Figure 3: Illustration of the experimental model that shows the regions where MDR is expected to evolve under different antibiotic treatments. The region to the right of the dashed line is the region where MDR is expected to be hindered when a strong antibiotic is administered first . In contrast, the region on the left-side, below the solid line, is the region where MDR is expected to evolve when a weak antibiotic is administered first. The C x and C y referring to the axes in the planes relate to C Y ≤ Bf(C X) andC X ≤ Bf(C Y) and divide to plane to have separate regions. Regarding the two formulas, variables C x and C y refer to the fitness costs of the treated bacteria and the Bf variable refers to the strength of the antibiotic.
This aforementioned diagram illustrates the differences between the formulas C Y ≤ Bf(C X) and C X ≤ Bf(C Y) divides the grey plane into three separate regions. Note that only the area under the grey plane is relevant regarding the data whereC X> C Y. In region A, both equations are satisfied which implies that MDR is tends to evolve independent of the order in which antibiotic treatments are applied to AIEC. However, in region B, C Y ≤ Bf(C X) is satisfied but not C Y ≤ Bf(C X), which means that MDR tends to evolve when a weak antibiotic treatment is administered before a strong antibiotic. Lastly, region C shows that both equations are violated, meaning that MDR tends not to evolve under either treatment. In summation, the model shows that MDR is less likely to evolve under sequential therapy when a strong antibiotic is applied before a strong antibiotic, meaning that the cost of fitness resistance is ultimately represented by region B.
Discussion/Conclusion: In an attempt to determine whether or not antibiotics would be a useful method of therapy for disrupting the growth of AIEC-LF82 bacteria and treating Crohn’s disease and IBD, I observed that the lb agar plates, that had two combined types of antibiotics were able to completely stop the colonization and growth of AIEC bacteria. I found that each of the aforementioned antibiotics would had a much weaker negative effect on the growth and colonization of AIEC bacteria when used singularly rather than when used in conjunction. When used singularly, most of the antibiotics only hindered the growth of AIEC bacteria to a relatively small degree, with AMPs, seeming to be the most effective antibiotics. It also appeared that using two different antibiotics combined may be a viable future treatment for patients suffering from Crohn’s disease, since they appeared to completely prevent the growth of AIEC bacteria and destroy any plasmids that the bacteria may have obtained in the process. Thus, Penicillin-Streptomycin and Kanamycin-Chloramphenicol antibiotic solutions appear to be a substantially potent treatment for patients suffering from Crohn’s disease and IBD regarding combination therapy.
The results of the experiment also showed that strong-weak sequential therapy is more effective overall than weak-strong sequential therapy and that the former type of sequential therapy significantly hinders MDR evolution in AIEC-LF82 bacteria. The population dynamics model in the experiment showed that the order of when the antibiotic is given affects the fitness cost and that strong-weak sequential therapy is also more effective than combination therapy but not weak-strong sequential therapy (7). In addition, it was shown that MDR evolution was reduced by first using the antibiotic for which resistant mutation confers the highest fitness cost and vice versa. The results from the experiment show that MDR evolution overall tends not be that affected when antibiotics are given simultaneous as opposed to being given at different times as seen in the model. Given that results from the experiment, it also appears that specific antibiotic combinations tend to be more effective at lowering MDR evolution and affect the fitness costs of AIEC bacteria than other combinations.
The time scale between the use of the two antibiotics tends to determine the frequency of resistance mutations found at each phase in the experiment. In the pre-antibiotic phase the population size post-treatment must remain low relative to carrying capacity; and it must be long enough for the population to reach the mutation-selection balance regarding resistance in order for the assumptions in the model to be true, which was the case. The experiment was ultimately successful because of the ease and effectiveness of switching antibiotics in a short period of time, despite the potential difficulty of controlling the exact concentration of antibiotics that reach the site of infection (2). When the antibiotics are used in conjunction, I also made sure we that there was no cross-resistance, no recombination between the resistance mutations, no possibility of compensatory mutations, and no epistasis between resistance mutations, hence the antibiotic combinations were chosen carefully. If cross-resistance between the two antibiotics was present it the experiment then the order effect would be cancelled the since evolution of resistance to one antibiotic would ultimately lead to the resistance to the following antibiotic. Hence, for the future of combination therapy and especially sequential therapy, medical institutions should avoid the use of two drugs that are known to lead to cross-resistance. Horizontal gene transfer is also another factor that can lead to the assembly resistance genes in bacterial lineages and ultimately can lead to MDR evolution (8) (10).
In summation, the order in which antibiotics are given can affect bacterial populations is because there is a large amount of competition in AIEC-LF82 bacterial combinations and because of that the frequency of resistant mutants is limited and lowered by the rate at which they form. Ultimately, resistance mutations will arise at a low rate and resistance mutations that incur a large cost on fitness will be less frequent in a population that is not treated with antibiotics, which is a process known as mutation-selection balance (3). In strong-weak sequential therapy the first antibiotic greatly reduces population density and the competition between bacterial cells is lowered as a result. With extremely small competition, the rate of resistance evolution against the second antibiotic is limited primarily by the rate at which resistance mutations arise. Resistance mutations against the two antibiotics incur different costs, therefore the order of antibiotics treatment will greatly determine and effect MDR evolution.
Since multidrug therapy is a clinical practice that is growing in popularity to treat bacterial infections (6), it is crucial to understand the full evolutionary consequences associated with drug deployments. The results from my experiment not only demonstrate the influence cost of resistance has on the evolution of MDR but also mentions approaches that will ultimately improve multidrug therapy, particularly regarding AIEC bacteria. For example, even though combination therapy is overall less effective than sequential therapy due to the fact that combination therapy tends to be more timely and can cause adverse and/or effects in the human body depending on the combination of antibiotics used, it is actually more effective at eliminating AIEC bacterial colonies in a short period of time if antibiotic solutions composed of two antibiotics such as Penicillin-Streptomycin and Kanamycin-Chloramphenicol solutions are used simultaneously. Regarding sequential therapy, even though strong-weak sequential therapy was shown by the results to be the most successful therapy and weak-strong sequential therapy was shown by the results to be the least effective, the most effective combinations in both type of sequential therapy types were LB + P/S+ AMP and the least effective combinations were LB + Kan + Spe. Since combination therapy was also shown to be the most effective only when Penicillin-Streptomycin and Kanamycin-Chloramphenicol antibiotics solutions are used and these specific combinations are not known to cause uncomfortable nor adverse effects in the human body, using these antibiotic solutions via combination therapy for the future treatment of AIEC holds promising potential. Even though weak-strong antibiotic sequential therapy was demonstrated to be much more effective than initially hypothesized, strong-weak sequential antibiotic therapy particularly treated has significant potential for treating Crohn’s Disease and IBD given the fact that it has the potential to severely hinder and/or completely halt the colonization of AIEC-LF82 bacterial colonies.
Keywords: AIEC; E. Coli; Crohn’s disease; IBD; antibiotics; Penicillin; Streptomycin; Kanamycin; Chloramphenicol; resistance