Thomson, Neal S.
Improved Culture Technique for Mycobacterium ulcerans
Faculty Mentor: Dr. Richard Robison, Department of Microbiology
Mycobacterium ulcerans is a pathogenic bacterium and is the causative agent of
Buruli Ulcer, a common mycobacterial disease similar to tuberculosis and leprosy. Buruli
Ulcer is endemic to Western Africa and produces a deforming and sometimes lethal skin
disease. One major obstacle for research is the slow growth of the bacterium in batch
culture. M. ulcerans currently grows in vitro with a doubling time of about 4.8 days,
resulting in an average culture time of 8-10 weeks before the appearance of colonies.
This evidence makes it difficult to study M. ulcerans in the laboratory. The goal of my
research was to improve the media and conditions used to culture M. ulcerans bacterium
to perhaps shorten the doubling times. My research partner and I believed that upon
identifying the proper conditions we could cut the average culture time to about 5 weeks,
which is similar to the growth rate of Mycobacterium tuberculosis (a close relative).
Obtaining a faster growth rate for this pathogen would double the research progress on
M. ulcerans in laboratories worldwide.
In a review of the current literature we found several research studies suggesting
that aquatic algae enhanced the growth rate of M. ulcerans in culture. The algae,
Hydrodictyon reticulatum, was said to be similar to an endemic species found in Western
Africa where M. ulcerans is present. Other research showed that lower oxygen
concentrations yielded faster growth rates. I decided to reference these studies and design
an experiment that would establish a procedure to utilize the algae in standard culture
techniques. I also planned to determine the optimal CO2 and O2 concentrations for the
growth of M. ulcerans. I hypothesized that with the ideal combinations of these two
variables (algae concentration/CO2 levels) that M. ulcerans generation time would be
greatly reduced during exponential growth.
The first step in my experimentation was to design an apparatus that would serve
as a growth environment for the algae. This task was accomplished successfully using our
laboratory supplies. The algae grew well in vitro, but it took almost two months to obtain
sufficient algae to begin the research study. Growing and maintaining the algae was a
challenging task. As a result I was unable to begin the experiment according to my initial
plans. Nevertheless, after several months of growth I was able to harvest and extract
enough algae to run two experimental trials of the study.
I used pure algal extract to supplement the bacterial media (Middlebrook 7H11)
and I plated two different strains of M. ulcerans on each plate, (ATCC 19423 and Africa
0551). The alga was added to the media at different concentrations to see if there was an
optimal range for growth. Concentrations ranged from 20% - 30% as I also had a 0%
plate for a negative control. The plated bacteria were incubated at 33C in the CO2
incubator. After four weeks of incubation I noticed that several of the plates had been
contaminated with a fungus. Unfortunately all of the plates were affected and were
discarded. We determined that the contamination was caused by an experimental error
during one of the preparatory steps of the experiment. Therefore we had to start the
experiment once again from the beginning.
During the second trial I was resolved to eliminate all of the previous human
errors that caused the contamination. I also realized that there were elements of the
experimental design that needed to be revised. Consequently, I designed the experiment
so that there was a more inclusive range of algal concentrations. Concentrations ranged
from 5%- 30% as the 0% negative control was again included. After only two weeks of
incubation at 33C in the CO2 incubator I observed very faint bacterial growth on each of
the plates. This was a surprising observation and I was very excited. Never before had we
observed such a rapid growth of M. ulcerans in the laboratory. The two different strains
had also displayed different morphological features. The strain that was obtained from
Africa was a yellowish color and the ATCC strain was a light cream color. I was very
careful not to disrupt the growth of the bacteria and focused on strict aseptic technique
when working with the plates. Careful examination of the plates consisted of a daily
analysis of growth to see if the bacterial colonies were consistent with those of M.
ulcerans. In approximately four weeks of culture time I observed that the bacterial
colonies began to raise and clump together, a characteristic of most mycobacteria. To
determine if the bacteria were indeed M. ulcerans I performed two acid-fast bacterial
stains on each of the plates. I concluded with my mentoring professor that the results
were positive for the presence of M. ulcerans.
This research study has been an important part of the research efforts in our
laboratory. It will hopefully improve other experiments that we have designed for M.
ulcerans. I have personally been able to understand better the aspects of experimental
design and have experienced that patience and persistence are valuable scientific
attributes. The experimental results have produced many questions regarding the
enhancement of bacterial growth rates using aquatic plants. How the algae specifically
interact with M. ulcerans on a metabolic level is yet to be determined. We are interested
also in the possibility of a synergistic influence of the algae with other known growth
factors. The ORCA Grant has provided me with an opportunity to dedicate more time to
science and has given me a clearer understanding of research. I am very grateful to have
received the award and expect that it will be a building block for other future projects.