Thanks for the question, we believe the high toxicity levels of piperidine may have played a role in the high variation of our data. In some instances even a small change in the concentration of piperidine would cause a drastic change in absorbance results, so perhaps something like an imperfect pipette measurement could cause this. The significant difference between the dose response data and bactericidal/bacteriostatic data is due to different starting concentrations of piperidine. The dose response test started with a diluted sample of piperidine while the bactericidal/bacteriostatic started with a sample of straight 100% piperidine, so after accounting for these differences in concentrations the results of these tests do support each other. As far as the variation in negative control data seen in the first column of the bactericidal/bacteriostatic data, we know our negative control DMSO was correctly placed in the well after having a TA observe our process. With this in mind, our current theory is that the high concentration of piperidine present in column 1 somehow contaminated or ” jumped” to the nearby negative control well.
Based on our bactericidal/bacteriostatic data, concentrations between 10% and 0.078% show inhibition of salmonella typhimurium growth, so to isolate an ideal concentration we would perform another, more extensive dose response test with more intervals of concentrations between the two concentration values listed above.
How do you think you could have utilized mutagenesis towards antibiotic resistant bacteria to cause the bacterial cell to lose the markers that allowed for it to evade antibiotics?
This is an interesting question, our research didn’t consider the use of mutagenesis but I think you’re right that it could be used to prevent bacterial cells from evading antibiotics. For example Salmonella Typhimurium possesses a type III secretion system which aids it in infection of host cells and evasion of antibiotics, so perhaps mutagenesis could be used to inhibit that function. However, I’m not familiar enough with the workings of mutagenesis to know how exactly that inhibition might be enacted on a large scale outside of a laboratory setting. In terms of our compound piperidine, maybe mutagenesis could be used to counteract the toxic effects associated with the compound, this could prove useful for future tests on macrophage cells and determination of a therapeutic index.
How did you get the idea to use fire ant venom? I know you mentioned why you used it, but I am wondering how the idea came about. Do you think the fire ant venom might work even better for a different kind of bacterial infection?
Thanks for the question! When searching for potential sources of antibiotics we came across a study on the antimicrobial effects of certain alkaloids secreted from the Crematogaster ant, which is most abundant in tropical and subtropical climates. Unfortunately we weren’t able to obtain the secretions of these ants or even the ants themselves, however fire ants are closely related to the Crematogaster ant so that was the next best option for testing. Concerning its effects on different bacterial infections, we encountered a few studies which found fire ant venom was effective at inhibiting a number of gram-negative and gram-positive bacteria like E coli, S. pneumoniae and S. pyogenes. Interestingly, the ant venom was more effective at inhibiting gram-positive bacteria. Salmonella Typhimurium is gram negative so further testing against gram-positive bacteria would likely be more effective.
Finding this range would mean finding the therapeutic index, to do that, we would first need to test piperidine against human macrophage and/or liver cells in vitro. If those tests proved successful, we could then move on to testing against live organisms.
Thank you for your question and yes there are! One that we used was the Mueller-Hinton agar well diffusion test. This test uses an agar plate and a bacterial spread to observe potential inhibition of the bacteria. Instead of measuring absorbance, distance of inhibition is measured by use of 3 holes filled with either the compound tested, negative control, or positive control. You can see an example of this in the bottom left of the results section on our poster. Unfortunately due to time constraints we were only able to test a low concentration of our compound. Running more of these tests at higher concentrations would have allowed us to more easily confirm the results of our experiments.
why do you think there was so much variation in your data? do you think it could have something to do with the drug, or environment, or anything else?
Thanks for the question, we believe the high toxicity levels of piperidine may have played a role in the high variation of our data. In some instances even a small change in the concentration of piperidine would cause a drastic change in absorbance results, so perhaps something like an imperfect pipette measurement could cause this. The significant difference between the dose response data and bactericidal/bacteriostatic data is due to different starting concentrations of piperidine. The dose response test started with a diluted sample of piperidine while the bactericidal/bacteriostatic started with a sample of straight 100% piperidine, so after accounting for these differences in concentrations the results of these tests do support each other. As far as the variation in negative control data seen in the first column of the bactericidal/bacteriostatic data, we know our negative control DMSO was correctly placed in the well after having a TA observe our process. With this in mind, our current theory is that the high concentration of piperidine present in column 1 somehow contaminated or ” jumped” to the nearby negative control well.
At what concentration do you believe Piperidine would inhibit the growth of Salmonella Typhimurium? How would you go about testing this?
Based on our bactericidal/bacteriostatic data, concentrations between 10% and 0.078% show inhibition of salmonella typhimurium growth, so to isolate an ideal concentration we would perform another, more extensive dose response test with more intervals of concentrations between the two concentration values listed above.
How do you think you could have utilized mutagenesis towards antibiotic resistant bacteria to cause the bacterial cell to lose the markers that allowed for it to evade antibiotics?
This is an interesting question, our research didn’t consider the use of mutagenesis but I think you’re right that it could be used to prevent bacterial cells from evading antibiotics. For example Salmonella Typhimurium possesses a type III secretion system which aids it in infection of host cells and evasion of antibiotics, so perhaps mutagenesis could be used to inhibit that function. However, I’m not familiar enough with the workings of mutagenesis to know how exactly that inhibition might be enacted on a large scale outside of a laboratory setting. In terms of our compound piperidine, maybe mutagenesis could be used to counteract the toxic effects associated with the compound, this could prove useful for future tests on macrophage cells and determination of a therapeutic index.
How did you get the idea to use fire ant venom? I know you mentioned why you used it, but I am wondering how the idea came about. Do you think the fire ant venom might work even better for a different kind of bacterial infection?
Thanks for the question! When searching for potential sources of antibiotics we came across a study on the antimicrobial effects of certain alkaloids secreted from the Crematogaster ant, which is most abundant in tropical and subtropical climates. Unfortunately we weren’t able to obtain the secretions of these ants or even the ants themselves, however fire ants are closely related to the Crematogaster ant so that was the next best option for testing. Concerning its effects on different bacterial infections, we encountered a few studies which found fire ant venom was effective at inhibiting a number of gram-negative and gram-positive bacteria like E coli, S. pneumoniae and S. pyogenes. Interestingly, the ant venom was more effective at inhibiting gram-positive bacteria. Salmonella Typhimurium is gram negative so further testing against gram-positive bacteria would likely be more effective.
How can research be furthered to find a range in which ant venom dose can be effective as an antibiotic but not toxic to other cells?
Finding this range would mean finding the therapeutic index, to do that, we would first need to test piperidine against human macrophage and/or liver cells in vitro. If those tests proved successful, we could then move on to testing against live organisms.
Are there any other forms of observation other than the absorbance data you could use to confirm the results of that experiment?
Thank you for your question and yes there are! One that we used was the Mueller-Hinton agar well diffusion test. This test uses an agar plate and a bacterial spread to observe potential inhibition of the bacteria. Instead of measuring absorbance, distance of inhibition is measured by use of 3 holes filled with either the compound tested, negative control, or positive control. You can see an example of this in the bottom left of the results section on our poster. Unfortunately due to time constraints we were only able to test a low concentration of our compound. Running more of these tests at higher concentrations would have allowed us to more easily confirm the results of our experiments.