February | 2016 | Aednet Project

Romuald Gryko – Military Institute of Hygiene and Epidemiology – Poland

Milestone 7: A germanant/phage lysin decontamination formulation suitable for field trials- (WiHE)- progress report April 2015

Colleagues at the Military Institute of Hygiene and Epidemiology in Poland (WiHE) are working to identify bacteriophages which contain lysins capable to targeting the cell wall of B.anthracis. They first isolated bacteriophages from environmental samples with activity against B.anthracis 34F2.

Environmental isolation method

In brief, 20 gram of environmental samples (agricultural soils, sewages) was added to 40ml of Tryptose Soy Infusion supplemented with 5 mM MgSO₄ (TSIMg). The suspension was then inoculated with 100 µl of a 24 h. culture of B. anthracis 34F2 and incubation for 24 hr at 37⁰C. Following incubation the sample was treated with chloroform and centrifuged at 4000rpm for 30 min (2952 x g).

To determine if any phages were present sample supernatant was dropped onto the surface of a Tryptose Soy Infusion Agar plate supplemented with 5 mM MgSO₄ (TSAMg) in which the upper layer of agar had been inoculated with B. anthracis 34F2.

Following overnight incubation at 37⁰C clear zones of inhibition were harvested from the agar and placed in 8 ml of TSIMg. Following agitation the solution was treated with chloroform and centrifuged after which the supernatants were diluted and plated onto a bilayer TSAMg as described above. Following overnight incubation clear plaques were again harvested and the process repeated on a further four occasions. The host range of the resulting phages and there lysins were determined using the methods described below.

Method to determine the bacterial host range

A 5 ml of suspension of test cells of Bacillus spp. strains in 0,7% TSAMg was laid over the surface of 15ml of TSAMg. Once the agar had solidified tested phage lysates were dropped (about 25 µl) on the surface of plates and were incubated for 24 h in 37⁰C and zones of lysis were noted. The gamma and Fah phages were included as positive controls.

Screening method to determine of phage produced lysins

A 5 ml 1% agarose overlay contained heat inactivated cultures of the test bacteria. This was achieved by harvesting the growth from 50ml of a 24 hr culture of the test bacteria at 37⁰C and resuspending it in 5ml of water. This was then mixed with 10 ml of 1.5% agarose and boiled for 15 mins. The agarose was then overlaid onto TSAMg and after solidifing 6 mm diameter holes were cut and the bottom of each hole was sealed with a drop molten TSAMg. For each hole 25 µl of 24h liquid culture of B. anthracis 34F2 (host strain for all phages) were dropped. After 3 h of incubation in 37⁰C lysates of tested phages (25 µl) in the rate 1.8 – 3.2 x 10⁶pfu were dropped to individual holes. Dishes were incubated for 24 – 48 h in 37⁰C and zones of brightening around holes were noted. The gamma and Fah phages were included as positive controls.

In the case of poorly visible of the zones plates were dipped on 15 min in the water containing 1 – 5 µg/ml of active chlorine.

Results

An example of the results achieved using this method can be seen in figure 1 and a summary of all of the results is presented in table 1. As expected the control phages, gamma and Fah were only able to infect the B.anthracis isolates include in this study. They also produced lysins which were specific to B.anthracis. While the majority of environmentally isolated bacteriophages were highly specific for B.anthracis there were exception such B.cereus ATCC 23261 and B.sp. Ba 813. We saw a different picture for the lysins and observed that a number of phages produced lysins with activity against B.anthracis other members of the Bacillus test panel.

Figure 1. A example of the activity of phage lysates on the lysis of dead cells of B.thuringiensis T7-030 and Bacillus sp. B.a. 813. #7. K1- is a negative control in that it only contains B.anthracis 34F2 and no phage

Table 1. The activity of 15 environmentally isolates bacteriophages and two control bacteriophages (gamma and Fah) propagated in B.anthracis 4342 against living and dead cells of test bacteria. A= activity of phages against living bacteria, B= activity of lysins against dead cells.

Strains

Bacteriophages

F12

F13

F14

F15

F16

F17

F18

F19

F20

F22

Gamma

Fah

B. anthracis

1584

B. anthracis

211

B. anthracis

SL 1809

B. anthracis

Sterne 34F2

B. thuringiensis

ATCC 33679

–

B. thuringiensis

ATCC 35646

–

+/-

–

B. thuringiensis

ATCC 10792 ^T

–

B. thuringiensis

T7 – 128

–

+/-

–

B. thuringiensis

T7 – 019

–

+/-

–

B. cereus

ATCC 19637

–

+/-

–

B. cereus

UW 85

–

+/-

–

B. cereus

F 17202

–

+/-

–

B. cereus

ATCC 14579 ^T

–

+/-

–

B. cereus

F 16959

–

B. cereus

F – 1728 S

–

+/-

–

B. cereus

ATCC 23261

–

+/-

–

B. mycoides

ATCC 21929

–

+/-

–

B. subtilis

ATCC 6633

–

B. sp. Ba 813

15 (11614-2)

–

B.sp. Ba 813 #

6 (I/2)

–

To further understand the nature of the environmental bacteriophages three isolates, F9, F15 and F17 were selected for further characterization by electron microscopy, PFGE and restriction enzyme digestion. Analysis of the images of F15 and F17 (figures 2 and 3) revealed that these phages belonged to the Siphoviride family. The morphology of F9 has yet to be determined.

Figure 2. An electron Microscopy image of the F15 bacteriophage

Measurement of the phage revealed a head size of 36.9-38nm and a tail of 172 nm.

Figure 3. An electron Microscopy image of the F17 bacteriophage

Measurement of the phage revealed a head size of 43.3-41nm and a tail of 154.35 nm.

To further characterize these phages their DNA was extracted and subjected to pulse field gel electrophoresis (PFGE) and to restriction enzyme digestion.

Pulsed field gel electrophoresis (PFGE)

Following dialysis a 50 µl suspension of phage was mixed with 50 µl of 2 % (w/v) plug agarose (CleanCut, Bio-Rad) and dispended into a plug mold were it was left to solidified. The plug was then removed from the mold, suspended in phage lysis buffer (50mM EDTA, 50mM Tris pH 8.0, 1% w/v SDS and 1mg/ml of proteinase K) and incubated overnight at 54 ºC, with shaking. The digestion buffer was then decanted, and the samples were washed three times using TE buffer (10mM Tris, 1 mM EDTA, pH 8) after which it was incubated in TE buffer (4 ºC, 1 h). The plug was then placed into a well cut into a 1% Pulsed Field Certified agarose (Bio-Rad) made using 0.5 % TBE. Lambda Ladder PFG Markers (New England Biolab) were used as the MW standards. The samples were electrophoresed using a CHEFDRII System (Bio-Rad) at 6Vcm/1 with pulse ramps from 1 to 25 s for 20 h at 14 ºC in 0.5 % TBE buffer. Following electrophoresis, nucleic acids were stained with ethidium bromide (1 µg/ml) for 30 min [Clokie MRJ, Kropinski AM (2009) Bacteriophages: Methods and Protocols.Volume 2: Molecular and Applied Aspects. Humana Press, Totowa, NJ.]

Figure 4. The DNA extracted from the phages F9, F15 and F17 were subjected to pulse field gel electrophoresis (PFGE)

Isolation of the phage DNA for restriction digestion

Following incubation the phage infected culture was treated with chloroform and then centrifugation at 25 000 rpm ( max 115900 x g) for 2 hours at 4 ºC in Sorvall T-890 rotor the phage pellet was suspended in SM buffer and sodium dodecyl sulfate (SDS) (final concentration of 0.5 %) and proteinase K (final concentration 50 µg/ml) were then added. The suspension was then incubated at 55 °C for 1 hour. An equal volume of phenol-chloroform (1:1) was then added to remove the proteinaceous material. This extraction was repeated twice, and the DNA was precipitated according to the standard procedures [Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.] Restriction enzyme digestions of the phage DNA were carried out according to the instructions provided by the suppliers. Restriction endonucleases tested for digestibility of the F9,F15 and F17 phages DNA were: EcoRV, HaeIII and Bsp. After the enzymatic digestion electrophoresis of the samples in 0.4 % (w/v) agarose with addition of ethidium bromide (1 µg/ml) was performed.

As can be seen in Figure 5 the restriction digestion profiles on all three phages differed suggesting that they were unrelated phages.

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