The Efficiency of Tetracycline on Escherichia coli in Varying pH Environment
Caleb Wilson, Daniel Björkman
North Carolina State University, Department of Microbiology, 4510 Thomas Hall, Campus Box 7615 Raleigh, NC 27695
MB 495 Fall 2012, Instructor: James Brown
Overview
Results
Our experiment was designed to determine if the effectiveness of
tetracycline against Escherichia coli (E.coli) is dependent on the
pH environment. This knowledge would benefit clinical practices
in the context of endogenous infections, in particular when dealing
with the variable pH of the GI tract. The tetracyclines (TET) are a
group of antibiotics that binds to 30S ribosome and interferes with
protein synthesis by blocking the A-site [3]. TETs mechanism of
uptake within the cytoplasm is not fully understood but occur
through passive diffusion affected by transmembrane pH gradients
or active transport affected by phosphate bond energy [1]. Therefore
the ionization state of TET in its most protonated form results in
the highest concentration of intracellular TET.
The baseline of [0.32] TET inhibition that was defined as
normal for mathematical comparison in our experiment was
created with two biological replicates, each with 9 technical
replicates of E.coli growth at pH ≈ 6.5, both in the presence and
absence of antibiotic. The calculated average showed a standard
9.86% inhibition of E.coli growth in the presence of TET
antibiotic. This is shown in the REF row on table 1.
Figure 1: Fully protonated tetracycline ionization state [3].
Other experiment have made use of the susceptibility of E.coli
toward TET [2]. We determined that a concentration of [0.32] TET
gave the most conclusive results. This was based on the finding of
E.coli using the ethanol used in the TET mixture as a growth
source, and needing a [TET] that was measurable but not
detrimental to the culture. We used an optimized pH of 6.5 in our
culture of E.coli strain B when establishing a baseline of
effectiveness for TET for comparison. The method used is
described in the results. Cultures were grown in pH 6.5 1xLB
medium. We ran two series of E.coli at different pHs (4.0, 5.0, 7.0,
8.0. 9.0, 10.0); one with 0.32 [TET] and one without. Our
calculations are based off of three biological replicates in
triplicate, each conducted with this manner.
Figure 2 shows our final results from the biological replicates to
determine if the effectiveness of TET was dependent on or
correlated with pH. Table 1 shows the raw calculations. The
experimental series testing pH ≈ 4.0 was excluded from the final
results as no E.coli growth occurred, and the mathematical
percentages were meaningless. Figure 1 shows that the biological
replicates at different pHs resulted in a different rate of inhibition
of E.coli growth, confirming that the effectiveness of TET is
dependent on the pH environment [1]. With the ionization state of
TET in the lowest pH environment, we would expect that the
highest rate of microbial inhibition to be seen in the lowest pH
environment able to accommodate E.coli growth. However, our
data shows that the highest rate of inhibition occurs at a pH ≈ 6.5
instead of pH ≈ 5.0 . A loose trend can still be seen as the media
becomes more basic.
To create a more accurate depiction on tetracyclines dependency
on the pH environment to be effective, much more experiments
should be repeated. The presence of multiple variables in the
biological replicates allows from a lot of measurements that do not
bare meaning in a calculation attempting to isolate a percentage of
TET inhibition.
References
[1]
Figure 2: Tetracyclines percent of inhibition on E.coli in various pH
environments measured by optical density in comparison to the
percent of inhibition at a control of pH 6.5.
Yamaguchi, A., Ohmori, H., Kaneko-Ohdera, M., Nomura, T., &
Sawai, T. (1991). Δph-dependent accumulation of teracycline in
ischerichia coli. Antimicrobial Agents and Chemotherapy, 35(1),
53-56.
[2]
Sanders, C. R. (2011). Sanders lab protocol. Retrieved from
<http://structbio.vanderbilt.edu/sanders/ dgk_protocols.pdf>
[3]
Jin, L., Amaya-Mazo, X., Apel, M. E., Sandisa, S. S., Johnson,
E., Zbyszynska, M. A., & Han, A. (2007). Ca2 and mg2 bind
tetracycline with distinct stoichiometries and linked
deprotonation. Biochysical Chemisry, 128(2-3), 185-96. doi:
10.1016/j.bpc.2007.04.005
[4]
Delcour, A. H. (2009). Outer membrane permeability and
antibiotic resistance. BBA: Proteins and Proteomics, 1794(5),
808-16. doi: 10.1016/j.bbapap.2008.11.005