Reactive oxygen species or ROS are reactive chemicals that are said to be able to contain the ability to able to speed up the process of bacterial death of different types of antimicrobials. This experiment sought to find out if resveratrol, a type of antioxidant dietary supplement, actually has the ability to suppress the lethality of antimicrobials and decrease the activity of ROS on bacteria. Various antimicrobials such as: Kanamycin, Moxifloxacin, Oxolinic acid, and Daptomycin were used to study the bactericidal activities on bacteria like E. coli and S. aureus. Testing resveratrol as an inhibitory factor for cell death had been tested with several tests with different conditions.
Table 1 shows the effects of resveratrol on minimal bactericidal concentrations with E. coli and S. aureus. The table shows a list of antimicrobials with or without the addition of resveratrol. The minimal concentration to inhibit (MIC) and minimal bacterial concentration (MBC) were taken into consideration for cultures of E. coli and S. aureus. Only three antimicrobials, Moxifloxacin, Kanamycin, and Oxolinic acid, show significant number. These MIC numbers represent the concentration of antimicrobial that is needed in order to work in the presence of resveratrol so that the bacteria is not able to survive or grow. The MBC numbers refer to the concentration of the bacteria that would have the ability to be killed, essentially, by these antimicrobials. The purpose of this table is to show the measurements of MIC in both lone antimicrobial and combined microbial/resveratrol treatments in plates of E. coli and S. aureus and to see what concentration work successfully against the resveratrol and its inhibitory activity.
Figure 1 contains two graphs showing the effect of resveratrol on bacterial growth rate. Graph A shows the results of E. coli cultures while graph B shows S. aureus cultures. The x-axis refers to the time of incubation for growth, while the y-axis measures the optical density or cell growth for the two cultures. The filled dots represent the absence of resveratrol and the empty dots signify the presence of resveratrol. The blue arrow indicates when resveratrol was added to the plate and the red arrow signifies at what absorbance the antibiotic was added. Figure 1a shows that resveratrol is added to the E. coli culture at 100 minutes. The cultures continue to grow with resveratrol, but the addition of the antimicrobial to the culture at about 0.5 OD slowed down the growth rate. The rate is much lower in comparison to the culture without the treatment of antibiotics, but there is still growth, which shows that resveratrol can help the growth rate of bacteria even when antibiotics are present. Figure 1b shows data for growing culture of S. aureus. An antimicrobial was added at less than 0.5 OD to help slow down the growth rate again. The initial addition of resveratrol, again, helped the cultures grow. In comparison to figure 1a, figure 1b has a lower bacterial growth rate. The purpose of these two figures is to show that resveratrol has the ability to help the growth rate of bacteria even in the presence of antibiotics since resveratrol serves to protect bacteria.
Figure 2 shows four different graphs that represent the effect of resveratrol on ciprofloxacin and kanamycin lethality with E. coli. Filled circles indicate the absence of resveratrol and only the presence of antibiotics, while empty circles show the presence of resveratrol. Figure 2a represents the data for E. coli cultures incubated at a set time, which is 45 minutes. Treatment with only Ciprofloxacin shows that the number of cells recovered drops when higher concentration amounts of Ciprofloxacin are added to the culture. The cultures with resveratrol treatment continue to grow, but the rate does decrease towards the end of the 45-minute mark, showing that some cells may or may not be alive. Figure 2b shows cell recovery with set concentration of Ciprofloxacin. The longer the incubation period, the less cell recovery there is when a set concentration of the antimicrobial is added to the culture. Resveratrol treatment by itself kept a steady number of cells recovered then decreased a bit towards the 120-minute incubation mark. Figure 2c shows treatment with the antimicrobial, kanamycin. The antimicrobial, alone, decreases the number of cells recovered as the concentration increases. The cultures with only resveratrol have a slight increase at about 3 ug/mL, but then start to decrease as the 45-minute incubation period goes by. This shows that there is a much higher number of cells recovered in comparison to the culture treated with only Kanamycin. Figure 2d shows the results of the number of cells recovered when time varies and the amount of concentration of Kanamycin is set. The number of cells recovered decreases drastically over the 120-minute incubation period with only Kanamycin. Over time, the number of cells recovered increases for the treatment with resveratrol. The purpose of these figures is to show that antibiotics, alone, can kill off bacterial cells while resveratrol is a factor for bacterial survival.
Figure 3 contains four graphs showing the resveratrol-mediated inhibition of rapid killing of E. coli by Oxolinic acid and moxifloxacin. Figure 3a, b, c, and d are like figure 2 where varying and set concentrations and incubation periods are used, but with different antibiotics. Just like figure2, the filled circles signify the absence of resveratrol, while the empty circles signify the presence of resveratrol. Figure 3a shows cultures mixed with Oxolinic acid. The number of cells recovered drops as the concentration of Oxolinic acid increases, while resveratrol helps maintain the availability of some cells. Figure 3b shows the treatment with Moxifloxacin at varying concentrations. With a very low concentration, the number of cells are very similar. Increasing concentrations drastically decrease the number of cells that can be recovered. The cultures treated with resveratrol decrease and fluctuate, but there are still cells more cells that are present in comparison to the culture treated with only Moxifloxacin. Figures 3c shows the treatment with Oxolinic acid at a fixed concentration but at varying incubation times. The longer the incubation period, the more the number of cells drop when treated with only the antimicrobial. The cultures with resveratrol have a slight increase, then decrease over time going back to almost the same amount of cultures that were present at the beginning. Figure 3d, like figure 3c, uses varying times, but a set concentration of Moxifloxacin. Just like figure 3b, the number of cells recovered stays the same for a bit then decreases. Over time, the culture treated with only Moxifloxacin decreases. The resveratrol treated culture also decreases over time, but there is still a good number of cells present. The purpose of these figures is to show that antibiotics have a drastic effect on the killing of cells. Resveratrol does its best to protect the cell growth, but longer incubation times also has a negative factor with decreasing the amount of cells present.
Figure 6 has two graphs that shows the effect of resveratrol concentration on lethality of moxifloxacin and kanamycin with E. coli. The x-axis goes in accordance to the concentration of resveratrol, while the y-axis refers to the percent of cells recovered. Figure 6a shows the effect of resveratrol with cultures mixed with moxifloxacin. Higher concentrations of resveratrol increase the number of cell recovery. Figure 6b shows cultures mixed with Kanamycin and the treatment of increasing concentrations of resveratrol. Just like figure 6a, higher concentrations of resveratrol increase the cell recovery. The purpose of these figures is to show that cell recovery will increase and be protected if higher concentrations of resveratrol are added into the cultures even in the presence of antibiotics.
Figure 7 shows four bar graphs comparing the effect of resveratrol on the recovery of antimicrobial-induced rifampicin-resistant mutants. Each graph shows three different treatments. Striped bars refer to cultures being treated with only resveratrol, solid black bars only have cultures induced with an antimicrobial, and white bars show treatment with both resveratrol and the antimicrobial. The x-axis indicates the time of the extended incubation for a course of three days. The y-axis refers to the relative mutation frequency, or how often mutation occurs during the three-day incubation period. Figure 7a shows that the presence of resveratrol alone provides very little mutation recovery over the course of three days. Ciprofloxacin, alone, show little mutation recovery, while both treatments combined show an increase in mutation recovery. The treatment for figure 7b is similar to figure 7a, except the amount of concentration for MIC used had been 3x instead of 5x. For the first two days, the resveratrol only treatment shows some mutation recovery, but slightly decreases by the third day. The number of mutations that occur during ciprofloxacin treatment is a slow increasing process for the first two days, but a rapid increase by the third day. Figure 7c shows the results of mutation recovery using kanamycin as the choice of antimicrobial. Resveratrol alone shows mutation recovery at around 2 for the first day, with a slight increase by the second but no significant change in comparison between the first and third days. Kanamycin treatment, alone, had no changes at all in the amount of mutation recovery. The combined treatment, however, shows an increase over the three-day incubation period. Figure 7d shows mutation recovery using cultures of S. aureus instead of E. coli. The antimicrobial chosen for this graph is Daptomycin. Resveratrol treatment, alone, does not show any significant changes, the mutation recovery remains at about 1.5 for the span of the three-day incubation. Daptomycin treatment shows little change in mutation recovery while the combined treatment shows more mutation recovery during the first and second days, but no significant change for the third day. The purpose of these graphs is to show that resveratrol combined with an antimicrobial has the ability to increase the viability of bacterial growth.
In conclusion, resveratrol can inhibit the bactericidal activity of antimicrobials and help maintain the viability. Figures 1, 2, 3, and 6 show these conclusions by positive results. Cultures that had resveratrol added had success with cell growth and recovery. Cultures that did not receive any treatment, in these figures, either did not have any cell survival or growth, which shows that antimicrobials are bactericidal. If there is high concentration of resveratrol present in a culture combined with antibiotic, resveratrol should be able to decrease the likelihood of cell death and a decrease in cell growth. Figure 7 showed how likely mutation would occur in different cultures with either resveratrol, antimicrobials, or a combination. These results show that combined treatment is more likely to form mutations. Also, resveratrol represented slight growth of mutations. Overall, resveratrol, in this experiment, lowers the activity of ROS on bacteria.