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Isolating Bacteriophages against Renibacterium salmoninarum, Aeromonas salmonicida, and Arthrobacter sp. KY 3901 (ATCC 21022)Valeria Chacon & Jediael Desir. Sponsor: Dr. James DalyMethods

Preparation of BacteriaInoculate fresh agar plates with bacterial samplesAdd colony with sterile loop to appropriate growth medium Shake and incubate at room temperature for 24 hoursNeed to achieve the late exponential/early stationary stage bacteriaCollection of SamplesPrepare phage buffer (PB) in separate flasksSterilize by autoclavingCollect different soil, sewage, and water samples from around campus and from fish hatcheriesAdd PB to sample, shake and let settleCentrifuge at 6,000 rpm at 4C for 30 minsPour off supernatant into tissue culture flasksAdd LB to each tissue culture flaskPass through micro-filter using disposable filter and add culturePour 50 mL into separate culture flaskAdd varying concentrations of CaCl2Phage IsolationAdd culture and phageSet up 2 control tubes and add appropriate growth mediumPour contents of all tubes into designated agar plates, gently swirl to distribute evenly and incubate at necessary temperature for allotted timeCheck for plaque formationsPhage PurificationDilute 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 formationsPerform plaque streaking procedure. Pour solution of top agar, corresponding bacteria and CaCl2 on topLet 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 3901SUNY RF for supporting this researchSUNY at Purchase College for allowing us to use their laboratory and resources Monica Furman and Talibah Stephenson for being our lab partnersDr. James Daly for overseeing this researchThis 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 areRenibacterium salmoninarumandAeromonas 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 infectArthrobacter sp. KY 3901, which is a type of bacteria found in soil.Arthrobacteris thought to be closely related toR. salmoninarum. If we can find phages that infectArthrobacter,theoretically, those same phages should be able to infectR. salmoninarumwhich would provide us with the basis for analternative 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 samplesFigure 1: Results of phage isolation from third collection of Arthrobacter sp. KY 3901 soil samplesFigure 4: Charted results of phage isolation of A. salmonicida water samples

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 presenceA supplementary test was performed to determine if the bacteriophage that infectedArthrobactersp. KY 3901would also infectArthrobacterpsychrolactophilus. Top agar was made using theArthrophageforArthrobactersp. 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 theArthrobactersp. KY 3901 plates.

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AerobiofilmSAMPLECALCIUM (mM)ENRICHMENT (L)PLAQUE SIZECOLONY DENSITY10100nonemostly clear10500nonemostly clear125100nonecloudy lawn125500nonecloudy lawn150100poss. air bubblecloudy lawn150500nonecloudy lawn20100poss. contaminantspotty 20500poss. contaminantspotty 225100nonecloudy lawn225500nonecloudy lawn250100poss. air bubblecloudy lawn250500nonecloudy lawn30100entire plateclear30500entire plateclear325100noneslightly cloudy325500noneslightly cloudy350100minimalcloudy lawn350500nonecloudy lawn

SoilSOILCALCIUMENRICHMENTPHAGE DENSITYPLAQUE SIZECOLONY DENSITY125mM100LLowsmthick lawn125mM500LMedsmthick lawn150mM100LMedn/aclear150mM500LHighn/aclear225mM100Lnoneposs air bubblethick lawn225mM500Lnonen/athick lawn250mM100Lnoneposs air bubblethick lawn250mM500Lnonen/athick lawn325mM100LLowposs 1 plaquethick lawn325mM500Lnonen/athick lawn350mM100LLow>6 sm plaquesthick lawn350mM500LMedsmthick lawn425mM100Lnonen/athick lawn425mM500Lnonen/athick lawn450mM100Lnonen/athick lawn450mM500Lnonen/athick lawn

Sheet2Low100Lthin lawn0mM1Med500Lthick lawn25mM2Highcloudy lawn50mM3noneminimal growth4distinct plaquesclearcloudy

Aeromonas WaterSAMPLESCALCIUMENRICHMENTPHAGE DENSITYPLAQUE SIZECOLONY DENSITY125 mM100Llow small thick lawn125mM500Llow small thick lawn225mM100Llow small thick lawn225mM500Llow smallthick lawn

AerobiofilmSAMPLECALCIUM (mM)ENRICHMENT (L)PLAQUE SIZECOLONY DENSITY10100nonemostly clear10500nonemostly clear125100nonecloudy lawn125500nonecloudy lawn150100poss. air bubblecloudy lawn150500nonecloudy lawn20100poss. contaminantspotty 20500poss. contaminantspotty 225100nonecloudy lawn225500nonecloudy lawn250100poss. air bubblecloudy lawn250500nonecloudy lawn30100entire plateclear

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