Bacteriophages: The Search to Find Bacteria’s Enemy

Juan E Maldonado Weng Primary Article

Gustavo A Martínez

Abstract

Mycobacteriophages are viruses that infect bacteria, specifically bacteria called mycobacteria. They exist in two different stages: the lytic, where the phage reproduces within the bacteria and afterwards kills the cell; and the lysogenic stage, where the phage’s DNA replicates within the bacteria. The experiment involved gathering soil; the soil sample was to be taken from a certain point, recording factors such as: air temperature, moisture content, GPS location, etc. Afterwards, 0.5 g of the sample is enriched with M. smegmitis in order to ensure there are phages during the purification step and that the phages receive proper nutrition. The solution is isolated in filtrate for placing it on agar plates that will allow the contained phage to grow. Three plaque purifications follow this step in the process. These steps were interrupted and slowed by complications with the samples, the bacteria used, and fungi that happened to grow on the agar plates. Despite the setbacks that have been faced, the work continues in identifying the newly found mycobacteriophage. The identification process currently stands in the third plaque purification step. After purifying the phage, the process would continue with the web pattern of the plaques. This process is to continue using a a high titer lisate in order to analyze the DNA sequence of the phage to afterwards process it and determine whether it is a new phage or previously identified one. After it is analyzed the phage’s gene sequence is to be stored in a national genomic database specialized in bacteriophages.

Introduction

Bacteriophages are numerous and diverse life forms which can be found in most locations where bacteria can be found. Similar to fingerprints, finding two of the same can be nearly impossible. These creatures are all unique to one another with different behaviors and habits. The bacteriophages serve as great examples of evolution since they have a high rate of reproduction as they replicate as fast as bacteria. With each new generation, they could easily have new mutations or adapt to new conditions.

The most efficient method to identify the bacteriophage would be by the way they

reproduce which could be Lytic or Lysogenic. Lytic would mean that the bacteriophage would enter the bacteria, reproduce, and then destroy the host. The lysogenic reproduction would involve inserting itself into the DNA of the host and reproduce along with it. The lifestyle the bacteriophage could be lytic or temperate, which means it could be both lytic and lysogenic.

Based on Hanlon’s review, the bacteriophages could be helpful to humans in many ways. Phages were used to treat bacterial infection before the discovery of penicillin. Since they only affect bacteria, there is still a possibility that bacteriophages can be used to cure diseases

and other illnesses caused by bacteria. Most of the capabilities for phages still are very mysterious and a good way to understand them is by seeing their genetic code. An advantage that modern science has is the ability to decode their genome to understand or to at least have them collected in an open database. A database would help globalize the search for understanding these creatures.

The problem is the act of collecting a vast amount of the bacteriophages to then be able to identify, study, and publish the information about them for the rest of the world to learn. Collecting samples of soil in search for bacteriophages will help to better understand their behavior, abilities, and function.

Materials and Methods

The process of identifying a phage is complex yet simple. The first important step was to collect soil to be able to extract the phages if there were any. The soil could be from almost anywhere. The soil could be moist, dry or damp. The soil should have had a proper amount of bacteriophages since there are bacteria everywhere.

The next step was to isolate the phage. From the soil sample, only .5 grams was used, placed in a tube (250mL flask) and was added several compounds. 40mL of water, 5mL of 10X 7H9/Glycerol media, 5mL of AD supplement and 0.5mL of 100mM CaCl2 were added to the soil sample, which enriched it.

The enrichment gave the sample bacteria to feed the phages. The next day, the sample was centrifuged and the phages that collected at the bottom of the flask were collected with a micropipette.

The step afterwards is confirming that there is a bacteriophage and then, purifying the phage. The extraction was spread into a plate with top agar, which restricts the phage’s movement. If the phage were there, in the next day there would be spots or plaques on the agar on the plate. From here, the phage was purified three times. The purpose of this was to isolate a single phage population from the potentially mixed community from the sample. This was the last step before time became a factor that constrained the continuation.

The collections that were made are indicated in the table below, with Juan’s collection in blue and Gustavo’s collection in red. The table puts into account the moisture level, depth at which soil was collected, and features of the area.

Results

Nine individual soil samples were taken and evaluated for the project of mycobacteriophages. Four of which were from Luquillo, one from Cayey, and the remaining four from Gurabo. Gathering these many samples was due not being able to find phages after the enrichment phase of the process.

Juan & Gustavo: Collections

# Date Time Depth Moisture Features Location1 2/9/2013 6:06 P.M. Superficial Dry/Moist Urban Luquillo2 2/18/2013 12:03 P.M. Superficial Dry-moist Urban Luquillo3 3/3/2013 3:29 P.M. Superficial Dry Urban Luquillo4 3/11/2013 6:36 P.M. Superficial Dry Urban Cayey5 3/17/2013 1:59 P.M. Superficial Moist Urban Luquillo6 2/18/2013 5:44 P.M. 2-2.5 cm Dry/Moist Rural Gurabo7 2/28/2013 4:13P.M. 3cm Saturated Rural Gurabo8 3/10/2013 6:25 P.M. 10 cm Damp Rural Gurabo9 3/17/2013 7:16 P.M. 1 cm Moist Rural Gurabo

The soil samples collected from Luquillo, a town near the north east coast of Puerto Rico, were dry and almost humid. The one soil sample gathered from Cayey was dry and collected from a tall height in Gurabo, town located around mountains. The four samples from Gurabo were also dry.

The testing of these soil samples never passed the enrichment phase due to technical complications with the bacteria being used. Under recommendation of Dr. Rubin, we re-tested one of the samples taken in the past with a better batch of bacteria.

Only one of the previous soil samples was tested. This sample had come from Gurabo, in a dry canal in a closed-access neighborhood. The re-testing resulted in the growth of plaques on the agar. This allowed us to eventually move on to the plaque purification process.

Currently, the process within this project is still in the plaque purification process. This is due to the fact that a few of the previous plates had been infected with fungi, or the solution for the third purification did not show any phages. Continued attempts would involve taking a plaque from the plate of the second purification and attempting another third purification.

Discussion

This experiment has been an experience in laboratory work that involves: field work such as gathering samples; hands on work in the laboratory in the attempt of finding and purifying the mycobacteriophages; and finally processing the newly discovered phages to then afterwards store it in a database. This process

has shown us to be tedious and at times frustrating due to negative results or complications in the growth of the phages. The work has been interrupted and slowed down by several complications. Currently, the process has yet not passed the third plaque purification.

Future works would involve completing the entire process of identifying the phage to afterwards be sent for recording. Important details to be comprehended are the conditions the phage was discovered and how this can be lead to discover more phages. As occurred in this lab, only one soil sample had traces of phages, which could mean various interesting clues. In the soil samples from Luquillo, the soil might have been exposed to coastal area conditions. In the

The implications of utilizing phages and understanding their unique behavior could lead into interesting developments in the field of medicine. There have been many recent research articles based on phage therapy, which could lead into new promising discoveries. Based on Clark and March’s review paper, many other labs have deemed phages the potential to be transporters of vaccines and even as alternatives to antibiotics and are testing it.

Literature cited

Clark J., and March J. 2006. Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Trends in Biotechnology [Internet] [Cited 2013 May 10] 10.1016/j.tibtech.2006.03.003 24:5 Availble at: http://www.sciencedirect.com.uprcdb.cayey.upr.edu:2048/science/article/pii/S0167779906000746?np=y

Hanlon G. 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. International Journal of Antimicrobial Agents [Internet] [Cited 2013 May 9] 10.1016/j.ijantimicag.2007.04.006 30:118-128 Available at: (http://www.sciencedirect.com/science/article/pii/S0924857907002038)