Isolation and Purification of Enterocin E-760 with Broad Antimicrobial Activity against Gram-Positive and Gram-Negative Bacteria

Abstract

ABSTRACT Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans , Enterococcus faecium , or Enterococcus hirae , was initially identified as antagonistic to Campylobacter jejuni . The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica , Citrobacter freundii , Klebsiella pneumoniae , Shigella dysenteriae , Pseudomonas aeruginosa , Proteus mirabilis , Morganella morganii , Staphylococcus aureus , Staphylococcus epidermidis , Listeria monocytogenes , Campylobacter jejuni , and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies. The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically, the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100°C and was stable at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated feed significantly ( P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log 10 CFU. Enterocin E-760 also significantly ( P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.