1. The Affects of Urban Environments on Mycobacteriophage Activity
Katherine Milliken, Madeline Gerwig, Jeffrey Migliozzi and Hanna Cioffi
SEA-PHAGES Program at the University of Pittsburgh
Abstract
The goal of our research was to investigate the effects of urban environmental
influences on Mycobacteriophage propogation. We hypothesized that the prescence of urban
enivronmental substances would decrease the propogation of phages in soil. We plated and
harvested lysate from Mycobacteriophage Cambiare. Then group members analyzed and
established the titer for plaque forming units of Mycobacteriophage Cambiare under
controlled laboratory setting. We then compared the titer of the phage when exposed to
varying concentrations of common urban environmental influences found in soil, such as road
salt, fertilizer and pesticide. Using our results, we were able to determine that the presence of
these substanceslowered the titer of Mycobacteriophage Cambiare.
Introduction
Bacteriophages are specific viruses that infect bacteria. The
phages then use the host they infected to reproduce. Phages are the
most diverse population of life on this Earth. There are more
bacteriophages than all other living organisms combined. There are 1031
bacteriophage particles in the biosphere
The purpose of our research is to investigate the influences of
urban environments on phage propagation through:
• Identification of uninfluenced phage qualities
• Incubation with urban environmental solution
• Comparison of phage propagation via titer with and without urban
influences
Methods
Conclusions
Future Direction
Acknowledgements and References
In future experiments, we would like to analyze the effects of urban
environmental influences further. We would like to analyze and compare plaque
morphologies as well as phage morphologies through the use of an
electromagnetic microscope. Additionally, we would like to analyze and compare
the phage genome to investigate influence on a genetic level.
Figure 1: Basic structure
of a mycobacteriophage
On a much broader scale, bacteriophage research is
important for the medical community. New research suggests
that using bacteriophages for treatment of bacterial infections, or
phage therapy, has the potential to be effective for patients.
Because many bacteria are becoming resistant to current
antibiotics, the need for effective phage therapy is more
important than ever. Examples of infections that could potentially
be treated by bacteriophages include Mycobacterium
tuberculosis and Mycobacterium avium.
Fig. 2: Bacteriophages infecting their host bacterium
Webbed
Plate
Serial Dilution Quick and
Dirty Titer
Repeat Dilutions/
Quick and Dirty
Titers with Urban
Solutions
Observations from
Plates with Urban
Solutions
Concentration in
Plaque forming
units/ml (PFU/ml)
Concentration in
Plaque forming
units/ml (PFU/ml)
% mass sample in
solution
Trial 1 Trial 2
Control 2.0E8 2.4E8
pesticide
1% 1.6E8 4.8E7
5% 8.0E6 1.2E7
25% 1.4E7 2.0E7
Fertilizer
1% N/A N/A
5% 1.4E7 1.2E7
25% 1.0E7 8.0E6
Salt
1% 2.2E7 2.0E7
5% 1.2E7 1.4E7
25% 1.8E7 1.6E7
Figure 6: This table illustrates the results
from the trials in quantitative values. All titer
quantities were evaluated from a 10^-4
dilution of Cambiare lysate.
1) "Group # 1 Mycobacteriophages Presentation." Group # 1 Mycobacteriophages
Presentation. N.p., n.d. Web. 23 Nov. 2014.
2) "UW Biology ENews - Fall 2007." UW Biology ENews - Fall 2007. N.p., n.d.
Web. 23 Nov. 2014.
3) Hatfull, Graham. BioSci 0058/0068 Lab Manual 2014-2015. Science Education
Alliance’s Phage Hunters Advancing Genomics and Evolutionary Science., n.d.
9) Electron micrograph of Cambiare unaltered from phagesdb.org
Figure 7: This photograph
represents the quick and dirty
titer plate for Cambiare without
substances applied. The titer
from this sample served as the
control for our results.
Figure 8: This table
represents titer
plates from phage
Cambiare
incubated in
different
concentrations of
the three urban
samples, and then
plated in different
dilutions up
through 10-4.
Road Salt Pesticide Fertilizer
1% urban
solution by
mass
5% urban
solution by
mass
25% urban
solution by
mass
Special acknowledgements to Dr. Graham Hatfull, Dr. Welkin Pope, Dr. Sarah
Grubb, Dr. Marcie Warner and the rest of the University of Pittsburgh SEA-
PHAGES team for the support and instruction over the course of this experiment.
Due to M. smegmatis
contamination, results
were not conclusive.
It appears that each urban solution has the same effect on
Cambiare’s titer. Statistical significance was not found between
the urban solutions at any concentration. The results hint at a
concentration threshold. It appears that any concentration of
urban solution between 5% and 25% will equally inhibit phage titer;
however once the concentration is lowered to 1%, the titer
significantly increases. Significant difference was found between
the 1% and 5% salt solution. This correlation does not hold true to
the pesticide solutions, but is perhaps due to the large variation of
the 1% titer reading. To further demonstrate the threshold, no
statistical significance was found between the control titer and that
of the 1% urban solutions. Due to contamination of the 1% f
fertilizer solution, it could not be
included in the analysis
Figure 9: Electron micrograph of phage Cambiare
in an unaltered state.
Results