Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages

Enterocin A and B in Enterococcus faecium CTC492 were co-induced by the different factors assayed in this study (r = 0.93) and followed primary metabolic kinetics. Enterocin production was significantly inhibited by sausage ingredients and additives, with the exception of nitrate. The addition of sodium chloride and pepper decreased production 16-fold. The temperature and pH influenced enterocin production, with optima between 25 and 35 degrees C, and from 6.0 to 7.5 of initial pH. The maximum activity was achieved, under favourable growth conditions, with MRS supplemented with sucrose (2%) plus glucose (0.25%) and Tween-80 (1%). MRS concentration, NaCl plus pepper addition, absence of Tween-80 in the growth medium, incubation at 45 degrees C and an initial pH under 5.5 were detrimental to bacteriocin production. Stress conditions did not favour enterocin production. Desadsorption was Tween-dependent. Enterocin A activity in the crude extracts stored at -80 degrees C was better preserved than enterocin B (when tested against their specific indicator strain), but anti-listerial activity remained intact. Applied as anti-listerial additives in dry fermented sausages, enterocins significantly diminished Listeria counts by 1. 13 log (P < 0.001), while Enterococcus faecium CTC492 added as starter culture did not significantly reduce Listeria counts (P > 0. 1) compared with the standard starter culture (Bac-). Enterocins A and B could be considered as extra biopreservative hurdles for listeria prevention in dry fermented sausages.     

Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density

The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system. The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway. The culture supernatants of C. piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C. piscicola LV17A or E. faecium CTC492. The cbaX fusion product induced bacteriocin production by Bac(-) C. piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E. faecium CTC492. This represents a relatively simple method of confirming the role of presumptive IPs. The transformation of C. piscicola LV17A with the CbaX gene under expression of the P32 promoter from L. lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.     

Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media

Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorusDCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faeciumCTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification.     

Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faecium T136

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.     

Purification and characterization of enterocin FH 99 produced by a faecal isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> FH 99

Enterococcus faecium FH 99 was isolated from human faeces and selected because of its broad spectrum of inhibitory activity against several Gram-positive foodborne spoilage and pathogenic bacteria. Ent. faecium FH 99 accumulates enterocin in large number in early stationary phase of the growth. The enterocin FH 99 was stable over a wide pH range (2–10) and recovered activity even after treatment at high temperatures (10 min at 100°C). The enterocin was subjected to different purification techniques viz., gel filteration, cation exchange chromatography and reverse-phase high-performance liquid chromatography. The activity was eluted as one individual active fraction. SDSPAGE revealed a molecular weight of less than 6.5 kDa. Studies carried out to identify the genetic determinants for bacteriocin production showed that this trait may be plasmid encoded as loss in both of the plasmids (size>chromosomal DNA) led to loss in bacteriocin production by Ent. faecium FH 99. Ent. faecium strain FH 99 is a newly discovered high bacteriocin producer with Activity Units 1.8 × 10⁵ AU ml⁻¹ and its characteristics indicate that it may have strong potential for application as a protective agent against pathogens and spoilage bacteria in foods.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

Comparison of Two Methods for Purification of Enterocin B,a Bacteriocin Produced by Enterococcus faecium W3

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption–desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.     

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.     

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Phenotypic and genotypic characterization of bacteriocins in clinical <Emphasis Type="Italic">enterococcal</Emphasis> isolates of Tunisia

The presence of bacteriocin structural genes (entA, entB, entP, entQ, entAS-48, entL50A/B, bac31, and cylL) encoding different bacteriocins (enterocin A, enterocin B, enterocin P, enterocin Q, enterocin AS-48, enterocin L50A/B, bacteriocin 31 and cytolysin L, respectively), and the production of bacteriocin activity were analysed in 139 E. faecalis and 41 E. faecium clinical isolates of Tunisia. Forty-eight of 139 E. faecalis isolates (34%) and 7 of 41 of E. faecium isolates (17%) were bacteriocin producers. Sixty-two per cent of the bacteriocin-producing enterococci showed inhibitory activity against L. monocytogenes. Different combinations of entA, entB, entP, and entL50A/B genes were detected among the seven bacteriocin-producer E. faecium isolates, and more that one gene were identified in all the isolates. The entA gene was associated in most of the cases with entB gene in E. faecium isolates. Cyl _LS were the unique genes detected among E. faecalis (in 24 of 48 bacteriocin-producer isolates, 50%). A β-hemolytic activity was demonstrated in 19 of the 24 cyl _LS -positive E. faecalis isolates (79%), this activity being negative in the remaining five isolates. The presence of different bacteriocin structural genes and the production of antimicrobial activities seems to be a common trait of clinical enterococci.     

Biochemical and Genetic Evidence of Enterocin P Production by Two Enterococcus faecium-Like Strains Isolated from Fermented Sausages

Two bacteriocin-producing Enterococcus faecium-like strains were independently isolated from fermented sausages. Bacteriocins were purified to homogeneity by ammonium sulfate precipitation, gel filtration, cationic exchange, hydrophobic interaction, and reverse-phase liquid chromatography. Two peptide inhibitory fractions were purified from each strain, denominated A and B for E. faecium AA13, and C and D for E. faecium G16. Fraction B was blocked for amino acid sequencing by Edman degradation, while the amino acid sequences obtained from peptides A, C, and D contained the YGNGV consensus motif in positions 5 to 9, and the ATRS sequence in positions 1 to 4. By use of PCR techniques and nucleotide sequencing, the structural gene of enterocin P was found both in E. faecium AA13 and E. faecium G16. Metabolic and genetic features of the two strains suggest that they are slightly different, they may produce more than one bacteriocin, and both produce enterocin P. Received: 13 May 1999 / Accepted: 22 June 1999     

Production of enterocin A by Enterococcus faecium MMRA isolated from ‘Rayeb’, a traditional Tunisian dairy beverage

Aims: Characterization and purification of a bacteriocin produced by a wild Enterococcus faecium strain, isolated from a Tunisian traditional fermented milk. Methods and Results: Enterococcus faecium MMRA was selected on the basis of its strong anti‐Listeria activity. The antibacterial activity was sensitive to proteases, confirming its proteinaceous nature. It was extremely heat stable (15 min at 121°C), remained active over a wide pH range (2–12), and also after treatment with lipase, amylase, organic solvents, detergents, lyophilisation and long‐term storage at ?20°C. Production of the bacteriocin occurred throughout the logarithmic growth phase, it did not adhere to the surface of the producer cells and the mode of action was bactericidal. After partial purification of the active supernatants, a 4‐kDa band with antibacterial activity was revealed by SDS‐PAGE electrophoresis and bioassay. Tryptic digestion followed by MALDI‐TOF mass spectrometry identified the peptide as enterocin A. Conclusions: The inhibitory activity of Ent. faecium MMRA, a wild strain isolated from the artisan dairy beverage ‘Rayeb’, is due to the synthesis of an enterocin A. Significance and Impact of the Study: Traditional fresh Tunisian fermented dairy products are generally manufactured with raw milk that can be used as a source of uncharacterized wild lactic acid bacteria strains. To our knowledge, this is the first report on the isolation of an enterocin A producing Ent. faecium from ‘Rayeb’. This bacteriocin or the producing strain might have a promising potential in biopreservation to enhance the hygienic quality of this dairy product.     

Anti-Listeria bacteriocin-producing bacteria from raw ewe's milk in northern Greece

Aims: The isolation and partial characterization of anti‐Listeria bacteriocin producing strains present in milk from areas of northern Greece in view of their potential use as protective cultures in food fermentations. Methods and Results: Three hundred and thirty‐two isolates were obtained from milk samples intended for Feta cheese production and gathered from 40 individual producers in Northern Greece. Isolates with anti‐Listeria activity were identified by multiplex PCR as Enterococcus faecium and grouped by (GTG)₅‐PCR. The genomes of the anti‐Listeria isolates were examined for the presence of known enterocin genes and major virulence genes by means of specific PCR. At least three known enterocin encoding genes were present in the genome of each of the 17 isolates. None of the 17 isolates harboured any of the virulence genes tested for or exhibited haemolytic activity. Conclusions: Enterococcus faecium was the dominant anti‐Listeria species in the milk samples. The isolates had the potential of multiple bacteriocin production and did not exhibit some important elements of virulence. Significance and Impact of the Study: Enterococci present in milk of this area of northern Greece may be partly responsible for the safety of Feta cheese and could be useful for the production of anti‐Listeria protective cultures.     

Purification and Characterization of a Novel Bacteriocin Produced by Enterococcus faecalis Strain RJ-11

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.     

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C₂/C₁₈ reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.     

The production in vivo of microcin E492 with antibacterial activity depends on salmochelin and EntF

Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation.     

Identification and Heterologous Expression of the <Emphasis Type="Italic">sec</Emphasis>-Dependent Bacteriocin Faerocin MK from <Emphasis Type="Italic">Enterococcus faecium</Emphasis> M3K31

Genome sequencing of Enterococcus faecium M3K31, a strain isolated from griffon vultures, previously revealed the presence of three sequences encoding for bacteriocins, namely enterocin P, enterocin HF, and a SRCAM 602-like bacteriocin. In this work, we describe the SRCAM 602-like bacteriocin that we named faerocin MK. Mature faerocin MK consists of 43 residues and contains an YGNGV-motif and two cysteine residues at positions 10 and 15, consistent with other class IIa bacteriocins. Faerocin MK and SRCAM 602 were chemically synthesized and their scope of activity was tested. Faerocin MK is active against a wide range of Gram-positive organisms while SRCAM 602 was not active against any tested organism. Although both peptides are more structured in trifluoroethanol, faerocin MK has an α-helical character nearly twice that of SRCAM 602. Nucleotide sequence analysis revealed that the faerocin MK precursor is produced with a 28-amino acid signal peptide and that an immunity gene follows the structural faerocin MK gene. Heterologous expression of the faerocin MK operon showed that faerocin MK and its immunity protein were successfully expressed in other organisms. This indicates that the bacteriocin is secreted through the sec pathway.     

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.