7 antonio8hernánphages report corrected

Isolation of Bacteriophages in Tropical Soils of Puerto Rico using Mycobaterium smegmatis and Bacillus cereus as host bacteria, a bacilophage was isolated and it was named FirgaroAnthony Maldonado Castro, Hernán G. Méndez Colón RISE ProgramDepartment Biology University of Puerto Rico-Cayey. Bacteriophages are viruses that infect bacteria, and can be found in various environments, including tropical soils. The question was if bacteriophages could be isolated from tropical soils of Puerto Rico; for which we hypothesized that indeed they can be isolated. Therefore the purpose of this experiment is to effectively isolate a novel bacteriophage from the tropical soils of Puerto Rico, in order for it to be characterized and annotated its genome. By isolating and characterizing phage that infect bacteria such as Bacillus cereus and Mycobacterium smegmatis information is acquired about other related bacteria like Mycobacterium tuberculosis that are pathogenic. For the methodology, the first part was the “phage hunt” in which various soil samples were collected from the environment and taken to the lab for analysis and see if a phage could be isolated from the sample. Once taken to the lab the samples were enriched and incubated with the two host bacteria and nutrient medium to allow the phages, if any, to replicate. The supernatant from the enrichment was used to do streak protocols using agar plates; if a phage was isolated from the sample, clear spots would develop on the plates. These clear spots, called plaques, are areas of phage growth, then the phage was purified three times to ensure that it is truly a bacteriophage and that it is only one type. After various samples a bacilophage, which was called Figaro, was isolated. This supports our hypothesis that bacteriophages can be isolated from tropical soils of Puerto Rico. Future work for this experiment includes characterization and annotation of the novel phage that was effectively isolated.

Bacteriophages are viruses that infect bacteria in order to use the bacterial replication mechanism for its own reproduction. They can be found in practically every place in the environment, representing the majority of all biological entities in the biosphere (Suttle 2005). The typical structure of phages consists of a capsid and the tail. The capsid stores the genetic code, and the tail assists in the injection of the genetic material into the bacteria.

Bacteriophages have two different life cycles namely, the lytic and the lysogenic cycles. A lytic phage infects the bacteria, replicates and immediately kills it through cell lysis. Within 2 minutes after the phage

DNA is injected into the host bacterium, directs the synthesis of proteins that shut off the transcription, translation, and replication of bacterial genes, allowing the virus to take control of the metabolic machinery of the host (Snustad 2012). Lysogenic phages infect the bacteria and integrate into the bacterial chromosome, where they continue to divide as the bacteria replicates. If conditions are favorable, the phage enters the lytic phase and proceeds to behave like a lytic bacteriophage (Snustad 2012).

Bacteriophages have specific relationships with their host bacteria, meaning that they are dependent on that bacterium for replication. Mycobateriophages are specific phages that infect bacteria from the genus

Mycobacterium spp., and Bacilophages are bacteriophages that infect bacteria from the genus Bacillus spp. This specific relationship can be useful to learn about the characteristics of both, the host bacteria and the phage that infects it. There are various uses for bacteriophages, which include therapeutics applications like vaccines and drug delivery and control of multi drug resistant bacteria (Pendleton 2013).

The study of bacteriophages and their host bacteria provides information about related bacteria. For example, while using Mycobacterium smegmatis, which is relatively harmless, other related bacteria like Mycobacterium tuberculosis or Mycobacterium leprae are indirectly being studied (Rubin & Vázquez 2012). The same mechanism works for the bacteria Bacillus cereus. Bacteria are abundant in all ecosystems, and where there are bacteria there should be bacteriophages, due to their dependence on their host for replication. Therefore, we hypothesize that bacteriophages should be present in tropical soils of Puerto Rico and can be effectively isolated. The purpose of this research is to first isolate and then characterize a newly discovered bacteriophage with the goal of contributing to the bacteriophage gene bank.

Materials and Methods

A soil sample was collected using a plastic spoon wrapped in plastic and a plastic bag to contain the sample. The sample was collected at a superficial depth. The following data about the sample was recorded and saved: date of collection, time, depth, moisture content, temperature site description and GPS location. The sample was then taken to the laboratory for its enrichment. All of the following procedures

where done according to the aseptic techniques described in the SEA-Phages Resource Guide. For the sample enrichment two ~0.5g amounts of soil were measured using a weighing scale. Both amounts were transferred to two 15mL centrifuge tubes. Next to one of the tubes 10mL of TSB Bacillus and 1mL of Bacillus cereus culture where added using a pipette and a micropipette. Similarly, to the other tube 10mL Master Mix smegmatis with 1mL of Mycobacterium smegmatis culture was added. The two enrichments were incubated in a shaker at 37°C for at least 24 hours. The two enrichments were then centrifuged for 15 minutes at 3000 RPM. The supernatants were filtered using a 5mL syringe and a .2μm filter, and 5mL was then added to the syringe. This mix was then filtrated into another plastic 15mL tube. The filtrate was then used to strike two agar petri dish. The two petri dishes were labeled with date, name of bacteria (Bacillus and Smeg), initials and numbers from 1-3. For streak protocol a wooden sterile swab was dipped into one of each enrichment filtrates; and was then used to streak the plate starting from number “1”, then, using a new swab, continuing to number “2”, and finally to number “3”. Next 4.5mL of Triptic Soy Agar with .5mL of B. cereus were added to plate #1 and 10mL of Top Agar with .5ml of M. smegmatis culture were added to plate #2. The plates were incubated at 37°C for 24 hours. After the incubation period, the plates were examined for plaque formation indicating bacterial death due to phage infection and the presence of bacteriophage growth. This same procedure was followed in order analyze all the 9 soil samples collected.

Once a bacilophage was detected in soil sample number 5, three phage purifications

were done for that plate. For this a single plaque was selected from the plate using a micropipette, and was added to a microtube containing 50μl of phage buffer. A petri dish labelled as “PP1” was streaked following the same procedure described above to do the filtrate streaking, but this time the sterile swab was dipped into the plaque and buffer solution. The plate was then incubated at 37°C for 24 hours. After the incubation period, if there was a sign of phage growth, a second and then a third purification was done, following the same procedure as for “PP1”. After the third purification, a second enrichment was made, adding a plaque from “PP3” to 10mL of TSB Bacilllus and .5mL of Bacillus cereus culture, and incubating it at 37°C for ~24 hours. After the incubation period, the filtrate was centrifuged for 10 minutes at 3000 RPM. Again, the supernatant was filtered as described before. From the filtrate, 10μl were extracted and added to a microtube containing 90-μmL. Dilutions were made by extracting 10μL from the first tube, adding them to the second tube, vortexing and centrifuging. Then 10μL from the second tube were added to the third one, until eight dilutions were made.

The dilutions were then used to do a spot test, using a labeled plate in which nine squared numbered zones were drawn. To the plate 4.5ml of Top Agar with 0.5mL of Bacillus cereus were added, without any streaking. After the agar solidified, 10μL of each dilution were placed in its corresponding zone: dilution 1 in zone “1”, dilution 2 in zone “2”, until reaching zone 8. The plates were incubated at 37°C. for ~24 hours. After the incubation period, the plates were examined to look for the best dilution to use for further characterization.

Using the filtrate from the second enrichment, an analysis of bacteriophage capsids proteins was done. First, 1mL of bacteriophage High titer Phage Lysate (HTPL) (filtrate) was transferred to a microtube and centrifuged at 10, 000 Xg for 1 hour at 4ºC. Next, 950µL of the supernatant was extracted leaving the pellet in the tube. Into the pellet, 50µL of LSB and BME were added, and the solution was boiled for 2 minutes, cooled for 2 minutes, and centrifuged. The samples in the tube were stained using Coomassie Blue G-250. A photograph was taken using a white light box.

Results

Sample Coordinates Description Location DateAMC - #1a

18.2° 12’ 40’’ N;

66.1° W

Rural, next to houses and close to farm yard with chickens, surrounded by trees and other plants; usually a site of organic material composting; slightly moist.Temperature: 26°CDepth: ~2cmTime: 17:43

Bo. Tomás de Castro, carr. 788, km. 2.9, Caguas, Puero Rico, 00725

February 2, 2014

AMC - #2a

18.2°12’ 67’’ N;

66.0° 0’ 41.46’’ W

Low moisture, Rural, fairly away from houses, used to be a farmyard, area within a tropical forest.Temperature: 26°CDepth: ~1cmTime: 13:23


February 17, 2014

AMC - #3a

18.2° 12’ 9’’ N;

66° 1’ 11.9’’ W

Moderate moisture, Rural, close houses, next to an asphalt road, surrounded by vegetation.Temperature: 26°CDepth: ~3cmTime: 18:20


February 23, 2014

AMC - #4a

18.2° 12’ 1.35’’ N; 66° 2’ 13.7’’ W

Wet, bank of a water stream, houses close by, and also a road, surrounded by vegetation.Temperature: 26°CDepth: ~3cmTime: 12:03


March 2, 2014

AMC - #5a

18.2° 12’ 58’’ N; 66° 1’ 9.8’’ W

Very moist, rural, close to houses, next to an asphalt road, an area of garbage disposal.Temperature: 26°CDepth: ~3 cmTime: 17:06


March 9, 2014

HMC –

1b

18° 7' 2.553" N;

66° 6' 58.72" W

Moist, granulated soil, dark in color, under a mango treeTemperature: 24°CDepth: ~1cmTime: 8:50

205 Avenida Antonio R.

Barceló

Cayey, PR 00736

February 4,

2014

HMC –

2b

18° 7' 7.172" N;

66° 6' 58.33" W

Dry, brown color, near a sidewalk and fountain.Temperature: 26°CDepth: ~2cmTime: 15:49


Barceló

Cayey, PR 00736

February

18, 2014

HMC –

3b

18° 7' 6.686" N;

66° 9' 46.61" W

Moist, dark color, under moss, covered by vegetation and next to a buildingTemperature:26°CDepth: ~1cmTime: 12:14


Barceló

Cayey, PR 00736

February

24, 2014

HMC –

4b

18° 7’ 8” N;

66° 9’ 45” W

Moist, dark brown soil.Temperature: 27°CDepth: ~3cmTime: 16:51


Barceló

Cayey, PR 00736

March 6,

2014

Table 1. Soil samples collected in Cayey and Caguas, Puerto Rico. The data was recorded for each soil sample collected. The only sample in which a bacteriophage was found and isolated was in sample 5a.

The samples were divided in two groups, four of them were collected in Cayey, and five of them were collected in Caguas, and labeled as “a” and “b”, as shown in Table 1. A total of nine soil samples were collected. The only sample in which a bacteriophage was found and isolated was sample number 5a. This was a plate containing Bacillus cereus, so it is a bacillophage.

The results from the protein polyacrilamide electrophoresis can be observed in figure 2 and figure 3.

Figure 3. Gel 2 form the protein electrophoresis. After running for 30 minutes the samples travelled all the way through the gel. The band patter for Figaro can be observed in row eight.

Discussion

Figure 1. Third purification plate showing clear spots: plaque formation due to phage growth.

All three phage purifications were done with positive results. After the third purification, shown in figure 1, three spot test were done, but with negative results in the three of them due to no phage growth. After this, a fourth purification was made, but using the filtrate instead of a plaque from “PP3” and incubated at 37°C for ~24 hours. The plate showed no plaque formation; there was no phage growth.

Figure 2. Gel 1 from the protein electrophoresis. After running for 30 minutes the samples only travelled halfway through the gel. The band patter from Figaro can be observed in row number eight.

The soil samples from Cayey did not showed phage growt; no bacteriophages could be isolated from those samples. The first soil samples collected from Caguas, as the ones from Cayey, did not showed sign of phage growth after the analysis was performed. At the time of the first two sample collections, problems with the bacterial culture at the labs arose, probably affecting our results. In this samples there could have been bacteriophages, but since the bacterial cultures were defective, there was no phage growth. From the third sample onwards, however, the bacterial cultures were working properly, but there was no phage growth until sample #5. Possible reasons for this lack of phage formation could be contamination, experimental errors during the procedure, or simply that the samples did not contain phages. One thing that has to be mentioned is that the samples were always collected a day before the analysis. The soil had to remain in a sealed plastic bag for approximately 12-24 hours,

during which organisms living in the samples could be affected by the isolated environment. All of these could be causes for the negative results obtained in the first samples. Sample #5 was collected from under a trash can, were the soil was in contact with decomposing organic material, and bacilophage Figaro was isolated. This unlike the previous soil samples, which were isolated from natural, pristine sites yet no phages were isolated from those. With this we can conclude that bacilophages can be more easily isolated from sites containing decomposing, or even rotting, organic material. It appears to be that that is the type of environment in which the host bacteria will develop and, since bacteriophages depend on their host for replication, it is more likely for them to also thrive in such environments. We can also conclude that indeed novel bacteriophages can be isolated from tropical soils of Puerto Rico.

The isolated bacteriophage is a bacilophage, since it grew in the Bacillus cereus plate. We decided to name it Figaro in honor of the famous character of the same name from the opera “Il Barbiere di Siviglia”, by Gioachino Rossini. In figure 1 the third purification done after the phage isolation can be observed. The white round spots are the plaques formed due to cell lysis, which means that the phage has effectively developed.

Now that we finally isolated a bacilophage, future work includes characterization and annotation of the bacteriophage genome.

Acknowledgements

Special thanks to:RISE ProgramHoward Hughes Medical InstitutePHAGES ProgramDr. Michael RubinGiovanni CruzGustavo MartínezNicolle RosaCristopher Qintanal

Reference

Pendleton JN, Gorman SP, Gilmore BF. 2013. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther 2013; 11:297-308; PMID:23458769; <http://dx.doi.org/10.1586/eri.13.12>

Rubin M, Vázquez E. 2012. Mycobateriophage Proteomics: From Genotype to Phenotype (There and Back Again)! Universty of Puerto Rico at Cayey, Puerto Rico.

Suttle CA. 2005. Viruses in the sea. Nature 437: 356–361.

Snustad P and Simmons M.2012. Principles of Genetics, 6th ed. John Wiley & Sons, Inc. Missouri, USA.

Howard Hughes Medical Institute. 2012 SEA-Phages Resource Guide. Maryland, USA.

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7 antonio8hernánphages report corrected