Isolation and characterization of Bacteriophages against Staphylococcus aureus

A dissertation report

1. Dissertation Report (January - April 2018) “ Isolation and characterization of Bacteriophages against Staphylococcus aureus ” Presented By: Shivraj Singh Supervisor:Prof . Gopal Nath

2. Introduction Staphylococcus aureus is a highly versatile gram positive bacterium residing on the skin and in the nasopharynx of approximately 30% of individuals. Nasopharyngeal colonization is a risk for acquiring S. aureus infections, which can cause a range of clinical symptoms that are commonly associated with skin and soft-tissue infections. The emergence of Drug resistant strains of S. aureus (MRSA,VISA,VRSA) has recently become a major public health concern.

3. In the United States, S. aureus infection accounts for approximately 3,00,000 hospitalizations per year. Two vaccine candidates have previously been evaluated in late-stage clinical trials but have not demonstrated efficacy. Currently, There is no vaccine available to prevent invasive Staph. aureus disease or MRSA. Thus , there is an urgent need to explore some alternative therapy for the treatment of such multi-drug resistant infections.

4. Bacteriophages are natural antibacterials able to regulate bacterial populations by the induction of bacterial lysis . They are active against gram-positive as well as gram-negative bacteria including MDR pathogens . Structure of bacteriophage

5. Aim and objectives 1. Collection and identification of S. aureus strains from different clinical samples . 2. Isolation and propagation of bacteriophages against antibiotic resistant isolates of S. aureus . 3. Characterization of S. aureus bacteriophages

6. MATERIALS AND METHODS

7. Identification of Staphylococcus aureus Colony Morphology Microscopic Observation Biochemical Observation Antibiotic Susceptibility Test

8. Colony Morphology Growth on MHA Growth on Blood Agar

9. Microscopic Observation Gram-positive cocci , arranged in grape like cluster. Non-motile and non-spore forming. Fig: Showing gram staining of Staphylococcus aureus

10. Bio-chemical Characteristics S . aureus ferments a number of sugars, producing acid but no gas. Catalase positive. Hydrolyse urea. Produce clear hemolysis on blood agar. Produce a golden yellow pigment. Coagulase positive

11. Biochemical characterization of S.aureus Biochemical reaction of staphylococcus aureus

12. Antibiotic susceptibility test

13. Isolation of bacteriophages Different water samples have been collected from different sites such as Assi ghat , Ravidas nala , and other different Ghats of Ganga River, sewage water of S.S Hospital, BHU and Umang Pharmacy Varanasi.

14. Isolation protocol Sample was taken in 15 ml falcon tube. 1% chloroform was added in the above sample and then it was shaked for 10-20 min. It was centrifuged at 10,000 RPM for 10 min. 1ml supernatant was collected in micro-centrifuge tube.

15. 200 microliter 2x LB broth and 10 microliter host suspension of 2 O.D. was added in the above micro-centrifuge tube . It was incubated at 37 o C for overnight. Again 1% chloroform was added in the above sample and then it was shaked for 10-20 min. It was centrifuged at 10,000 RPM for 10 min. 200 microliter supernatant was collected in micro-centrifuge tube.

16. Now 890 microliter TMG buffer and 10 microliter host suspension of 2 O.D. was added in above glass tube . Then 4 ml soft agar (precooled to 50 o C) was added in the glass tube . It was overlayed on MHA plate. MHA plate was incubated at 37 o C for overnight . Next day the plaque was observed on the plate.

17. Fig: Initial Plaque formation by Bacteriophages on the Lawn of Staphylococcus aureus host Plaque formation by Bacteriophages

18. Purification and Bulk production of bacteriophages  The s ingle plaque was picked from MHA plate.  A plate lysis procedure was used for phage propagation to get a higher titre of phages .  The bacteriophages was purified by using dialysis membrane and filter of 0.22 mm pore size.

19. Table 1: Activity of bacteriophages on various strains of S. aureus

20. Activity of phages on different bacteria

21. Table 3: Effect of different pH on phages -

22. Table 2: Effect of different temperature on phages

23. Relative tire of phages were calculated using the following table The maximum relative titer would be the burst size for the phage

24. Phage no.17 Incubation Time ( Min No. of Plaques observed Actual count (pfu/ml) Relative titre 60 4 4*10 5 2.67 70 1 1*10 5 0.67 80 90 1 1*10 5 0.67 100 110 120 4 4*10 5 2.67 130 1 1*10 5 0.67 140 10 10*10 5 6.67 150 72 72*10 5 48.00

25. Phage no.18 Incubation Time (Min) No. of Plaques observed Actual count (pfu/ml) Relative titre 40 50 1 1*10 5 0.67 60 3 3*10 5 2.00 70 2 2*10 5 1.33 80 90 3 3*10 5 2.00 100 6 6*10 5 4.00 110 12 12*10 5 8.00 120 79 79*10 5 52.67

26. Phage no. 20 Incubation Time (Min) No. of Plaques observed Actual count (pfu/ml) Relative titre 40 3 3*10 5 1.71 50 2 2*10 5 1.14 60 70 2 2*10 5 1.14 80 6 6*10 5 3.43 90 8 8*10 5 4.57 100 3 3*10 5 1.71 110 89 89*10 5 50.86

27. Conclusion Bacteriophages are widely distributed in nature. Phages exist for most of bacteria. With the use of proper techniques and method these phages can be isolated in the laboratory. Therefore phage treatment has extended from medical field to others such as agriculture, fisheries, food industry and waste water treatments. In our study, we were able to isolate the bacteriophages against S. aureus . This pioneer study concluded that sewage water is the chief source for the bacteriophages with a lot of diversity. This study provides a pathway for collection of bacteriophages from different inexpensive sources. Therefore by using this concept, bacteriophages can be used as revitalized therapy against growing antibiotic resistant bacteria .

28. Acknowledgement First and foremost, I would like to express my deep sense of gratitude to my supervisor Prof. Gopal Nath , M.D., Ph.D., F.A.M.S, Professor, Department of Microbiology, Institute of Medical Sciences, BHU, for his critical supervision advice and guidance. He provided me a lot of encouragement and support throughout my stay in the laboratory and I learnt a lot from him. It is proud privilege for me to express my profound regards and deep sense of gratitude to Prof. R.K. Asthana , Coordinator, Applied Microbiology, Prof. N.K. Dubey , Former Coordinator, Applied Microbiology, Prof. R.P. Sinha , Department of Botany, Prof. Ragini Tilak , Department of Microbiology, IMS, BHU and all non-teaching staff of Applied Microbiology course for their valuable advice, pertinent suggestion and ever willing help which definitely helped me in my research paper. I sincerely record my thanks to Ms. Pooja Gupta (CSIR-SRF) and Mr. Virendra Bahadur Yadav (DBT-JRF) for their kind advices that helped me to execute my work along with their expertise assistance . I equally grateful to Mr. Alakh Narayan Singh, Mr. Deepak Kumar, Mr. Rajesh Kumar, Mr. Dilip Kumar Prajapati . I would like to express my special thanks to sister and love to my beloved parents for their constant encouragement, moral and eternal support and patience .