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International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

Purification and characterization of Enterocin OS13 α & β; novel bacteriocins from a food isolate Enterococcus faecalis OS13 with potent activity towards antibiotic resistant nosocomial enterococci. Ahmed Osama Elgendy (PhD) Department of Microbiology and Immunology

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International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

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  1. Purification and characterization of Enterocin OS13 α & β; novel bacteriocins from a food isolate EnterococcusfaecalisOS13 with potent activity towards antibiotic resistant nosocomialenterococci Ahmed Osama Elgendy (PhD) Department of Microbiology and Immunology Faculty of Pharmacy, Beni-Suef University Egypt International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  2. Overview Background Attempted efforts Concluding Remarks International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  3. Background Antibiotic Resistance Multi Drug Resistance (MDR) http://www.hsri.or.th/en/media/948 International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  4. Enterococci Enterococci Enterococci One of the major causes of nosocomial infections especially at ICUs Enterococci Enterococci Enterococci Enterococci International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  5. Suggested solutions Rational and/or restricted antibiotic use Search for new non conventional sources of antimicrobials International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  6. Bacteriocins Antimicrobial peptides produced by bacteria that inhibit or kill closely related species or different strains of the same species. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  7. Efforts attempted 65 isolates Level I : 12 isolates Level II : 1 isolate Level III : International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  8. 65 Enterococus isolates 43 Food isolates22 Clinical isolates Urine, Stool, Vaginal and Pus. Efforts attempted • I. Identification • Gram stain • Bile esculin agar • PYR test • Sequencing of 16s rRNA gene • II. Phenotypic Characterization of some virulence factors • Test for Blood hemolysis • Test for Gelatinase enzyme • Bile salt hydrolase activity • III. Investigation for Bacteriocin Production • Spot on lawn assay • Diffusion zone International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  9. 31.5 % 13% 55% 55% 58% International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  10. 12 strains with relevant antimicrobial activities was detected. (3 isolated from food samples and 9 from clinical samples). International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  11. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  12. 65 isolates Level I : 12 isolates Level II : 1 isolate Level III : International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  13. 12 bacteriocinogenic isolates • Cont. Phenotypic Characterization: • Fermentation profile • Antimicrobial susceptibility testing Genotypic Characterization: Multi Locus Sequence Typing (MLST) PCR Screening for different known bacteriocin genes International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  14. Fermentation Profile (API 50 CH): All the 12 isolates have the same fermentation profile where they truly ferment the following : Glycerol, Ribose, Galactose, Glucose, Fructose, Mannose, Mannitol, Sorbitol, N-Acetyl Glucosamine, Amygdaline, Arbutine, Esculine, Salicine, Celibiose, Maltose, Saccharose, Trehalose, Melezitose, Gentibiose, Tagatose, Potassium Gluconate. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  15. Antimicrobial susceptibility testing: International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  16. ** Genotypic Characterization: Multi Locus Sequence Typing (MLST) • A multilocus sequence typing (MLST) scheme based on seven housekeeping genes. • MLST is used to investigate the epidemiology and population structure of Enterococcus faecalis. • Clonal Complex 2 (CC2) and Clonal Complex 9 (CC9) mostly spread in the hospital environment. • (CC2) include these Sequence types [ ST6, ST2, ST51] • (CC9) include these Sequence types [ ST9, ST17, ST18, ST42, ST52] • Enterococcus faecalis V583 belongs to ST6 (CC2). International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  17. Published E. faecalis strains with ST116 International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  18. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  19. PCR Screening for presence of different known bacteriocin genes: (18 genes) Revealed absence of any of these genes except Cytolysin and Enterolysin A genes inside only strain OS6 while the antimicrobial activity in other strains may be regarded to a bacteriocin different from those checked by PCR or due to effects of other metabolites or they could be potentially novel bacteriocin. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  20. 38 isolates Level I : 12 isolates Level II : 1 isolate Level III : International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  21. 1 bacteriocinogenic isolate • Bacteriocin purification and characterization We choose the bacteriocin from strain OS13 for these purification steps. (This strain is a highly bacteriocin producer) E. faecalis OS13 was shown to produce antimicrobial activity against some of closely related bacteria: (Lactobacillus sakei, Lactobacillus plantarum and E. faecium) Other species of gram positive bacteria as Listeria and Staphylococcus were resistant. All tested gram negative bacteria and fungi were resistant. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  22. Bacteriocin purification and characterization: • These Steps were followed in our work: • Protein precipitation by Ammonium sulphate. • Ion exchange chromatography (SP-Sepharose Cataion Exchange) • SDS – PAGE • First Reverse phase chromatography (RPC1 column) • Second Reverse phase chromatography (Sephasil peptide C8 column) • MALDI – TOF / MS analysis International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  23. SDS-PAGE : International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  24. Reverse Phase Chromatography: • - Sephasil C8 International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  25. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  26. Mass spectrometry: (MALDI-TOF) Enterocin OS13α Enterocin OS13β International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  27. Peptide mass similarity search was done at ExPASy using TagIdent tool (http://web.expasy.org/tagident/) and revealed that there was no molecular weight similarity with any other antimicrobial peptides except holotricin-2 (7858 Da) purified from larvae of a Coleopteran insect, Holotrichiadiomphalial (Lee at al., 1994) indicating that they could be novel bacteriocins especially when compared their molecular weights with all those recovered from Enterococcus spp. and published online International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

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  29. Effect of heat and Proteinase K enzyme on detected bacteriocin Crude enterocin OS13 was sensitive to both heat and proteinase K enzyme Purified enterocin OS13α (1,024,000 BU/ml) and Enterocin OS13β (128,000 BU/ml) became (zero BU/ml) after heat treatment while became 512,000 BU/ml (50 %) and 128,000 BU/ml (100 %) respectively after Proteinase K treatment. International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  30. Concluding Remarks • Bacteriocin peptides like the enterocin OS13 α & β represent novel antimicrobial agents perfectly designed to combat multiresistant nosocomial Enterococcal infections, without disturbing commensal microflora. • However, several safety criteria should be taken into consideration for E. faecalis producing bacteriocins before being used in any proposed application. • Further work is needed for all possible biotechnological applications of the detected bacteriocin International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  31. Special Thanks International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  32. Special Thanks International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  33. Thank You International Scientific Conference on Bacteriocins and Antimicrobial Peptides (Kosice 2013)

  34. Egypt Ancient Civilizations

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