Two mycobacteriophages, Incognito and Serotinus, were isolated from tropical soil samples in Puerto Rico. Incognito was isolated from a soil sample taken 10 cm deep under a plantain tree in Hatillo, Puerto Rico on March 8, 2014. After purification, Incognito was determined to likely be a temperate phage. Serotinus was isolated from a soil sample taken 2.3 cm deep underneath a decaying fruit in Cayey, Puerto Rico on March 20, 2014. Serotinus was purified and determined to likely be a lytic phage. Both phages were purified to obtain medium titer phage lysates.

1. Group 1:
Paola G. Caballero León
Kenny Colón Colón
Alejandra M. De Jesús Soto
Anthony Hernández Rivera
Isolation and Purification of
Mycobacteriophages named as Incognito and
Serotinus from Tropical Soils of Puerto Rico
Dr. Michael Rubin
RISE Program
University of Puerto Rico at Cayey

2. Introduction:
Bacteriophages:
• viruses that infect bacteria.
• cannot replicate or propagate outside
their host (bacteria)
Mycobacteriophages:
• Viruses that infect a specific type of
bacteria (mycobacteria genus)
• Harmless agents:
• Mycobacterium smeggmatis
• Infectious disease agents:
• Mycobacterium tuberculosis
• Mycobacterium leprae
Mycobacteriophages are a potential
alternative in the treatment of bacterial
infections and related diseases: phage
therapy.
http://www.genome.ou.edu/3653/Lecture16-9_29_06_files/image002.jpg

3. Experimental Design
www. Phages.org
www. Phages.org

4. Collect soil sample Enrichment Filtrate
Isolation Purification
3 plaque purifications
2nd Enrichment
MTPL
HTPL
Characterization
EM
DNA
Sequencing
Electrophoresis
Proteomics
http://upload.wikimedia.org/wikibo
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http://www.dna-sequencing.org/graph_graphics.gif
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5. 1- A
Paola G. Caballero León
Anthony Hernández Rivera
Incognito
http://24.media.tumblr.com/0a049264fba0072a818f733a6c533578/tumblr_mqvlz4t5FK1
qcnibxo1_500.png

6. Analyzed Samples
Sample Place Coordinates Soil Depth Date
1
PCL
Manatí, PR 18.37964,
-66.47183
Underneath
goat
excrement
~5 cm February 2,
2014
2
AHR
Cayey, PR 18.11785,
-66.13971
Near plants ~ 7cm February 3,
2014
3
PCL
Manatí, PR 18.37964,
-66.47183
Underneath
goat
excrement
~ 7 cm February 16,
2014
4
AHR
Vega Baja,
PR
18°26’14” N
66°24’21” W
Under a
plantain tree
~7 cm February 23,
2014
5
PCL
Salinas, PR 17.9599,
-66.22941
Under a
“guayaba”
tree
~10 cm March 2,
2014
6
PCL
Salinas, PR 17.97166,
-66.24925
Under a tree ~ 10 cm March 2,
2014

7. Analyzed Samples
Sample Place Coordinates Soil Depth Date
7
AHR
Vega Baja,
PR
18°27’38” N
66°25’32” W
Near
“Laguna
Tortuguero”
~ 5 cm March 9,
2014
8
AHR
Vega Baja,
PR
18°23’30” N
66°23’29” W
Underneath
rabbit’s
excrement
~ 6 cm March 9,
2014
9
PCL
Hatillo, PR 18.473341,
-66.785332
Under a
plantain
tree
~ 10 cm March 8,
2014
10
PCL
Hatillo, PR 18.474114,
-66.788401
Near a
water
trough
~ 10 cm March 8,
2014
11
PCL
Hatillo, PR 18.473911,
-66.787264
Under a tree
and horse
excrement
~10 cm March 8,
2014

8. Positive Phage Results
Sample 10 PCL
• Hatillo, PR
• Depth 10 cm
• Soil under a plantain
tree
• March 8, 2014

9. Mycobacteriophage Isolation

10. Plaque Purification 3x

11. Incognito
http://24.media.tumblr.com/0a049264fba0072a818f733a6c533578/tum
blr_mqvlz4t5FK1qcnibxo1_500.png

12. • A mycobacteriophage named as Incognito was isolated
from tropical soil under a plantain tree in Hatillo, Puerto
Rico.
• Depth ~10 cm
• Coordinates:18.473341, -66.785332
• Incognito was purified and Medium Titer Phage Lysates
were obtained.
• Incognito is most likely a temperate phage.
Conclusions

13. 1- B
Kenny J. Colón Colón
Alejandra M. De Jesús Soto
Serotinus

14. Samples Analyzed
http://phagesdb.org
/phages/Nephalae/

15. Analyzed Samples
Sample Date Coordinates Temperature (ºC) Depth
(cm)
Moisture
content
Area Proximity
1
AMDS
Feb / 4 / 14 Lat. 18.119793
Long. -66.15790
23.33 2.54 Moist Urban
UPR Cayey
Campus
Tree
Dead Leafs
2
KJC
Feb / 4 / 14 Lat. 18.119506
Long. -66.157878
23.33 4.4 Dry Urban
UPR Cayey Campus
Dead tree
Trunk
3
AMDS
Feb / 18 / 14 Lat.
Long.
26.11 3.3 Dry Urban.
Coca Navas Street Cayey,
P.R. 00736
Palm leaf
House
Cement
4
KJC
Feb / 18 / 14 Lat. 18.187409
Long. -66.140466
19.44 2.54 Dry Rural
Urb. Campo Lago, Cidra,
P.R. 00739
Sewage
5
AMDS
Feb / 23 / 14 Lat.
Long.
27.78 5.3 Saturated Urban
Coca Navas Street Cayey,
P.R. 00736
Under plant
Roots
6
KJC
Feb / 23 / 14 Lat. 18.119251
Long. -66.161560
27.22 2.5 Dry Urban
UPR Cayey Campus
Mango Tree
7
AMDS
Feb / 24 / 14 Lat. 18.11524
Long. -66.16313
25.7 2.0 Moist Urban
Antonio R. Barceló Street
Cayey, P.R. 00736
Underneath a decaying
fruit
Fruit – tree
8
KJC
Feb / 24 / 14 Lat.18.115079
Long. -66.155562
24.44 3.8 Moist Urban
Los Veteranos Avenue,
Cayey, P.R. 00739
Garbage
Dump
9
AMDS
March / 9 / 14 Lat. 18.11529
Long. -66.14004
27.06 3.1 Dry Urban
Ciaprian Ortiz Rodriguez
Avenue Cayey, P.R. 00736
Underneath sheep
excrement
Tree
10
AMDS
Mar / 9 / 14 Lat. 18.12745
Long. -66.12056
26.2 5.2 Saturated Rural
Vegas Cayey, P.R. 00736
Dairy
Cows excrement
Cows

16. 11
KJC
Mar / 9 / 14 Lat. 18.187424
Long. -66.140468
17.78 4.4 Saturated Urban
Urb. Campo
Lago, Cidra, P.R. 00739
Water
Drainage
12
KJC
Mar / 9 / 14 Lat. 18.187581
Long. -66.140479
25.56 3.17 Dry Urban
Urb. Campo
Lago, Cidra, P.R. 00739
Cement
Drainage
13
AMDS
Mar / 16 / 14 Lat. 18.11402
Long. -66.16907
28.33 2.5 Saturated
José De Diego Avenue
Cayey, P.R. 00736
Septic tank
Plantain trees
14
AMDS
Mar / 16 / 14 Lat. 18.11746
Long. -66.17972
28.61 2.6 Moist Rural
“Buena Vista Sur” Cayey,
P.R. 00736
Stagnant water
Plants
15
AMDS
Mar / 16 / 14 Lat. 18.1174
Long. -66.17993
28.33 2.3 Moist Rural
“Buena Vista Sur”
Cayey, P.R. 00736
Under a plantain
tree
16
KJC
Mar / 16 / 14 Lat. 18.187940
Long. -66.140350
23.33 3.04 Moist Rural
Lake shore, Cidra, P.R.
00739
Plantain
Crop
17
AMDS
Mar / 20 / 14 Lat.
Long.
26.11 1.5 Dry Urban.
Coca Navas Street, Cayey
00736
Coffee plant
Cement
House
18
AMDS
Mar / 20 / 14 Lat. 18.11524
Long. -66.16313
25.0 2.3 Moist Urban
Antonio R. Barceló Avenue,
Cayey, P.R. 00736
Underneath a
decaying fruit
Fruit – tree
19
KJC
Mar / 20 / 14 Lat. 18.119730
Long. -66.157776
28.89 5.71 Dry Urban
UPR Cayey Campus
Under plant
Roots
20
KJC
Mar / 20 / 14 Lat. 18.119564
Long. -66.158065
28.33 5.01 Dry Urban
UPR Cayey Campus
Under Rotten
Fruit

17. Positive Phage Results
Sample 18
• Date: Mar / 20 / 14
• Coordinates: 18.11524ºN
66.16313ºW
• Temperature: 25.0ºC
• Depth: 2.3 cm
• Moisture content: Moist
• Area: Urban. Antonio R. Barceló Avenue,
Cayey, P.R. 00736
• Proximity: Underneath a decaying fruit, fruit-
tree

18. Positive Phage Results
Mycobacteriophage Isolation
Process soil samples
(Enrichment,
Filtrate)
Streak protocol after 24 – hour
incubation
Plaques appeared
after 48 hours of
incubation
Are there plaques?

19. Positive Phage Results
Purifications 3x 1st Purification
2nd Purification 2nd Purification
3rd Purification 3rd Purification

20. • A mycobacteriophage named as Serotinus was isolated
from tropical soil underneath a decaying fruit in Cayey,
Puerto Rico.
• Depth ~2.3 cm
• Serotinus was purified and Medium Titer Phage Lysates
were obtained.
• Serotinus is most likely a lytic phage.
Conclusions

21. Acknowledgements
 Dr. Michael Rubin and Dr. Edwin Vázquez
 Mr. Joseph Perez and Ms. Myrielis Rivera
 Mr. Giovanni Cruz and Mr. Christopher
Quintanal
 Howard Hughes Program and RISE
Program at UPR-Cayey

22. http://jhuphagehunters.wordpress.com/2013/10/3
1/view-from-up-ahead/
After a total of 31 samples…

23. Group 1:
Paola G. Caballero León
Kenny Colón
Alejandra M. De Jesús
Anthony Hernández
Isolation and Purification of
Mycobacteriophages named as Incognito and
Serotinus from Tropical Soils of Puerto Rico
Dr. Michael Rubin
RISE Program
University of Puerto Rico at Cayey