Isolation and biochemical characterisation of enterocins produced by enterococci from different sources

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.     

Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish

Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4 Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D.     

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.     

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in G?ttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium

AIMS: Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s). METHODS AND RESULTS: LAB were present in high numbers in final products as well as during processing. Isolates, Enterococcus faecium B1 and E. faecium B2 (E. faecium LMG 19827 and E. faecium LMG 19828, respectively) inhibited Gram-positive indicators, including Listeria monocytogenes. Partially purified bacteriocins showed a proteinaceous nature. Activity was stable after heat-treatment except at alkaline pH values. Both strains displayed a bacteriostatic mode of action. Bacteriocin production was associated with late exponential/early stationary growth. Molecular mass, calculated by SDS-PAGE, was 3.4 kDa for B1 bacteriocin, and 3.4 kDa and 5.8 kDa for B2 bacteriocins. PCR screening of enterocin-coding genes revealed three amplified fragments in total genomic DNA that may correspond with PCR signals for enterocin P, enterocin L50A and enterocin L50B. Both B1 and B2 contained a 42-kb plasmid. No differences in bacteriocinogenic capacity were found between wild type strains and plasmid-cured strains. CONCLUSIONS: It was possible to isolate bacteriocinogenic E. faecium active against various Gram-positive bacteria from final products of tempeh. SIGNIFICANCE AND IMPACT OF THE STUDY: A first step in applying biopreservation to fermented South-east Asian foods is to obtain bacteriocinogenic LAB from this source. Such isolates may also be used for biopreservation of mould-fermented foods in general, including various types of mould-ripened cheese.     

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.     

Bacteriocinogenic Potential of <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated from Wine

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.     

Description of durancin TW-49M, a novel enterocin B-homologous bacteriocin in carrot-isolated Enterococcus durans QU 49

Aims:  To characterize the novel bacteriocin produced by Enterococcus durans .
Methods and Results:  Enterococcus durans QU 49 was isolated from carrot and expressed bactericidal activity over 20–43°C. Bacteriocins were purified to homogeneity using the three-step purification method, one of which, termed durancin TW-49M, was an enterocin B-homologous peptide with most identical residues occurring in the N-terminus. Durancin TW-49M was more tolerant in acidic than in alkali. DNA sequencing analysis revealed durancin TW-49M was translated as a prepeptide of the double-glycine type. Durancin TW-49M and enterocin B expressed similar antimicrobial spectra, in which no significant variation due to the diversity in their C-termini was observed.
Conclusions:  Durancin TW-49M, a novel nonpediocin-like class II bacteriocin, was characterized to the amino acid and genetic levels. The diverse C-terminal parts of durancin TW-49M and enterocin B were hardly to be suggested as the place determining the target cell specificity.
Significance and Impact of the Study:  This is the first and comprehensive study of a novel bacteriocin produced by Ent. durans . The high homology at the N-terminal halves between durancin TW-49M and enterocin B makes them suitable to study the structure-function relationship of bacteriocins and their immunity proteins.     

Clinical streptococci and their sensitivity to enterocins produced by different strains of the species Enterococcus faecium (short communication)

In children, acute otitis media (AOM) is one of the most frequently occurring infections caused by Streptococcus pneumoniae and Str. pyogenes. The standard treatment of AOMis provided by antibiotics; however, an increased resistance of the causative agents to antibiotics requires the need to search for innovations. This study was focused on in vitro testing sensitivity of streptococci isolated from AOM to enterocins produced by 9 different origin strains of E. faecium. Enterocins (Ent) represent ribosomally synthesized proteinaceous substances with antimicrobial activity against Gram-positive and/or Gram-negative bacteria which are produced mostly by strains of the species Enterococcus faecium. Str. pneumoniae were sensitive at least to 1 Ent. Str. pneumoniae SPn 754 was sensitive to 5 Ent. Five Str. pyogenes were sensitive to enterocins. Ent A (P) inhibited the growth of 3 Str. pneumoniae, and 4 Str. pyogenes (activity between 100 and 3,200 AU/ml). Most of Ent inhibited the growth of streptococci tested (100-3,200 AU/ml). Str. pyogenes were more sensitive to Ent than Str. pneumoniae. Although more detailed further studies are required, our results indicate a new possibility for enterocin use.     

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil

AIMS: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. METHODS AND RESULTS: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. CONCLUSIONS: Enterococcus durans L28-1 produced a new bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.     

Gene Cluster Responsible for Secretion of and Immunity to Multiple Bacteriocins,the NKR-5-3 Enterocins

Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.     

Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13

AIMS: The partial characterization of bacteriocins produced by an environmental strain Enterococcus faecium EK13, isolated from cattle dung water. METHODS AND RESULTS: A bacteriocin was partially purified by ammonium sulphate precipitation, followed by a SP-Sepharose column, reverse-phase chromatography and N-terminal region sequenced. The anti-microbial substance produced was found to be a heat-stable polypeptide with molecular mass 4.83 kDa, which was determined by N-terminal amino acid sequencing to be enterocin A. A second substance was specified by PCR as enterocin P. Bacteriocins were stable at 4 and -20 degrees C for long storage periods. The optimum of bacteriocin production was observed in the range of pH 5.0-6.5 at 30 and 37 degrees C. The most active substances are produced by strain EK13 in logarithmic growth phase and bacteriocins are produced after 1 h of fermentation. The highest activity detected in fermentation experiments was 51 200 AU ml(-1) and the most sensitive indicator strain was found to be Listeria innocua LMG 13568. Differences in bacteriocin activity against two indicators could be explained by more than one type of enterocin production by strain EK13, or with different mode of action or in different sensitivity of strains. CONCLUSION: Enterococcus faecium strain EK13 isolated from cattle dung water produces two bacteriocins, enterocin A and P, with an inhibitory effect against the strain of the genera Enterococcus, Leuconostoc, Lactobacillus, Streptococcus, Staphylococcus, Bacillus and Listeria (in different origin). SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecium EK13 environmental strain is a new producer of enterocin A and P. The E. faecium EK13, isolated from cattle dung water, is presented with the further aim to utilize it for waste treatment by biotechnological processes.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.     

Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157

Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON-157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity.     

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco

AIMS: Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. METHODS AND RESULTS: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. CONCLUSION: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus, were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry. In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum, originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus , were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry.
In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum , originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.