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Report on secondment from UHOH at the Eliava Institute (Tbilisi, Georgia)

Researcher details:

Details of the secondment

Home institute:Institute of Animal Sciences, Department of Environmental and Animal HygieneUniversity of Hohenheim, 70599 Stuttgart, Germany

Host institute:G. Eliava Institute of Bacteriophages, Microbiology and VirologyTbilisi, Georgia

Dates of the secondment: 20.09.2015 – 20.10.2015

Research activities

Laboratory work was aimed at:

- Cultivation and propagation of 4 Bacteriophage samples isolated from Bacillus anthracis- Determination of phage titers after propagation and spot test for viability- Stable infection and propagation of each phage in B. anthracis Sterne (34F2)- Phage-DNA-extraction- Restriction analyses of the phage-DNA for differentiation

Table 1 represents the characteristics of the examined phages and results of previous experiments.

First name Carina Last name RohmerAddress Maienfeld 17, 88477 Weihungszell, GermanyContact number +4915221774795Email address carina.rohmer@gmail.comGender FemaleDate of birth 03.12.1990Nationality Germany

Table 1: phage characteristics

Sample Viral family Sporulation test Microscopy Biofilm formation

SA 60 Myovirus -

stable infected B. anthracis A58 did not build chains yes

SA 156 Siphovirus

Inhibition of sporulation in stable infected B. anthracis A58 - no

AF 137 Siphovirus

Increased sporulation in stable infected B. anthracis A58 - no

SA 137 initial 2 phages mixed -

stable infected B. anthracis A58 did not build chains yes

SA 137 turbid ? - - ?SA 137 clear ? - - ?

1. Spot test for viability of the phages

Susceptible host strains, B. anthracis Sterne (34F2) and B. anthracis cmu, from the laboratory at EIG were cultivated and each phage sample tested via spot test for viability. To this purpose 200 µl of each host strain in liquid medium was mixed with 3 ml top agar and plated on BHI-agar plates. After solidification 10 µl of each phage sample was dropped on the plate surface. Plates were incubated over night at 37°C.

Large plaques were visible on each plate the next day (fig. 1), proving the presence of viable phages in all phage preparations.

Fig. 1: Spot test for viability on BHI agar (Host: B. anthracis Sterne (34F2))

2. Propagation and determination of phage titers

Serial dilutions of the phage suspensions were made in 5 ml of BHI-broth (10 -1 to 10-8). For the propagation of the phages, 1 ml each of the 10-4, 10-5, 10-6 and 10-8 dilution steps was mixed with 100 µl of a liquid overnight culture of the host strain 34F2. Three ml of top agar was added and the mixture plated each onto one BHI-plate. Plaque counting was done after overnight incubation at 37°C.

Next day the top agar-layer of the 10 -4, 10-5 and 10-6 dilutions, where bacteria were growing in a net-like form due to the lytic activity of the phages, of each sample was collected in a fresh tube, 1 ml of BHI-broth for each plate (3 ml total) was added and the tubes were centrifuged at 3100 x g for 45 minutes. The supernatant was filtered through a 0,22 µm pore size filter and plaque counting by serial dilution was performed again.

Unexpectedly, at plates of SA 137 with single plaques two different plaque forms were identified, one turbid and one clear form. The turbid plaques were rare but bigger in size than the little clear ones. A turbid and one clear plaque were picked and propagated separately. For further differentiation the sample containing both phages will be labelled “SA 137 initial”, the propagated sample originating from the clear plaque “SA 137 clear”, and the turbid one “SA 137 turbid”.

Table 2: Results of the phage titers after propagation

Sample No. Phage Titer after propagation1 SA 60 4,0 x 1010

2 SA 156 6,5 x 1010

3 AF 137 2,7 x 1010

4 SA 137 initial 1,5 x 1010

5 SA 137 turbid 5,3 x 1010

6 SA 137 clear 2,3 x 1010

3. Phage-DNA extraction

At first the DNA Extraction QIAamp® DNA Mini Kit (250) (QIAGEN) was used, but the resulting DNA-content was too low. Therefore a classical “Phenol-Chloroform-Extraction method” was performed (please see suppl. materials for protocol). Amount and quality of the DNA was checked by gel-electrophoresis (fig. 2) and the DNA finally stored at -20°C until usage.

Fig. 2: DNA content after Phenol-Chloroform-Extraction. M = marker (Lambda-DNA-HindIII-Digest; Amersham Biosciences); 1 = SA 60 (2 µl); 2 = SA 156 (2 µl); 3 = AF 137 (2 µl); 4 = SA 137 initial (2 µl); 5 = SA 137 turbid (2 µl)

4. Restriction analysis of phage-DNAPhage DNA was digested with Enzymes (New England Biolabs Inc., Ipswich, MA, United States). Digestion was performed in 20 µl containing 1 µl of the appropriate enzyme and buffer as recommended by the supplier for 16 hours at 37 °C (except Sma I, incubation at 25°C for 16 hours). DNA from the phage preparations was added as follows: SA 60 (2,5 µl), SA 156 (3 µl), AF 137 (4,5 µl), SA137 initial (4,5 µl), SA 137 turbid (2,5 µl) and SA 137 clear (3,5 µl).

5. Agarose gel electrophoresis

Gels with 0,8 or 1% agarose were used depending on the fragment sizes expected. Electrophoresis was performed for 2 to 3 hours at 80 V and 150 mA.

Fig. 3: Digest with restriction enzymes HindIII and XbaI in 1% Agarose-gel

Fig. 4: Digest with restriction enzymes PstI and SmaI in 0,8% Agarose-gel

Fig. 5: Digest with restriction enzyme Pvu II in 1% Agarose-gel

The resulting electrophoresis images (fig. 3-5) show the appearance of at least 4 different phage types. SA 156, AF 137 and SA 137 clearly show different digestion patterns whereas SA 60 and SA 137 turbid reveal the same restriction pattern with all enzymes used, thereby preventing the differentiation between these two phage isolates. Whether or not they are identical indeed would need more detailed investigations. SA 60 had earlier been characterized via TEM as a Myovirus, so one can speculate that SA 137 turbid should present itself also a member of the Myoviridae family.

SA 137 initial and SA 137 clear also show the same restriction patterns in all digests. This can be explained by the fact that SA 137 initial consist of a mixture of SA 137 clear and turbid, where the titer of the clear form is much higher than those of the turbid one.

Supplemental material

Protocol Phenol-Chloroform-Extraction

Prepare your phage lysate by plate lysate or liquid culture methods. Lysates should be purified by filter sterilization (0,22 µm). Lysates may be in broth (BHI, LB, TSB etc) or in saline. High-titer phage lysate is used for DNA extraction.

Reagents required

Phage lysate (0,5 ml; >10 PFU/ml)Nuclease solution (20 mg/ml DNase I; 20 mg/ml RNase A)Proteinase K10% SDS0,5 M EDTA pH 8,0PhenolChloroformIsoamyl alcohol70% ethanolSterile molecular biology-grade water5 M NaCl96% ethanol

Materials required

Sterile 1,5 ml microcentrifuge tubesMicrocentrifugeHeat block (65 °C)

Lysis buffer

100 µl 10% SDS (final concentration 1% SDS)100 µl 0,5 M EDTA pH 8,0 (final concentration 50 mM EDTA)800 ml dH20

Protocol- Place the 500 µl lysate into a clean centrifuge tube. Add 0,5 µl of nuclease solution per ml of lysate

(10 µg/ml DNase & RNase).- Incubate the lysates at 37 °C for 30 min.- To each 500 µl aliquot of resuspended phage solution add proteinase K to a final concentration of

100 µl/ml and incubate at 56 °C for 15 min.- Add 50 µl lysis buffer and incubate at RT for 15 minutes.

- Add phenol 1/1 (vol/vol) to the sample. Mix by inverting the tube for 10 minutes and centrifuge at 10.000 x g for 10 minutes. Transfer supernatant into a new tube.

- Add phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol). Mix by inverting the tube for 10 minutes and centrifuge at 10.000 x g for 10 minutes. Transfer supernatant into the new tube.

- Add chloroform/isoamyl alcohol 24/1 (vol/vol). Mix by inverting the tube for 10 minutes and centrifuge at 10.000 x g for 10 minutes. Transfer supernatant into a new tube

- Add 20 µl 5 M NaCl and 1980 µl 96% ethanol to the sample. Mix by inverting the tube and incubate for 30 min at -20°C.

- Centrifuge the tubes at 10.000 x g for 10 minutes. Discard supernatant carefully.- Wash the pellet with 1 ml 70% ethanol.- Centrifuge tubes at 10.000 x g for 10 minutes.- Let pellet dry.- Resuspend pellet in 50 – 100 µl sterile TE buffer.