screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of...

Screening for enterocins and detection of hemolysin and

vancomycin resistance in enterococci of different origins

L. De Vuysta,*, M.R. Foulquie Morenoa, H. Revetsb

aResearch Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Vrije Universiteit Brussel (VUB-IMDO),

Pleinlaan 2, B-1050 Brussels, BelgiumbDepartment of Immunology, Parasitology and Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie,

Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium

Received 19 April 2002; accepted 12 September 2002

Abstract

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different

sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of

indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis

were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition

of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria.

No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering

of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria,

Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin

structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of

known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely

distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The

vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA

gene only occurred among E. faecium strains. The incidence of h-hemolysis was not restricted to E. faecalis strains, but

among the E. faecium strains the structural genes of cytolysin were not detected. h-Hemolysis occurred in strains both

from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic

activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or

probiotics.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bacteriocin; Enterocin; Vancomycin; Enterococcus

1. Introduction

Enterococci are an important group of the generally

recognized as safe (GRAS) lactic acid bacteria (LAB)

(Devriese and Pot, 1995). Enterococcus faecium and

Enterococcus faecalis are the most frequently occur-

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1605(02)00425-7

* Corresponding author. Tel.: +32-2-629-32-45; fax: +32-2-

629-27-20.

E-mail address: [email protected] (L. De Vuyst).

www.elsevier.com/locate/ijfoodmicro

International Journal of Food Microbiology 84 (2003) 299–318

ring enterococcal species in the human GIT (Murray,

1990), they persist in the extraenteral environment,

and they are ubiquitous in food processing establish-

ments. Hence, they can become an important part of

the food microflora. However, the status of enter-

ococci as harmless bacteria in foods is being chal-

lenged by their growing importance in nosocomial

infections. E. faecalis is the most frequent enterococ-

cal species isolated (Jett et al., 1994). Consequently,

the control of enterococci in foods has assumed a new

level of importance in food processing and food

microbiology. However, enterococci display desirable

metabolic activities such as lipolytic and esterolytic

activities, citrate utilization and bacteriocin (enter-

ocin) production (Tsakalidou et al., 1993; Giraffa,

1995; Sarantinopoulos et al., 2001). Hence, enter-

ococci may play an important role in the ripening

and the development of aroma and flavor of certain

traditional cheeses from Mediterranean countries

(Coppola et al., 1988; Litopoulou-Tzanetaki and Tza-

netakis, 1992; Tzanetakis and Litopoulou-Tzanetaki,

1992; Litopoulou-Tzanetaki et al., 1993; Torri Tarelli

et al., 1994; Freitas et al., 1995; Macedo et al., 1995;

Centeno et al., 1996). In situ bacteriocin production

may further improve the competitiveness of strains

that naturally occur on the one hand, and deliver a

microbiologically safe end product on the other hand.

Hence, bacteriocin-producing enterococci may be

exploited as commercial starter cultures, provided

they can be considered as safe (Giraffa et al., 1997).

For a long time, some enterococci have been used as

probiotic cultures in human and animal health promo-

tion (Lewenstein et al., 1979; Bellomo et al., 1980;

Underdahl, 1983).

Most enterococci are known to produce class II

bacteriocins, i.e. bacteriocins that are small (4–6

kDa), heat-stable, cationic, hydrophobic, and antibac-

terial peptides (De Vuyst and Vandamme, 1994; Nes

et al., 1996). They consist of between 30 and 60

amino acid residues, and are in particular inhibitory

towards related, Gram-positive bacteria. Currently,

three subclasses can be distinguished among class II

bacteriocins (Ennahar et al., 2000). Enterocin A

(Aymerich et al., 1996), enterocin P (Cintas et al.,

1997), and enterocin CRL35 (Farıas et al., 1996) from

E. faecium, bacteriocin 31 from E. faecalis (Tomita et

al., 1996), and mundticin from Enterococcus mundtii

(Bennik et al., 1998) belong to class IIa. Class IIa

antibacterial peptides are characterized by the pres-

ence of a consensus sequence motif YGNG in their

N-terminal amino acid sequence, the presence of

cysteine residues involved in one (or more) disulfide

bond(s), as well as by their strong inhibitory effect

towards Listeria (Eijsink et al., 1998; Ennahar et al.,

2000). They are synthesized ribosomally as prepep-

tides with an N-terminal leader peptide of the double-

glycine type that is cleaved off during translocation,

and they are secreted out of the cell by dedicated

transport machinery, except for enterocin P (Cintas

et al., 1997). The latter bacteriocin is secreted via

the general secretory pathway of the cell, a property

that was first used to distinguish another subclass

among class II bacteriocins; however, the former

class IIc has been eliminated (Ennahar et al., 2000;

Cleveland et al., 2001). Class IIb bacteriocins are

composed of two polypeptide chains. Class IIc (for-

merly sometimes referred to as class IId) groups

bacteriocins that do not belong to the other subclasses

(Moll et al., 1999; Herranz et al., 2001), e.g. enterocin

B (Casaus et al., 1997) and enterocins L50A and

L50B (Cintas et al., 1998) from E. faecium, and

enterocins 1071A and 1071B from E. faecalis (Balla

et al., 2000). Enterocin B and the enterocins 1071A

and 1071B share the characteristics of class IIa

bacteriocins but do not contain a YGNG motif

(Casaus et al., 1997). Enterocins L50A and L50B

are synthesized without an N-terminal leader sequence

or signal peptide (Cintas et al., 1998). Furthermore,

the enterocins L50A and L50B share structural

homology with the staphylococcal hemolysins,

although they do not show hemolytic activity. Finally,

enterocin AS-48 is a cyclic peptide with broad anti-

bacterial activities against Gram-positive and Gram-

negative bacteria, and therefore it was considered a

peptide antibiotic (Galvez et al., 1989; Martınez-

Bueno et al., 1994).

An example of a class I bacteriocin or lantibiotic

is the two-component cytolysin (bacteriocin/hemoly-

sin) from E. faecalis (Booth et al., 1996). Cytolysin

is produced by a significant number of clinical E.

faecalis isolates (Gilmore et al., 1994; Booth et al.,

1996), and has been associated with virulence in

animal models (Jett et al., 1992). In addition, some

enterocins are encoded on pheromone-responsive,

conjugative plasmids (Gilmore et al., 1994; Martı-

nez-Bueno et al., 1994; Tomita et al., 1996). Pher-

L. De Vuyst et al. / International Journal of Food Microbiology 84 (2003) 299–318300

omones, hemolysin or cytolysin production, as well

as resistance to phagocytosis and adherence may

potentially contribute to virulence in enterococci

(Libertin et al., 1992; Jett et al., 1994; Franz et al.,

1999a; Gasson and Eaton, 2001). Moreover, vanco-

mycin resistance is encoded by transferable genetic

elements (Arthur and Courvalin, 1993). In recent

years, there has been an emergence of antibiotic-

resistant enterococci, E. faecium being the predom-

inant enterococcal species in the subset of vancomy-

cin-resistant enterococcal (VRE) isolates (Morrison

et al., 1997).

The aim of the present study was to assess the

bacteriocin production capacity of 426 Enterococcus

spp. of geographically widespread origin and from

different sources. The vancomycin resistance pheno-

type, the presence of vancomycin resistance genes as

well as the incidence of hemolysis was also checked.

Finally, the relationship between bacteriocin produc-

tion, hemolysis, and vancomycin resistance was eval-

uated to estimate their implications for the safe

commercial application of bacteriocin-producing

enterococci in both food and health care.

2. Material and methods

2.1. Bacterial strains

A collection of 426 Enterococcus strains, that is

part of a European joint research project (FAIR CT97-

3078), is available at the BCCM/LMG Bacteria Cul-

ture Collection (Laboratory of Microbiology, Gent,

Belgium). This FAIR-E collection consists of 173

strains of E. faecium, 175 strains of E. faecalis, 34

strains of Enterococcus durans, 19 strains of Enter-

ococcus hirae, 12 strains of Enterococcus gallinarum,

6 strains of Enterococcus casseliflavus, 2 strains each

of Enterococcus malodoratus, E. mundtii and Enter-

ococcus avium, and 1 strain of Enterococcus pseu-

doavium randomly collected from food and feed as

well as animal and clinical and nonclinical human

isolates. Source and strain information is available

in the catalogue of this FAIR-E collection of enter-

ococci (Vancanneyt et al., 1999). Fifty-two strains

were used as indicators for their sensitivity towards

bacteriocins produced by the enterococci (Table 1).

Control strains for PCR detection of eight entero-

cin structural genes were obtained from different

research groups (Table 2). Four control strains for

the detection of the vancomycin resistance genotype

(vanA, vanB, vanC1, and vanC2) were kindly pro-

vided by Dr. Denis Pierard (University Hospital,

VUB, Brussels, Belgium): E. faecium Iowa 1, E.

faecium Iowa 2, E. gallinarum LMG 13129, and E.

casseliflavus 110.

2.2. Media, growth and maintenance conditions

All LAB strains were grown in MRS (de Man,

Rogosa and Sharpe) medium (Oxoid, Basingstoke,

UK) at 37 jC. Staphylococcus carnosus, Bacillus

cereus and Escherichia coli were grown in Brain

Heart Infusion (BHI) (Oxoid) at 37 jC, while Listeriaspp. were grown in the same medium at 30 jC.Propionibacterium spp., Clostridium spp., and Bifi-

dobacterium spp. were grown in MRS at 30 jC,Reinforced Clostridium Medium (RCM) (Oxoid) at

37 jC, or Beerens Medium (Beerens, 1990) at 37 jC,respectively, in an anaerobic cabinet containing 80%

N2, 10% CO2 and 10% H2 (Don Whitley Scientific,

West Yorkshire, UK). Stock cultures were stored at

� 80 jC in the same medium as used for strain

cultivation, supplemented with 25% (v/v) of glycerol.

Fresh cultures were prepared by inoculation of 10 ml

of the appropriate medium with 5 Al of the frozen

stock followed by incubation for 16 to 24 h at the

temperature indicated above.

2.3. Bacteriocin assay

Enterococcus strains that were selected for their

inhibitory activity towards Listeria, Clostridium and

Propionibacterium during a prescreening were further

screened for their capacity to produce bacteriocins by

testing cell-free culture supernatants against a series of

indicator strains (Table 1). Cell-free culture super-

natants of the Enterococcus strains were obtained by

microcentrifugation (10,000 rpm, 10 min) of 1.5 ml of

culture twice, and were adjusted to pH 6.5. Bacter-

iocin activity was detected by the agar spot assay (De

Vuyst et al., 1996), except for Clostridium spp. as

indicator strains for which an agar well diffusion

assay was performed (Lopez-Lara et al., 1991). Plates

were incubated for 16 to 20 h at the appropriate

growth temperature. Growth inhibition was visually

L. De Vuyst et al. / International Journal of Food Microbiology 84 (2003) 299–318 301

detected either by observing clear inhibition zones on

top of the agar (agar spot assay) or clear halos around

the wells (agar well diffusion assay), and further

referred to as Bac+. All bacteriocin assays were done

in duplicate.

2.4. PCR detection of enterocin structural genes

PCR amplification of known structural genes of

enterocin A (Aymerich et al., 1996), enterocin B

(Casaus et al., 1997), enterocin P (Cintas et al.,

1997), enterocin L50A/ L50B (Cintas et al., 1998),

and bacteriocin 31 (Tomita et al., 1996) was per-

formed with the specific primers listed in Table 3. In

general, primers were constructed based on the C-

and N-terminal amino acid sequence of the mature

bacteriocin, and an internal nested primer was in some

cases used in conjunction with the forward primer to

further confirm the specificity of the PCR reaction.

For enterocin AS-48, the forward primer was con-

structed based on a sequence within the N-terminal

leader peptide of the bacteriocin precursor (Galvez

et al., 1989). In the case of cytolysin that consists of

two polypeptides (Cyl LL and Cyl LS), the forward

primer was constructed based on the Cyl LL sequence,

while the reverse primer was constructed based on

the Cyl LS sequence (Booth et al., 1996). No specific

primer could be designed for enterocin CRL35 (5

out of 21 amino acids are not identified; Farıas et

al., 1996).

PCR amplification was performed using total DNA

isolated by the rapid alkaline lysis method as described

in detail by Dutka-Malen et al. (1990). PCR was

performed on a DNA thermal cycler (Biometra Ther-

mocycler T3; Westburg, Leusden, The Netherlands) in

a final volume of 50 Al containing 1� PCR buffer (20

mM Tris–HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2,

200 AM each of the four dNTPs, 0.5 AM of each

Table 1

Indicator organisms used during the screening for bacteriocin production

Bacillus cereus LMG 13569 Lactobacillus helveticus LMG 13555

Bifidobacterium bifidum LMG 11041T Lactobacillus plantarum LMG 13556

Bifidobacterium breve LMG 11042T Lactobacillus reuteri LMG 13557

Bifidobacterium gallinarum LMG 11596T Lactobacillus sakei LMG 13558T

Bifidobacterium longum LMG 13197T Lactococcus lactis subsp. cremoris LMG 6897

Bifidobacterium infantis LMG 8811T Lactococcus lactis subsp. cremoris LMG 9460

Clostridium sporogenes LMG 13570 Lactococcus lactis subsp. cremoris LMG 13563

Clostridium tyrobutyricum LMG 13571 Lactococcus lactis subsp. lactis LMG 6890

Clostridium tyrobutyricum LMG 1285* Lactococcus lactis subsp. lactis LMG 7949

Enterococcus faecalis LMG 13566 Lactococcus lactis subsp. lactis LMG 8522

Enterococcus faecalis LMG 7937 Leuconostoc cremoris LMG 13562

Enterococcus faecalis LMG 8222 Listeria innocua RZS

Enterococcus faecalis LMG 11395 Listeria innocua CTC 1012

Enterococcus faecalis LMG 16216 Listeria innocua CTC 1014

Enterococcus faecalis LMG 16337 Listeria innocua LMG 13568*

Enterococcus faecium LMG 11397 Listeria innocua LMG 11387*

Enterococcus faecium LMG 11423 Listeria ivanovii LTH 3097

Enterococcus faecium LMG 14203 Pediococcus pentosaceus LMG 13560

Enterococcus faecium LMG 14255 Pediococcus pentosaceus LMG 13561

Enterococcus faecium LMG 15877 Propionibacterium acidipropionici LMG 13572

Enterococcus faecium CTC 492 Propionibacterium sp. LMG 13573

Lactobacillus acidophilus LMG 13550 Propionibacterium sp. LMG 13574

Lactobacillus delbrueckii subsp. bulgaricus LMG 13551 Propionibacterium freudenreichii subsp. shermanii LMG 16424*

Lactobacillus casei LMG 13552 Staphylococcus carnosus LMG 13567

Lactobacillus curvatus LMG 13553 Streptococcus thermophilus LMG 13564

Lactobacillus fermentum LMG 13554 Streptococcus thermophilus LMG 13565

CTC=Centre de Tecnologia de la Carn, Institut de Recerca i Tecnologia Agroalimentaries,Monells, Spain; LMG=LaboratoryMicrobiology Gent

(BCCM/LMG Bacteria Culture Collection), Gent, Belgium; LTH= Institut fur Lebensmitteltechnologie, Universitat Hohenheim, Hohenheim,

Germany; RZS=Rijkszuivelstation, Melle, Belgium.

* Used in prescreening experiment.


primer and 1.25 units of Taq DNA polymerase (Gibco

Life Technologies, Paisley, UK). The cycles used were

95 jC for 5 min for the first cycle, 95 jC for 30 s, 58

jC (for the primers of enterocin A, bacteriocin 31 and

cytolysin) or 56 jC (for the primers of the enterocins

B, P, L50A, L50B, and AS-48) for 30 s, and 72 jC for

30 s for the next 30 cycles; 72 jC for 5 min were used

for the last cycle. PCR products were resolved by

electrophoresis on a 8% polyacrylamide–Tris–ace-

tate–EDTA gel.

Initial PCR experiments carried out with total DNA

of the control strains (Table 2), using the eight sets of

primers separately, exclusively revealed the presence

of the expected enterocin in each strain, confirming

the specificity of the primers used. In the case of

enterocins P, L50A and L50B, no control strains were

available. Interestingly, the primers designed for the

enterocins P, L50A and L50B used in the PCR

reactions with total DNA of the strains, mentioned

in Table 2, did not give any amplification signal. This

Table 3

Specific terminal and nested primers for the PCR detection of enterocin structural genes

Enterocin Forward primer Nested primer Reverse primer

A 5V-GGT ACC ACT CAT

AGT GGA AA-3V5V-AAT GTA CGG TCG

ATT GGG CCA-3V5V-CCC TGG AAT TGC

TCC ACC TAA-3VB 5V-CAA AAT GTA AAA

GAA TTA AGT ACG-3V5V-AAC TTA TCT AAA

GGT GGA GCA-3V5V-AGA GTA TAC ATT

TGC TAA CCC-3VP 5V-GCT ACG CGT TCA

TAT GGT AAT-3V– 5V-TCC TGC AAT ATT

CTC TTT AGC-3V31 5V-CCT ACG TAT TAC

GGA AAT GGT-3V5V-TGG GTA GAC TGG

AAT AAA GCT-3V5V-GCC ATG TTG TAC

CCA ACC ATT-3VAS48 5V-GAG GAG TAT CAT

GGT TAA AGA-3V5V-GCA GTT GCA GGA

ACT GTG CT-3V5V-ATA TTG TTA AAT

TAC CAA-3VL50A 5V-ATG GGA GCA ATC

GCA AAA TTA-3V– 5V-TTT GTT AAT TGC

CCA TCC TTC-3VL50B Idem L50A-F – 5V-TAG CCA TTT TTC

AAT TTG ATC-3VCyl 5V-GGC GGT ATT TTT

ACT GGA GTN-3V– 5V-CCT ACT CCT AAG

CCT ATG GTA-3V

Table 2

Control strains used for the detection of known enterocin structural genes through PCR

Control strain Enterocins Bacteriocin data Origin

Enterococcus faecium

CTC492

A 47 aa, pediocin-like,

GG-leader (18 aa)

M. Hugas, IRTA, Monells, Spain

B 53 aa, GG-leader (18 aa)

Enterococcus faecalis

FA2-2 (pYI17)

31 43 aa, pediocin-like,

sec-dependent leader (24 aa)

Y. Ike, Gumma University School of Medicine,

Gumma, Japan

Enterococcus faecium

CRL35

CRL35 21 aa, pediocin-like,

no leader reported

F. Sesma, INSIBIO, Tucuman, Argentina

Enterococcus faecalis X1 AS-48 70 aa, leader M. Nunez, CIT-INIA, Madrid, Spain

Enterococcus faecalis X2

Enterococcus faecalis X3

Enterococcus faecalis 7C5

Enterococcus faecalis

OG1X (pAM714)

Cyl LL 38 aa, lantibiotic,

secretion signal (30 aa)

D.B. Clewell, University of Michigan,

Ann Arbor, Michigan, USA

Cyl LS 21 aa, lantibiotic,

secretion signal (42 aa)


indicates that the latter primers were sufficiently

specific to distinguish the structural genes of the

enterocins P, L50A and L50B from the other enter-

ocins investigated.

For reproducibility, PCR reactions were repeated

on freshly isolated total DNA from 27 enterococci

that were randomly chosen. Three strains were

included as negative controls, namely E. mundtii

W6 (producer of mundticin), E. faecium FAIR-E 20,

and E. faecalis FAIR-E 256. E. faecium CTC 492

(enterocins A and B), E. faecalis FA2-2 carrying the

plasmid pYI171 (bacteriocin 31), E. faecalis X1

(enterocin AS-48), and E. faecalis OG1X carrying

the plasmid pAM714 (cytolysin) were used as positive

controls.

Confirmation of specific enterocin sequences

(enterocins P, L50A and L50B for which no controls

were available, and bacteriocin 31) or sequences from

strains carrying multiple enterocin structural genes, in

both cases chosen randomly, was done by sequencing

of PCR amplicons with an automated DNA sequencer

(ALFk DNA Sequencer; Amersham Pharmacia Bio-

tech, Uppsala, Sweden).

2.5. Hemolysis

The Enterococcus strains were grown overnight in

MRS medium at 37 jC, and then transferred onto

Blood Agar Base (Oxoid) plates containing 7% of

human blood (University Hospital Gent, Gent, Bel-

gium). The plates were incubated overnight at 37 jC.The hemolytic reaction was recorded by observation

of a clear zone of hydrolysis around the colonies

(h hemolysis), a partial hydrolysis and greening zone

(a hemolysis) or no reaction (g hemolysis).

2.6. Vancomycin and teicoplanin susceptibility test

Susceptibility of the Enterococcus strains to the

glycopeptide antibiotics, vancomycin and teicopla-

nin, was checked using a broth dilution technique.

A glycopeptide standard solution was prepared

by dissolving 6.4 mg of either vancomycin (Lilly

Deutschland, Hessen, Germany) or teicoplanin

(Hoechst Marion Roussel Deutschland, Bad Soden,

Germany) in 100 ml of Trypticase Soy Yeast Ex-

tract (TSYE) medium. TSYE contains per liter of

distilled water, adjusted to pH 7.1: 30.0 g of trypticase

soy broth (Merck) and 3.0 g of yeast extract (Merck).

A two-fold dilution series of the antibiotics was

prepared by transferring 5 ml of the standard solution

into 5 ml of sterile TSYE resulting in tubes with a

concentration of 64, 32, 16, 8, 4, and 2 Ag of the

antibiotic per ml. The Enterococcus strains were

grown overnight in TSYE. They were subsequently

transferred to each tube (1 Al). After an incubation

period of 24 h at 37 jC, the Minimum Inhibitory

Concentration (MIC) values were determined as the

lowest antibiotic concentration that did not allow

growth.

2.7. Detection of the glycopeptide resistance genotype

by multiplex PCR

Detection of glycopeptide resistance genotypes

was performed by multiplex PCR as described pre-

viously (Dutka-Malen et al., 1995). Primers for

vanA, vanB, vanC1, and vanC2 were used in the

assay.

3. Results

3.1. Screening for bacteriocin production

A prescreening of 426 strains of Enterococcus

spp. that was carried out by different European

research groups in the frame of a European joint

research project (FAIR) revealed 122 Enterococcus

strains inhibitory towards Listeria, Clostridium and/or

Propionibacterium: 63 E. faecium, 41 E. faecalis, 8

E. durans, 5 E. hirae, 2 E. gallinarum, 1 E. casseli-

flavus, 1 E. mundtii, and 1 E. avium. These inhibitory

Enterococcus strains were subjected to an extended

screening for bacteriocin production using 52 indi-

cator bacteria, under carefully controlled conditions.

The results are shown in Tables 4 and 5. Fifty strains

(41.0%) did not produce a bacteriocin (Bac�). Sev-

enty-two strains (59.0%) were bacteriocinogenic

(Bac+): 58.7% of the E. faecium strains and 68.3%

of the E. faecalis strains. Other Bac+ strains were

found among E. durans (3 of 8 tested), E. hirae (2

of 5 tested), one E. casseliflavus (only one tested),

and one E. mundtii (only one tested). E. gallinarum

(two tested) and E. avium (only one tested) were

Bac�.


The Bac+ E. faecium and E. faecalis strains did not

show significantly different inhibitory spectra. Almost

half of the Bac+ Enterococcus strains displayed a very

narrow inhibitory spectrum. For instance, eight en-

terococcal strains exclusively inhibited strains of

Listeria spp., and the inhibitory spectrum of one

Enterococcus strain was limited to the genera Listeria

and Clostridium; two Enterococcus strains inhibited

only Lactococcus spp. Eighteen strains inhibited five

or more of the tested genera. The latter so-called

broad-spectrum inhibitors were equally divided

among the E. faecium and E. faecalis strains; the

only E. mundtii strain (FAIR-E 95) tested also dis-

played a broad inhibitory spectrum. Inhibition of

Listeria was the most prevailing type of antagonism

(59.0% of the Bac+ strains). This was followed by

inhibition of lactobacilli, of which Lactobacillus sakei

was the most sensitive. Related enterococci were

inhibited by 44.0% of the Bac+ strains. Lactococci

and Bifidobacteria were inhibited by 29.0% and

40.0% of the Bac+ strains, respectively. The majority

of the Bac+ strains (71.0%) did not inhibit Lactococ-

cus. Nine enterococci (13.0%) inhibited one or two

Lactococcus strains, while only 12 strains (16.0%)

were able to inhibit all lactococci tested. Among the

spoilage and/or pathogenic bacteria other than Liste-

ria, Clostridium was most sensitive (inhibited by

36.0% of the Enterococcus strains), followed by

Propionibacterium (26.0%) and Staphylococcus

(9.0%). Pediococcus and Bacillus strains were

inhibited only by 8.0% and 1.0% of the Bac+ strains,

respectively. Strains of Leuconostoc and Streptococ-

cus were not inhibited.

3.2. Detection of enterocin structural genes

PCR reactions were performed on total DNA of

the 122 inhibitory Enterococcus strains resulting

from the prescreening. The PCR results are shown

in Tables 4 and 5. They revealed the presence of at

least one enterocin structural gene in 64 strains

(52.5%), further referred to as PCR+ strains:

79.4% of the E. faecium strains, 31.7% of the E.

faecalis strains, and 12.5% of the E. durans strains.

The enterocins A, B, L50A and L50B were exclu-

sively found among E. faecium strains, while enter-

ocin P and bacteriocin 31 structural genes were

detected in strains of both E. faecium and E. durans.

The enterocin AS-48 gene was found exclusively

among E. faecalis strains, whereas cytolysin was

found in E. faecalis strains and in one E. faecium

strain.

Among the PCR+ enterococci, 25 strains (39.1%)

showed a positive signal for one single enterocin, 31

strains (48.4%) were positive for two different enter-

ocins, 7 strains (10.9%) for three distinct enterocins,

and 1 strain (1.6%) was positive for four enterocins.

These data indicate the presence of different enter-

ocin structural genes among several enterococci

(Tables 4 and 5). Sequencing of the PCR amplicons

from the randomly chosen strains FAIR-E 27 (L50A/

L50B, P), FAIR-E 195 (Bac31, P), FAIR-E 210 (P),

FAIR-E 211 (P), FAIR-E 213 (Bac31, P) and FAIR-

E 219 (Bac31, P) confirmed these findings.

The structural gene of enterocin A was detected in

46 E. faecium strains, of which it was unique for only

11 strains. The combination of enterocins A and P

was most frequent (17 strains), followed by the

combination of the enterocins A and B (6 strains).

Other combinations with enterocin A were found for

bacteriocin 31 (4 strains), enterocin P and bacteriocin

31 (4 strains), enterocins B and P (3 strains), and

enterocins B, P, L50A and L50B (1 strain). The

enterocin B structural gene was detected in 9 E.

faecium strains, and was always accompanied by

the enterocin A structural gene. The enterocin P

structural gene was found in 27 E. faecium strains

(twice as the sole enterocin gene) and in E. durans (1

strain). The bacteriocin 31 structural gene never

occurred singly and was found in 8 E. faecium strains

and in E. durans (1 strain). The structural genes of

the enterocins L50A and L50B were detected in 2 E.

faecium strains, once in combination with enterocin P

and once with the enterocins A, B, and P. The

structural gene of enterocin AS-48 was found in 5

E. faecalis strains, twice in combination with the

cytolysin structural gene. The cytolysin structural

gene was detected in E. faecalis (10 strains) and once

in E. faecium, either appearing single (9 strains) or in

combination with enterocin AS-48 (2 strains). Eight

of the broad-spectrum inhibitors mentioned above

contained both the enterocin AS-48 and cytolysin

structural genes.

Among the 64 PCR+ Enterococcus strains, 50

strains were considered Bac+ based on the screening

test, indicating that in 14 PCR+/Bac� strains either no


Table 4

Classification of the 72 Bac+ FAIR-E isolates based upon their inhibitory spectra, mentioning the origin and source of the strains, and prevalence of enterocin structural genes and glycopeptide

resistance genes as well as the MIC-values for vancomycin and teicoplanin resistance

Enterococcus Source/origin Inhibitory spectrum*

strain FAIR-E

numberLactococcus

lactis subsp.

lactis (3)a

Lactococcus

lactis subsp.

cremoris (3)b

Lactobacillus

spp. (9)cPediococcus

pentosaceus

(2)d

Leuconostoc

cremoris

(1)e

Streptococcus

thermophilus

(2)f

Enterococcus

faecium (6)g

Inhibitory spectrum restricted to Enterococcus spp.

Enterococcus

faecium 212

Greek Feta

cheese

1

Enterococcus

faecium 213

Greek Feta

cheese

1

Enterococcus

faecium 218

Greek Feta

cheese

4

Enterococcus

faecium 219

Greek Feta

cheese

1

Enterococcus

faecium 220

Greek Feta

cheese

1

Enterococcus

faecalis 255

Italian

Crescenza

cheese

Enterococcus

faecalis 257

Italian

cheese

1

Inhibitory spectrum restricted to other lactic acid bacteria

Enterococcus

faecium 13

Brine of

Greek

Feta curd

1

Enterococcus

faecalis 29

Minipig

feces

Enterococcus

faecalis 43

Italian

shellfish

4

Enterococcus

faecalis 45

Poultry

offals

1

Enterococcus

faecalis 113

Human skin

Enterococcus

faecalis 114

Human

surgical

wound

1

Enterococcus

faecium 202

Greek Feta

cheese

1

Enterococcus

faecium 206

Greek Feta

cheese

1

Enterococcus

faecalis 377

Tuna

intestines

1

Enterococcus

faecalis 381

Poultry

offals

1 1

Enterococcus

hirae 390

Italian

cheese

1

Inhibitory spectrum restricted to Enterococcus spp. and other lactic acid bacteria

Enterococcus

faecium 196

Greek Feta

cheese

1 1

Enterococcus

durans 389

Italian Valtellina

cheese

1 1

Inhibitory spectrum restricted to Enterococcus spp. and spoilage or pathogenic bacteria

Enterococcus

faecium 197

Greek Feta

cheese

6

Enterococcus

faecium 198

Greek Feta

cheese

6

Inhibitory spectrum restricted to Enterococcus spp., other lactic acid bacteria and spoilage or pathogenic bacteria

Enterococcus

faecium 23

Ostrich cecum 1 1 2

Enterococcus

faecium 26

Black olives 1 1

Enterococcus

faecium 27

Minipig feces 1 1 3


Enterocin Hemolysis Glycopeptide resistance

Enterococcus

faecalis

(6)h

Bifidobacterium

spp. (4)iListeria

spp. (6) jClostridium

spp. (3)kPropionibacterium

spp. (3)lStaphylococcus

carnosus

(1)m

Bacillus

cereus (1)nEscherichia

coli (1)o

structural

genesPhenotype

(MIC vancomycin/

MIC teicoplanin)

Genotype

A, P g

A, P, 31 g

A, 31 g

A, P, 31 g

A, 31 g

2 – g 8/2

1 – g 8/2

A, 31 g

1 – g

– g

– g

1 – g

1 – g

A, P h

A, P h

– g 4/2

2 cytolysin g 4/2

– g

A, P h

1 – g

2 A, P g

3 A, P g

1 5 1 1 A h

1 6 1 A, B g

1 2 3 P, L50A/B g

(continued on next page)


Enterococcus Source/origin Inhibitory spectrum

strain FAIR-E

numberLactococcus

lactis subsp.

lactis (3)a

Lactococcus

lactis subsp.

cremoris (3)b

Lactobacillus


pentosaceus

(2)d

Leuconostoc

cremoris

(1)e

Streptococcus

thermophilus

(2)f

Enterococcus

faecium (6)g

Enterococcus

faecium 31

Minipig feces 1 3

Enterococcus

faecium 34

German sausage 1 2

Enterococcus

faecalis 88

Italian cheese 3 3 5 1

Enterococcus

faecalis 90

Italian cheese 3 3 6 1 3

Enterococcus

faecalis 92

Italian cheese 3 2 6 1 3

Enterococcus

mundtii 95

Italian grass

silage

1 4

Enterococcus

faecium 152

Japanese quail 1

Enterococcus

faecium 170

Belgian silage 1 3

Enterococcus

faecium 171

Belgian cheese 1 2

Enterococcus

faecium 172

Slovakian cow’s

rumen

1

Enterococcus

faecalis 177

Minipig feces 3 3 6

Enterococcus

faecium 178

Minipig feces 1 4

Enterococcus

faecalis 179

Minipig feces 3 3 1 6

Enterococcus

faecalis 256

Italian cheese 3 2 6

Enterococcus

faecalis 259

Italian cheese 1

Enterococcus

faecalis 309

Argentinian

cheese

3 2 6

Enterococcus

faecium 386

Italian Fontina

cheese

1

Enterococcus

faecalis 404

Italian raw

milk

3 2 4

Inhibitory spectrum restricted to other lactic acid bacteria and spoilage or pathogenic bacteria

Enterococcus

faecium 20

Swiss

cheese

1

Enterococcus

faecium 39

Human clinical

isolate

1 1

Enterococcus

faecalis 77

Italian goat

cheese

3 3 5

Enterococcus

faecalis 103

Pig isolate 5

Enterococcus

hirae 110

Human urine 1 3 2

Enterococcus

faecalis 116

Human urine 4

Enterococcus

faecium 119

Human feces 1

Enterococcus

faecalis 124

Human feces 5

Enterococcus

faecalis 125

Human feces 1 5

Enterococcus

faecium 131

Human urine 1

Enterococcus

faecium 132

Human rectum

Enterococcus

faecium 134

Human bile 1 1

Enterococcus

faecium 135

Human blood 1 1

Table 4 (continued)



Enterococcus

faecalis

(6)h

Bifidobacterium

spp. (4)iListeria




carnosus

(1)m

Bacillus


coli (1)o

structural

genesPhenotype

(MIC vancomycin/

MIC teicoplanin)

Genotype

1 6 1 A g

1 1 3 A g

3 3 2 3 1 AS-48 g

2 4 2 3 1 cytolysin, AS-48 h

4 4 2 3 cytolysin, AS-48 h

5 1 6 1 3 – g

2 5 1 A, P g

1 6 1 A, B, P g

1 6 A, B, P g

1 6 A g

6 2 2 cytolysin h

2 6 1 A, B, P g

6 1 2 2 cytolysin g

6 2 1 1 3 1 – g

3 2 – g 4/2

6 2 1 2 3 1 – g

1 5 A, B g

2 2 4 2 1 1 1 AS-48 g

5 1 1 – g

1 1 1 cytolysin g >64/>64 vanA

1 3 2 3 1 AS-48 g

2 1 cytolysin h 2/4

1 1 – g 2/4

2 3 1 cytolysin h

2 2 1 A, B, P, L50A/B g

3 1 cytolysin h

3 3 1 cytolysin h

1 5 1 A, B g >64/32 vanA

1 1 A g >64/32 vanA

1 5 1 A, B g >64/64 vanA

2 5 1 A, B g >64/32 vanA

(continued on next page)


or a too low phenotypic expression of the enterocin

genes occurred or resistance genes were present. In

addition, among the 58 PCR� strains, 22 strains were

found Bac+ during the screening, including 4 of the 18

broad-spectrum inhibitors.

3.3. Detection of hemolysis

h-Hemolysis of human blood was observed for E.

faecalis FAIR-E strains 90, 92, 103, 116, 124, 125,

146, and 177, all of which displayed a broad inhibitory

Enterococcus Source/origin Inhibitory spectrum

strain FAIR-E

numberLactococcus

lactis subsp.

lactis (3)a

Lactococcus

lactis subsp.

cremoris (3)b

Lactobacillus


pentosaceus

(2)d

Leuconostoc

cremoris

(1)e

Streptococcus

thermophilus

(2)f

Enterococcus

faecium (6)g

Enterococcus

faecium 137

Human bile 1 1

Enterococcus

faecalis 146

Horse

Enterococcus

faecium 153

Bizon 1

Enterococcus

faecalis 299

Italian natural

whey

2

Enterococcus

faecalis 335

Irish Cheddar 1 2 1

Enterococcus

faecium 362

Italian Ricotta

cheese

1

Inhibitory spectrum restricted to spoilage or pathogenic bacteria

Enterococcus

durans 1

Greek Feta

curd

Enterococcus

durans 2

Greek yoghurt

Enterococcus

faecium 3

Greek Feta

cheese

Enterococcus

faecium 84

Italian soft

cheese

Enterococcus

faecalis 100

Tick’s blood

Enterococcus

faecalis 109

Human urine

Enterococcus

faecium 121

Human feces

Enterococcus

faecium 201

Greek Feta

cheese

Enterococcus

casseliflavus

323

Irish raw

milk

Enterococcus

faecium

366

Italian

Scarmorza

cheese

a The Lc. lactis subsp. lactis strains were LMG 6890, LMG 7949 and LMG 8522.b The Lc. lactis subsp. cremoris strains were LMG 6897, LMG 9460 and LMG 13563.c The Lactobacillus spp. were Lb. acidophilus LMG 13550, Lb. delbrueckii subsp. bulgaricus LMG 13551, Lb. casei LMG 13552, Lb. curvatus LMG 13553, Lb. fermentum LMG

13554, Lb. helveticus LMG 13555, Lb. plantarum LMG 13556, Lb. reuteri LMG 13557 and Lb. sakei LMG 13558.d The Pediococcus pentosaceus strains were LMG 13560 and LMG 13561.e The Leuconostoc cremoris strain was LMG 13562.f The Streptococcus thermophilus strains were LMG 13564 and LMG 13565.g The Enterococcus faecium strains were LMG 11397, LMG 11423, LMG 14203, LMG 14255 and LMG 15877.h The Enterococcus faecalis strains were LMG 7939, LMG 8222, LMG 11395, LMG 13566, LMG 16216 and LMG 16337.i The Bifidobacterium spp. strains were B. infantis LMG 8811, B. bifidum LMG 11041, B. breve LMG 11042 and B. longum LMG 13197.j The Listeria spp. strains were L. innocua RZS, CTC 1012, CTC 1014, LMG 11387, LMG 13568 and L. ivanovii LTH 3097.k The Clostridium spp. strains were C. sporogenes LMG 13570, C. tyrobutyricum LMG 13571 and C. tyrobutyricum LMG 1285.l The Propionibacterium spp. strains were P. acidipropionici LMG 13572, P. sp. LMG 13573 and P. freudenreichii subsp. shermanii LMG 16424.m The Staphylococcus carnosus strain was LMG 13567.n The Bacillus cereus strain was LMG 13569.o The Escherichia coli strain was LMG 2092.

* Number of sensitive strains out of the total of strains (between brackets) tested.

Table 4 (continued)


spectrum and also contained the cytolysin structural

gene (Table 4). In the case of E. faecium strains, h-hemolysis was observed for the FAIR-E strains 23,

196, 201, 202, and 206. The four last strains were

derived from Greek Feta cheese and displayed a

narrow inhibitory spectrum. The first strain was of a

nonfood origin and had a broad inhibitory spectrum.

None of these strains contained the cytolysin structural

gene. No hemolytic activity was observed for any of

the other strains tested.


Enterococcus

faecalis

(6)h

Bifidobacterium

spp. (4)iListeria




carnosus

(1)m

Bacillus


coli (1)o

structural

genesPhenotype

(MIC vancomycin/

MIC teicoplanin)

Genotype

1 3 1 A, B g >64/32 vanA

2 1 cytolysin h

5 1 A g

2 1 1 – g 4/2

2 3 3 1 1 – g

5 A, P g 4/2

6 P, 31 g

6 – g

6 A, 31 g

1 A, P g >64/>64 vanA

1 – g 2/4

1 – g

1 1 A, P g

1 A, P h

1 – g vanC2

5 A, P, 31 g 4/2


Table 5

Characteristics of the 50 FAIR-E strains (Bac�) that showed no inhibition towards any of the 52 indicator strains used, presence of structural

genes and glycopeptide resistance genes as well as the MIC-values for vancomycin and teicoplanin resistance

Enterococcus strain Source/origin Enterocin Hemolysis Glycopeptide resistance

FAIR-E number structural

genesPhenotype (MIC vancomycin/

MIC teicoplaninn)

Genotype

Enterococcus faecium 4 Greek Feta cheese A g

Enterococcus faecium 24 Pharmaceutical product – g

Enterococcus avium 28 Minipig feces – g

Enterococcus faecium 56 Reygrass silage – g 4/2

Enterococcus faecium 76 Italian soft cheese A g

Enterococcus faecalis 112 Urine – g 2/4

Enterococcus faecalis 115 Urine – g

Enterococcus faecalis 117 Urine – g 2/4

Enterococcus faecium 118 Feces A, P g

Enterococcus faecalis 122 Feces – g 4/2

Enterococcus faecalis 123 Feces – g

Enterococcus durans 139 Chicken intestine – g

Enterococcus durans 140 Belgian Cheddar cheese – g

Enterococcus durans 143 Unknown – g >64/32 vanA

Enterococcus faecalis 144 Pig’s tonsil – g

Enterococcus faecalis 145 Animal waste – g

Enterococcus faecalis 147 Horse – g

Enterococcus faecalis 148 Pig – g

Enterococcus faecium 149 Pig’s heart – g

Enterococcus faecium 150t1 Belgian goat cheese A, P g

Enterococcus faecium 150t2 Belgian goat cheese A g

Enterococcus hirae 165 Pigeon – g 32/16

Enterococcus gallinarum 187 Faeces – g 8/2 vanC1

Enterococcus gallinarum 194 Unknown – g 8/2 vanC1

Enterococcus faecium 195 Greek Feta cheese A, P, 31 g

Enterococcus faecium 203 Greek Feta cheese A, P g




Enterococcus faecium 210 Greek Feta cheese P g

Enterococcus faecium 211 Greek Feta cheese P g

Enterococcus faecium 214 Greek Feta cheese – g




Enterococcus faecium 222 Greek Feta cheese A g


Enterococcus faecium 227 Irish Cheddar cheese – g

Enterococcus durans 231 Irish Cheddar cheese – g

Enterococcus hirae 240 Greek cheese – g

Enterococcus faecium 244 Sardinian cheese – g

Enterococcus faecium 246 Unknown A g

Enterococcus faecalis 260 Italian Fontina cheese – g

Enterococcus faecium 266 Starter culture – g 4/2

Enterococcus faecalis 298 Natural whey – g 4/2

Enterococcus durans 332 Italian Fontina cheese – g

Enterococcus hirae 360 Irish semi-soft cheese – g

Enterococcus faecalis 375 Italian Venaco cheese – g

Enterococcus faecalis 378 Unknown – g 4/2

Enterococcus faecium 402 Tuna intestine – g


3.4. Detection of glycopeptide resistance

Using the broth dilution technique, MIC values for

vancomycin and teicoplanin were determined. The

results are represented in Tables 4 and 5, together

with the results of the multiplex PCR reactions. The

vanA genotype has been detected in 8 strains of which

7 strains were E. faecium (six of human origin and one

food isolate) and 1 strain was E. durans. None of the

41 E. faecalis strains was positive for vanA. In all

cases, the vanA genotype resulted in a vancomycin

and teicoplanin resistance phenotype with MIC values

above 64 Ag/ml. None of the Enterococcus spp.

isolates was positive for vanB. Both E. gallinarum

strains tested were positive for the vanC1 gene cluster.

Phenotypically, these strains showed moderate resist-

ance to vancomycin (8 Ag/ml) but they were sensitive

to teicoplanin (2 Ag/ml). The only E. casseliflavus

strain tested, which was derived from Irish raw milk,

was positive for the vanC2 gene cluster. This strain

did not show any phenotypical resistance towards

either vancomycin or teicoplanin. All other strains

had MIC values of 2 Ag/ml for both antibiotics and no

van genes were detected.

4. Discussion

Enterococci play an important role in the ripening

of certain traditional cheeses from Mediterranean

countries, and are also marketed as probiotic prepa-

rations in health care (Franz et al., 1999a). Further-

more, enterococci are well known for their production

of bacteriocins, so-called enterococcins or enterocins

that inhibit other bacteria, including several food

spoilage and pathogenic bacteria, e.g. Listeria and

Clostridium (Giraffa, 1995). Enterococci are therefore

interesting additional starter cultures or protective

cultures for cheese manufacture and for the prepara-

tion of novel probiotics (Parente et al., 1989; Centeno

et al., 1996; Coppola et al., 1988; Elmer, 2001;

Foulquie Moreno et al., 2002; Sarantinopoulos et al.,

2002). However, severe safety criteria must be estab-

lished to guarantee a safe, commercial use of enter-

ococci. Considering their clinical involvement in

infections and antibiotic resistance (Murray, 1990;

Jett et al., 1994; Morrison et al., 1997), the safety of

E. faecium and E. faecalis strains associated with food

fermentations and probiotics is being questioned.

Therefore, the bacteriocin type produced and the

presence of hemolytic substances and vancomycin

resistance must be evaluated, in particular with respect

to the introduction of novel, commercial cultures.

Bacteriocin production was mainly found among

the strains of E. faecalis (68.3%) and E. faecium

(58.7%) included in this study. Both species encom-

passed strains of different and geographically well

spread origin covering food isolates (38 strains,

mainly from cheese) and 32 strains of animal origin

(from at least 10 different animals) and human origin

(both clinical and nonclinical isolates). These findings

corroborate other studies showing that, among Enter-

ococcus strains of nonfood origin, E. faecalis displays

the highest bacteriocinogeny (Du Toit et al., 2000; Del

Campo et al., 2001). A remarkable variation of

inhibitory spectra occurred among the E. faecium

and E. faecalis strains tested (Table 4), including

inhibition of, for instance, only closely related enter-

ococci, other LAB or food spoilage and/or pathogenic

bacteria. No correlation was found between the origin

of the strains and the inhibitory spectrum. This may be

due to the ubiquitous nature and persistence of enter-

ococci. Indeed, fecal and environmental contamina-

tion leads to a widespread prevalence of enterococci

making it difficult to trace back the origin of a strain.

In general, strains involved in food fermentations

probably originate from the human or animal GIT,

which is believed to be the principal source of enter-

ococci (Murray, 1990). Remarkably, a clustering of

human isolates from both fecal and clinical origin was

observed in the group of strains inhibiting LAB,

Listeria, and either Staphylococcus or Clostridium.

The variation in inhibitory spectra may be due to

several antagonistic factors, among which the produc-

tion of the known enterocins A and B (Aymerich et

al., 1996; Casaus et al., 1997), P (Cintas et al., 1997),

31 (Tomita et al., 1996), and CRL35 (Farıas et al.,

1996), as well as the production of AS-48 (Martınez-

Bueno et al., 1994), or cytolysin (Booth et al., 1996;

Cintas et al., 1998). Remarkably, the chromosomally

located enterocins A and B were only found among

the E. faecium strains tested. The plasmid-encoded

enterocins AS-48 (Martınez-Bueno et al., 1994; Joos-

ten et al., 1997) and L50A and L50B (Cintas et al.,

1998) were detected among various strains of E.

faecalis and E. faecium, respectively. Chromosomal


localization of enterocin genes can be described as an

intrinsic feature, while plasmid localization will be the

result of acquired antibacterial potential through con-

jugal gene transfer.

The structural gene of enterocin A is widely dis-

tributed among E. faecium strains, whereas that of

enterocin B always occurs in the presence of enterocin

A. This is most probably due to the fact that no

transport genes have been found for enterocin B

production (Franz et al., 1999b). Assuming that these

genes were lost as a result of several mutations, one

may hypothesize that the structural gene of enterocin

B was lost by several strains as well, resulting in the

observation that the structural gene of enterocin A

occurs solely in some strains. This had no significant

impact on the inhibitory spectrum as observed in this

study. In contrast, Casaus et al. (1997) report on the

different target specificity and synergistic action of

enterocins A and B. In addition, all E. faecium strains

possessing the enterocin B structural gene belonged to

the same subcluster of the genomic group I of E.

faecium, except for E. faecium FAIR-E 119 (Vancan-

neyt et al., 2002). Besides enterocin A, a high prev-

alence was seen for enterocin P and bacteriocin 31

among E. faecium and E. durans strains. Whereas

cytolysin (Booth et al., 1996) and enterocin AS-48

(Galvez et al., 1989) were originally found in clinical

isolates of E. faecalis, they are also found in strains

from other sources including foods (Joosten et al.,

1996, 1997; Maisnier-Patin et al., 1996) as was

confirmed by this study. Hence, no relationship can

be established between the presence of enterocin

structural genes and the origin of the strain, given

their widespread distribution, probably due to their

possible transfer and acquisition between strains.

Moreover, no correlation seemed to exist between

the presence of known enterocin genes and the activ-

ity spectra of these enterococci.

The detection of enterocin structural genes does

not imply the production of the corresponding bacter-

iocin. Lack of gene expression, for instance due to the

absence of (the induction of) transcription (Nes et al.,

1996), novel enterocins, and the occurrence of multi-

ple bacteriocin resistance genes (Eijsink et al., 1998)

may further contribute to wide differences in inhib-

itory spectra among strains of the same genus as

observed in this study. As an example, in the case

of the enterocins A and B, both constitutive and

regulated production is reported (Nilsen et al., 1998;

Franz et al., 1999b). It is also noteworthy that a clear-

cut relationship between the structure of an enterocin

and its inhibitory spectrum is not clearly understood.

For instance, an anti-Listeria activity is hypothetically

explained by the presence of a conserved amino acid

sequence motif among the class IIa bacteriocins

(Nieto-Lozano et al., 1992). In addition, the presence

of one or more disulfide bonds could be responsible

for the nature of the inhibitory spectrum (Callewaert et

al., 1999; Ennahar et al., 2000). However, other

researchers have shown that the C-terminal part of a

bacteriocin is likely responsible for variations in the

inhibitory spectrum (Fimland et al., 1996), whereas

others indicate the importance of electrostatic inter-

actions between the bacteriocin molecules and the cell

envelope of the target strains (Bhugaloo-Vial et al.,

1996; Chen et al., 1997).

Importantly, enterocins show a strong activity

towards Listeria, which can be of practical use in

the food industry (Parente and Hill, 1992; Giraffa,

1995; Giraffa et al., 1995; Nunez et al., 1997; Galvez

et al., 1998). Therefore, Enterococcus strains display-

ing a limited inhibitory spectrum due to the produc-

tion of enterocins targeted towards Listeria and/or

Clostridium (Torri Tarelli et al., 1994; Giraffa, 1995;

Franz et al., 1996; this study) would be interesting as

protective cultures for cheese manufacture, given their

very limited antagonistic activity towards dairy starter

cultures such as Lactococcus and Streptococcus

(Foulquie Moreno et al., 2002; Sarantinopoulos et

al., 2002). This would also be of interest when

considering Enterococcus strains as cocultures for

enhanced ripening of cheeses.

Finally, these Enterococcus strains, in particular

those with broad-spectrum activity, must be checked

carefully for the presence of hemolysins/cytolysins.

Cytolysin activity is seen to be a potent virulence

factor in several animal model studies (Ike et al.,

1984; Jett et al., 1992; Chow et al., 1993), and it

plays an important role in the severity of human

infections (Huycke et al., 1991; Jett et al., 1994).

Cytolysin expression goes hand in hand with hemol-

ysis. The incidence of h-hemolysis was not restricted

to E. faecalis strains, but also occurred in E. faecium.

It occurred in strains both from food and nonfood

origin. However, in h-hemolytic E. faecalis strains the

cytolysin structural gene was always present, while in


E. faecium this gene could not be detected. So, the

hemolysis of the latter strains must be caused by

another cytotoxic component. In two strains of E.

faecalis and in one strain of E. faecium, the cytolysin

structural gene was present, but no h-hemolysis

occurred. It can be hypothesized that cytolysin was

not expressed and that instead another (novel) enter-

ocin or antibiotic substance was responsible for the

inhibition. In general, the incidence of h-hemolysis is

lower for E. faecium strains than for E. faecalis

strains. However, absence of hemolytic activity

should be a selection criterion for (bacteriocin-pro-

ducing) starter strains for dairy use (Giraffa, 1995);

nevertheless, absence of hemolytic activity in enter-

ococci isolated from food does not mean that these

bacteria are nonvirulent (Franz et al., 1999a). A

thorough understanding of all characteristics will

certainly contribute to the safety evaluation of enter-

ococci. Interestingly, apparently no single virulence

factor in Enterococcus by itself can convert a harmless

strain into a pathogen (Gasson and Eaton, 2001).

However, strains that are used in the food industry

as starter cultures might acquire virulence factors in

vitro from strains isolated as nosocomial pathogens in

clinical settings (Gasson and Eaton, 2001).

In this study, the incidence of the vanA gene among

E. faecium strains was low (11.1%) and only occurred

in one food isolate. It did not occur in E. faecalis. In a

study of European cheeses, Teuber et al. (1999) also

reported a low (4%) incidence of VRE. Giraffa and

Sisto (1997) did not find VRE among strains isolated

from Italian cheeses in the period 1980–1990;

remarkably, high frequencies of VRE occurred in

cheeses produced more recently (Giraffa et al.,

2000). In contrast, high frequencies of VRE are found

in meat (Wegener et al., 1997; Quednau et al., 1998;

van den Braak et al., 1998; Son et al., 1999). Food of

animal origin is thought to be the most likely route of

transmission of VRE from the animal reservoir to

humans (Klare et al., 1995).

In conclusion, whether interesting Enterococcus

strains, in particular bacteriocin producers, are safe

for use as starter or protective cultures in the produc-

tion of fermented foods and as probiotics is clearly

strain specific. Each strain has to be tested for the

different traits that contribute to its safety evaluation

such as hemolysin/cytolysin activity and vancomycin

resistance. However, based on their lower incidence

among E. faecium strains in this study, it would be

advisable that bacteriocin-producing strains of this

species are used rather than E. faecalis strains. This

is especially valid taking into account the fact that

pheromone-responsive plasmids, which often encode

virulence genes, and antibiotic resistance genes are

mainly carried by E. faecalis (Clewell, 1990; Chow et

al., 1993; Wirth, 1994; Singh et al., 1998).

Acknowledgements

This study was carried out with the financial support

from the Commission of the European Communities’

Agriculture and Fisheries (FAIR) specific RTD pro-

gramme (CT97-3078, Enterococci in Food Fermenta-

tions: Functional and Safety Aspects). It does not

necessarily reflect its views and in no way anticipates

the Commission’s future policy in this area. The

authors are grateful to the different groups of the EU

consortium for their involvement in the prescreening of

the FAIR-E collection of enterococci, in particular Nele

Berthels (VUB, Brussels, Belgium), Prof. Dr. G.

Kalantzopoulos and Prof. Dr. E. Tsakalidou (Agricul-

tural University of Athens, Athens, Greece), Prof. Dr.

K. Kersters and Dr. M. Vancanneyt (University of

Gent, Gent, Belgium), Prof. Dr. J. Huis in’t Veld and

Dr. S. Biesterveld (University of Utrecht, Utrecht, The

Netherlands), Prof. Dr. W. Holzapfel and Dr. C. Franz

(Federal Research Center for Nutrition, Karlsruhe,

Germany), Prof. Dr. F. Dellaglio and E. Knijff

(University of Verona, Verona, Italy), Dr. T. Cogan

and Dr. M. Rea (Teagasc, Fermoy, Ireland), and Dr. A.

Lombardi and C. Andrighetto (Istituto per la Qualita e

la Tecnologie Agroalimentari, Thiene, Italy). The

authors further acknowledge their finances from the

Research Council of the Vrije Universiteit Brussel and

the Fund for Scientific Research Flanders. M.R.

Foulquie Moreno was recipient of a Marie Curie

Fellowship from the European Commission (grant

FAIR CT97-5013).

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