1. 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 , Haryana
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).

5. Bacteriocins
BACTIBASE dataset (version 2, July 2009)
Ribosomally synthesized small peptides antimicrobial activity

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

7. 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 infection in a mouse model (Lopez et al. 2007).
Fermenticin HV6b and nisin ZP inhibit wide range of pathogens, spermicidal and anticancerous activity as reported to induce apoptosis in cancerous cells (Kaur et al. 2013; Kamrajan et al. 2015).

8. 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

9. Mode of action of bacteriocins

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

11. Design and Synthesis of Hybrid Bacteriocins

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

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

14. Comparison of MIC of WT and hybrid bacteriocins

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

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

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

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

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

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

21. 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

22. Acknowledgements ICMR CSIR 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

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