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Title: Diverse antimicrobial activity from Enterococcus faecium NRRL B-30746 bacteriocin

Author
item SVETOCH, EDWARD - ST RES CTR RUSSIA
item ERUSLANOV, BORIS - ST RES CTR RUSSIA
item PERELYGIN, VLADIMIR - ST RES CTR RUSSIA
item VITSEVICH, EVGENI - ST RES CTR RUSSIA
item MITSEVICH, IRINA - ST RES CTR RUSSIA
item BORZENKOV, VALERY - ST RES CTR RUSSIA
item LEVCHUK, VLADIMIR - ST RES CTR RUSSIA
item SVETOCH, OLGA - ST RES CTR RUSSIA
item KOVALEV, YURI - ST RES CTR RUSSIA
item STEPHANSHIN, YURI - ST RES CTR RUSSIA
item Siragusa, Gregory
item Seal, Bruce
item Stern, Norman

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/2007
Publication Date: 6/1/2008
Citation: Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Vitsevich, E.V., Mitsevich, I.P., Borzenkov, V.N., Levchuk, V.P., Svetoch, O.E., Kovalev, Y.N., Stephanshin, Y.G., Siragusa, G.R., Seal, B.S., Stern, N.J. 2008. Diverse antimicrobial activity from Enterococcus faecium NRRL B-30746 bacteriocin. Journal of Agric Food Chem. 56(6), 1942-1948.

Interpretive Summary: Poultry borne Campylobacter and Salmonella continue to cause human disease. Antibiotic resistance of these bacteria has encouraged development of alternative therapeutic treatments. We isolated a bacterium that inhibited these pathogenic bacteria in an agar plate assay. Subsequently, we purified and characterized a protein produced by this inhibitory bacterium, termed a “bacteriocin”. This protein had an impressive range of effectiveness against a wide variety of disease causing bacteria. We demonstrated the effectiveness of this bacteriocin as a therapeutic approach in live chickens which were colonized with Campylobacter jejuni and Salmonella enteritidis. We reduced these two agents by more than 100,000-fold in both the intestine and the liver of the bacteriocin-treated birds. The poultry industry may use this approach to control Salmonella and Campylobacter during chicken production. Technical Abstract: Antibiotic therapy to resolve bacterial disease has been compromised by the increased prevalence and magnitude of bacterial antibiotic resistance. In our efforts to identify new effective antimicrobials, bacteria isolated from poultry intestinal contents were screened for bacteriocin synthesis against Campylobacter jejuni NCTC 11168. From this iterative screening process the bacteriocin producing bacterium, Enterococcus faecium (E 50-52) NRRL B-30746, was selected for detail study. The isolate was grown in broth culture, the cell-free supernatant was precipitated and, multiple steps were used to purify the antimicrobial peptide to single band homogeneity. Genetic and biochemical traits indicated the peptide belonged to Class IIa bacteriocin and was named E 50-52. The peptide had a molecular weight of 3339.7 and pI of 8.0. The MIC of the purified E 50-52 peptide against 96 isolates of C. jejuni ranged from 0.025 to 6.4 µg/ml; for 24 isolates of Yersinia enterocolitica from 0.156 to 1.6; for 29 isolates of Staphylococcus aureus and 11-Listeria monocytogenes from 0.2 to 0.8 µg/ml. The MICs of 31 additional bacterial isolates (Salmonella, E. coli, Shigella, Citrobacter, Klebsiella, Shigella, Pseudomonas, Proteus and Morganella) were <1.6 µg/ml. In therapeutic broiler trials, oral treatment with E 50-52 reduced both Campylobacter jejuni and Salmonella enteritidis by >100,000-fold in the ceca and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens traverses the Gram-negative and Gram-positive barrier and may serve as a promising novel alternative to currently used antibiotics.

Technical Abstract: Antibiotic therapy to resolve bacterial disease has been compromised by the increased prevalence and magnitude of bacterial antibiotic resistance. In our efforts to identify new effective antimicrobials, bacteria isolated from poultry intestinal contents were screened for bacteriocin synthesis against Campylobacter jejuni NCTC 11168. From this iterative screening process the bacteriocin producing bacterium, Enterococcus faecium (E 50-52) NRRL B-30746, was selected for detail study. The isolate was grown in broth culture, the cell-free supernatant was precipitated and, multiple steps were used to purify the antimicrobial peptide to single band homogeneity. Genetic and biochemical traits indicated the peptide belonged to Class IIa bacteriocin and was named E 50-52. The peptide had a molecular weight of 3339.7 and pI of 8.0. The MIC of the purified E 50-52 peptide against 96 isolates of C. jejuni ranged from 0.025 to 6.4 µg/ml; for 24 isolates of Yersinia enterocolitica from 0.156 to 1.6; for 29 isolates of Staphylococcus aureus and 11-Listeria monocytogenes from 0.2 to 0.8 µg/ml. The MICs of 31 additional bacterial isolates (Salmonella, E. coli, Shigella, Citrobacter, Klebsiella, Shigella, Pseudomonas, Proteus and Morganella) were <1.6 µg/ml. In therapeutic broiler trials, oral treatment with E 50-52 reduced both Campylobacter jejuni and Salmonella enteritidis by >100,000-fold in the ceca and systemic S. enteritidis was reduced in the liver and spleen. The wide range of antibacterial activity of bacteriocin E 50-52 against pathogens traverses the Gram-negative and Gram-positive barrier and may serve as a promising novel alternative to currently used antibiotics.