Swenson, David W by wfq74180


									                            Swenson, David W.
        Evaluation of The Activity of Cationic Steroid Antibiotics
                           Paul B. Savage, Chemistry and Biochemistry

Relevance and Introduction
Food-borne illnesses are a continuing problem in the US [1]. Gram-positive and Gram-negative
bacteria pose significant threats to the integrity of the food supply and to the health of the public.
For example, according to statistics from the Centers for Disease Control, an estimated 1 million
infections and 500 deaths are associated with food-borne Salmonella infections each year [2].
Additional concern is caused by the fact that many patients who die from food-borne illnesses
are infected with drug-resistant bacteria, and resistant organisms are increasingly found in the
food supply [3].

Cationic steroid antibiotics (CSAs), developed in Paul B. Savage’s laboratory, have proven to be
very active, broad-spectrum antibacterial agents [4]. They have been shown effective against
some drug-resistant bacteria [4]. For this reason CSAs are a promising new form of antibiotic
for protecting food (and thus the food-consuming public) from bacteria. My research was
focused specifically on evaluating the activity of CSAs against Salmonella. The reasons for my
focus on Salmonella were its prevalence in meats and its serious disease-causing potential. I
expected that conclusions gathered from research on this Gram-negative bacterium could be
generalized (at least to some degree) to other common disease-causing Gram-negative bacteria.

Most of my time was spent determining the optimal conditions for reducing Salmonella
populations on the surface of chicken; the purpose of this study was to provide sufficient data to
suggest that CSAs would be useful antibacterial agent for use in the food industry. A side
project (which took much more time than I originally expected) was to determine the site of
activity of the CSAs in/on bacteria. This project required use of a thiol-tagged CSA, gold
nanoparticles, and electron microscopy. The bacteria of choice were E.Coli. and Staph. Aureus
since they had been used in previous microscopy work in the laboratory.

For the studies of CSA activity against Salmonella on chicken breast, I used methods that I had
previously determined (by referencing relevant literature and through trial and error) with
another student in the lab. The specific trial conditions of temperature, time, and CSA
concentration were noted and manipulated to determine what conditions are optimal for CSA
activity against Salmonella.

For each trial I prepared circular discs of chicken breast of 4 cm diameter and 0.5 cm thickness.
A double control was needed to make the data useful. One control was neither inoculated with
Salmonella, nor treated with any compound. The second set of controls was a group of chicken
discs that was inoculated and then treated only with distilled water, instead of being treated with
CSAs in water.
The trial conditions that were intentionally altered include temperature, time of treatment with
CSAs, and concentration of CSAs in water. In addition, another variable was introduced
unintentionally: the concentration of the Salmonella in the inoculum. Although I tried to prepare
the inoculum the same each time, the concentration varied significantly during the various trials.
Therefore, the collective data must be analyzed with this in mind. In order to have publishable
data (as in publishable in an academic journal) the work I performed would need to be replicated
on a much larger scale. Unfortunately my man power was limited.

For the studies on the CSAs’ site of activity, I prepared the samples for electron micrsoscopy and
participated in, but did not perform, the microscopy work (the lab technician performed the
essential microscope work and developed the images). The method for preparing microscopy
samples of Salmonella-treated-with-CSAs was relatively easy, but took some trial and error to
get it right. I won’t go into detail about it, however, because the results only proved that the data
we hoped to gather was not obtainable with the technique we were using.


For the chicken breast studies, in addition to various botched trials, I had two large trials that
were very consistent with one another. The most effective conditions I found were a temperature
of 55°C and a CSA concentration in water of 200ug/mL. Due to time considerations (and after
considering realities of industrial food preparation) I decided to limit the CSA treatment time to
60 seconds and tried to maintain a bacterial inoculum concentration of no higher than 10^6/mL.
Under these conditions I observed approximately a two log reduction in bacterial counts from the
chicken discs that were only inoculated (but not treated with CSAs) and those that were treated
with the CSAs at 200ug/mL. Less impressive was the difference between treatment with
distilled water and treatment with CSAs: about a one log reduction in bacterial counts. Still, in
such a complex system, with so many variables, even a consistent one log reduction in bacteria is
encouraging. As I mentioned previously, I think this study would need to be standardized and
run with more replication in order to determine optimal conditions for the efficacy of these
CSAs, however the initial results seem consistent and promising if not astounding.

The results from the microscopy study initially appeared very exciting. I obtained photographs
of E. Coli. that clearly had gold-nanoparticles localized within the bacteria. However after
difficuly replicating the results, and eventual success in doing so, I conducted a control run that
proved that the gold-nanoparticles were not specifically binding our CSA compound, but rather
were binding some unidentified component of the E.Coli. itself. This essential control showed
that a different method would need to be developed to visually identify the site of activity of
these CSAs.

[1]    Mead, P.S. Emerg. Infect. Dis. 1999, 5, 607-625. Food related illness and death in US.
[2]    Hileman, B. C & E. News 2001, 47-48.
[3]    Sidhu, M.S. Microb. Drug Resist. 2001, 7, 73-84. Disinfectant and Antibiotic Resistance
       of lactic Acid Bacteria Isolated from the Food Industry.
[4]    Savage, P.B. J. Antimicrobial. Chemotherapy. 2001, 47, 671-674. Activities of cholic
       acid-derived antimicrobial agents against multidrug-resistent bacteria.

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