Mycobacteriophage isolation from tropical soil sample: Serotinus

Alejandra M. De Jesús-Soto1, Kenny J. Colón-Colón2

1Department of Mathematics, University of Puerto Rico at Cayey, Puerto Rico2Department of Biology, University of Puerto Rico at Cayey, Puerto Rico

A B S T R A C T

Mycobacteriophages are viruses that infect a specific type of bacteria. This abundant microorganism can be easily obtained from soil samples. Using Mycobacterium smegmatis as a host, a mycobacteriophage was isolated and purified from a soil sample. The goal is to obtain a pure phage population in order to analyze the information and then include it in the Mycobacteriophages Database. The first step was to collect a soil sample in order to make the enrichment and filtrate. Three plaque purifications followed this step in the process. A pure phage population was isolated from a tropical soil in Puerto Rico and named Serotinus. Future work includes performing the spot test, a process to identify the phage’s web pattern. Knowing the phages genetic information allows the identification of characteristics that may lead to important discoveries in modern medicine. Phages therapy seems to be an alternative medical treatment.

1. Introduction

Mycobacteriophages are DNA viruses that infect a specific type of bacteria belonging to the mycobacteria genus. These are the most abundant type of microorganism in the biological universe. Mycobacteriophage infections may or may not lead to the death of the bacterium. This depends on its life style, which can be lytic or lysogenic. Lytic cycles results in the destruction of the infected bacteria and its membrane. It is used by temperate or virulent phages to achieve reproduction. The virulent phage infects bacteria with genetic material in order to undergo cell lysis. On the other hand, temperate phages undergo lysogenic cycle, in which the phage DNA first integrates into the bacterial chromosome to produce the prophage and make the host a lysogen. When the bacterium reproduces, the prophage is also

copied. The prophage can be copied or activated, a process in which the prophage exits the chromosomal material to enter the lytic cycle.

The findings of existent phage research have traversed many arenas of genetics and have even instigated the conception of new therapies and medical treatments (George 2013). A critical problem in heath arose due to the emergence of pathogen bacteria resistant to most currently available antimicrobial agents. The development of alternate anti-infection modalities is one of the highest priorities of modern medicine. Therefore, phage therapy seems to be a solution for this problem.

Mycobacteriophages have been isolated from a variety of environments around the world. They can be easily obtained from soil samples. Using

Scientific Manuscript

Mycobacterium smegmatis as a host, we sought to determine the presence of mycobacteriophages in a soil sample. Therefore, we hypothesized that we can isolate a mycobacteriophage population from a tropical soil sample in Puerto

Rico. The objectives of this research are to isolate, purify, and harvest a novel phage population while the ultimate goal is to identify the characteristics of the phage that may serve modern medicine.

2. Materials and Methods

Figure 1.Flow chart of the experimental procedure. Steps to isolate, purify and characterize a mycobacteriophage population from a soil sample.Each of the stages of the experiment was carried out by using the Sea-phages Resource Guide (2012) of the Science Education Alliance. There were some adjustments on procedures that had to be executed throughout the process to ensure the isolation and harvest of the bacteriophage. For this reason, alterations made during the procedure are included.

Note that all materials used underwent a sterilization process, either by chemical methods using ethanol at 70% and Lysol® or by physical method using autoclave in aseptic technique during their use. It is recommended not to talk while doing any of the procedures to further avoid contamination.

2.1. Sample Collection

Soil sample was collected from optional locations such as in wet places or where there was decomposition. After digging a few centimeters the collected samples were maintained in a sterile container. Data regarding localization coordinates, environmental temperature, excavation depth and moisture content was recorded.

2.2. Enrichment

In a 50 mL conical tube, 0.500 g of the soil sample was mixed with Master Mix, substance that contained 8 mL of sterilized water, 1 mL of 10x 7H9/glycerol broth and 1 mL of AD supplement and 0.1 mL of CaCl (Science Education Alliance. 2012). Finally, 1 mL of Mycobacterium smegmatis was added and the solution was incubated at 37°C and aerated at 220 rpm for 16-24 hours (Science Education Alliance 2012).

2.3. Filtration

After completion of the incubation period, solution was centrifuged at 3,000 rpm for 15-20 minutes. Afterwards, supernatant was filter-sterilized and poured into a 15mL conical tube. Both the pellet and the supernatant were stored.

2.3.1 Centrifuge

Another option to obtain a purer sample from the enrichment protocol after centrifuging the conical tube of the enrichment protocol was to take 1 mL of the supernatant from the conical tube and transfer it to a microtube. The microtube was centrifuged at 10,000 rpm for 10 minutes. Then, 500 µL of the supernatant of the centrifuged microtube was transferred to another microtube to obtain the filtrate.

2.4 Plate Streaking

A sterile utensil (wooden stick) was moistened with the filter-sterilized supernatant once. It was then used to gently swipe streaks on the Luria medium agar plate in quadrants. Figure 2 shows the correct way of streaking a plate. The segment labeled as (1) was first streaked, and then, another sterile swab was used to drag part of sample segment (1) to segment (2). The process of taking a sample from segment (2) and dragging it to segment (3) was repeated. Then, 4.5 mL of ~55.0oC Top Agar and 0.5 mL of the Mycobacterium smegmatis were poured into the plate from quadrant 3 to quadrant 1. The plate was closed by placing the cap on top. Finally, after agar solidification the plate was placed in an incubator at 37°C for ~24 hours (Science Education Alliance 2012). After this part of the procedure, the presence of mycobacteriophages in the collected sample was finally determined.

2.5. Plaque purification

Figure 2. Streaking Technique. Available source: http://faculty.mc3.edu/jearl/ML/streak1.gif

After confirming the presence of mycobacteriophages, the next step consisted

in the extraction of an isolated plaque using the wooden tip of a sterile swab. Part of the plaque was transferred to a sterile microtube that contained 25 µL (microliters) of Phage Buffer (PB) which would help with the dilution and sustain the mycobacteriophages on the plaque. The plaque sample was streaked in a “smeg plate” (2.4 plate streaking protocol) and was left to rest from 16-24 hours until the next day. This plaque extraction procedure was progressively repeated with each plate until the third (3) plate was obtained. This was done to assure the pureness of a single type of phage from each plaque sample. The plaque purification samples of each three repetitions were placed in an environment temperature of 4°C (Science Education Alliance. 2012).

2.6 Phage-Titer Assay

The third plaque purification sample went through another enrichment protocol (2.2). This was made for supplementing the purified mycobacteriophage. Eight (8) microtubes were labeled from 1 to 8. A volume of 90 µL (microliters) of PB was added to each microtube. In the microtube labeled (1), 10 µL (microliters) of the supernatant of the enrichment of the third plaque purification sample was added. Later, 10 µL (microliters) were extracted from microtube (1) to microtube (2). The same process was done going from the microtube (2) to microtube (3). This step was repeated successively until all 8 microtubes were diluted.

2.7 Empirical Test

A smeg plate was prepared after labeling it and drawing eight (8) square divisions on the bottom of it with a number corresponding to the division on an edge for better plaque view. Before going to the important part of the empirical test, 4.5 mL

of TA and 0.5 mL of Mycobacterium smegmatis were mixed and spread on top of the Luria medium Agar. Next, 10 µL of the sample of microtube (1) from the phage-titer dilutions was added to the square segment labeled with the same number. The process was repeated for each microtube sample on the corresponding numbered segment. The plate is then placed in an incubator at 37°C for 16-24 hours (Science Education Alliance 2012).

2.8 Mycobacteriophage Proteomics with Electrophoresis

The Medium Titer Phage Lysate (MTPL) was used for conducting the proteomics part of the experiment. 1 ml of the MTPL was transferred to a sterile microtube and centrifuged at 1x104 rpm for 1 hour. Then, 950 μl were aspirated from the supernatant. Next, 50 μl of a mix of 25 μl of BME with 475 μl of LSE buffer was added to the pellet of the microtube. The microtube was placed on a heat-block for 2 minutes of heat exposure and then was moved to a cooler with ice for at least 2 minutes. For the electrophoresis procedure, the gel used was a precast gel. The running buffer was prepared by adding 100 ml of 10X TGS buffer to 900 ml of distilled water. The gel was rinsed with distilled water and placed on the electrophoresis device, following that the running buffer was poured in the wells. The sample was added in one of wells of the gel and it was run at 200 volts for about 30 minutes. After the 30 minutes had passed, the gel was transferred to a rocker where it was washed with distilled water for 5 minutes, later stained with Bio-Rad BioSafe Coomassie Blue G-250 Stain and left shaking for 1 hour. After finishing, the gel was washed with water for 30 minutes. The gel was taken for subsequent protein identification by mass spectroscopy after

excising the bands and placing them in microtubes.

2.9 Preparing phage sample for Electron Microscopy

A plastic-faced paper was placed over a counter for working in a clean area. Parafilm was placed in a petri dish lid. A double-sided tape was pasted in the parafilm of the petri dish lid. After removing the liner from the tape, using tweezers, a new grid (shiny side up) was placed along the edge of the double-sided tape. 10 μl of the phage preparation was added to the grid without spilling it outside the edge, passed that, it was left during 2 minutes for that the phage could retain to the grid. The excess of phage substance was absorbed (edge of the grid) using the tip of a filter paper. The grid was washed two times pipetting 10 μl of sterile water on top of the grid without touching the surface of it or spilling outside the edge. After waiting 2 minutes for the water to wash the grid, the excess was absorbed using the tip of another clean filter paper. Finally, the same process was repeated adding 10 μl of 1% uranyl acetate to the grid. The grid is left to air-dry. The samples were kept by the mentor, Dr. Rubin, for future analysis using the electron microscope of the Central and Caribbean University at Bayamón, Puerto Rico.

3. ResultsTwenty individual soil samples were

collected and analyzed (Table 1). Positive results were obtained from sample #18 which was collected from an urban area in the city of Cayey. Location coordinates were: 18.11524 North, 66.137 West. The sample was collected 2.3 centimeters beneath the surface in an environment whose temperature skirted 25.0º Celsius.

After collecting and enriching the sample, the presence of mycobacteriophages was confirmed. Then, after three plaques purifications, a phage with clear plaques was isolated (Figure 2). From these plaques was obtained the MTPL, Medium Titter Phage Lysate. The next step performed was a spot test in order to identify the mycobacteriophage’s web pattern to do ten plates from the dilutions but time made it difficult to continue so the electrophoresis assay phage sample was done with the MTPL and without doing the ten plate cultivation. The result of the electrophoresis assay showed that the protein bands were similar to other previous isolated phage which means that it is probable that we had been working with a previously sequenced mycobacteriophage, but either way, the bands were excised for a spectroscopy test for measuring the absorbency of the phage solution. As for the analysis of the phage sample using electron microscope, the grid prepared was left with the mentor to be taken for a visual evaluation later on.

4. DiscussionThis research on the collection and

analysis of a soil sample for the isolation of a phage has been a worthwhile laboratory experience. Although the process was somewhat tedious and frustrating at the

Figure 3: Clear plaques obtained after third purification

beginning, due to all the negative results it was a learning experience that taught us patience, dedication and perseverance. Finally, after analyzing 20 samples, a mycobacteriophage was isolated and named Serotinus. After three purifications, the phage formed clear plaques on his host Mycobacterium Smegmatis, indicating that Serotinus is most likely a lytic phage.

Future work would include completing the entire process of identifying and characterizing the phage in order to record and send the information to the Mycobactoriophages Database. The implications of isolating phages could lead to new and interesting developments in modern medicine. Currently, much research is being done related to phage therapy, and the study of novel phage populations could lead to promising discoveries.

5. Acknowledgments:

This work was funded by the Howard Hughes Program and the RISE Program at the University of Puerto Rico at Cayey. The authors would like to thank Dr. Michael Rubin, Dr. Edwin Vazquez, Mr. Joseph Perez, Mr. Giovanni Cruz, Mr. Gustavo Martínez and Mr. Christopher Quintanal for their support during the entire process.

6. Literature Cited

Alvarado EJ, Cruz-Arzón JA. 2013. Mycobacteriophage isolation from tropical soil sample: Mikriplithari and Ususindagari. Department of Mathematics-Physics, University of Puerto Rico at Cayey, Puerto RicoDepartment of Biology, University of Puerto Rico at Cayey, Puerto Rico.

George MP. 2013. Mycobacteriophage Meru: Isolation and Characterization of a Novel

Mycobacteriophage. [Internet]. [cited 2014 May 26], 5(10):1-3. Available from http://www.studentpulse.com/articles/770/mycobacteriophage-meru-isolation-and-characterization-of-a-novel-mycobacteriophage

Rubin M, Vázquez E. 2012. Mycobacteriophage Proteomics: From Genotype to Phenotype (There and Back Again)!.Howard Hughes Program, Department of Biology, University of Puerto Rico at Cayey.

Science Education Alliance. 2012. SEA-PHAGES Resource Guide. Howard Hughes Medical Institute. Chevy Chase, Maryland

Table1. Soil samples tested in order to verify the presence of mycobacteriophage population. Table includes sample information: date of sampling, coordinates, ambient temperature, depth, moisture content, area and proximity.

Sample

Date Coordinates Temperature (ºC)

Depth(cm)

Moisture content

Area Proximity

1AMDS

Feb / 4 / 14 Lat. 18.119793Long. -66.15790

23.33 2.54 Moist UrbanUPR CayeyCampus

TreeDead Leafs

2KJC

Feb / 4 / 14 Lat. 18.119506Long. -66.157878

23.33 4.4 Dry UrbanUPR Cayey Campus

Dead treeTrunk

3AMDS

Feb / 18 / 14

Lat. 18.11801Long. -66.13711

26.11 3.3 Dry Urban.Coca Navas Street Cayey, P.R. 00736

Palm leafHouseCement

4KJC

Feb / 18 / 14

Lat. 18.187409Long. -66.140466

19.44 2.54 Dry RuralUrb. Campo Lago, Cidra, P.R. 00739

Sewage

5AMDS

Feb / 23 / 14

Lat. 18.11797Long. -66.13706

27.78 5.3 Saturated UrbanCoca Navas Street Cayey, P.R. 00736

Under plantRoots

6KJC

Feb / 23 / 14

Lat. 18.119251Long. -66.161560

27.22 2.5 Dry UrbanUPR Cayey Campus

Mango Tree

7AMDS

Feb / 24 / 14

Lat. 18.11524Long. -66.16313

25.7 2.0 Moist UrbanAntonio R. Barceló Street Cayey, P.R. 00736

Underneath a decaying fruitFruit – tree

8KJC

Feb / 24 / 14

Lat.18.115079Long. -66.155562

24.44 3.8 Moist UrbanLos Veteranos Avenue, Cayey, P.R. 00739

GarbageDump

9AMDS

March / 9 / 14

Lat. 18.11529Long. -66.14004

27.06 3.1 Dry UrbanCiaprian Ortiz Rodriguez Avenue Cayey, P.R. 00736

Underneath sheep excrementTree

10AMDS

Mar / 9 / 14 Lat. 18.12745Long. -66.12056

26.2 5.2 Saturated RuralVegas Cayey, P.R. 00736

DairyCows excrementCows

11KJC

Mar / 9 / 14 Lat. 18.187424Long. -66.140468

17.78 4.4 Saturated UrbanUrb. CampoLago, Cidra, P.R. 00739

WaterDrainage

12KJC

Mar / 9 / 14 Lat. 18.187581Long. -66.140479

25.56 3.17 Dry UrbanUrb. CampoLago, Cidra, P.R. 00739

CementDrainage

13AMDS

Mar / 16 /14

Lat. 18.11402Long. -66.16907

28.33 2.5 Saturated José De Diego Avenue Cayey, P.R. 00736

Septic tankPlantain trees

14AMDS

Mar / 16 / 14

Lat. 18.11746Long. -66.17972

28.61 2.6 Moist Rural“Buena Vista Sur” Cayey, P.R. 00736

Stagnant waterPlants

15AMDS

Mar / 16 / 14

Lat. 18.1174Long. -66.17993

28.33 2.3 Moist Rural“Buena Vista Sur”Cayey, P.R. 00736

Under a plantain tree

16KJC

Mar / 16 / 14

Lat. 18.187940Long. -66.140350

23.33 3.04 Moist RuralLake shore, Cidra, P.R. 00739

PlantainCrop

17AMDS

Mar / 20 / 14

Lat. 18.1179Long. -66.137

26.11 1.5 Dry Urban.Coca Navas Street, Cayey 00736

Coffee plantCementHouse

*** 18AMDS

Mar / 20 / 14

Lat. 18.11524Long. -66.16313

25.0 2.3 Moist UrbanAntonio R. Barceló Avenue, Cayey, P.R. 00736

Underneath a decaying fruitFruit – tree

19KJC

Mar / 20 / 14

Lat. 18.119730Long. -66.157776

28.89 5.71 Dry UrbanUPR Cayey Campus

Under plantRoots

20KJC

Mar / 20 / 14

Lat. 18.119564Long. -66.158065

28.33 5.01 Dry UrbanUPR Cayey Campus

Under RottenFruit