1. Isolating Bacteriophages against Renibacterium
salmoninarum, Aeromonas salmonicida, and
Arthrobacter sp. KY 3901 (ATCC 21022)
Valeria Chacon & Jediael Desir. Sponsor: Dr. James Daly
Methods
• Preparation of Bacteria
• Inoculate fresh agar plates with bacterial samples
• Add colony with sterile loop to appropriate growth medium
• Shake and incubate at room temperature for 24 hours
• Need to achieve the late exponential/early stationary stage bacteria
• Collection of Samples
• Prepare phage buffer (PB) in separate flasks
• Sterilize by autoclaving
• Collect different soil, sewage, and water samples from around campus and
from fish hatcheries
• Add PB to sample, shake and let settle
• Centrifuge at 6,000 rpm at 4°C for 30 mins
• Pour off supernatant into tissue culture flasks
• Add LB to each tissue culture flask
• Pass through micro-filter using disposable filter and add culture
• Pour 50 mL into separate culture flask
• Add varying concentrations of CaCl2
• Phage Isolation
• Add culture and phage
• Set up 2 control tubes and add appropriate growth medium
• Pour contents of all tubes into designated agar plates, gently swirl to
distribute evenly and incubate at necessary temperature for allotted time
• Check for plaque formations
• Phage Purification
• Dilute each culture tube with a 1:10 ratio (1mL enrichment to 9ml Phage
Buffer)
• Conduct a serial dilution and inoculate a gridded plate. Check for plaque
formations
• Perform plaque streaking procedure. Pour solution of top agar,
corresponding bacteria and CaCl2 on top
• Let incubate at room temperature overnight
Results
Conclusion
We were able to isolate bacteriophages that corresponded to two of the bacteria that we worked with:
Arthrobacter sp. KY 3901 & Aeromonas salmonicida. These findings corroborated both our general and
specific hypothesis. Due to time constraints, we were unable to test if the bacteriophages we found for
Arthrobacter sp. KY 3901 would bind to R. salmoninarum. We were then able to conclude that the
Arthrophage for Arthrobacter sp. KY 3901 were specific to that particular bacterial strain & most likely
would not bind to R. salmoninarum. We are unsure if we have isolated Reniphages from the bio-film &
water samples we have acquired from the BKD water; we need more time to see if plaques will appear.
Even though phage therapies have been used for fish infected with the bacterial pathogen A. salmonicida,
finding new bacteriophages that bind more effectively to this bacteria can eventually lead to more
effective treatment of such pathogenic diseases. Even though we were unable to find Arthrophages that
where genus specific, that doesn't mean that such phages do not exist. There might actually be phage out
there with such characteristics and that could possibly bind to and infect R. salmoninarum. All we have
to do is look.
References
Cross, T., Schoff, C., Chudoff, D., Graves, L., Broomell, H., Terry, K., et al. An Optimized Enrichment
Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates. J.
Vis. Exp. (98), e52781, doi:10.3791/52781 (2015).
Acknowledgements
• We would like to thank:
• David Dunbar from Cabrini College for providing us with samples of Arthrobacter sp. KY 3901
• SUNY RF for supporting this research
• SUNY at Purchase College for allowing us to use their laboratory and resources
• Monica Furman and Talibah Stephenson for being our lab partners
• Dr. James Daly for overseeing this research
• This research was supported by NIH Bridges to the Baccalaureate grant #R25GM062012-14
Abstract
The goal of this research was to isolate bacteriophage that correspond to the
bacteria we are looking to infect. We have worked with primarily three types
of bacteria: Arthrobacter sp. KY 3901 (ATCC 21022), Aeromonas
salmonicida, and Renibacterium salmoninarum. The bacteriophage isolates
that infect Arthrobacter have been extracted from soil in many disparate
environments. Bacteriophage that infect A. salmonicida have been isolated in
areas where recent outbreaks of furunculosis have occurred. The samples we
have acquired were taken from both biofilm and water from a fish hatchery
that had experienced a recent outbreak of furunculosis. R. salmonicida is the
only bacteria that we have worked with that lacks a corresponding
bacteriophage. We also have acquired sewage, water, and bio-film samples
from a hatchery where an outbreak of bacterial kidney disease occurred. This
bacterial disease is lethal to salmonid fish. Because R. salmoninarum is most
closely related to Arthrobacter, theoretically, bacteriophage that bind and
infect Arthrobacter should do the same to R. salmoninarum. We have isolated
phages for Arthrobacter taken from 12 different samples of soil on campus.
After isolating the phage from the soil, we added different concentrations of
phage enrichment, as well as either 25 mM CaCl2 to one set of samples and 50
mM CaCl2 to another set of samples. We compared the difference in efficacy
of phage binding between the samples containing different concentrations of
CaCl2. By looking at the plaques on the agar plates, we were able to determine
the CaCl2 concentrations that helped the bacteriophage bind better to the
bacteria. The same procedure was carried out for Aeromonas phages taken
from water and biofilm samples. The procedure in which sewage, water, and
bio-film samples were taken for R. salmoninarum yielded no corresponding
phage.
Introduction
Our research group is looking at pathogenic bacteria that affect fish. The
pathogenic bacteria we are primary focused on are Renibacterium
salmoninarum and Aeromonas salmonicida; these bacterial pathogens are
responsible for Bacterial Kidney Disease (BKD) and Furunculosis,
respectively, in many species of fish. Because of increased bacterial resistance
to traditional antibiotics, there has been a resurgence in alternative treatment
methods such as phage therapy. Phage therapy is the use of bacteriophages
administered either orally or by injection to quell the proliferation of the
virulent bacteria within an infected organism. We are also looking at phage
that infect Arthrobacter sp. KY 3901, which is a type of bacteria found in
soil. Arthrobacter is thought to be closely related to R. salmoninarum. If we
can find phages that infect Arthrobacter, theoretically, those same phages
should be able to infect R. salmoninarum which would provide us with the
basis for an alternative treatment method for BKD in fish.
Hypotheses
Where a particular bacterium is found there will also be corresponding
bacteriophage in the same general proximity.
Since bacteriophage are found in close proximity to their corresponding
bacteria, then A. salmonicida phages, Arthrobacter sp. KY 3901 phages, and
R. salmoninarum phages should be able to be found within the samples
Figure 2: Results of phage isolation from
collection of A. salmonicida biofilm samples
Figure 1: Results of phage isolation from third
collection of Arthrobacter sp. KY 3901 soil samples
Figure 4: Charted results of phage isolation of A. salmonicida
water samples
SAMPLE CALCIUM (mM) ENRICHMENT (µL) PLAQUE SIZE COLONY DENSITY
1 0 100 none mostly clear
1 0 500 none mostly clear
1 25 100 none cloudy lawn
1 25 500 none cloudy lawn
1 50 100 poss. air bubble cloudy lawn
1 50 500 none cloudy lawn
2 0 100 poss. contaminant spotty
2 0 500 poss. contaminant spotty
2 25 100 none cloudy lawn
2 25 500 none cloudy lawn
2 50 100 poss. air bubble cloudy lawn
2 50 500 none cloudy lawn
3 0 100 entire plate clear
3 0 500 entire plate clear
3 25 100 none slightly cloudy
3 25 500 none slightly cloudy
3 50 100 minimal cloudy lawn
3 50 500 none cloudy lawn
SAMPLE CALCIUM (mM) ENRICHMENT (µL) PLAQUE SIZE COLONY DENSITY
1 25 100 small thick lawn
1 25 500 small thick lawn
1 50 100 none clear
1 50 500 none clear
2 25 100 poss. air bubble thick lawn
2 25 500 none thick lawn
2 50 100 poss. air bubble thick lawn
2 50 500 none thick lawn
3 25 100 poss. 1 plaque thick lawn
3 25 500 one thick lawn
3 50 100 >6 sm. plaques thick lawn
3 50 500 small thick lawn
4 25 100 none thick lawn
4 25 500 none thick lawn
4 50 100 none thick lawn
4 50 500 none thick lawn
SAMPLES CALCIUM (mM) ENRICHMENT (µL) PLAQUE SIZE COLONY DENSITY
1 25 100 small thick lawn
1 25 500 small thick lawn
2 25 100 small thick lawn
2 25 500 small thick lawn
Figure 4: Example of phage isolation results from
third collection of Artobacter SP. Ky 3901 soil
samples. Note abundant presence of plaques due to
phage
Figure 3: Example of phage isolation results from second
collection of Arthrobacter sp. KY 3901 soil samples.
Note clear plate due to abundant phage presence
Figure 5: Example of phage isolation results
from second collection of Arthrobacter sp. KY
3901 soil samples. Note plaques on plate due to
phage presence
Figure 6: Example of phage isolation results from
third collection of Arthrobacter sp. KY 3901 soil
samples. Note almost clear plate due to abundant
phage presence
A supplementary test was performed to determine if the bacteriophage that infected Arthrobacter sp.
KY 3901 would also infect Arthrobacter psychrolactophilus. Top agar was made using
the Arthrophage for Arthrobacter sp. KY 3901, as well as the two different bacterial strains. After
leaving the agar plates to incubate at room temperature for approximately four days, we noticed that
plaques only formed on the Arthrobacter sp. KY 3901 plates.