“Isolation and Characterization of Mycobacteriophages Isolated From Tropical Soils ofPuerto Rico”Michael Rubin, Giovanni Cruz, Carla Figueroa, Crystal ColónPhage Hunters Advancing Genomics and Evolutionary Science Program, ScienceEducation Alliance of the Howard Hughes Medical InstituteAbstract:Mycobacteriophages are viruses that infect bacteria, specifically Mycobacteriumsmegmatis. These viruses are commonly found in soil. By collecting soil samples fromdifferent towns of Puerto Rico and enriching them with a nutrient media containingMycobacterium smegmatis, Mycobateriophages can be identified and eventually purifiedand characterized. Eleven soil samples from Las Piedras, Cidra, Cayey, Barranquitas,Caguas, and Aibonito were studied. None of them showed positive results. This researchcontributes to the study in the way to understand and treat different diseases liketuberculosis.Introduction:Bacteriophages are viruses that infectbacteria, rughly exist worldwide(Serwer et al.2007). Mycobacteriophages areviruses that infect bacteria likeMycobacterium smegmatis(Rubin M.,Vázquez E.2012). These are common andcan be found in soil. Their sizes range from100 to 200 nm and they cannot replicate norpropagate outside of their host bacteria, butthey can be found inside or outside ofbacterial cells which means that it has twotypes of replication. The temperate and lyticcycle. The temperate cycle infects thebacteria and merge its DNA without causingbacterial lysing. The lyctic cycle causescellular lysis. ( López-Cuevas O.,et al. 2011)Since they are an abundant simplelife form, they are used in genetics forcloning or mutations, in epidemiology theyare studied because some phages can maketheir host bacteria more deadly to humans,and also they can be used as therapeutics tokill specific bacteria. One of the cases wherea Mycobateriophages can be used is in thesearch to treat tuberculosis. They haveproven to be a rich resource for its geneticsand provide a general strategy forrecombineering and mutagenesis ingenetically naïve systems (van Kessel J.,Marinelli L. and Hatfull G.2008).The main goal of the research is tocharacterize a novel Mycobacteriophagefrom Puerto Rico using genomic andproteomic approaches.Since Mycobacteriophages aresusceptible life-forms to bacteria and toobtain a pure phage requires an excellentaseptic technique that is different for eachstep of the process. The most common wayto study phages is to collect soil samples.These samples undergo a process that
includes enrichments, harvesting, plating,purification, empirical tests, web pattern,and a high Titer phage lysate (HTPL). At theend of this process the obtained solution isready for DNA purification and eventually itwill be characterized. Most of the phagesfrom which their DNA’s are characterizedare named and inserted in a genome bancwhere they are shared with the world.Materials and MethodsThe procedure started in February 18 andended in April 3. It was divided in severalsteps: soil sample collection andenvironmental data, preparing theenrichment, harvesting and preparing theenriched sample, plaque streak protocol,plaque purification (3x), empirical test andweb pattern, ten plate preparation and hightiter phage lysate, and characterization of thephage. A negative plaque was prepared withphage buffer was prepared.Soil Sample Collection and EnvironmentaldataAfter taking two photos the soil wascollected using a wrapped spoon. Theenvironmental data was recorded and alsothe date and time of sampling, location, airtemperature, depth, moisture content, andthe features of the site.EnrichmentUsing a clean spatula, 0.5 grams ofthe sample were added to a 50 ml tube. Tothis flask it was added: 8 mL of sterile H2Ousing 2 x 25- mL pipettes, 1 mL of sterile 10x 7H9/glycerol broth, 1 mL of ADsupplement, 0.1 mL of 100 mM CaCl2, and1mL of late long/early stationary phase M.smegmatis culture. At the end it wasincubated at 37° C, shaking at 220 rpm, for24 hours.Harvesting and Preparing the EnrichedSampleThe tube with the sample was balanced and itwas spin at 3,000 rpm for 10 minutes to pelletparticulate matter, including most of thebacterial cells. The liquid from the centrifugesample was poured into a fresh 50 mL conicaltube. The enrichment sample was filtersterilized.Plaque Streak ProtocolAfter removing a sterile wooden stickfrom its package and opening the tube with thecentrifuged filtered enrichment it was gentlystreak across the top third of the agar plate. Thisstep was repeated three times from the streakedarea to the un-streaked portion. Then 4.5 mL ofTop Agar was added to 0.5 mL aliquot of M.smegmatis. At the end it was dispensed onto themost dilute area of the streaked plate. Themixture was spread across the plate from themost dilute point to the more concentrated areasby titling the plate. After the Top Agar washard, the plates were put in an incubator at 37˚ Cfor 24 hours. If at the other day the plate showssigns of the presence of a phage please continuewith the instructions, if not, start againcollecting new a new sample from anotherlocation.Plaque PurificationFirst the Petri dish with the plaque was openedand touching the center of the plaque with asterile blue pipette tip it was transferred to asterile microtube with 100 µL of Phage Buffer.After returning the cover to the Petri dish andusing the aseptic technique, a sterile woodenstick was removed from its package to performthe Streak Technique mention in the previousstep. At the end the Top Agar and the bacteria(M. smegmatis) were added to the plate and itwas incubated at 37˚C. This process should bemade three times.
Figure 1.Shows the result of theharvest of the first soil sample.Empirical Test and Web PatternBy drawing a circle around the area,there were designated a label to the plaque.For all of the founded plaques, there wasmade plaque purification. Then, four microcentrifuge tubes were labeled from 1 to 4.To each one of them it was added; 90 µL ofPhage Buffer and 10 µL of the 10 ˚ phagesample (to the “1” tube), 10 µL of the “1”tube to the “2” tube, and successively untilthe “4” tube. At the end the Top Agar andthe bacteria (M. smegmatis) were added tothe plate and it was incubated at 37˚C. If ithas plaques proceed.Ten Plate Preparation and High TiterPhage Lysate (HTPL)With the appropriate volume anddilution of lysate, a 50 mL flask wasinfected with M. smegmatis. There wereadded 45 mL of Top Agar to the flask thatwas previously contaminated with thebacteria. After pulling 25 mL of the mixture5 mL per agar plate should be dispense. Toeach plate there were added 8 mL of PhageBuffer. After letting the plates sit for 2 to 4hours the plates were stored at 4˚C. Then thePhage Buffer was removed and transferredto a 50 mL conical tube. The tubes werecentrifuged at 2500xg for 20 minutes topellet cell debris. The supernatant wasmoved into a 50 mL filter sterilization unit,using suction the lysate was filter-sterilized.At the end the tube containing the solutionwas stored at 4˚ C.At this point the solution is ready topurify its DNA and eventually to becharacterized eventually.ResultsDuring the research eleven soilsamples were studied. Each one of them wastaken from a different location and thereforedifferent properties were observed. Thesecan be perceived in the Figure 2. Threewere from Cidra, one from Cayey, one fromBarranquitas, five from Las Piedras, and onefrom Caguas. The air temperature fluctuatedfrom 80˚ F to 91.4˚F. Most of the soilsamples were obtained during the afternoon.The earlier sample collection was at 11:16a.m. and the latest sample collection was at6:37 p.m. Most of the soil samples wereobtained from farms and under citrus trees.Any of the soil samples showed thepresences of phages. Most of the PetriDishes looked like Figure1, without anyplaque.Some of the reasons that can explainthese results can be: the places were the soilsamples were collected were not appropriateor that the time that the samples spend untilitwas enriched was to prolonged and affectedthe presence of phages.
.DiscussionAt first none of the soil samplescollected had the presence of phages. Thiscan be explained by different factors thatinclude the temperature, time, moisture, andlocation.The average temperature was 84˚F.The highest temperature was 91. 4˚F and thelowest was 80˚F. This is a normal range forour island’s air temperature meaning thattemperature may probably not be the mainreason for the absence of phages in thesamples.The time at which samples werecollected varied during the day. Some werecollected during the morning, the earliest at11:16 am, and the rest during the afternoonand the evening, the latest at 6:37 pm.The moisture content of the samplesvaried with location and with the depth atwhich the sample was extracted. Theaverage depth of the samples was 1.17 in.This means that the samples were fairlysuperficial where there was a constantcirculation of liquid, solid or gaseous debris,animals, etc.The soil samples were collected from5 different cities or towns of Puerto Rico.25% were obtained from Cidra (3 samples),8% from Barranquitas (1 sample), 8% fromCaguas (1 sample), 17% from Cayey (2samples) and 42% from Las Piedras (5samples), for a total of 12 samples. None ofthe samples were taken from the same spotor nearby area. The samples from LasPiedras were picked from the sameneighborhood but from differentLocation Date andtimeAirtempe-ratureDepth Moisture PlaquesCidra,P.R.18˚8’ 26’’N66˚8’ 10’’ W02/18/134:15 pm83˚F 3cm dry NoCayey, P.R.18˚7’30.12’’N66˚ 7’8.01’’W03/02/131:20 pm85˚F 2.5cmdry NoCidra, P.R.18˚8’24.13’’N66˚8’10.09’’W03/03/136:37 pm80˚F 3cm wet NoCidra, P.R.18˚8’26.99’’N66˚8’11.12’’W03/13/1311:16 am83˚F 4cm Dry NoBarranquitas,P.R.18˚11’22.65’’N66˚19’25.07’W03/16/133:05 pm82˚F 4cm Wet NoLas Piedras,PR18.20693 N65.87793 W02/18/1311:40p.m80˚F 1.27cmDry NoLas Piedras,PR18.20554 N65.87694 W03/03/134:05 p.m.91.4˚F 3.14cmDry NoCaguas, PR18.16853 N65.07780 W03/08/139:42 p.m.82.4˚F 2.57cmDry NoLas Piedras,PR18.20459 N65.87794 W03/10/133:45 p.m82.4˚F 1.27cmDry NoLas Piedras,PR18.20509 N65.87780 W03/17/134:08 p.m.84.2˚F 2.58cmDry NoLasPiedras,PR18.20657 N65.87817 W03/17/134:32 p.m.89.6˚F 4.50cmMoist NoFigure 2.Shows final results and soil properties.
environments. Samples were from underfruit trees, from horse and cow stables, froma swap’s shore, from plain areas. Some wereurban, like the samples from Cayey, andothers, like the one from Barranquitas aremore rural.ConclusionAfter seven weeks studying diversesamples from the soil of different towns inPuerto Rico, there was any type of plaquethat indicated the presence ofbacteriophages. This shows that in thespecific places where the soil samples werepicked there is low degradation of organicmaterial. The results obtained leads us tokeep searching for more soil samples inthis specific towns of Puerto Rico anddetermine what is affecting the growth ofbacteriophages in them. Hopefully lateron, this research will be retaken.ApendixAfter nineteen soil samplecollections and enriched samples, twoplates showed the presence of plaques,which means that bacteriophages were beingexpressed.The soils samples were collected inLas Piedras and Cidra. Figure 3 shows theplate with the plaques from the soil samplecollected at Cidra and Figure 4 from LasPiedras. Location and other details areshown in Figure 5.Both plaques were purified and theyare being prepared to the next step of theprocedure in order to determine their DNA.The bacteriophage isolated from Las Piedraswill be call Minhaand the one from Cidra,Koralia.References:1. Asai D., Bailey C.,Barker L.,Bradley k., Khaja R., LewisM.2012.Sea-Phages.Maryland(United States): HHMI;.Location Date andtimeAirtempe-ratureDepth Moisture PlaquesCidra,P.R.18˚8’ 26.45’’N66˚8’10.44’’W04/13/1311:30 pm90˚F 6 cm moist YesLas Piedras,P.R.18˚12’30’’N65˚52’21” W04/15/136:40 am79˚F 10 cm moist YesFigure 3. Plate with the first harvest of Cidra’s soil sample.Figure 4. Plate with the first harvest of Las Piedras’ soil sample
2. López-Cuevas O., Castro-delCampo N., León-Félix J.,González-Robles A., Chaidez C.2011.Characterization ofbacteriophages with lytic effecton various Salmonella serotypesand Escehrichia coliO157:H7.NRCP.57:1042-1051.3. Serwer P., Hayes S., Thomas J.,Hardies S.2007.Propagating themissing bacteriophages: a largebacteriophage in a newclass.VJ.doi:10.1186/1743-422X-4-21.4. VanKessel J., Marinelli L.,Hatfull G. 2008. Recombineeringmycobacteria and their phages.NRM.6:851-857.