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 of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

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 two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).     

Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus

Enterococcus faecium T8, isolated from vaginal secretions of children with human immunodeficiency virus, produces a class IIa sec-dependent bacteriocin that is structurally different from three other class IIa sec-dependent bacteriocins, i.e., enterocin P and an enterocin P-like bacteriocin, produced by Enterococcus faecium, and bacteriocin 31, produced by Enterococcus faecalis, and from a class III bacteriocin produced by E. faecalis. The genes encoding the bacteriocin, immunity protein, mobilization protein, and relaxase nuclease are located on a 7-kb plasmid. Bacteriocin T8 has a molecular mass of 5.1 kDa based on its DNA sequence, similar to the 5.0 kDa recorded for bacteriocin 31 but larger than the 4.6 kDa reported for enterocin P. At the amino acid level, bacteriocin T8 is 69% homologous to bacteriocin 31 and 47% homologous to enterocin P. Bacteriocin T8 is active against E. faecalis isolated from patients diagnosed with vaginosis, against Lactobacillus sakei, and against a Propionibacterium sp. The peptide is heat stable (60 min at 100 degrees C) and remains active in phosphate buffer from pH 4.0 to 10.0. The mode of activity is bactericidal, as determined with E. faecalis.     

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis

AIMS: To assess the inhibitory activity on Gram-positive and Gram-negative bacteria of several species of enterococci recovered from a natural corn silage. METHODS AND RESULTS: The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone-responsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 degrees C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. CONCLUSIONS: The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.     

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.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

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.     

Bacteriocin production by Enterococcus faecium NA01 from 'wara'—a fermented skimmed cow milk product from West Africa

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100°C and active at pH 2.0–6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.     

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.     

Isolation and characterization of bacteriocin‐producing bacteria from the gastrointestinal tract of broiler chickens for probiotic use

Aims: To isolate and characterize the bacteriocin‐producing bacteria (BPB) from the gastrointestinal tract of broiler chickens for probiotic use. Methods and Results: In total, 291 bacterial strains were isolated from broilers and screened for bacteriocin‐producing ability. The bacteriocins produced by Enterococcus faecium SH 528, Ent. faecium SH 632 and Pediococcus pentosaceus SH 740 displayed inhibitory activity against pathogens including Clostridium perfringens and Listeria monocytogenes. Activity of the bacteriocins remained unchanged after 30 min of heat treatment at 60°C or exposure to organic solvents, but diminished after treatment with proteolytic enzymes. PCR was used to detect the structural genes enterocin A and B in SH 528, enterocin L50 and P in SH 632, and pediocin PA‐1 in SH 740. Most of them were resistant to 0·5% bile salts and remained viable after 2 h at pH 3·0. Ent. faecium SH 528 exhibited the highest amylase activity among the strains tested. Conclusions: We selected Ent. faecium SH 528 and SH 632 and Ped. pentosaceus SH 740 by probiotic selection criteria including inhibition activity against pathogens. Significance and Impact of the Study: The isolated BPB could potentially be used in the poultry industry as probiotics to control pathogens.     

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.     

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.     

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.     

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 .     

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.     

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 (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.     

Isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in Taiwan

AIMS: To isolate, characterize and identify lactic acid bacteria (LAB) in dochi (fermented black beans), a traditional fermented food in Taiwan. METHODS AND RESULTS: A total of 30 samples were collected from three different dochi producers and analysed after different periods of storage. Fifty-two cultures of LAB were isolated from dochi samples and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified six different bacterial groups (A-F) and showed that the majority of the isolates were homofermentative LAB. Enterococcus faecium was the most abundant of the dochi-isolated LAB. All isolated LAB were able to grow in MRS broth containing 6% NaCl, but only Enterococcus, Pediococcus and Tetragenococcus species could grow in MRS broth containing 10% NaCl. Furthermore, antibacterial activities of isolates were determined, and four isolates showed inhibitory activities against the indicator strain Lactobacillus sakei JCM 1157(T). CONCLUSIONS: These results suggest that Ent. faecium is the main LAB present during the fermentation of dochi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the dochi fermentation process.     

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.