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

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.Microorganisms produce a variety of compounds which demonstrate antibacterial properties. One group of these compounds, the bacteriocins, consists of relatively small bactericidal peptides. The widespread occurrence of bacteriocins in bacterial species isolated from complex microbial communities, such as the intestinal tract, oral surfaces, or other epithelial surfaces, suggests that they may have a regulatory role in terms of population dynamics within bacterial ecosystems. Bacteriocins are defined as compounds produced by bacteria that have a biologically active protein moiety and bactericidal action (51). In recent years, a renewed interest in bacteriocinlike activities has led to the discovery, isolation, and purification of bacteriocins from both gram-negative and gram-positive organisms (28). They are now being considered for a variety of antimicrobial uses in foods and medicine (17, 41).Lactic acid bacteria (LAB) are among the most well known and investigated producers of microbial antagonists. These include the well-characterized bacteriocins (6, 30, 55), potential bacteriocinlike substances (57), and other antagonists not necessarily related to bacteriocins (24,36,44). The LAB are gram-positive, non-spore-forming, catalase-negative organisms devoid of cytochromes. They are anaerobic...

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…Recently, some class IIa bacteriocins have been found to be effective against both G + and G -bacteria, such as Campylobacteri jejuni and Escherichia coli O157:H7 (Drider et al 2006;Stern et al 2006;Line et al 2008;Svetoch et al 2008).…”

Characterization of bacterial antimicrobial peptides active against Campylobacter jejuni

“…While many of the bacteriocins typically produced by enterococci are inactive against C. jejuni, 74 novel bacteriocins with this activity have been described. 75,76 There are also reports of anti-Campylobacter bacteriocins wherein the active component has not been characterized. 73 As with some Lactobacillus spp., there is evidence that some enterococci exert antagonism towards C. jejuni through means other than bacteriocins.…”

“…This bacteriocin showed promise in vivo, decreasing the Campylobacter counts of broiler chickens artificially challenged with C. jejuni, in addition to decreasing the levels of naturally acquired C. jejuni. 75 The propensity for C. jejuni to develop resistance to enterocin E-760 was recently studied. 7 Following the administration of E-760 to chickens, C. jejuni isolates only showed the development of low levels of resistance.…”

“…NRRL B-30745, an isolate from chicken ceca, was found to display strong inhibitory activity towards both Grampositive and Gram-negative bacteria including S. enterica and C. jejuni. 75 After several chromatographic steps, this broad spectrum activity was assigned to enterocin E-760. This bacteriocin showed promise in vivo, decreasing the Campylobacter counts of broiler chickens artificially challenged with C. jejuni, in addition to decreasing the levels of naturally acquired C. jejuni.…”

Identification of Three Novel <i>B. thuringiensis</i> Strains that Produce the Thuricin S Bacteriocin

“…They are Grampositive, non-spore-forming, catalase-negative, acid tolerant, and strictly fermentative, with lactic acid as the major end product of sugar fermentation (Line et al, 2008). The best known and most fully studied bacteriocin from LAB is nisin, a lantibiotic produced by Lactococcus lactis which has been accepted by the world Health Organization as a preservative in food industry (Abee, 1995;Jack et al, 1995;Delves-Brougnton et al, 1996).…”