at each top and bottom should not be calculated due to the problem of evaporation and there would be 6 rows left. 3 rows at the top should be non-infected medium control which means there must be nothing growing. The next 3 rows are infected control which means 100% growth of test organism. Then, we need to introduce the extracts or chemicals that we want to test and incubate this plate. From 128μg/ml to 0.25μg/ml or 128μg/ml to 2μg/ml depending on how many serial dilutions we use. This method is a very basic way to check the antimicrobial ability of the extract or compounds.

 

When choosing the bacteria that we are going to test, the panel should at least consist of a Gram-positive and a Gram-negative bacterium. Plus, we need to experiment with antibiotics resistant bacteria, as we are doing this study to find a way to deal with them. It is important to kill them, however, these days, instead of killing them, lots of researchers try to inhibit their biofilm formation. As they form biofilms, they make thick layers and it prevents antibiotics from directly affecting bacteria right beneath them. So maybe we can turn our eyes to antibiofilm activity.

 

Before we move on to in vivo test, we have to check cytotoxicity. I guess most of us want to terminate only bacteria, not our cells. Therefore, we must clarify their cytotoxicity. If all the in vitro tests are finished, then we can conduct in vivo test. The goal of in vivo test is to check whether it is effective in animal models and not harming their cells. Plus, through this process, we need to obtain pharmacokinetics, pharmacodynamics, and toxicological information.

Until now, I summarized the paper I read. There is more information about how to test antifungal, antiviral, and antiparasites agents. However, if I explain all of them, it would be too long. So maybe next time, I’m going to write more information about them.