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Santosh Kumar Tiwari, PhD Assistant Professor Department of Genetics Maharshi Dayanand University

Design of synthetic-hybrid bacteriocins from enterocin E50-52 and pediocin PA-1 for therapeutic applications. Santosh Kumar Tiwari, PhD Assistant Professor Department of Genetics Maharshi Dayanand University Rohtak-124001, Haryana Email: santoshgenetics@gmail.com.

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Santosh Kumar Tiwari, PhD Assistant Professor Department of Genetics Maharshi Dayanand University

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  1. Design of synthetic-hybrid bacteriocins from enterocin E50-52 and pediocin PA-1for therapeutic applications Santosh Kumar Tiwari, PhD Assistant ProfessorDepartment of Genetics Maharshi Dayanand University Rohtak-124001, HaryanaEmail: santoshgenetics@gmail.com 6th World Congress on Biotechnology, October 05-07, 2015, New Delhi

  2. Antibiotics Vs Resistance

  3. World Health Organization (WHO) foreseen: • Almost one billion people will be infected with Mycobacterium tuberculosis between the years 2000 and 2020. • About 35 million humans will die till 2020 as a result of tuberculosis in antibiotic-resistant form . • Over 70% of bacterial pathogens that cause fatal infections are likely to be resistant to at least one of the drugs (Infectious Disease Society of America, IDSA). • Several preventive measures have been taken to avoid the microbial resistance development, but still there is an urgent need for new antimicrobial agents and new strategies to overcome problematic resistant pathogens. • Antimicrobial peptides (AMPs), particularly bacteriocins produced by bacteria, may be an important contributor in this context as they often have a relatively narrow killing spectrum (Nes et al. 2007).

  4. Bacteriocins BACTIBASE dataset (version 2, July 2009) Ribosomally synthesizedsmall peptidesantimicrobial activity

  5. Diversity BACTIBASE dataset (version 2, July 2009) Total bacteriocins :177 Gram-positive bacteria : 156 (113 from lactic acid bacteria) Gram-negative bacteria : 18 Archaea domain : 3

  6. Therapeutic potential of bacteriocins • Thuricin CD isolated from Bacillus thuringiensis DPC6431, specifically eliminates Clostridium difficile without disrupting the beneficial microbial community (Rea et al. 2010). • Nisin, mersacidin and lacticin 3147 can eradicate infections caused by Streptococcus pneumoniae, MRSA in mice, tooth diseases in dogs and bovine mastitis in dairy cows (Òkuda et al. 2013). • Microcin J25 has been shown to drastically reduce Salmonella infectionin a mouse model (Lopez et al. 2007). • Fermenticin HV6b and nisin ZP inhibitwide range of pathogens, spermicidal and anticancerous activity as reported to induce apoptosis in cancerous cells (Kaur et al. 2013; Kamrajan et al. 2015).

  7. Nisin: A bacteriocin produced by Lactococcus lactis The residues in red have positive net charge, blue are hydrophobic. Dha, dehydroalanine; Dhb, dehydrobutyrine; Lan, lanthionine; Mla, methyllanthionine; S, thioether bridge NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | JULY 2006 | 531

  8. Mode of action of bacteriocins

  9. Design and Synthesis of Hybrid Bacteriocins Pediocin PA-1 TTKNYGNGVCNSVNWCQCGNVWASCNLATGCAAWLCKLA Enterocin E50-52

  10. Design and Synthesis of Hybrid Bacteriocins

  11. Producer strain Indicator strain Spot assay plate method Agar Well Diffusion Assay (AWDA) AU/ml OR MIC Percentage inhibition of indicator strain Methods for detection of antimicrobial activity

  12. Minimum Inhibitory Concentration (MIC) Enterocin E50-52 (B) Pediocin PA-1 EP PE (C) (D)

  13. Comparison of MIC of WT and hybrid bacteriocins

  14. ATP Efflux Control PA1 E50 EP PE E. coli O157:H7 Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090

  15. Dissipation of membrane potential Micrococcus luteus ATCC 10420 Cells Fluorescence (au) Bacteriocin Valinomycin DiSC3(5) Probe Nigericin Glucose Time (s)

  16. E. coli untreated 1000 950 900 Dissipation of membrane potential 850 800 750 700 E. coli treated with EDTA only 650 600 Fluorescence (au) (au) E. coli treated with lysozyme and EDTA 550 500 450 400 350 300 250 Vancomycin 200 Glucose Nigericin E50-52 150 100 DiSC3(5) 50 0.1 0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 Time (s)

  17. Inhibition pattern of target bacteria by wildtype and hybrid bacteriocins Control PA1 E50 EP PE E. coli O157:H7 Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090

  18. Tiwari et al. 2015. Applied and Environmental Microbiology 81: 1661-1667.

  19. Bacteriocins in our laboratory UGC CSIR Plantaricin LD1 Enterocin LD3 Halocin HA1, 3 Pediocin LB44 Wisellicin LM85 DST ICMR Bacteriocins Mode of Action Hybrid bacteriocins HTP screening Halocin HA3 DBT IUSSTF

  20. Research Group MSc Dissertation Aabha Komal Gitika Anu Gita Parul Nidhi Karishma Monica Nandita, Jyoti Ritu Bhawana Sonia Pooja Naveen PhD completed Aabha PhD completed Ramanjeet PhD Student Vijay PhD Student Manoj Project Fellow Poonam Project Fellow

  21. Acknowledgements Prof. Sheela Srivastav Department of Genetics University of Delhi South Campus, New Delhi Prof. Michael L. Chikindas School of Environmental and Biological Sciences Rutgers State University, New Jersey ICMR CSIR

  22. Felicitated for Indo-US Research Fellow by IUSSTF, New Delhi.

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