CURRENT MICROBIOLOGY Vol. 41 (2000), pp.

410 – 416
DOI: 10.1007/s002840010159 Current
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2000

Combined Effect of Bacteriocins on the Survival of Various Listeria

Species in Broth and Meat System
Graciela Vignolo,1 Jorge Palacios,1 Marı́a E. Farı́as,1 Fernando Sesma,1 Ulrich Schillinger,2 Wilhelm Holzapfel,2
Guillermo Oliver1
1
Centro de Referencia para Lactobacilos, CERELA, CONICET, Chacabuco 145, 4000 Tucumán, Argentina
2
IHT, Fed. Res. Centre for Nutrition, Engesserstr. 20, 76131 Karlsruhe, Germany

Received: 17 March 2000 / Accepted: 26 June 2000

Abstract. The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705
(produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35,
17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against
Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in
viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin.
A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal
inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used,
no survivors were observed after 24 h of incubation. Similar results were obtained when the
bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB
bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacte-
riocin-resistant Listeria population.

The ability of Listeria strains to grow at temperatures
ranging from 1° to 45°C, their high salt tolerance, and
their ability to initiate growth at a relatively low pH
make these pathogens particularly difficult to control in
food. A novel approach to the control of Listeria mono-
cytogenes and L. innocua in food is the use of antimi-
crobial bacteriocins from lactic acid bacteria [4, 14].
Several bacteriocins are bactericidal to foodborne patho-
genic strains of Listeria. However, certain features, such
as the level of inhibition generally observed, the mode of
bacteriocin action, and the development of bacteriocin-
resistant strains, require further examination.
One of the concerns regarding the use of bacterio-
cins is the development of highly tolerant and/or resistant
strains. It has been observed that Listeria develops tol-
erance towards nisin and pediocin-like bacteriocins at
relatively high frequency in both laboratory media and
foods [18, 23]. Depending on the strain and conditions
used, including the bacteria/bacteriocin ratio, mutation
frequencies of 10⫺6 have been reported [8, 10]. Although
bacteriocins, either produced in situ or added to the
Correspondence to: G. Vignolo: E-mail: vignolo@cerela.org.ar
product, are effective in reducing Listeria populations
and other spoilage or pathogenic microorganisms in
model systems, the occurrence of relatively high num-
bers of survivors or regrowth in food systems is gener-
ally observed. This induction of bacteriocin-resistant or
tolerant strains and mutants [14, 15] may pose further
problems in the use of bacteriocins in biopreservation. In
foods with a long shelf life, even a small number of these
variant cells can multiply to high concentrations. How-
ever, improved control of the target microorganisms and
inhibition of bacteriocin-resistant strains and species can
be obtained by using a combination of one or more
bacteriocins. It is generally believed that the increased
resistance of the mutants is caused by alterations in the
cell envelope, including changes in the fatty acid com-
position of the membrane [2, 9, 11]. The antilisterial
activity of nisin has been reported, and the sensitivity of
L. monocytogenes to this bacteriocin has been observed
to be strain dependent [21].
This paper reports that bacteriocins can be combined
to produce a more effective antibacterial effect against
Gram-positive bacterial cells. Two non-nisin bacterio-
cins, lactocin 705 and enterocin CRL35, were tested
G. Vignolo et al.: Bacteriocins and Listeria Species
together with nisin on the survival of L. monocytogenes
and L. innocua in Tryptic soy broth ⫹0.6% yeast extract
and in a meat system.
Materials and Methods
Bacterial strains and culture conditions. Listeria monocytogenes
FBUNT (Facultad de Bioquı́mica, Quı́mica y Farmacia, Universidad
Nacional de Tucumán, Argentina), ScottA, WR129, SR215, and 4ab; L.
seeligeri WS2253 (Institute of Hygiene and Toxicology, IHT,
Karlsruhe, Germany); and L. innocua 7, 11, 12 and L1PE (Unité de
Recherches Laitiéres et Génétique Appliqueé, INRA, France) were
used. They were stored at ⫺20°C in TSBYE (Tryptic soy broth,
supplemented with 0.6% yeast extract) plus 10% glycerol. Before use,
they were grown twice in TSBYE (pH: 6.7) at 30°C. Listeria strains
used in this work were isolated from different food sources except L.
monocytogenes FBUNT, which was a clinical isolate.
Preparation of bacteriocins. Lactocin 705 was prepared according to
the procedure described by Palacios et al. [16]. Briefly, an overnight
culture in MRS broth of L. casei CRL705, isolated from dry sausages,
was heated to inactivate proteases and kill cells, and the adsorption-
desorption pH-depending methodology developed by Yang et al. [24]
was applied. The active extract was further subjected to RP-HPLC and
SDS-PAGE. Enterocin CRL35, produced by Enterococcus faecium
CRL35 isolated from cheese, was obtained after removing cells from
LAPTg broth [17] and precipitated with (NH4)2SO4 (60%) at 4°C. A
Biogel-P6 column was used to separate ammonium sulfate, and the
bacteriocin preparation was then applied to a CM Sephadex cation
exchanger column. The pooled concentrated fractions were then loaded
on a C18 reverse-phase column [5]. Nisin (Nisaplin, 1 ⫻ 106 IU/g) was
kindly provided by Aplin & Barret Ltd. (Trowbridge, UK). Stock
solutions (105 IU/ml) were prepared by solubilizing appropriate
amounts of powder in a 0.02 N HCl solution. The pH was adjusted to
2.0 with 1 N NaOH; the solution was then filter-sterilized (pore size
0.22 ␮m, Millipore) and stored at ⫺20°C.
Sensitivity of Listeria strains to bacteriocins. The well diffusion
assay in TSAYE (Triptic soy agar supplemented with 0.6% yeast
extract) was used to determine the sensitivity of the strains to lactocin
705 (17,000 AU/ml), enterocin CRL35 (17,000 AU/ml), and nisin at
increasing concentrations (100 to 5000 IU/ml). Bacteriocin activity
(AU/ml) was tested as described below. Frequency of bacteriocin
resistants was determined by comparing the number of colonies arising
after 1 h with the original inoculum size. Insensitive variants of Listeria
strains were isolated by growing the strain in broth containing high AU
of a bacteriocin (17,000 AU/ml of lactocin 705 and enterocin CRL35
and 2000 IU/ml of nisin) for three to four transfers and isolating
colonies on an agar plate containing the bacteriocin. The presence or
absence of a zone of growth inhibition against 10 ␮l (about 170 AU of
lactocin 705 and enterocin CRL35 and 4 AU of nisin) of each prepa-
ration was determined.
Determination of bacteriocin activity. The activity of the purified
stock solutions of lactocin 705 and enterocin CRL35 was determined
by a spot-on-lawn method. Thirty microliter of each twofold dilution
was spotted onto an indicator lawn of L. monocytogenes FBUNT and
L. innocua 7 and incubated for 16 –18 h at 30°C. The indicator lawn
was prepared by adding 70 ␮l of an overnight culture to 7 ml of overlay
TSBYE soft agar. The titer was defined as the reciprocal of the highest
dilution giving a visible zone of inhibition of the indicator lawn and
was expressed in AU/ml. By this method, one IU of nisin was equiv-
alent to 0.16 AU.
411
Kinetics of cell destruction by bacteriocins. Approximately 106
cells/ml of L. monocytogenes FBUNT and L. innocua 7 in stationary
phase were inoculated in TSBYE (pH:6.5) containing either 17,000
AU/ml of lactocin 705, 17,000 AU/ml of enterocin CRL35, or 2000
IU/ml of nisin. These bacteriocins were used individually or in com-
bination; the mixture contained equal AU/ml of each bacteriocin. At
time intervals the survivors were enumerated on TSAYE medium after
appropriate dilutions in peptone water, and colonies were counted after
48 h of incubation at 30°C.
Meat preparation and inoculation. Fresh lean meat was obtained
from a local abattoir, aseptically minced, and stored at ⫺20°C. Ten-
gram portions in saline solution (0.85% NaCl) were placed into stom-
acher bags, and the final volume was then adjusted to 20 ml by the
separate addition of 106 CFU/ml of L. monocytogenes FBUNT and the
purified bacteriocins individually or in combination. Individual con-
centrations of the bacteriocins were 17,000 AU/ml for lactocin 705 and
enterocin CRL35, and 2000 IU/ml for nisin. When they were assayed
in combination, the final concentration were the addition. The bags
were stomached for 3 min and further incubated at 20°C for 24 h. The
survivors of Listeria were ennumerated in PALCAM agar at different
time intervals. Each test was run in duplicate, and mean values were
calculated.
Results
Ten strains of Listeria were tested for sensitivity against
lactocin 705, enterocin CRL35, and differents concen-
trations of nisin (Table 1). Results indicated species and
strain differences when the well assay was employed,
and the presence or absence of zones of inhibition was
recorded. While all Listeria strains were resistant to nisin
concentrations of up to 2000 IU/ml, only L. monocyto-
genes Scott A, WR129, SR215 and L. innocua 12 was
shown to be resistant to both non-nisin bacteriocins (lac-
tocin 705 and enterocin CRL35) under the test condi-
tions. The strains of L. innocua tested were as resistant to
nisin as the most resistant L. monocytogenes, whereas the
strain of L. seeligeri was more sensitive. Moreover, the
sensitivity to bacteriocins was found to be dependent on
the culture medium. When MRS and TSBYE were com-
pared, higher numbers of survivors were counted in
TSBYE after 1 h of exposure to increasing concentra-
tions of nisin than in MRS (data not shown). Out of the
different pH of the media, one explanation for this is the
presence of some components in MRS medium as so-
dium acetate, which may synergistically inhibit Listeria,
with nisin increasing the overall effect. These results
suggest the importance of the specific conditions used to
determine the bacteriocin levels that allow good efficacy
in foods.
L. monocytogenes FBUNT, Scott A and L. innocua
7 were selected to be tested with lactocin 705, enterocin
CRL35, and nisin, either individually or combined, in
TSBYE broth. The results in Table 2 show that the action
of bacteriocins on growing cells of Listeria was charac-
terized by a two-phase response: an initial decrease in
412 CURRENT MICROBIOLOGY Vol. 41 (2000)

Table 1. Sensitivity of Listeria strains to different bacteriocins

Nisin (IU/ml) Lactocin 705 Enterocin CRL35

(AU/ml) (AU/ml)
Strains 100 500 1000 2000 5000 17,000 17,000

L. monocytogenes FBUNT ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹
L. monocytogenes ScottAa ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫺
L. monocytogenes WR129a ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫺
L. monocytogenes 4ba ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹ ⫺
L. monocytogenes SR215a ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺
L. innocua 7b ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺
L. innocua 11b ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫺
L. innocua 12b ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺
L. innocua L1PEb ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫺
L. seeligeri WS 2253a ⫺ ⫺ ⫺ ⫺ ⫹ ⫹⫹ ⫹

a
Strains from IHT, Karlsruhe, Germany.
b
Strains from INRA, France.
Inhibition of growth was expressed as follow: [⫺] negative, inhibition zone ⱕ5 mm; [⫹] between 6 and 12 mm; and [⫹⫹] between 13 and 20
mm.

Table 2. Bactericidal effectiveness of nisin, lactocin 705, and enterocin CRL35 to Listeria strains after incubation at 30°C in TSBYE broth
during 24 h

cfu/ml in TSBYE containing

Incubation Enterocin
time None Lactocin 705 CRL35 Nisin
Listeria species (h) (control) (17,000 AU/ml) (17,000 AU/ml) (2000 IU/ml)

L. monocytogenes FBUNT 0 8.8 ⫻ 105 8.0 ⫻ 105 6.2 ⫻ 105 6.5 ⫻ 105
1 1.4 ⫻ 106 8.2 ⫻ 102 9.3 ⫻ 102 6.5 ⫻ 102
24 2.5 ⫻ 109 8.0 ⫻ 103 7.5 ⫻ 104 2.5 ⫻ 105
L. monocytogenes Scott A 0 2.2 ⫻ 106 8.1 ⫻ 105 7.8 ⫻ 105 7.4 ⫻ 105
1 3.0 ⫻ 106 9.0 ⫻ 102 9.0 ⫻ 102 8.0 ⫻ 102
24 5.2 ⫻ 109 4.4 ⫻ 104 2.2 ⫻ 105 4.2 ⫻ 105
L. innocua 7 0 1.4 ⫻ 106 1.0 ⫻ 106 1.6 ⫻ 106 1.2 ⫻ 106
1 3.5 ⫻ 106 1.5 ⫻ 103 8.6 ⫻ 103 1.3 ⫻ 103
24 1.5 ⫻ 109 2.5 ⫻ 104 8.0 ⫻ 105 5.0 ⫻ 105

viable counts after 1 h, followed by a resurgence of
growth to high cell numbers after 24 h. Lactocin 705
(17,000 AU/ml) proved to be more effective in inhibiting
the three Listeria strains than enterocin CRL35 and nisin.
Exposure of 8.0 ⫻ 105 viable cells of L. monocytogenes
FBUNT to lactocin 705 showed less than one log cycle
of growth after 1 h. Although regrowth was observed in
all cases, this strain was more sensitive to the action of
bacteriocins than L. innocua. Nisin displayed the fastest
rate of bactericidal action on L. monocytogenes FBUNT,
showing a maximum viability loss of 3 log units (i.e.,
99% cell destruction) achieved in 1 h, followed by lac-
tocin 705 and enterocin CRL35 with a viability loss of
approximately 2.9 and 2.7 log units in 1 h, respectively.
The survivors to each of these bacteriocins resumed
growth, reaching high cell counts at 24 h, these being
higher in the presence of nisin and enterocin CRL35 than
with lactocin 705.
As shown in Table 3, when bacteriocins were tested
in combination, the mix of lactocin 705 ⫹ nisin and
enterocin CRL35 ⫹ nisin was observed to be more
effective in preventing regrowth than lactocin 705 ⫹
enterocin CRL35. Bactericidal effectiveness was ob-
served only when lactocin 705 ⫹ enterocin CRL35 ⫹
nisin combined together were used. In this case, a dra-
matic inhibition of viable cells at 1 h after inoculation of
the mix was detected, with no regrowth of any of the
studied strains. However, when the two non-nisin bacte-
riocins were used together, a higher number of survivors
was detected at 24 h than with the pairs containing nisin.
L. monocytogenes FBUNT decreased from 1.0 ⫻ 106 and
9.8 ⫻ 105 cells/ml to less than 10 cells/ml when lactocin
G. Vignolo et al.: Bacteriocins and Listeria Species 413

Table 3. Bactericidal effectiveness of nisin, lactocin 705, and enterocin CRL35 combinations to Listeria strains after incubation at 30°C in
TSBYE broth during 24 h

cfu/ml in TSBYE containing

Lact 705
Lact. 705 Ent ⫹
Incubation None ⫹ Lact 705 CRL35 ent CRL35
Listeria species time (h) (control) ent CRL35 ⫹ nisin ⫹ nisin ⫹ nisin

L. monocytogenes FBUNT 0 8.8 ⫻ 105 1.0 ⫻ 106 1.0 ⫻ 106 9.8 ⫻ 105 1.0 ⫻ 106
1 1.4 ⫻ 106 8.0 ⫻ 101 2.0 ⫻ 101 4.0 ⫻ 101 ⬍1.0 ⫻ 101
24 2.5 ⫻ 109 3.2 ⫻ 102 ⬍1.0 ⫻ 101 ⬍1.0 ⫻ 101 ⬍1.0 ⫻ 101
L. monocytogenes Scott A 0 2.2 ⫻ 106 7.8 ⫻ 105 8.5 ⫻ 105 8.2 ⫻ 105 6.0 ⫻ 105
1 3.0 ⫻ 106 1.5 ⫻ 102 3.2 ⫻ 101 7.5 ⫻ 101 ⬍1.0 ⫻ 101
24 5.2 ⫻ 109 8.6 ⫻ 102 ⬍1.0 ⫻ 101 6.6 ⫻ 101 ⬍1.0 ⫻ 101
L. innocua 7 0 1.4 ⫻ 106 9.5 ⫻ 105 1.0 ⫻ 106 9.8 ⫻ 105 9.0 ⫻ 105
1 3.5 ⫻ 106 3.4 ⫻ 102 7.8 ⫻ 101 1.8 ⫻ 102 ⬍1.0 ⫻ 101
24 1.5 ⫻ 109 6.8 ⫻ 102 5.0 ⫻ 101 2.0 ⫻ 102 ⬍1.0 ⫻ 101

Table 4. Frequencies of resistant Listeria variants to bacteriocins added individually or in combination after 1 h at 30°C in TSBYE broth
during 24 h

L. monocytogenes L. monocytogenes
Bacteriocin FBUNT Scott A L. innocua 7

lact 705 1.0 ⫻ 10⫺3 7.0 ⫻ 10⫺4 1.5 ⫻ 10⫺3

Ent CRL35 1.5 ⫻ 10⫺3 7.0 ⫻ 10⫺4 1.0 ⫻ 10⫺2
Nisin 1.0 ⫻ 10⫺3 1.0 ⫻ 10⫺3 1.0 ⫻ 10⫺3
Lact 705 ⫹ ent CRL35 2.0 ⫻ 10⫺5 8.0 ⫻ 10⫺5 1.5 ⫻ 10⫺4
Lact 705 ⫹ nisin 4.0 ⫻ 10⫺5 9.6 ⫻ 10⫺5 3.5 ⫻ 10⫺4
Ent CRL35 ⫹ nisin 8.0 ⫻ 10⫺3 8.3 ⫻ 10⫺4 1.8 ⫻ 10⫺4
Lact 705 ⫹ ent CRL35 ⫹ nisin ⬍1.5 ⫻ 10⫺6 ⬍1.1 ⫻ 10⫺5 ⬍1.6 ⫻ 10⫺5

705 and enterocin CRL35 plus nisin respectively were
added. On the other hand, a final count of 3.2 ⫻ 102
cells/ml at 24 h was obtained when the two non-nisin
bacteriocins were assayed together.
Table 4 shows the frequencies of resistant Listeria
variants to bacteriocins after 1 h at 30°C in TSBYE
broth. Mutants of Listeria strains resistant to the mix
containing the three bacteriocins showed a resistance
frequency of less than 1.0 ⫻ 10⫺5, whereas, when indi-
vidual bacteriocins were used, Listeria-resistant variants
were observed to occur at a higher frequency (10⫺2–
10⫺4).
When the sensitivity of variants of the three Listeria
strains to the bacteriocins under study in media contain-
ing lactocin 705, enterocin CRL35, or nisin was re-
corded, the variants, depending upon the growth condi-
tions, showed cross-resistance to lactocin 705 and
enterocin CRL35, but not to nisin (Table 5). The survi-
vors isolated from broth containing nisin were resistant
only to this bacteriocin.
When the same experiment was carried out in meat
slurry, a reduction of viable cells of L. monocytogenes
FBUNT was obtained, a maximum being reached when
a combination of the three bacteriocins was added (Table
6). A bacteriostatic effect was observed when the bacte-
riocins were used individually in the meat system, while
a decrease in viable counts of approximately 2 log cycles
after 3 h of incubation was produced with a reduction in
cfu/g at 24 h of the pathogen between 49% and 60% of
the initial population. No viable counts of L. monocyto-
genes FBUNT after 3 h of incubation were obtained
when the three bacteriocins were added together.
Discussion
The Listeria strains studied in this work differed consid-
erably in their sensitivity to nisin, lactocin 705, and
enterocin CRL35. Similar results have been reported [3,
21], indicating important strain differences in the degree
of inhibition, showing that some Listeria strains are more
sensitive to nisin than others. Moreover, the differences
in sensitivity showed no correspondence with either
strain or serotype [6]. Regarding the kinetics of cell
destruction, it was observed that population decreased
414 CURRENT MICROBIOLOGY Vol. 41 (2000)

Table 5. Sensitivity of variants of Listeria strains to lactocin 705, enterocin CRL35, and nisin at 30°C in TSBYE broth

Isolated from broth

Listeria strains containing Lactocin 705 Enterocin CRL35 Nisin

L. monocytogenes FBUNT none ⫹⫹ ⫹⫹ ⫹

lactocin 705 ⫺ ⫺ ⫹
enterocin CRL35 ⫺ ⫺ ⫹
nisin ⫹ ⫹ ⫺
L. monocytogenes Scott A none ⫹ ⫹ ⫹
lactocin 705 ⫺ ⫺ ⫹
enterocin CRL35 ⫺ ⫺ ⫹
nisin ⫹ ⫹ ⫺
L. innocua 7 none ⫹ ⫹ ⫹
lactocin 705 ⫺ ⫹ ⫹
enterocin CRL35 ⫹ ⫺ ⫹
nisin ⫹ ⫹ ⫺

Sensitivity (⫹) and resistance (⫺) to bacteriocins were determined from the presence or absence of zone of growth inhibition against 10 ␮l
(about 170 AU of lactocin 705 and enterocin CRL35 and 4 AU of nisin).

Table 6. Effectiveness of lactocin 705, enterocin CRL35, and nisin added individually or in combination to Listeria monocytogenes FBUNT in
meat slurry incubated at 20°C during 24 h

log cfu/g in meat

0h 3h 10 h 24 h

Control 5.89 7.30 8.65 9.96

Lactocin 705 5.70 4.80 4.90 5.11
Enterocin CRL35 5.00 4.20 4.50 4.95
Nisin 4.95 3.50 4.54 4.80
Lact 705 ⫹ ent CRL35 5.10 2.50 2.60 2.50
Lact 705 ⫹ nisin 4.90 2.70 2.80 2.80
Ent CRL35 ⫹ nisin 5.00 2.60 3.00 3.00
Lact 705 ⫹ ent CRL35 ⫹ nisin 4.50 0.00 0.00 0.00

initially and increased upon further incubation, these
results being in agreement with those of Schillinger et al.
[19], who reported a regrowth of survivors of L. mono-
cytogenes Scott A after exposure to nisin concentrations
between 10 and 500 IU/ml as well as with those of Song
and Richard [20], who observed that survivors of L.
innocua resumed growth after the addition of nisin, pe-
diocin AcH, and enterococcin EFS2 to TSBYE broth.
Cross-resistance between bacteriocins has been ob-
served when the sensitivity of Listeria variants to lacto-
cin 705, enterocin CRL35, and nisin was tested. Similar
results were obtained by Rekhif et al. [18], who reported
that mutants of L. monocytogenes ATCC 15313 resistant
to one of three bacteriocins tested (mesenterocin 52,
curvaticin 13, and plantaricin C19) diplayed more resis-
tance to the two other, but not to nisin. Insensitivity of a
variant to lactocin 705 and enterocin CRL35 while re-
taining sensitivity to nisin, and vice versa, may be asso-
ciated with the mechanism by which a bacteriocin enters
the cell following binding to the cell surface, as well as
with the ability to form pores in bacterial membranes.
Additional work is currently under way in our laboratory
to determine the mechanism of cooperation between
lactocin 705 and enterocin CRL35, as well as their mode
of action.
From a practical point of view, considering the dif-
ference in the kinetics of inhibition and cross-resistance
of the survivors, it is to be expected that the combined
use of nisin plus one of these bacteriocins would result in
more efficient inhibition of Listeria. Even when nisin
displayed the most rapid inhibitory activity at 1 h, the
survivors resumed growth, reaching the highest cell
counts at 24 h. These results are similar to those found
with the kinetics of cell destruction of L. innocua, where
nisin proved to be the most effective inhibitor in com-
parison with pediocin AcH and enterococcin EFS2, but
nisin was also the bacteriocin that allowed the highest
cell counts at 24 h [19].
Spontaneous resistant mutants to individual bacte-
riocins were observed to occur at a high frequency
G. Vignolo et al.: Bacteriocins and Listeria Species
(10⫺3–10⫺4), this occurrence being lower when combi-
nations of two or three bacteriocins were used. These
results are in accordance with Schillinger et al. [19], who
observed variants of L. monocytogenes surviving nisin
action at a frequency of 10⫺3 to 10⫺4, but contradict the
findings of Harris et al. [8], who detected mutants of L.
monocytogenes resistant to 2000 IU/ml of nisin at a
frequency of 10⫺6 to 10⫺8. Nisin resistance response of
L. monocytogenes Scott A was reported to be correlated
with membrane fatty acid composition, a significant re-
duction in phospholipid content being observed [9, 11,
22]. This resistance could be due to the inability of nisin
to form pores in more rigid membranes [1]. The results
presented here indicate that bacteriocins of lactic acid
bacteria in combination have a higher antibacterial action
against Listeria strains than when used individually.
Hanlin et al. [7], studying the antibacterial efficiency of
pediocin AcH and nisin against several Gram-positive
bacterial strains, assumed that a mixture containing more
than one bacteriocin would be bactericidal to more cells
in a sensitive population, since cells resistant to one
bacteriocin would be killed by another. Moreover,
mainly synergistic effects were reported when the inter-
actions between pairs of bacteriocins from lactic acid
bacteria were tested [13]. One possible explanation for
the different effectiveness of bacteriocin pairs would be
that the bacteriocins used in this study belong to different
classes, which vary considerably in the nature and se-
quence of amino acid residues: nisin, lactocin, and ente-
rocin are a lantibiotic (class I), a two-peptide bacteriocin
(class IIB), and a pediocin-like (class IIA), respectively
[12]. To avoid an inefficacious action of bacteriocins
over a long time, our results suggest that a mixture
containing bacteriocins that belong to different classes
would be more effective in preventing regrowth of Lis-
teria in foods.
The combined effect of lactocin 705, enterocin
CRL35, and nisin against L. monocytogenes FBUNT in
meat slurry showed no viable counts after incubation for
3 h. These results indicate that even when the effective-
ness of bacteriocins may be antagonized by many factors
acting simultaneously (adsorption on meat components
and degradation by proteolytic enzymes), the industrial
application of combined, partially purified, and/or puri-
fied bacteriocins in meat products may be a useful ap-
proach to reduce the frequency at which resistant popu-
lations develop, to improve the hygiene standards, and to
extend the shelf life of meat and meat products. Although
the use of nisin is permitted in more than 50 countries in
a variety of foods, other bacteriocins with different
and/or more effective antimicrobial activity may be con-
sidered as new biopreservatives.
415
ACKNOWLEDGMENTS
This study was supported by funds from PICT 98 N°09-04632 (AN-
PCyT), Argentina. The authors would like to thank A.Y. Borchia for
her excellent technical assistance, and Aplin and Barret Ltd. for the gift
of a nisin sample.
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