1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

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 α-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.


  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

The beneficial role of lactic acid bacteria (LAB) and their safety in food fermentation are well documented. First and foremost, by their metabolic activities in fermented food products, shelf-life and safety are increased and in addition, the aroma, texture and flavour may be improved ( Lindgren & Dobrogosz 1990; Hammes & Tichaczek 1994; Stiles 1996). The preservative action of LAB in foods results from the formation of metabolites with antimicrobial activity, e.g. organic acids (lactic, acetic and formic), hydrogen peroxide (in the presence of oxygen), diacetyl, aldehydes (e.g. β-hydroxypropionaldehyde) and bacteriocins ( Lindgren & Dobrogosz 1990; Gould 1992; Hammes & Tichaczek 1994 ; Holzapfel et al. 1995 ). Bacteriocins are produced by some strains of LAB; they are antimicrobial peptides with activity against strains closely related to the producer micro-organism ( Klaenhammer 1993). Some bacteriocins are also active against Gram-positive food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis and spores of Clostridium perfringens. For this reason, they have received much attention for use as natural, or so-called ‘bio-preservatives’ in foods in recent years ( Klaenhammer 1993; Holzapfel et al. 1995 ; Stiles 1996).

In view of the potential of LAB bacteriocins for improving food safety and preservation, a major focal point of research has involved the characterization and production of bacteriocins from LAB of food origin. Bacteriocin production by LAB is believed to confer an ecological advantage over competitors present in the same ecosystem ( Damelin et al. 1995 ). At present, little is known about bacteriocin production of LAB from intestinal origin. Speculatively, production of bacteriocins by intestinal LAB may play an important role in the establishment of bacteriocin producers, thereby enabling them to compete in an environment of great abundance and complexity of micro-organisms.

Bacteriocins are divided into three classes ( Nes et al. 1996 ). class I consists of the lantibiotics, which are small, membrane-active peptides that contain the unusual amino acids lanthionine and β-methyllanthione. Class II bacteriocins are small (4–6 kDa), heat-stable, non-lanthionine containing peptides and are subdivided into three subgroups: (a) Listeria-active peptides which are ‘pediocin-like’ and which are characterized by a well conserved YGNGVXC consensus motif at their N-terminal ends; (b) two-peptide bacteriocins; and (c) bacteriocins which are secreted by the bacterial preprotein translocase (sec-pathway). Class III bacteriocins are large (>30 kDa) and heat-labile proteins ( Nes et al. 1996 ).

The best characterized bacteriocins from enterococci (enterocins) were described from strains originating from food sources. These include the class II enterocins A, B, P, CRL35 and bacteriocin 31 ( Aymerich et al. 1996 ; Farías et al. 1996 ; Tomita et al. 1996 ; Casaus et al. 1997 ; Cintas et al. 1997 ; Franz et al. 1999a ; O'Keeffe et al. 1999 ). Enterocins L50A and L50B, also produced by an enterococcal food isolate, do not belong to any of the three classes of bacteriocins as defined by Nes et al. (1996) , but show homology to the staphylococcal peptide toxins ( Cintas et al. 1998 ). Well characterized enterocins from strains isolated from clinical sources are the cyclic antibiotic peptide, AS-48 ( Gálvez et al. 1989 ), and the class I bacteriocin, cytolysin ( Gilmore et al. 1994 ). Interestingly, the bacteriocin phenotype is frequently associated with pheromone-responsive conjugative plasmids of Ent. faecalis, as is the case for bacteriocin 31, cytolysin and AS-48 bacteriocins. There are only a few reports of bacteriocin production by enterococci from an intestinal source, and the amino acid sequences of these enterocins were generally not determined ( Krämer & Brandis 1975; Laukováet al. 1993 ).

Lactic acid bacteria may be applied as animal feed supplements and directly as probiotic preparations ( Pollman et al. 1980 ; Loguercio et al. 1987 ; Fuller 1989). As Enterococcus spp. form part of the autochthonous flora of the intestinal tract of humans and animals ( Fuller 1989), and have also been isolated from fermenting plant and dairy products ( Devriese et al. 1991 ; Villani et al. 1993 ; Maisnier-Patin et al. 1996 ), they show potential for application as probiotics. Indeed, enterococci (e.g. strain SF68) have been used as probiotics for the successful treatment of colibacillosis in animals and gastroenteritis in humans ( Lewenstein et al. 1979 ; Bellomo et al. 1980 ; D'Apuzzo & Salzberg 1982; Underdahl 1983). In a previous study ( Du Toit et al. 1998 ), LAB were isolated from pig faeces and potential probiotic characteristics, such as bile tolerance, acid resistance and production of antimicrobial substances, were investigated. The aim of this study was to isolate and presumptively identify enterococci from pig faeces for potential use as probiotics and to determine whether they produce bacteriocins. It was also aimed at characterizing such bacteriocins in terms of their activity spectrum and their approximate molecular weight, and using PCR to assess whether these bacteriocins are novel or similar to previously described enterocins produced by strains of Enterococcus spp. isolated from food.


  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

Media and culture conditions

Enterococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp. and Streptococcus spp. were grown aerobically in MRS broth (Merck). Bacillus spp., Staphylococcus spp. and Listeria spp. were grown aerobically in Brain Heart Infusion broth (Biolab, Merck). Propionibacterium spp. were cultivated anaerobically in GYP (glucose 5 g, yeast extract 3 g, peptone 10 g, meat extract 10 g and NaCl 5 g l−1; pH 7·0) at 30 °C. Clostridium spp. were grown anaerobically in Reinforced Clostridial Medium (Merck). All strains were incubated at 37 °C.

Isolation of Enterococcus strains

Enterococcal isolates were obtained from the faeces of Göttingen minipigs subjected to an in vivo feeding trial ( Du Toit et al. 1998 ). Serial 10-fold dilutions of the faeces were made in quarter-strength Ringer solution and 100 μl of each dilution were spread-plated onto CATC agar (Merck), a selective growth medium for enterococci. The plates were incubated aerobically at 37 °C for 48 h. Typical red colonies were isolated from the plates of highest dilutions showing growth, and purified by repeated streaking onto MRS agar (Merck).

Screening for bacteriocin-producing strains

As CATC medium is selective for enterococci, all Gram-positive, catalase-negative cocci isolated from this medium were presumptively identified as Enterococcus spp. and screened for antimicrobial activity using the agar spot test method ( Schillinger & Lücke 1989) and the bacteriocin screening medium of Tichaczek et al. (1992) without catalase. The indicator strains used were Lactobacillus sakei DSM 20017, Ent. faecium DSM 20477, Streptococcus mutans DSM 6187 and Clostridium difficile DSM 1296. Isolates that showed antagonistic activity against at least one of these indicator strains were selected for further studies.

Presumptive identification of bacteriocin-producing strains

Sugar fermentation reactions were performed using API 50CH test strips and 50CHL medium, according to the manufacturer's instructions (BioMérieux). Growth of the strains was studied, at pH 9·6, in 6·5% (w/v) NaCl and at 10 °C and 45 °C in MRS broth ( Schleifer & Kilpper-Bälz 1984; Devriese et al. 1991 ). The cultures were incubated for 5 d at 10 °C and 3 d at 45 °C. The configuration of lactic acid, hydrolysis of arginine and production of CO2 from glucose were determined according to the methods described by Schillinger & Lücke (1987).

Antimicrobial activity assays

Cell-free supernatant fluid was obtained by growing the bacteriocin-producing strains in MRS broth for 18 h at 37 °C. Cultures were centrifuged at 9400 g for 10 min in a minicentrifuge; the cell-free supernatant fluid was then adjusted to pH 6·5–7·0 with 1N NaOH, and heated at 100 °C for 5 min to inactivate any remaining cells.

The antimicrobial activity spectrum of each strain was determined by spotting neutralized supernatant fluid onto soft (0·7% w/v) agar inoculated with Gram-positive indicator strains ( Table 1). Soft agar was seeded with 100 μl of an overnight culture of the indicator strain (107 cfu ml−1) and used to overlay MRS agar plates. Plates were incubated for 24 h at 37 °C and examined for clear zones of inhibition.

Table 1.  Inhibitory spectrum of the pH neutralized cell-free supernatants of the enterococcal strains isolated from pig faeces, as determined with the agar spot test of Uhlman et al. (1992)
 Inhibitory activity *
Indicator organismEnt. faecalis BFE 1071Ent. faecium BFE 1072Ent. faecalis BFE 1113Ent. faecium BFE 1170Ent. faecium BFE 1228Ent. faecalis BFE 1229Ent. faecalis BFE 1263
  • *

    +, clear inhibition zone >1 mm; (+) weak inhibition 0·5–1 mm; –, no inhibition zone.

  • † 

    LMG: Culture collection of the Laboratory of Microbiology, University of Gent, Belgium; DSM: Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; RK: Isolates from Red Cross Childrens Hospital, Cape Town, South Africa; WS: Technical University, München-Weihenstephan, Germany; SLCC: Seeliger Listeria Culture Collection, Würzburg, Germany.

Lactobacillus acidophilus LMG 13550 (+)
Lactobacillus casei LMG 13552
Lactobacillus curvatus LMG 13553(+)
Lactobacillus fermentum LMG 13554
Lactobacillus helveticus LMG 13555+
Lactobacillus plantarum LMG 13556
Lactobacillus reuteri LMG 13557
Lactobacillus sakei LMG 13558+++
Pediococcus pentosaceus LMG 13560(+)++
Pediococcus pentosaceus LMG 13561
Leuconostoc cremoris LMG 13562
Leuconostoc cremoris LMG 13563+++++
Streptococcus thermophilus LMG 13564
Streptococcus thermophilus LMG 13565
Enterococcus faecalis LMG 13566, RK++/(+)++/(+)+/(+)++
Enterococcus faecium RK+++(+)(+)++
Enterococcus durans DSM 20633, RK(+)(+)(+)
Enterococcus avium DSM 20679(+)(+)(+)
Enterococcus dispar DSM 6630+++(+)(+)++
Enterococcus cecorum DSM 20682(+)(+)(+)
Enterococcus gallinarum DSM 20628(+)(+)(+)
Enterococcus mundtii DSM 4838(+)(+)(+)
Staphylococcus carnosus LMG 13567
Listeria monocytogenes WS 2247, +++
WS2249, WS 2250
Listeria ivanovii WS 2255, SLCC 4769+++
Listeria innocua LMG 13568, WS 2257, WS 2258 +++
Listeria seeligeri WS 2253+++
Listeria welshimeri WS 2254+++
Bacillus cereus LMG 13569
Clostridium sporogenes LMG ·3570 +++
Clostridium tyrobutyricum LMG 13571+++
Propionibacterium acidopropionici LMG  13572 +
Propionibacterium sp. LMG 13573, LMG  13574 +++

Kinetics of bacteriocin production

For each bacteriocin-producing strain, 50 ml MRS broth (pH 6·5) was inoculated at a level of 104 cfu ml−1 and incubated at 37 °C. Viable cell counts were determined by spread-plating onto MRS agar, while bacteriocin activity was determined at 2 h intervals for the first 24 h and at 4 h intervals for the remaining 24 h. Bacteriocin activity was determined using the critical dilution method, as described by Schillinger et al. (1993) , with Ent. faecalis LMG 13566 as indicator strain. One arbitrary activity unit (AU) was defined as the reciprocal of the highest dilution showing a clear inhibition zone, and was multiplied by a factor of 100 to obtain AU ml−1.

Influence of medium pH on bacteriocin production

MRS broth was adjusted to pH 3·0, 4·0, 5·0, 6·0, 7·0, 8·0, 9·0 and 10·0, using 5N NaOH or 5N HCl, and inoculated with 1% (v/v) bacteriocin-producing culture (107 cfu ml−1). The cultures were incubated at 37 °C for 24 h, after which the viable count (cfu ml−1) and bacteriocin activity (AU ml−1) were determined as described previously.

Effect of enzymes, pH and heat on bacteriocin activity

Neutralized supernatant fluid (1600 AU ml−1) was incubated at 37 °C for 3 h, 60 °C for 30 min, 80 °C for 30 min and 100 °C for 30 min, and autoclaved (121 °C, 15 min). Enzyme inactivation of the antimicrobial activity in the supernatant fluid was done according to the method described earlier ( Franz et al. 1996 ). To determine the effect of pH on bacteriocin activity, neutralized cell-free supernatant fluid was adjusted to pH levels ranging from 2·0 to 11·0 (intervals of 1·0) with 5N NaOH or 1N HCl. The pH-adjusted supernatant fluid was kept at 4 °C for 24 h. As control, samples treated with proteinase K before adjusting the pH were tested against the same indicator. Bacteriocin activity was determined after completion of all tests using the critical dilution method and Ent. faecalis LMG 13566 as indicator strain.

Bactericidal activity

  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

Bactericidal activity was determined according to the methods of Franz et al. (1996) . For each of the seven bacteriocin-producing cultures, 1 ml neutralized cell-free supernatant fluid (3200 AU ml−1) was added to 8 ml MRS broth. Enterococcus faecalis LMG 13566 was grown for 24 h at 37 °C, after which it was diluted in MRS broth using a 10-fold dilution series. A 1 ml aliquot of a suitable dilution containing approximately 106 cfu ml−1 of indicator bacteria was inoculated into 9 ml bacteriocin-containing MRS (320 AU ml−1). As a control, 9 ml MRS broth were inoculated with 1 ml of the same diluted indicator organism in the absence of bacteriocin. Incubation was at 37 °C. Cell counts (cfu ml−1) of Ent. faecalis LMG 13566 were determined at different time intervals by spread-plating onto MRS agar.

Haemolytic activity

  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

Production of haemolysin was determined by plating actively growing cells of the seven strains of Ent. faecalis and Ent. faecium onto Columbia Blood agar (Oxoid) supplemented with 5% (v/v) human blood. Plates were incubated at 37 °C in an anaerobic jar (Oxoid) with a gas generating kit. Results were recorded at 24 and 72 h. A clear zone of βhaemolysis on blood agar plates was considered a positive result.

Partial purification of the enterocins

  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

For partial purification of the enterocins, cultures were grown for 18 h at 37 °C and the supernatant fluid obtained by centrifugation at 13 100 g at 4 °C for 10 min. The pH was adjusted to 6·5–7·0 and the fluid cooled in an ice-water bath. Ammonium sulphate was added slowly to the supernatant fluid while stirring to reach a final saturation of 80% (56·1 g (NH4)2SO4 100 ml−1 supernatant fluid). The suspension was centrifuged at 13 100 g for 30 min at 4 °C, and the precipitate resuspended in distilled water and dialysed for 24 h at 8 °C against distilled water. The molecular weight cut-off of the membrane was 1000 Da. The dialysate was freeze-dried, resuspended in distilled water and frozen at −20 °C until use.

Molecular size approximation

The molecular sizes of the bacteriocins were approximated by tricine SDS-PAGE, according to the method of Schägger & von Jagow (1987). Half of the gel was used for molecular weight determination and was fixed and stained with Coomassie blue. The other half was prepared for assaying antimicrobial activity by fixing it in 20% (v/v) iso-propanol and 10% (v/v) acetic acid for 30 min at 20 °C. The gel was then washed with distilled water for 2 h (with 30 min intermitted changes), left overnight in distilled water and washed for another 30 min. The gel was overlaid with MRS soft agar seeded with actively growing cells of Ent. faecalis LMG 13566 (107 cfu ml−1) and incubated at 37 °C for 18 h. The position of the active enterocin band was detected by an inhibition zone surrounding the peptide band.

Plasmid DNA isolation

Bacterial plasmid DNA was isolated according to a modified method of Birnboim & Doly (1979). After harvesting and washing, the cells were incubated in the presence of lysozyme (10 mg ml−1) for 2 h at 37 °C. Cells were then lysed and plasmids extracted according to the protocol of Birnboim & Doly (1979). Plasmid DNA was extracted using the method described by Burger & Dicks (1994), followed by CsCl density gradient centrifugation ( Sambrook et al. 1989 ). Gel electrophoresis was performed using a 0·8% (w/v) agarose gel and Tris-acetate buffer according to Sambrook et al. (1989) .

PCR amplification of known enterocin genes

PCR amplification of known enterocin genes with specific primers was done using total DNA isolated by the method of Dellaglio et al. (1973) . The specific primers used for the known enterocin genes are listed in Table 2. PCR (Biometra, Biomedizinische Analytik GmbH, Göttingen, Germany) reactions were carried out in a 50 μl volume containing 26·8 μl water, 5 μl of a 10× reaction buffer with MgCl2 (Boehringer Mannheim), 8 μl deoxynucleoside triphoshate mixture (1·25 mmol l−1), 2·5 μl of each primer, 0·2 μl (1 U) Taq DNA polymerase (Boehringer Mannheim) and 5 μl DNA template (20 ng). The PCR conditions consisted of an initial denaturing step of 5 min at 95 °C, followed by 30 cycles of 1 min denaturing at 95 °C, 1 min annealing at a temperature specific for the primers for each of the known enterocin genes as shown in Tables 2, and 1 min extension at 72 °C. The PCR products were separated on a 2·5% agarose gel and visualized by staining with ethidium bromide ( Sambrook et al. 1989 ). The total DNA of Ent. faecium BFE 900, which produces both enterocins A and B ( Franz et al. 1999a ), was used as a positive control for primers designed to detect enterocin A and enterocin B. Unfortunately, it was not possible to secure positive controls for the other known enterocins.

Table 2.  DNA homology of six known enterococcal bacteriocin genes with faecal strains of Ent. faecium and Ent. faecalis as detected by PCR
SpeciesStrainBacteriocinPrimerPCR result
  • *: 

    no PCR product with any of the isolates.

  • +: PCR product with the isolate named.

Ent. faeciumL50Enterocin L50A andf:5′-STGGGAGCAATCGCAAAATTAG-3′BFE 1072+
Ent. faecalisY1717Bacteriocin 31f:5′-TATTACGGAAATGGTTTATATTGT-3′ r:5′-TCTAGGAGCCCAAGGGCC-3′


  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

Seven Enterococcus strains isolated from pig faeces showed activity when tested against the sensitive indicators ( Table 1).

These bacteriocin-producing strains isolated from pig faeces were Gram-positive, catalase-negative cocci and produced l-(+)-lactic acid but no CO2 as the major end product of glucose fermentation. All strains were capable of growing at 10 and 45 °C, at pH 9·6 and in the presence of 6·5% NaCl ( Table 3). Based on these characteristics, as well as on carbohydrate fermentation patterns ( Table 3), the strains were presumptively identified as Ent. faecalis (BFE 1071, 1113, 1229 and 1263) and Ent. faecium (BFE 1072, 1170 and 1228). Strain BFE 1071 differed from the other strains by the ability to ferment starch but not lactose. Strains BFE 1071 and 1113 differed from Ent. faecalis BFE 1229 and BFE 1263 by their fermentation of inositol. Strain BFE 1072 was different from all the other isolates by fermenting α-methyl- d-mannoside and d-raffinose, while strain BFE 1228 differed by its ability to ferment d-xylose. The phenotypic characteristics of these isolates were typical of those reported for Ent. faecium and Ent. faecalis by Knudtson & Hartman (1992) and Devriese et al. (1993) . The results also indicated that the isolated strains were different.

Table 3.  Phenotypic characteristics of the bacteriocin-producing strains isolated from pig faeces
TestEnt. faecalis BFE 1071Ent. faecium BFE 1072Ent. faecalis BFE 1113Ent. faecium BFE 1170Ent. faecium BFE 1228Ent. faecalis BFE 1229Ent. faecalis BFE 1263
  1. All strains were Gram-positive, catalase negative, ovoid shaped cocci, and grew at 10°C, 45°C, pH 9·6 and in presence of 6·5% NaCl. l-(+)-lactic acid but no CO2 was produced as major fermentation product from glucose; arginine was hydrolysed by all strains, and the final pH reached in MRS broth after 3 days at 37°C ranged from 4·2 to 4·4. In addition, all strains fermented N-acetyl-glucosamine, amygdaline, arbutine, cellobiose, esculin, d-fructose, galactose, d-glucose, β gentiobiose, mannitol, d-mannose, maltose, ribose and salicine. No strains fermented adonitol, d-arabitol, l-arabitol, d-arabinose, 5-ceto-gluconate, dulcitol, erythritol, d-fucose, l-fucose, glucogene, inulin, 2-ceto-gluconate, d-lyxose, α-methyl- d-glucoside, β-methyl-xyloside, l-sorbose, d-turanose, xylitol and l-xylose.

Acid production from
l-Arabinose +++
α-Methyl- d-mannoside +
d-Raffinose +
d-Tagatose +++++
d-Xylose +

All strains identified as Ent. faecalis displayed a narrow range of antimicrobial activity, mainly towards other enterococci, whereas the strains of Ent. faecium inhibited a wider range of indicator bacteria, including Listeria monocytogenes, Listeria innocua, Clostridium sporogenes, Clostridium tyrobutyricum and Propionibacterium spp. ( Table 1).

Enterocin production started during early exponential growth (at a cell density of 108 cfu ml−1) and maximum production (1600 AU ml−1) was recorded at the beginning of the stationary phase, after which no further increase was detected (results not shown). The enterocins remained active for 48 h, with slight decreases in activity towards the end of the 48 h (results not shown).

The effect of initial medium pH on cell growth and enterocin production is shown in Table 4. All isolates grew to maximum density (approximately 109 cfu ml−1) in MRS broth adjusted to pH levels between 6·0 and 10·0. The viable cell counts were lower for enterococci grown in MRS broth adjusted to pH 4·0 and 5·0 ( Table 4). No bacteriocin activity could be detected in the supernatant fluid of MRS broth adjusted to pH 3·0 and 4·0, even though growth of enterococci occurred to levels of approximately 106 cfu ml−1. The highest bacteriocin activity was recorded for strains BFE 1113 and BFE 1229 grown in MRS broth adjusted to pH 6·0 and 7·0. However, above pH 7·0, bacteriocin production appeared to be inhibited, as shown by either no, or very low levels of activity in the medium supernatant fluid.

Table 4.  Influence of initial pH of MRS broth on viable cell counts and bacteriocin activity of bacteriocin-producing enterococci isolated from pig faeces, determined after 18 h at 37°C
Ent. Faecalis BFE 1071 Ent. faecium BFE 1072 Ent. faecalis BFE 1113 Ent. faecium BFE 1170 Ent. faecium BFE 1228 Ent. faecalis BFE 1229 Ent. faecalis BFE 1263
  1. *Log cfu ml −1.

  2. †AU ml −1.


The enterocins were sensitive to α-chymotrypsin, proteinase K, pronase, trypsin, papain and pepsin, but bacteriocin activity was not affected by lipase, lysozyme and catalase ( Table 5). Low levels of bacteriocin activity were retained in the supernatant fluid after 15 min at 121 °C ( Table 5), and bacteriocin levels were highest in supernatant fluids adjusted to pH values from 6·0 to 8·0 ( Table 6). Enterococcus faecalis and Ent. faecium isolates exhibited no β-haemolytic activity, as no zones of clearing were detected on blood agar plates (results not shown).

Table 5.  Effects of enzymes and heat treatment on inhibitory activity of the cell-free supernatant of enterococci isolated from pig faeces
TreatmentEnt. faecalis BFE 1071Ent. faecium BFE 1072Ent. faecalis BFE 1113Ent. faecium BFE 1170Ent. faecium BFE 1228Ent. faecalis BFE 1229Ent. faecalis BFE 1263
  • *

    +, indicates activity of bacteriocin.

  • –, indicates loss of bacteriocin activity.

Proteinase K
Heat (AU ml−1)
65°C 30 min1600160016001600160016001600
80°C 30 min160016001600800160016001600
100°C 30 min800160016004004001600800
37°C 3 h80016001600160016001600800
121°C 15 min400400200100100200200
Table 6.  Effect of pH on the bacteriocin activity of enterococci isolated from pig faeces
 AU (ml−1)
pHEnt. faecalis BFE 1071Ent. faecium BFE 1072Ent. faecalis BFE 1113Ent. faecium BFE 1170Ent. faecium BFE 1228Ent. faecalis BFE 1229Ent. faecalis BFE 1263

The effect of the enterocins on the growth of Ent. faecalis LMG 13566 is shown in Table 7. For the control culture, viable cell numbers increased from 106 to about 109 within 9 h at 37 °C. Addition of the cell-free neutralized supernatant (CFNS) fluid of BFE 1071 to the indicator culture inhibited growth of cells, as numbers did not change for the first 3 h of incubation at 37 °C and cell numbers remained at log 6·0 cfu ml−1. In the following 6 h, a decrease in viable count of 1 log unit was observed. For the CFNS fluid of BFE 1072, cell numbers of the indicator strain decreased from log 6·0 cfu ml−1 to log 3·0 cfu ml−1 within the first 3 h of incubation at 37 °C. However, during the next 6 h of incubation, cell numbers increased to about log 5·0 cfu ml−1. The CFNS fluid of BFE 1113 decreased numbers of the indicator bacteria by about 2 log units after 1 h of incubation, and then numbers increased by 1 log unit within the next 2 h of incubation. The numbers of indicator bacteria then decreased again from about log 5·0 cfu ml−1 to log 2·0 cfu ml−1 after 7 h of incubation, after which an increase of about 3 log units was observed in the next 2 h of incubation. The CFNS fluid of BFE 1170 and BFE 1228 acted similarly by decreasing cell numbers of the indicator bacteria from log 6·0 cfu ml−1 to about log 3·0 cfu ml−1 after 3 h of incubation at 37 °C. No cells could be detected for the following 6 h of the experiment with supernatant fluid from BFE 1170. This was similar to the case of CFNS fluid from BFE 1228, except that an increase of 3 log units was detected within the last 2 h of incubation with supernatant fluid of the latter isolate. Addition of CFNS fluid of BFE 1229 to the indicator cells led to a decrease from log 6·0 cfu ml−1 to below detection level during the first 2 h of incubation. Numbers of indicator strains then increased again from undetectable levels to log 5·3 cfu ml−1 after 7 h of incubation. When CFNS fluid of BFE 1263 was added to indicator bacteria, growth did not occur for the next 3 h of incubation, after which cell numbers decreased from log 6·0 cfu ml−1 to log 2 cfu ml−1 after further incubation for 4 h. Numbers of indicator bacteria then increased again to log 5·4 cfu ml−1 during the last 2 h of incubation at 37 °C.

Table 7.  Bactericidal effect of the enterocins of enterococci isolated from pig faeces, on Ent. faecalis LMG 13566 in MRS broth at 37°C
 Log cfu (ml−1)
Time (h) Control *Ent. faecalis BFE 1071 Ent. faecium BFE 1072Ent. faecalis BFE 1113Ent. faecium BFE 1170Ent. faecium BFE 1228Ent. faecalis BFE 1229Ent. faecalis BFE 1263
  • * 

    MRS broth with no bacteriocin added.

  • † MRS broth with bacteriocin (320 AU ml −1) added.


Tricine SDS-PAGE showed that the partially purified bacteriocins contained numerous protein bands ( Fig. 1). The specific band that displayed antibacterial activity was approximately 3·4 kDa in size ( Fig. 1).


Figure 1. Tricine SDS-PAGE of the partially purified enterocins. (a) Stained gel showing the proteins present after (NH4)2SO4 precipitation, with lanes 1–7 containing 1071, 1071, 1113, 1170, 1228, 1229 and 1263, respectively, and the molecular weight marker (lane 8). (b) Gel overlaid with Enterococcus faecalis LMG 13566 showing zones of inhibition.

Plasmid profiles of the isolates are displayed in Fig. 2. No plasmids were detected for strains BFE 1072, BFE 1179 and BFE 1228. Strains BFE 1071 and BFE 1263 exhibited an identical plasmid profile. Plasmids were also isolated from strains BFE 1113 and BFE 1229.


Figure 2. Plasmid profiles of the Enterococcus isolates. Lane 1: λ-DNA cut with EcoRI and HindIII; lanes 2–8: plasmid profiles of BFE 1071, BFE 1072, BFE 1113, BFE 1170, BFE 1228, BFE 1229 and BFE 1263, respectively

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Total DNA from the seven bacteriocin-producing enterococcal strains was subjected to a PCR assay with the specific primers encoding the 5′- and 3′-terminal regions of the genes that code for the mature part of the different enterocins as listed in Table 2. A PCR product of approximately 126 bp was obtained from DNA of strains BFE 1170, 1228 and 900 using the enterocin A primer, while a product of 159 bp was obtained from DNA of strain BFE 900 using the enterocin B primer. In addition, a product of 130 bp was obtained from DNA of strain BFE 1072 using the primers specific for enterocins L50A and L50B.


  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References

Enterococcus spp. have been reported to produce bacteriocins which inhibit Gram-positive food-borne bacteria and intestinal pathogens ( Krämer & Brandis 1975; Siragusa 1992; Laukováet al. 1993 ; Torri Tarelli et al. 1994 ; Vlaemynck et al. 1994 ; Aymerich et al. 1996 ; Franz et al. 1996 ).

In this study, seven Enterococcus strains, isolated from pig faeces, showed antimicrobial activity against Gram-positive bacteria including enterococci, lactobacilli, clostridia, propionibacteria and Listeria spp. The neutralized culture supernatant fluids were not inactivated by lysozyme and lipase, which suggests that the enterocins do not require a lipid or carbohydrate moiety for activity. The sensitivity spectrum of the enterocins to proteolytic enzymes corresponded well with that of known enterocins produced by Ent. faecalis and Ent. faecium ( Parente & Hill 1992; Arihara et al. 1993 ; Villani et al. 1993 ; Torri Tarelli et al. 1994 ; Franz et al. 1996 ).

Incubating the enterocins at 37 °C for 3 h slightly affected activity when compared with a control kept at 4 °C. This could be of importance if enterocins are produced in the human or animal intestinal tract, indicating that their activity level may decrease within a short time period. Enterocin 1170 was most sensitive to heat as its activity decreased after 30 min at 80 °C and 100 °C. However, all the enterocins retained various levels of activity after heating at 121 °C for 15 min and can therefore be considered as heat stable.

Activity of the enterocins was highest when the supernatant fluid of the cultures was adjusted to pH values between 6·0 and 8·0. The enterocins of the Ent. faecalis isolates remained active over a wide pH range from 2·0 to 11·0, whereas the activity of the Ent. faecium isolates was destroyed above pH 9·0. The activity of enterocins over a wide pH range may be advantageous when produced in the gastrointestinal tract, where pH levels are known to vary from pH 3·0 in the stomach to pH > 7·0 in the large intestine. Growth and bacteriocin production at low pH may be advantageous for survival in the upper intestinal tract in view of probiotic use.

The bactericidal mode of action of enterocins produced by the seven Enterococcus strains was indicated by a decrease in viable cell numbers of the indicator strain Ent. faecalis LMG 13566. However, cell numbers of indicator bacteria increased towards the end of the experiment in the presence of all the different enterocins. This type of inhibition kinetics has been observed before with Listeria as indicator and nisin, pediocin AcH and enterococcin EFS2 as antimicrobial substances ( Maisnier-Patin et al. 1996 ; Song & Richard 1997), and may indicate that sensitive bacteria become resistant to the bacteriocin. Based on the observed bactericidal mode of action and proteinaceous nature of the antimicrobial substances produced by the seven Enterococcus isolates, the antimicrobial compounds were classified as bacteriocins.

The enterocins studied displayed the same production kinetics as primary metabolites, being produced at the end of the logarithmic growth phase, and these production kinetics were comparable with those of most other bacteriocins produced by LAB ( De Vuyst & Vandamme 1994). Maximum production correlated with high cell densities, as was also reported for other enterocins ( Parente & Hill 1992; Torri Tarelli et al. 1994 ; Franz et al. 1996 ). Decreasing activity after prolonged incubation of the producer strain may be explained either by degradation due to culture proteases, low culture pH, or readsorption to the producer cell surface ( Parente & Hill 1992; Parente & Ricciardi 1994; Torri Tarelli et al. 1994 ).

All enterocins in this study exhibited low molecular weights of approximately 3·4 kDa and were heat stable; they could therefore be tentatively classified as group II bacteriocins according to Nes et al. (1996) . The low molecular weights of enterocins in this study were in accordance with enterocins reported by Parente & Hill (1992), Vlaemynck et al. (1994) , Aymerich et al. (1996) and Farías et al. (1996) , but they differed from the higher molecular weight enterocins described by Krämer & Brandis (1975), Villani et al. (1993) , Dallas et al. (1996) , Maisner-Patin et al. (1996) and Franz et al. (1999a) . However, the molecular weights for enterocins reported in this study were approximations, as size determination by SDS-PAGE is not very accurate. The results indicated that bacteriocinogenic isolates of Ent. faecalis all contained plasmids, while those of Ent. faecium did not. Absence of plasmids in the Ent. faecium strains was confirmed by large scale plasmid DNA isolation using CsCl gradient extraction (results not shown). Further studies are necessary to determine whether the genes encoding enterocin production are located on the chromosome or on plasmid DNA. Reports on other enterocins produced by Ent. faecium and Ent. faecalis indicate that genes for enterocin production can be either plasmid- or chromosomally-encoded ( Parente & Hill 1992; Olasupo et al. 1994 ; Aymerich et al. 1996 ; Franz et al. 1996 , ,1999a; Tomita et al. 1996 1997; Cintas et al. 1997 , 1998).

The food-borne pathogen Listeria monocytogenes has been associated with many cases of listeriosis ( Farber & Peterkin 1991). Anti-Listeria activity is a distinguishing characteristic for most enterococcal bacteriocins ( Giraffa 1995). Accordingly, all isolates identified as Ent. faecium inhibited the Listeria spp. in this study. However, the Ent. faecalis isolates did not show this ability. Nevertheless, it is conceivable that the enterocins from the Ent. faecalis isolates may inhibit Listeria strains other than those used as indicator strains in this study. PCR amplification with the different enterocin primers revealed that Ent. faecium BFE 1170 and BFE 1228 produced enterocin A, while Ent. faecium BFE 1072 produced enterocin L50A, L50B or both. The primers used for PCR amplification of enterocins L50A and L50B were designed to amplify either of these bacteriocins, and the size difference is too small to resolve by the agarose gel electrophoresis conditions applied in this study. However, it is known that the genes for enterocins L50A and L50B in the Ent. faecium 6T1a and L50 strains occur in close proximity and probably within an operon-type structure ( Cintas et al. 1998 ; Floriano et al. 1998 ). Moreover, these bacteriocins act synergistically and resemble two-peptide bacteriocins of class IIb ( Cintas et al. 1998 ). Therefore, BFE 1072 probably also produces both enterocins L50A and L50B. Further studies on the sequences of the PCR products will show whether they are identical or whether they are natural variants. The PCR technique has previously been used successfully in enterococci and lactobacilli ( Remiger et al. 1996 ; Joosten et al. 1997 ) to detect known bacteriocins. The present PCR amplification results also illustrate the inaccuracy of SDS-PAGE for determining the molecular weight of bacteriocins. The molecular weights for enterocins A, B, and L50A and B have previously been theoretically deduced from the nucleotide sequence, or accurately determined by mass spectrometry, and are greater than 3 kDa; they were reported to be 4829 Da for enterocin A, 5643 Da for enterocin B, 5190 Da for enterocin L50A and 5178 Da for enterocin L50B ( Aymerich et al. 1996 ; Casaus et al. 1997 ; Cintas et al. 1998 ; Franz et al. 1999a ).

The present study shows that enterococci from the intestinal tract may produce similar bacteriocins to those produced by enterococci from a food source. This finding may be explained by the fact that enterococci occurring in food production, especially as autochthonous flora associated with the ripening of Mediterranean cheeses ( Franz et al. 1999b ), originate from intestinal sources. In addition, the results suggest that the remaining four bacteriocinogenic enterococcal strains in this study may produce enterocins different to those previously reported for enterococci isolated from a food source. Further studies, including amino acid sequencing and/or DNA sequencing, are required to determine whether these enterocins from intestinal enterococci are indeed novel. The genes coding for the production of the bacteriocin produced by BFE 1071 are currently being sequenced.

Enterococci are part of the normal flora of humans and animals. Bacteriocins produced by intestinal Enterococcus isolates may help to control the autochthonous microflora and may be advantageous to the producing strain for its establishment and competition in the gastrointestinal tract. Some strains of Ent. faecium and Ent. faecalis are known probiotics and have been used with great success in commercial probiotic preparations ( Bellomo et al. 1980 ; Pollman et al. 1980 ; Loguercio et al. 1987 ; Fuller 1989; O'Sullivan et al. 1992 ; Mikešet al. 1995 ; Buydens & Debueckelaere 1996). Further studies on other potential probiotic characteristics (e.g. bile tolerance, acid resistance, adherence) of the enterococci used in this study are required to determine their suitability for probiotic application. However, as enterococci are also associated with human infections such as endocarditis, especially in a hospital environment and in the presence of underlying disease or immunosuppression ( Franz et al. 1999b ), they should also be evaluated for the presence of potential virulence factors before probiotic use.


  1. Top of page
  2. Abstract
  3. Introduction
  5. Bactericidal activity
  6. Haemolytic activity
  7. Partial purification of the enterocins
  8. Results
  9. Discussion
  10. References
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