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