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

ConclusionWe 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.

ReferencesCross, 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.

IntroductionOur 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 collected.

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 DENSITY1 0 100 none mostly clear1 0 500 none mostly clear1 25 100 none cloudy lawn1 25 500 none cloudy lawn1 50 100 poss. air bubble cloudy lawn1 50 500 none cloudy lawn2 0 100 poss. contaminant spotty 2 0 500 poss. contaminant spotty 2 25 100 none cloudy lawn2 25 500 none cloudy lawn2 50 100 poss. air bubble cloudy lawn2 50 500 none cloudy lawn3 0 100 entire plate clear3 0 500 entire plate clear3 25 100 none slightly cloudy3 25 500 none slightly cloudy3 50 100 minimal cloudy lawn3 50 500 none cloudy lawn

SAMPLE CALCIUM (mM) ENRICHMENT (µL) PLAQUE SIZE COLONY DENSITY1 25 100 small thick lawn1 25 500 small thick lawn1 50 100 none clear1 50 500 none clear2 25 100 poss. air bubble thick lawn2 25 500 none thick lawn2 50 100 poss. air bubble thick lawn2 50 500 none thick lawn3 25 100 poss. 1 plaque thick lawn3 25 500 one thick lawn3 50 100 >6 sm. plaques thick lawn3 50 500 small thick lawn4 25 100 none thick lawn4 25 500 none thick lawn4 50 100 none thick lawn4 50 500 none thick lawn

SAMPLES CALCIUM (mM) ENRICHMENT (µL) PLAQUE SIZE COLONY DENSITY1 25 100 small thick lawn1 25 500 small thick lawn2 25 100 small thick lawn2 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.