TITLE: An optimized and High Yielding Protocol for Isolation and Amplification of Bacteriophages Against Methicillin-resistant Staphylococcus aureus (MRSA)
AUTHORS: Atif  Khan

[DESCRIPTION]
Bacteriophages are bacteria-specific viruses having great potential as therapeutic agents against multidrug-resistant strains/bacteria.Though "phage therapy" is a century-old concept, there is very limited progress on its therapeutic application due to the rapid expansion in antibiotics portfolios in the last few decades. However, the emergence of multidrug-resistant organisms in the recent past has brought back our attention to bacteriophages. The first step towards developing effective phage therapy against multidrug-resistant bacteria is isolation, amplification, and purification of specific bacteriophages. There are many reported protocols for the isolation of host-specific bacteriophages from the environment. However, most of them are complex, multistep, low-yielding, resource-intensive protocols, requiring elaborate laboratory setup. We have demonstrated a simple two-step, high-yielding protocol for isolating and amplifying bacteriophages against Methicillin-Resistant Staphylococcus aureus (MRSA). We have shown that mixing various environmental samples (i.e. sample pooling) and phage amplification at two different temperatures significantly enhance the yield of MRSA phages.

[STEPS]
SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
1. Collect untreated sewage water samples from the domestic sewage treatment plant and hospital wastewater treatment plant.
 

SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
2. Collect solid fecal samples from cages of chicken poultry farm and store them at 4°C until further processing.
4 °C 
SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
3. Measure 40 mL of each sewage sample and centrifuge them in 50 mL centrifuge tubes at 7000 rpm for 15 min.
40 mL 7000 rpm, 25 °C 
 

 


SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
4. Filter the supernatant obtained through a 0.2 µm filter, and store the filtrate at 4 0C.
4 °C 
 


SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
5. Weigh 10 g of dry poultry farm fecal sample and mix it with 100 mL of sterile saline in a sterile 250 mL conical flask.
10 g 100 mL 
 



SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
6. Keep the suspension at 30 0C for 5 – 6 h at 120 rpm to detach the phages from solid particles.
30 °C   

SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
7. Collect 40 mL of this sample and centrifuge it at 7000 rpm for 15 min.
40 mL 7000 rpm, 25 °C 
 



SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
8. Filter the supernatant obtained through a 0.2 µm filter, and store the filtrate at 4 0C.
4 °C 

SECTION: STEP 1: PREPARATION OF WATER SAMPLE FILTRATE FOR ISOLATION OF BACTERIOPHAGES (CRITICAL STEP)*
9. Mix all three filtrates (Domestic sewage, hospital wastewater, and poultry farm) in a sterile environment to make a single filtrate suspension (pooled sample) for isolation of MRSA-specific bacteriophages.
 


SECTION: STEP 2: GROWTH OF BACTERIAL HOST
10. Thaw the glycerol stock of MRSA stored at -80 °C.
 

SECTION: STEP 2: GROWTH OF BACTERIAL HOST
11. Inoculate 5 µL of the thawed culture in 20 mL of TSB in a 50 ml sterile conical flask and incubate for 24 h at 37 0C and 120 rpm.
5 µL 10 mL 37 °C 

SECTION: STEP 2: GROWTH OF BACTERIAL HOST
12. The grown culture was centrifuged at 10,000 rpm for 10 min and resuspended the pellet in 10 mL of fresh TSB. Use this suspension for the isolation and enrichment of MRSA-specific bacteriophages.
10 mL 10000 rpm, 25 °C 

SECTION: STEP 3: ENRICHMENT/AMPLIFICATION OF MRSA BACTERIOPHAGES
13. Mix 10 mL of pooled sample filtrate (obtained Step 1) and 10 mL of sterile 2X TSB in a 50 mL sterile glass conical flask.
10 mL 

 

SECTION: STEP 3: ENRICHMENT/AMPLIFICATION OF MRSA BACTERIOPHAGES
14. Add 1 mL of an overnight grown pure culture of MRSA (obtained from Step-2) to the above suspension and mix gently. Incubate the suspension at 30 0C for 24 h at 85 rpm.
1 mL 30 °C 

 

SECTION: STEP 3: ENRICHMENT/AMPLIFICATION OF MRSA BACTERIOPHAGES
15. Following incubation at 30 0C, Incubate the suspension at 4 0C for 24-48 hr.
4 °C 

SECTION: STEP 3: ENRICHMENT/AMPLIFICATION OF MRSA BACTERIOPHAGES
16. After 24-48 hr, centrifuge the suspension at 14,000 rpm for 15 min and filter the supernatant through a sterile 0.2 µm syringe filter.
14000 rpm, 25 °C 

SECTION: STEP 3: ENRICHMENT/AMPLIFICATION OF MRSA BACTERIOPHAGES
17. Quantify the titer of bacteriophages in the filtrate using the drop cast method (as described in step 4).
SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
18. Quantification of bacteriophages in the filtrate obtained in any step of the experiment can be done using the drop cast method. 
 

 

SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
19. Take 100 µL of overnight grown pure culture of MRSA (from Step-2) and mix with 3 mL of molten soft agar (TSB + 0.6% Agar).
100 µL 3 mL 

SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
20. Pour the suspension on prepared hard agar (TSB + 2% Agar) and allow it to solidify.
 

SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
21. To quantify bacteriophages in the given filtrate, serially dilute the filtrate in SM buffer and spot 5 µL of each dilution on the bacterial lawn. Allow the plates to solidify completely.
5 µL 

SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
22. Incubate the solidified plates overnight at 37 0C for the development of clear visible plaques. 
37 °C 

SECTION: STEP 4: QUANTIFICATION OF BACTERIOPHAGES BY DROP CAST TEST
23. Calculate the phage titer (PFU/mL) by using the following formula:


Number of PFU/mL=     Number of plaques in a drop X Dilution factor
                                                              Volume of drop spotted