Journal of Applied Microbiology 2004, 97, 48–56
doi:10.1111/j.1365-2672.2004.02276.x
Effect of combined physico-chemical preservatives on
enterocin AS-48 activity against the enterotoxigenic
Staphylococcus aureus CECT 976 strain
S. Ananou1, E. Valdivia1, M. Martı́nez Bueno1, A. Gálvez2 and M. Maqueda1
1
Departamento de Microbiologı´a, Facultad de Ciencias, Univ. Granada, Granada, Spain, and 2Área de Microbiologı´a, Facultad de
Ciencias Experimentales, Univ. Jaén, Jaen, Spain
2003/1139: received 12 December 2003, revised 11 February 2004 and accepted 18 February 2004
ABSTRACT
S . A N A N O U , E . V A L D I V I A , M . M A R T Í N E Z B U E N O , A . G Á L V E Z A N D M . M A Q U E D A . 2004.
Aims: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory
cultures, and behaviour of the AS-48 activity in the presence of food preservatives.
Methods and Results: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species
tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected
for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and
temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration
(MBC) of AS-48, decreased from 15 lg ml)1 at 37C to 10 lg ml)1 at 15C. Sublethally injured cells showed an
increased sensitivity with a MBC of 5 lg ml)1. In this way, the highest effectiveness of Ent AS-48 against S. aureus
CECT 976 was obtained at 4C in combination with high concentrations of NaCl (6 and 7%). Interestingly,
enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of
Ent AS-48. These low concentrations also provoked a delay of bacterial growth.
Conclusion: The results presented indicated that Ent AS-48 has a potential for application as a protective agent
against S. aureus in foods.
Significance and Impact of the Study: In this study, we have established the conditions for an efficient
inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of
environmental factors and Ent AS-48.
Keywords: bacteriocin, Enterococcus faecalis, enterotoxin A, Staphylococcus aureus.
INTRODUCTION
In spite of modern technologies and safety concepts, the
reported numbers of food-borne illnesses and intoxications
are still on the increase. According to the Council for
Agricultural Science and Technology of the US, microbial
pathogens in food cause an estimated 6Æ5–33 million cases of
human illness and up to 5000 deaths annually, with the main
foods implicated including meat, poultry, eggs, seafood and
Correspondence to: M. Maqueda, Departamento de Microbiologı́a, Facultad de
Ciencias, C/Fuentenueva s/n, 18071 Granada, Spain (e-mail: [email protected]).
dairy products. The bacterial pathogens that account for
much of these cases include Salmonella, Campylobacter
jejuni, Escherichia coli O157:H7, Listeria monocytogenes,
Staphylococcus aureus and Clostridium botulinum (Buzby et al.
1996; Mead et al. 1999).
Staphylococcal intoxication rank as one of the most
prevalent causes of gastroenteritis worldwide, resulting from
ingestion of one or more preformed staphylococcal enterotoxins (SEs) in contaminated food (Igarashi et al. 1985;
Bennet 1986; Jablonski et al. 1997). Staphylococcal enterotoxins (SEA to SEE) constitute a family of nine major
serological types of heat stable proteins with a relative
ª 2004 The Society for Applied Microbiology
COMBINED ACTION OF DIFFERENT PRESERVATIVES ON AS-48 ACTIVITY AGAINST S. AUREUS CECT 976
molecular mass of 32 000 (Coultat 1996), produced in
extremely low quantities throughout the logarithmic growth
phase. The incidence of SE involvement in staphylococcal
food poisoning appears to change with time. However, SEA
is the most common toxin implicated in food poisoning
(Holmberg and Blake 1984) and is characterized by emesis
and diarrhoea following a short incubation period. Other
common symptoms include nausea, abdominal cramps,
headaches, muscular cramping and/or prostration.
With the increasing demand for more natural and safe
food products, there is a need for new preservation
techniques to avoid different forms of spoilage and food
poisoning. Traditional ways to control microbial spoilage
and safety hazards in foods are being replaced by new,
innovative techniques including mild heating, modified
atmosphere and vacuum packaging, high hydrostatic pressure, ultraviolet light and employment of natural antimicrobial systems such as bacteriocins (Montville and
Wikowski 1997; Hugas et al. 2002; O’Sullivan et al. 2002;
Ross et al. 2002). The US National Food Processors
Association recommends the use of combined preservation
methods to create a series of hurdles throughout the
process, each representing a barrier that must be overcome
by bacteria to initiate food spoilage (Kihm et al. 1994;
Leistner 1999, 2000). Nisin, employed for over 50 years, is
currently approved in more than 60 countries for use in
foods, and is the only bacteriocin licensed for use as a food
preservative (FDA 1988). However, many examples of
inclusion of bacteriocin-producing starter cultures in food
fermentation have been reported in the literature. Hence,
the use of bacteriocins either alone or in combination with
physicochemical treatments and chemical preservatives may
be an efficient way to preserve the nutritional quality of raw
materials and food products through an extended shelf life
and the inhibition of spoilage and pathogenic bacteria.
Enterocin AS-48 is a cyclic peptide of 7Æ14 kDa encoded
by the conjugative plasmid pMB2 (Martı́nez Bueno et al.
1990a, 1994, 1998; Samyn et al. 1994). It is produced by
Enterococcus faecalis subsp. liquefaciens S-48 and it is unique
in its cyclic structure and its broad antimicrobial spectrum
against Gram-positive bacteria, including the pathogens
S. aureus, L. monocytogenes, Bacillus cereus as well as some
Gram-negative bacteria (Gálvez et al. 1986, 1989a,b; Mendoza et al. 1999). This fact, together with its remarkable
stability and solubility over a wide pH range, suggest that
AS-48 could be a good candidate as a natural food
preservative (Abriouel et al. 2001). There is extensive
information about antimicrobial activity and physicochemical characteristics of this macromolecule (Gálvez et al.
1986, 1989a,b, 1991; Martı́nez Bueno et al. 1994; Samyn
et al. 1994; Abriouel et al. 2001). For instance, the primary
target of AS-48 is the bacterial cell membrane, where it
forms pores or channels of around 7 Å in diameter. This
49
leads to a membrane permeation not dependent on membrane potential (Gálvez et al. 1991). Currently, we are
performing a series of studies to understand how AS-48 acts
on the main foodborne pathogenic bacteria as well as the
influence of environmental factors concurring in foods on its
antimicrobial effectiveness. The present work describes the
antimicrobial activity of enterocin AS-48 against S. aureus
CECT 976 producer of enterotoxin A (SEA). The susceptibility of this strain to AS-48 in combination with the
treatments currently applied in foods processing such as pH,
temperature and sodium chloride has also been investigated.
Finally, we have verified the indirect action of AS-48 on
SEA production by staphylococci.
MATERIAL AND METHODS
Organisms and maintenance
Enterococcus faecalis A-48-32 was used as AS-48 producer
strain (Martı́nez Bueno et al. 1990b). Enterococcus faecalis
S-47 from our laboratory collection was used as standard
indicator strain. The staphylococcal strains used in this work
to evaluate the activity of AS-48 are listed in Table 1, and in
all cases were supplied by the Spanish Type Culture
Collection (CECT). Enterococci and staphylococci were
grown in brain heart infusion broth (BHI broth; Oxoid,
Table 1 Antimicrobial activity of concentrated preparations of AS-48
(0Æ125 lg ll)1) against different staphylococcal strains determined by
the agar well diffusion assay (100 ll). The indicator strain Enterococcus
faecalis S-47 has been also included for comparison
Indicator strain
Size zone of
inhibition (mm)
Enterococcus faecalis S-47
Staphylococcus epidermidis CECT 231
S. epidermidis CECT 4033
S. hominis CECT 234
S. xylosus CECT 273
S. carnosus CECT 4491
S. saprophyticus CECT 235
S. aureus CECT 239
S. aureus CECT 59
S. aureus CECT 82
S. aureus CECT 435
S. aureus CECT 86
S. aureus CECT 192
S. aureus CECT 826
S. aureus CECT 828
S. aureus CECT 827
S. aureus CECT 4465 (SEC)
S. aureus CECT 4466 (SED)
S. aureus CECT 4459 (SEB)
S. aureus CECT 976 (SEA)
18
12
12
0
10
16
16
16
15
11
0
12
11
12
14
14
15
14
12
14
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 48–56, doi:10.1111/j.1365-2672.2004.02276.x
50 S . A N A N O U ET AL.
Basingstoke, UK) at 37C without shaking. When solid,
BHI was supplemented with 1Æ5% agar (BHA).
AS-48 preparations
Concentrated preparations of Ent AS-48 were obtained as
follows: E. faecalis A-48-32 was propagated in 10 l of
buffered CM-G medium (Gálvez et al. 1986) inoculated
with overnight culture (4% v/v) and incubated at 37C for
8 h. Then, 1 volume of distilled water was added to the
culture and the pH adjusted to 6Æ5 with 0Æ5 N NaOH.
Enterocin recovery was carried out by adding Carboxymethyl-Sephadex CM-25 (Pharmacia, Uppsala, Sweden) gel
slurry equilibrated in distilled water to cultured broth (1/40,
v/v). The mixture was held under stirring for 30 min, and
decanted for another 30 min. The supernatant was discarded, and the gel slurry containing bacteriocin activity was
loaded on a 10 · 50 cm glass column. The gel was washed
with 5 bed volumes of distilled water and 2 volumes of
0Æ5 M NaCl, followed by 2 volumes of 1Æ5 M NaCl in
distilled water to elute enterocin activity. Eluted fractions
were dialyzed against deionized water using dialysis tubes of
2000 exclusion size and sterilized by filtration (0Æ22 lm pore
size cellulose filter; Millipore, Iberica S.A., Madrid, Spain).
The concentration of AS-48 in these preparations was
estimated in ca 0Æ25 lg ll)1 according to the standard curve
obtained using E. faecalis S-47 as sensitive strain.
The activity of Ent AS-48 against different staphylococcal
strains was determined by the agar well (9 mm) diffusion
assay (Gálvez et al. 1986). The diameter of inhibition zone
(well included) was measured and expressed in millimetres.
Antimicrobial activity assay
Inhibition of staphylococci growth on liquid media was
carried out as follows: cultures growing in BHI broth at
37C with an optical density at 620 nm ca 0Æ01
(1Æ3 · 106 CFU ml)1) were added of 10, 15, 20 or
30 lg ml)1 of Ent AS-48. At desired intervals, samples
were removed and serially diluted into ice-cold sterile saline
solution (NaCl 0Æ85%). The appropriate dilutions were
plated on triplicate trypticase soya agar plates, and the
average number of colonies (CFU ml)1) obtained after 24 h
incubation at 37C was used to establish the growth and
survival curves. Growth was monitored turbidimetrically at
620 nm by using a Spectronic-20 spectrophotometer (Bausch & Lomb, Inc., Rochester, NY, USA).
Determination of the minimum bactericidal
concentration (MBC)
The MBC of Ent AS-48 for S. aureus CECT 976 was
determined by addition of increasing concentrations of the
enterocin to tubes containing 5 ml of BHI broth inoculated
with ca 106 CFU ml)1 and incubated at 37C. The lowest
AS-48 concentration that produces death of 99Æ9% of the
initial culture was defined as the MBC. At appropriate
times, the survivors were enumerated by sampling and
spreading on three BHA plates.
Sublethal heat treatments
Different heat treatments (60, 65 and 70C for 1 and 5 min
each) were applied to 2-ml BHI logarithmic phase culture of
S. aureus CECT 976 (5Æ2 · 107 CFU ml)1) to produce a
sublethal cell injury. Treatment of 65C for 5 min produced
the 97Æ7% of death in the population, and the effect on cell
viability was determined following the concentration of
viable cells during the following 24 h of incubation. For Ent
AS-48 assays, aliquots of the cell suspension were added of
different enterocin concentrations (5, 7 and 10 lg ml)1)
before being heat treated, and the survivors enumerated by
sampling on BHA plates for triplicated at different incubation times.
Effect of incubation temperature, pH and sodium
chloride treatment
The effect of a subinhibitory concentrations (10 lg ml)1) of
Ent AS-48 against logarithmic-phase culture in BHI
(ca 106 CFU ml)1) of S. aureus CECT 976 was tested at
different incubation temperatures (4, 10, 15, 28 and 37C).
The influence of pH on cell sensitivity to AS-48 was
determined as follows: logarithmic-growing cells (ca 106
CFU ml)1) were suspended in the following buffers: 50 mM
ortho-phosphoric acid (pH 4 and 5) or 100 mM sodium
phosphate (pH 6, 7 and 8). Then, AS-48 in deionized water
was added to each cell suspension to a final concentration of
10 lg ml)1. The sensitivity to AS-48 of cultures of S. aureus
CECT 976 (ca 106 CFU ml)1) in the presence of different
concentrations of sodium chloride (Scharlau, Barcelona,
Spain) (0, 2, 4, 6 and 7%) was also determined. In all cases,
the evolution of the control and treated cultures was
investigated by sampling in triplicate BHA plates at 2 h
intervals to determine the number of remaining viable cells.
The logarithmic reduction factor (LRF) was calculated
according to Cutter and Siragusa (1995):
LRF ¼ log CFU ml1 in controls
log CFU ml1 in treated samples:
Effect of AS-48 on SEA production
Series of 4-ml BHI cultures of S. aureus CECT 976
producer of SEA and CECT 4441 as non-producer SEA
(used as negative control), with or without AS-48
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 48–56, doi:10.1111/j.1365-2672.2004.02276.x
51
10
9
8
7
6
5
4
3
2
1
0
10
1
0·1
D·O620
(10 lg ml)1), were incubated at 37C. Tubes of each culture
were removed at different intervals of time (6, 9, 24, 48, 72,
96 and 120 h) and the supernatants precipitated with 60%
saturation of ammonium sulphate for 12 h. Then, the
precipitate was resuspended in 40 ll of 10 mM sodium
phosphate buffer (pH 7) and 1 ll of each was adsorbed onto
nitrocellulose sheet (0Æ45-lm pore size BA85; Schleicher and
Schuell, Dassel, Germany) to detected the SEA by Dot-blot
immunoassay (Hawkes et al. 1982), carried out according to
the manufacturer’s instructions. This assay has a range of
sensitivity of 50–500 ng per dot, using anti-SEA, delipidized
whole serum (Sigma, St Louis, MO, USA). The approximate titre in units (U) was expressed as reciprocal of the
maximal dilution of precipitates that gave a positive reaction.
The results obtained were the media of triplicate-independent experiments.
Log CFU ml–1
COMBINED ACTION OF DIFFERENT PRESERVATIVES ON AS-48 ACTIVITY AGAINST S. AUREUS CECT 976
0·01
0
2
4
6
0·001
24
8
Time (h)
Fig. 1 Effect of AS-48 on viability (solid symbols) and optical
density (O.D.620 nm) (open symbols) of logarithmic-phase cultures
of Staphylococcus aureus CECT 976 incubated at 37C with
different enterocin concentrations: 0 (e), 10 lg ml)1 (() and
15 lg ml)1 (s)
RESULTS
Antistaphylococcal spectrum of AS-48
High-cell density, pH-controlled cultures were used for
partial purification of Ent AS-48 on cation exchange because
the yield of enterocin recovery was very high (95Æ99%)
(Abriouel et al. 2003). The strong positive charge of AS-48
facilitates its recovery from cultured broths by this procedure, while most other components of the growth medium
are not retained in the gel (Gálvez et al. 1989a).
The sensitivities of several staphylococcal strains, belonging to CECT including enterotoxigenic S. aureus ones,
against concentrated AS-48 preparations in solid medium is
shown in Table 1. AS-48 (0Æ125 lg ll)1) inhibited most of
the strains tested, although their sensitivities varied largely,
with zones of inhibition ranging from 10 to 16 mm.
Staphylococcal strains were, in general, less sensitive to
Ent AS-48 than E. faecalis S-47, from our laboratory
collection, used as indicator strain (inhibition halos of
18 mm). Staphylococcus carnosus CECT 4491, S. saprophyticus CECT 235 and S. aureus CECT 239 were the most
sensitive nonenterotoxigenic strains tested. However, we
chose the strain S. aureus CECT 976 as indicator for
subsequent experiments because it produces SEA, the
enterotoxin that causes food-borne intoxication at a greater
frequency.
Antimicrobial activity of AS-48 against S. aureus
CECT 976
Addition of AS-48 (10, 15, 20 and 30 lg ml)1 final
concentration) to exponentially growing cultures of
S. aureus CECT 976 resulted in a rapid decrease in cell
population (Fig. 1), being the effect proportional to the
amount of Ent AS-48 added. Accordingly, AS-48 has a
bactericidal and bacteriolytic mode of action on this strain,
with a MBC of 15 lg ml)1 for an initial cell number of ca
106 CFU ml)1.
Effect of different parameters on AS-48 activity
The results shown in Fig. 2a suggest that addition of Ent
AS-48 to staphylococcal cultures incubated at 28 and 37C
at subinhibitory concentration, caused a marked decrease in
the number of viable cells within the first 6 h of incubation,
but then cultures resumed the logarithmic growth. Interestingly, when the treatment was carried out at the
suboptimal temperature of 15C, the MBC was reduced
from 15 to 10 lg ml)1 during the 48 h of incubation
(Fig. 2b). However, the effect of Ent AS-48 was much lower
when the incubation temperatures were 10 and 4C, where
there was not possible the cell growth. At 10C, there was
only a slight reduction in the concentration of viable cells,
and cells incubated at 4C showed a greater resistance to Ent
AS-48, even at the highest concentrations tested of
20 lg ml)1 (Fig. 2b,c).
The pH of the medium is one of the most important
factors influencing the effectiveness of most food antimicrobials. Hence, its influence on the activity of subinhibitory
concentrations of AS-48 (10 lg ml)1) against logarithmicphase cells of S. aureus CECT 976 was investigated in a
wide range of pH 4–8. The results obtained demonstrated
that the numbers of survivors after 24 h of incubation with
AS-48 at pH 6–8 were similar (results not shown).
Nevertheless, incubation at pH 5, and to a minor extent at
pH 4, caused a remarkable reduction in the viability of the
cells, while survival of control cells was affected to a much
less extent by the acidic conditions.
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 48–56, doi:10.1111/j.1365-2672.2004.02276.x
52 S . A N A N O U ET AL.
10
Log CFU ml–1
Log CFU ml–1
(a) 10
9
8
7
6
5
4
3
2
1
0
6
8
24
0
4 8
24
48
2
4
6
8
Time (h)
24
Fig. 3 Combined effect of sublethal heat treatment of Staphylococcus
aureus CECT 976 exposed to AS-48. Cell cultures were heated at 65C
for 5 min without enterocin (controls r) or with increasing AS-48
concentrations: 5 lg ml)1 (d), 7 lg ml)1 (m) or 10 lg ml)1 (j). The
number of cells remaining viable right after the heat treatment
incubated at 37C was determined
Moreover, the number of survivors after 24 h of incubation
in samples treated with Ent AS-48 decreased markedly, and
the MBC of AS-48 for sublethally injured cells was reduced
to 7 lg ml)1.
Log CFU ml–1
(c) 10
9
8
7
6
5
4
3
2
1
0
5
4
3
2
1
0
0
Log CFU ml–1
(b) 10
9
8
7
6
5
4
3
2
1
0
2 4
9
8
7
6
AS-48 combined effect with variations in
temperature, pH and sodium chloride
0
4 8
24
Time (h)
48
Fig. 2 Effect of incubation temperature on the number of viable cells
of Staphylococcus aureus CECT 976 treated with AS-48 (solid symbols)
or without AS-48 (open symbols). (a) 37C (s) and 28C (n) treated
with 10 lg ml)1 of AS-48. (b) 15C (e) and 10C (() treated with
10 lg ml)1 of AS-48. (c) 4C (j) 10lg ml)1, (·) 15 lg ml)1 and ( )
20 lg ml)1 of AS-48
Effect of sublethal heat shock on AS-48 activity
The effect of sublethal injury of Ent AS-48 on S. aureus
CECT 976 cells in BHI was also investigated. First, we
established that a heat treatment of 65C for 5 min, reduced
the initial cell numbers of the culture by 97Æ7% (from
5Æ2 · 107 CFU ml)1 to 1Æ4 · 105 CFU ml)1). When logarithmic-phase cells of S. aureus (O.D.620 nm ¼ 0Æ07) were
sublethally heated in the presence of increasing concentrations (0, 5, 7 and 10 lg ml)1) semipurified AS-48, the
number of viable cells was markedly reduced in proportion
both to the amount of enterocin added and the time of
incubation. A noticeable decrease in the number of survivors
was found even when the cells treated with 7 and
10 lg ml)1 were plated at 2 h when compared with a
reduction caused on the controls by heat alone (Fig. 3).
The effect of NaCl (2, 4, 6 and 7%) combined with
variations in pH (5 and 7) and incubation temperatures
(4, 15 and 37C) on AS-48 activity (10 lg ml)1) against
S. aureus CECT 976 (5Æ3 · 105 cells ml)1) was also investigated. The results obtained showed the importance of the
incubation temperature: when the assay was carried out at
37C and neutral pH or at 15C and pH 5, the addition of
increasing amount of NaCl did not affect the AS-48 activity
on viability of this bacteria (LRF <1). However, addition of
NaCl (2, 4, 6 and 7%) at pH 5 on cultures growing at 37C
or alternatively on cultures at pH 7 incubated at 15C,
reduced the effectiveness of AS-48 for all NaCl concentrations tested (results not shown). Only when cultures were
incubated at refrigeration temperature (4C) in the presence
of 6 and 7% NaCl, the viability of cell population was
reduced approximately by 3Æ6 and 2Æ7 log units with respect
to the control at pH 5 or 7, respectively (Fig. 4).
Effect of AS-48 on SEA production
Enteroxin A (SEA) production by S. aureus CECT 976 was
investigated in untreated cultures (control) as well as in the
presence of AS-48, both growing in log phase. A marked
decay of enterotoxin titres was observed during prolonged
incubation of bacteriocin-treated cultures: in controls,
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 48–56, doi:10.1111/j.1365-2672.2004.02276.x
COMBINED ACTION OF DIFFERENT PRESERVATIVES ON AS-48 ACTIVITY AGAINST S. AUREUS CECT 976
53
results were also confirmed by comparing the maximum
SEA titres produced by staphylococcal populations with
equivalent cell densities: 128 U were produced by the
control after 96 h of incubation (19 · 108 CFU ml)1) while
the treated culture at 120 h of growth (15Æ6 ·
108 CFU ml)1) only produced 32 U, fourfold lower that
in the control.
LRF
8
7
6
5
4
3
DISCUSSION
2
1
0
I
II
III
IV
V
VI
48 h
24 h
8h
Fig. 4 Antimicrobial activity of AS-48 (10 lg ml)1) against Staphylococcus aureus CECT 976 cultures in combination with different
sodium chloride concentrations, carried out at different pHs and
incubated at refrigeration temperature (4C). LRF denotes the
logarithmic reduction factor, which was calculated as the log CFU/ml
in controls minus log CFU/ml in treated samples. (I) AS-48, (II) AS48 + NaCl 6% and (III) AS-48 + NaCl 7% at pH 5. (IV) AS-48, (V)
AS-48 + NaCl 6% and (VI) AS-48 + NaCl 7% at pH 7
10
144
9
128
8
112
7
96
6
80
5
64
4
48
3
32
2
16
1
0
SEA titre (U)
Log CFU ml–1
detectable titres of SEA were produced after 9 h of growth,
reaching a maximum at 96 h of incubation (Fig. 5),
however, addition of subinhibitory concentrations of AS48 (10 lg ml)1) at the beginning of the assay (1Æ35 ·
106 CFU ml)1) caused a delay in bacterial growth during
the first 24 h of incubation, as well as a marked inhibition on
SEA production. Nevertheless, in spite of the AS-48-treated
cells reach similar growth levels to the control culture, the
amount of SEA produced during the incubation period was
much lower (32 U vs 128 U in the control) and did not
increase during the incubation time (96–120 h). These
0
0
9
24
48
72
Time (h)
96
120
Fig. 5 Influence of AS-48 treatment on S. aureus CECT 976 growth
(lines) and SEA production (bars). Logarithmic-phase cultures (time
zero in the graph, 1Æ35 · 106 CFU ml)1) were incubated with
10 lg ml)1 of AS-48 (j—j, solid bars). Control culture ((—(,
clear bars)
The use of bacteriocins and/or bacteriocin-producing
strains of lactic acid bacteria is of great interest as they are
generally recognized as safe organisms, and their products as
natural biopreservatives (Cintas et al. 1995; Stiles 1996).
However, it is desirable to have a comprehensive understanding of the influences that environmental factors have on
the microorganisms in order to quantitatively estimate their
survival for future applications in food model systems. In
previous studies, the effectiveness of AS-48 to controller the
growth of L. monocytogenes, B. cereus and Salmonella in
laboratory systems have been reported (Abriouel et al. 1998,
2002; Mendoza et al. 1999). In this work, we have established the conditions for an efficient inhibition of growth
and SEA production by S. aureus CECT 976, by a
combination of AS-48 and physico-chemical factors (hurdle
technology).
AS-48 inhibited the growth of several strains of staphylococci tested, being highly specific against the enterotoxigenic
S. aureus CECT 4459, 4465, 4466 and 976 strains. Activity of
AS-48 on logarithmic-phase cultures of S. aureus CECT 976,
selected as producer of enterotoxin A, determined a decrease
of cell viability after 1 h of incubation with AS-48. The MBC
for this strain has been established in 15 lg ml)1 for ca
106 cells ml)1, the higher value reported for AS-48 against
Gram-positive bacteria (Mendoza et al. 1999; Abriouel et al.
2002). Partial loss of viability of S. aureus CECT 976 caused
by subinhibitory concentrations of AS-48 occurred in a wide
range of incubation temperatures (4–37C). Interestingly, the
greater efficacy of AS-48 was obtained at 15C, where
the MBC was reduced (15 to 10 lg ml)1) in spite of the
staphylococcal growth in the control. However, higher
concentrations of enterocin (20 lg ml)1) were necessary in
order to detect some inhibitory effect at 4C. Similar results
were obtained with AS-48 against the psychrotrophic bacteria
L. monocytogenes and B. cereus growing at low temperatures,
due to a modification in lipid composition with increase the
membrane fluidity (Mendoza et al. 1999; Abriouel et al.
2002). In spite of the fact that refrigeration temperatures do
not allow growth of staphylococci, AS-48 could act as an
invisible barrier to control bacterial growth under situations
of temperature abuse.
It is relevant that the antimicrobial action of AS-48 on this
strain can be enhanced greatly by sublethal heat treatment
ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 48–56, doi:10.1111/j.1365-2672.2004.02276.x
54 S . A N A N O U ET AL.
(65C for 5 min). The heat processing destroyed a large
population of these bacteria (99Æ7%) and rendered the
remaining viable cells more sensitive to AS-48, as the MBC
calculated experimented an important reduction (15 to
5 lg ml)1). These results were expected as sublethally
injured cells by different stressing conditions may become
sensitive to different physical and chemical agents to which
healthy cells are resistant (Ray 1989; Kalchayagand et al.
1992; Kihm et al. 1994).
Acidification is a usual practice in food processing
because low pH itself is inhibitory to many microorganisms, and it also reduces the thermal resistance of heat
resistant microorganisms. Staphylococcus aureus CECT 976,
as the majority of the Gram-positive bacteria tested,
exhibited an increased sensitivity to AS-48 at low pH
values (Mendoza et al. 1999; Abriouel et al. 2002). In a
similar way, activity of nisin, sakacin P and curvacin A is
also enhanced at low pH (Jaquette and Beuchat 1998;
Gänzle et al. 1999). This effect could be probably due to
the oligomerization of these cationic peptides (Abriouel
et al. 2001) as well as to changes in the surface charge of
the bacterial target.
Sodium chloride is probably the oldest known food
preservative against those pathogenic bacteria that are
inhibited by a water activity (aw) of 0Æ92 or less. However,
one of the distinguishing characteristics of S. aureus is its
considerable tolerance to NaCl, being routinely capable of
growth at aw values between 0Æ83 and 0Æ86 (2Æ6 M NaCl)
(Townsend and Wilkinson 1992; Yabu and Kaneda 1995;
Thomas et al. 1996). The results presented in this work
indicated that the activity of AS-48 on this strain of
staphylococci was influenced by the concentration of salt
employed, the incubation temperature and pH of the
medium. In this way, combinations of lower temperatures
(4C) and high NaCl concentrations (6 or 7%) increase the
efficacy of AS-48 against staphylococci synergistically with
values of logarithmic reduction between 3Æ6 and 2Æ7
according to the pH tested. This effect can be very useful
to reduce staphylococcal populations under refrigeration
conditions. However, in cultures carried out at growth
temperature (15 and 37C), the presence of NaCl has not a
pronounced effect on AS-48 activity in the assay conditions.
This behaviour is similar to those obtained with nisin
(Thomas et al. 1996) and could be explained in relation to
some physiological changes produced in this halotolerant
bacterium by high NaCl concentrations, such as a rapid
accumulation of compatible solutes (Townsend and Wilkinson 1992), the changes in the composition of the cytoplasmic
membrane (Hurst et al. 1984a,b).
Likewise, the conditions of production of enterotoxin A
by S. aureus CECT 976 has been investigated in control
cultures as well as in the presence of subinhibitory
concentrations of AS-48. The results obtained in this work
are encouraging because of an addition of AS-48 to
logarithmic growth cultures caused a delay on bacterial
growth during the first 24 h, as well as a marked inhibition
of SEA production in relation to the control, in a similar way
to those obtained with the enterotoxigenic strain B. cereus
LWL1 after treatment with AS-48 (Abriouel et al. 2002).
One way to avoid toxin production in foods is to keep
S. aureus population below its infective dose (5Æ105 organisms per gram of contaminated food) (Evenson et al. 1988)
and we have demonstrated that such reduction can be
accomplished by AS-48 either alone or in combination with
some physico-chemical preservatives. These features,
together with its capacity to inhibit efficiently proliferation
and viability of staphylococci at low temperature suggest
that Ent AS-48 may be an useful tool to prevent food
poisoning by this bacteria.
ACKNOWLEDGEMENTS
Samir Ananou has been beneficiary of a fellowship from
AECI (Spanish Ministry of Foreing Affairs). This work was
supported by a grant from the CICYT (BIO98-CO02-01) of
the Spanish Ministry of Education and Science.
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