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TM
Online Journal of
Veterinary Research
©
Volume 8:22-32, 2004.
Peptide AS-48 (Enterococcus faecalis)
for prevention and treatment of
mastitis in dairy cows
Davidse EKa, Balla Ea, Holzapfel WHb, Muller CJCc, Cloete
SWPc, Dicks LMTa
Department of Microbiology, Stellenbosch University, 7600 Stellenbosch, South
Africa, bInstitute of Hygiene and Toxicology, Federal Research Centre for
Nutrition, Haid-und-Neu-Str. 9, 76131, Karlsruhe, Germany, cDepartment of
Agriculture, Private Bag X1, 7607 Elsenburg, South Africa
a
ABSTRACT
Davidse ek, Balla E, Holzapfel WH, Muller CJC, Cloete SWP,
Dicks LMT Peptide AS-48, a cyclic antimicrobial peptide from
Enterococcus faecalis, may be used in the prevention and
treatment of mastitis in dairy cows, Online Journal of
Veterinary Research 8:22-32, 2004. Peptide AS-48, produced
by Enterococcus faecalis FAIRE 92, inhibited the growth of a
Staphylococcus aureus strain isolated from mastitic milk. Peptide
AS-48 was isolated from the cell-free supernatant by using a
combination of Triton X-114 phase partitioning and cation exchange
2
chromatography. The partially purified peptide was liposomeencapsulated at a yield of 400 AU (arbitrary units)/ml and injected
into infection-free quarters of healthy Holstein cows. These quarters
were then infected by injecting 2 ml of the S. aureus pathogen (3.3
x 103 cfu/ml) through the teat canals. Control udders not pretreated with AS-48 were also injected with the same level of S.
aureus. From the second day after milking to day 7, the somatic
cell count (SCC) in milk from udders that have been pre-treated
with liposome-encapsulated peptide AS-48 decreased by 60%. The
viable cell numbers of S. aureus in milk from pre-treated udders
remained more-or-less the same over the 7-day period (1 x 102
cfu/ml), whereas the S. aureus cell numbers in milk from untreated
udders increased to 8 x 102 cfu/ml. When S. aureus-infected udders
with a SCC higher than 5 x 105/ml were injected together with
liposome-encapsulated peptide AS-48 (6 400 AU/ml), the SCC in
milk from these animals decreased by ca. 85% and the number of
viable S. aureus by ca. 99%. Streptococcus agalactiae and
Streptococcus dysgalactiae, isolated from mastitic milk, were also
inhibited in in vitro tests, but not Escherichia coli.
KEYWORDS: Mastitis, Treatment, Peptide AS-48
INTRODUCTION
Staphylococcus aureus is one of the most common etiological
agents of bovine mastitis in lactating dairy cows and contributes to
significant economic losses in the dairy industry (Hébert et al 2000;
Twomey et al 2000). In the Western Cape of South Africa, S.
aureus is responsible for 40% of all mastitis cases reported. Despite
mastitis management programs to reduce the incidence of intramammary udder infections (IMI), S. aureus remains a major
pathogen (Twomey et al 2000). After entering the mammary gland
through the teat canal, the bacteria multiply rapidly, leading to
inflammation and tissue damage (Hébert et al 2000). Apart from
infection, S. aureus secretes various toxins and enzymes, which
may lead to food poisoning when the milk is consumed (Coetzer et
al 1994).
Staphylococci found in infected tissues are mainly located
extracellularly (Onyeji et al 1994). However, virulent strains of S.
aureus can penetrate phagocytic cells and survive within leukocytes
(Bramley & Dodd 1984; Onyeji et al 1994; Watson 1994; Hébert et
al 2000). Antibiotics are routinely administered at drying-off to
treat sub clinical cases of mastitis and prevent further infection
(Twomey et al 2000). However, animals infected with S. aureus
respond poorly to antibiotic treatment (Watts 1990), probably due
to the intracellular location of the bacterial cells in the alveoli
3
and/or macrophages (Bramley & Dodd 1984; Hébert et al 2000).
Although many of the antibiotics penetrate neutrophils, S. aureus
may survive (Francis 1989) and causes chronic intraphagocytic
infections (Onyeji et al 1994).
The emergence of antibiotic resistance in bacteria has led to a
considerable debate in the use of antibiotics for prophylactic
treatment, which in turn led to a search for alternative treatments
(Twomey et al. 2000). Immunization against S. aureus does not
prevent IMI, as evident from the lack of high concentrations of
antibody and phagocytic cells in the milk (Nickerson 1985).
Furthermore, inadequate knowledge of the pathogenesis of
staphylococcal mastitis and the immune mechanisms protecting the
mammary gland from infection has limited the scope for novel
approaches to vaccination (Watson, 1992).
Bacteriocins produced by lactic acid bacteria are generally
considered safe and may present a cost-effective alternative to
treat mastitis caused by strains of S. aureus, Streptococcus
agalactiae and Streptococcus dysgalactiae. In a previous study with
a combination of the bacteriocins nisin and lysostaphin, 66% of the
S. aureus-infections could be cured (Reichelt et al 1984). In
another study, which involved the incorporation of the bacteriocin
lacticin 3147 into a teat seal (1280 AU/ml), 99.9% of S. aureus
cells were killed (Ryan et al 1998).
In the present study we have encapsulated the cyclic peptide AS48, produced by E. faecalis FAIRE 92, into liposomes and evaluated
its effect against a pathogenic strain of S. aureus that causes
mastitis in dairy cows.
MATERIALS AND METHODS
Bacterial strains and growth conditions: Enterococcus faecalis
FAIRE 92, the producer of peptide AS-48, forms part of the EU
FAIR-E collection at the BCCMTM/LMG Culture Collection,
Laboratorium voor Microbiologie (LMG), University of Ghent (RUG)
(K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium). Strains of S.
aureus, S. agalactiae, S. dysgalactiae and Escherichia coli (Table 1)
were isolated from mastitic milk and were obtained from the
Diagnostic Veterinary Laboratory, Stellenbosch, South Africa. Other
indicator strains used in this study (Table 1) were from the LMG at
the University of Ghent. The Lactobacillus and Leuconostoc spp.
were grown in MRS broth (Biolab Diagnostics, Midrand, South
Africa). The other indicator bacteria were cultured in BHI broth
(Biolab).
4
Table 1. Spectrum of antimicrobial activity of peptide AS-48
Organism
Bacillus cereus
Escherichia coli
Lactobacillus acidophilus
Lactobacillus casei
Lactobacillus curvatus
Lactobacillus fermentum
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillus sakei
Leuconostoc cremoris
Pediococcus pentosaceus
Staphylococcus aureus
Staphylococcus carnosus
Streptococcus agalactiae
Streptococcus dysgalactiae
Strain(s)
LMG 13569
MKB EC1
LMG 13550
LMG 13552
LMG 13553
LMG 13554
LMG 13556
LMG 13557
LMG 13558
LMG 13562, 13563
LMG 13560, 13561
MKB 38
LMG 13567
MKB SA1
MKB SD1
Sensitivity*
++
++
++
++++
++
+
+
++
++
+++
+
++
++
++
*+, ++, +++, ++++ reflects the degree of sensitivity to peptide AS-48.
Production and inhibitory activity of peptide AS-48: An
overnight culture (10 ml) of E. faecalis FAIRE 92 was inoculated
into 1 litre of BHI-G broth (BHI broth supplemented with 1%,
wt/vol, glucose and 0.15 M NaH2PO4H2O) and incubated at 37oC
without aeration until stationary growth. On an hourly basis 1 ml
was sampled, serially diluted and plated onto BHI Agar (Biolab) and
the number of viable cells (CFU/ml) determined. At the same time 1
ml of the culture was centrifuged and the cell-free supernatant
adjusted to pH 6.0 to 7.0 with 1 N sterile NaOH. The sample was
concentrated ten-fold by freeze-drying and tested for activity
against S. aureus by using the spot-on-lawn method (Van Reenen
et al. 1998). One arbitrary unit (AU) of peptide AS-48 is defined as
the reciprocal of the highest dilution that produced an inhibition
zone of at least 2 mm in diameter. From the antimicrobial activity,
expressed as AU/ml, and the cell count (log10 cfu/ml), the specific
antimicrobial activity (AU/log10 cfu) was determined.
Isolation, purification and concentration: Peptide AS-48 was
isolated and purified according to the method described by Métivier
5
et al (2000). One litre of BHI-G broth was inoculated with 10 ml of
an actively growing culture of E. faecalis FAIRE 92 and incubated
for 7 h at 37oC. Cells were harvested by centrifugation (9000 x g,
4oC) and Triton TX-114 added to the supernatant to obtain a final
concentration of 2% (wt/vol). The sample was adjusted to pH 5.5
with concentrated HCl, heated to 25 to 30oC, and incubated at this
temperature for 1 to 2 h, after which the upper-phase was removed
and replaced with the same volume of cold Millipore Q water
(18.2) containing Triton TX-114 (0.2%, wt/vol). The Triton TX114 was dissolved by careful stirring. The mixture was heated to 25
to 30oC and left to separate into two phases. The lower detergentrich phase was recovered, diluted five-fold with cold Millipore Q
water, and loaded onto a 15 ml SP-Sepharose Fast flow column
(Amersham Pharmacia Biotech, Uppsala, Sweden). The column was
washed with cold Millipore Q water until a constant baseline
absorbance at 280 nm was reached. Loading and washing was done
at 8oC to avoid phase partitioning. The bacteriocin was eluted with
an ammonium acetate step-gradient of 0.1 to 1.6 mol liter-1 (pH
6.0). Fractions of 4 ml were collected, concentrated by freezedrying, dissolved in 100 l Millipore Q water and tested for activity
against S. aureus, S. agalactiae, S. dysgalactiae and E. coli, as
described before. The active samples were pooled and stored at 80oC.
Sensitivity to proteolytic enzymes: Purified peptide AS-48
(3200 AU/ml) was used in these tests. Resistance of peptide AS-48
to proteolytic enzymes was determined by incubation in the
presence of proteinase K (20 U/mg of peptide AS-48), pronase (7
U/mg of peptide AS-48), pepsin (2500 U/mg of peptide AS-48),
papain (30 U/mg of peptide AS-48), -chymotrypsin (90 U/mg of
peptide AS-48) and trypsin (110 U/mg of peptide AS-48) at 37oC
for 1 h. All enzymes were from Boehringer-Mannheim (Howard
Place, Midrand, South Africa) and tested at their optimum activity
pH. After incubation, the enzymes were heat-inactivated for 3 min
at 100oC and peptide AS-48 tested for antimicrobial activity against
S. aureus. The activity of the treated samples was compared to that
of a control sample, i.e. peptide AS-48 that had not been treated
with proteolytic enzymes.
Molecular mass determination: A sample collected after
separation in SP-Sepharose was subjected to tricine-SDSpolyacrylamide gel electrophoresis (SDS-PAGE), according to the
method described by Schägger & Von Jagow (1987). A protein
marker with sizes ranging from 2.35 to 46 kDa (Rainbow Marker;
Amersham Pharmacia Biotech, Uppsala, Sweden) was used. One
half of the gel was stained with Coomassie Brilliant Blue R250. The
position of the active peptide AS-48 in the gel was determined by
6
overlaying the other half of the gel, prewashed as described by Van
Belkum et al (1991), with cells of an overnight grown culture of S.
aureus, embedded in BHI agar (0.75% agar, wt/vol). In another
experiment, approximately 100 pmol of purified sample containing
the bacteriocin was diluted in 10 l of 10:90 acetonitrile-water
containing 0.01% formic acid and injected via the Rheodyne
injection port of a Quattro triple quadropole mass spectrometer
(Micromass, Manchester, United Kingdom). The carrier solvent was
10:90 acetonitrile-water at a flow rate of 20 l/min, delivered by a
Pharmacia-LKB 2249 high-pressure liquid chromatography pump.
The capillary voltage and the cone voltage were set at 3.5 kV and
60 V, respectively. Data were collected by scanning from 400 to
1500 m/z at 2 s/scan. The multiple charged spectra were
deconvoluted to obtain the accurate mass of the peptides.
Calibration was done by using horse heart myoglobin (Sigma, St.
Louis, Mo).
Preparation of liposomes and determination of encapsulation
efficiency: The method of Degnan & Luchansky (1992) was used.
Multilamellar vesicles were prepared using phosphatidyl choline
(Sigma) at 100 mg/ml chloroform. Four ml methanol/chloroform
(1:1) and 2 ml phosphatidylcholine/chloroform mixture was placed
into a 500 ml round-bottom flask and the solvent removed through
rotary evaporation (37oC, 60 rpm, 30 min, under vacuum). After
evaporation, a thin, opaque lipid film was formed on the inner
surface of the flask. Five ml of peptide AS-48 (6400 AU/ml) and
eight glass beads (4 mm in diameter; Fisher Scientific, Pittsburgh,
PA) were added to the contents of the flask and rotated for a
further 1 h at atmospheric pressure. This suspension was incubated
at 37oC for 2 h to complete swelling of the liposomes. The peptide
AS-48/liposome preparation was then transferred to a sterile 15 ml
polypropylene test tube and stored at 4oC for 7 d. The
encapsulation efficiency (E%) was determined by adding 45 l of
the peptide AS-48/liposome suspension to each of two
microcentrifuge tubes. Five l proteinase K (10 mg/ml) was added
to one of the tubes to inactivate free, i.e. non-encapsulated,
peptide AS-48. Sterile distilled water was added to a second tube.
Both samples were incubated at 37oC for 1 to 2 h and then heated
for 3 min at 100oC to inactivate proteinase K and, at the same time,
release encapsulated peptide AS-48 from the intact liposomes. The
antimicrobial activity of the released peptide AS-48 was determined
by using the spot-on-lawn method, as described before. The E% of
encapsulated peptide AS-48 was calculated as follows: E% =
activity (AU/ml) of peptide AS-48 in the liposome mixture treated
with proteinase K, divided by the activity (AU/ml) of peptide AS-48
in the liposome mixture to which sterile distilled water was added
(x100).
7
In vitro antimicrobial activity tests with encapsulated
peptide AS-48: One ml of encapsulated peptide AS-48 (3200
AU/ml) was added to a 100 ml culture of S. aureus at the beginning
of the lag and mid-exponential growth phases, respectively. Sterile
demineralised water (1 ml) was added to the control flask. Changes
in the turbidity of the cultures were recorded at 600 nm, and the
number of viable cells (cfu/ml) was determined by plating onto BHI
Agar (Biolab). The activity (AU/ml) of the encapsulated peptide was
determined by using the spot-on-lawn method, as described before.
Prevention and treatment of S. aureus-infections in udders:
For the prevention experiment, 10 quarters of five mastitis-free
cows with SCC of less than 500 000 per ml milk were selected. Milk
from these quarters contained no viable cells of S. aureus, as
determined by plating onto Baird-Parker Agar (Biolab). Five of the
quarters were infused with 1 ml encapsulated peptide AS-48 (6400
AU/ml), followed by 2 ml of S. aureus (3.3 x 103 cells/ml) injected
through the teat canal. The other five quarters served as controls
and received the same treatment, except that peptide AS-48 was
replaced by 1ml sterile saline (0.75%, w/v, NaCl). Injection was
immediately after milking, thus on day 1 of milk collection. The AS48 peptide, saline and S. aureus cells were gently massaged
upwards into the teat canal. Milk samples were collected daily, (up
to day 7), from which the SCC and viable cell numbers of S. aureus
were determined.
The number of replications depended on the number of available
mastitis-free cows at a specific stage of lactation. Since the
microbial challenge with S. aureus was expected to result in marked
changes in SCC and S. aureus counts, five replicates were regarded
as sufficient for the purposes of this study. Moreover, since only a
marked response to the treatment with peptide AS-48 would be of
therapeutic value, it was argued that a high number of replicates
was not needed.
All counts were expressed as log10 values. Where no S. aureus was
detected, a value of 100 was allocated to each count prior to
analysis. The analyses were also complicated by the fact that ten
different quarters of five individual cows were sampled. The data
could thus not be described as uncorrelated, as assumed for
analysis of variance. This complication was accounted for by the
estimation of the intra-class correlation depicting all possible
correlations between repeated samples obtained from the cows
(Harvey, 1990). Co-variation arising from the repeated samples
from specific cows was not only accounted for by this procedure,
but it was also possible to estimate the repeatability of the SCC and
8
S. aureus cell numbers (Turner & Young 1969). Repeatability (t)
was calculated as follows:
t = b2/ b2 + e2
= b2 the intra-class
correlation between cows
= e2 the residual
variation
Apart from the random effect of cows in the models used to analyse
the data, fixed effects of treatment and days post treatment were
included. The interaction between treatment and days post
treatment was also estimated.
For the treatment experiment, six quarters from the udders of three
lactating Holstein cows from the dairy herd of the Elsenburg
Research Center suffering from mastitis and with a SCC higher than
2.5 x 106/ml milk, were selected. Milk collected from these quarters
tested positive for the presence of S. aureus (≥ 1 x 104 CFU/ml).
Three of these quarters were injected with 1ml encapsulated
peptide AS-48 (6400 AU/ml), as described before. The other three
quarters served as controls and were injected with 1ml sterile saline
solution (0.75%, w/v, NaCl). As in the prevention experiment,
injection was done immediately after milking and the encapsulated
peptide and saline gently massaged upwards into the teat canals.
Milk samples were collected daily (up to day 7) from which the SCC
and number of viable S. aureus were determined.
The number of replications conducted in this experiment was also
relatively low. Again, the experiment depended on the number of
mastitic udders available at a specific stage of lactation. The
number of replicates was regarded sufficient for the purposes of
this experiment. The data were analysed as a 2 (treatments) x 7
(day) factorial design. As for the previous experiment, the analysis
was complicated by the fact that the same quarter was sampled
repeatedly. The same basic procedure was followed to account for
repeated sampling. The difference was that individual quarters were
confounded with treatments in this case. The appropriate analysis
was thus to nest individual quarters within treatments.
Repeatability was estimated from between quarter variance
components as described previously.
RESULTS
The highest antimicrobial activity levels recorded for peptide AS-48
was 1593 AU/ml during late exponential growth, i.e. after 7h at
37°C (Fig. 1). This corresponded to a specific antimicrobial activity
9
of 148 AU/log10 cfu (Fig. 1). Isolation and further purification of
peptide AS-48 with Triton TX-114 and SP-Sepharose resulted in an
increase of antimicrobial activity (3200 to 12800 AU/ml; data not
shown), which corresponds to a two to eight-fold increase in
specific antimicrobial activity (296 to 1184 AU/log10 cfu). In in vitro
tests, all strains of Bacillus, Lactobacillus, Staphylococcus and
Streptococcus were inhibited, but not E. coli (Table 1).
Figure 1. Production of peptide AS-48 during the
growth of E. faecalis FAIRE 92., Growth of E. faecalis
FAIRE 92, expressed as CFU/ml; and , specific
antimicrobial activity (AU/log10 CFU) of peptide AS-48
against S. aureus. = highest activity recorded as
1593 AU/ml.
11.5
10
9.5
80
9
log10 cfu/ml
100
8.5
60
7.5
20
7
8
40
0
0
1
2
3
4
5
6
7
8
9
Time ( hours)
Peptide AS-48 was completely inhibited by treatment with
proteinase K, pronase, pepsin, papain, -chymotrypsin and trypsin
(results not shown).
Mass spectrometry analysis indicated that the active samples,
collected from the SP-Sepharose column and pooled, contained a
single peptide with a molecular mass of 7.150 kDa (Fig. 2).
Figure 2. Molecular mass of peptide AS-48 calculated
from the electro-spray ionization-mass spectrometry
multiple charged spectra.
10
Specific antimicrobial activity
( AU/log 10 cfu)
11
120
10
140
10.5
160
11
7150.00
100
%
7476.26
7490.24
0
3000
4000
5000
6000
7000
8000
12
Separation on tricine-SDS-PAGE yielded only one active peptide
band in the range of 6.4 kDa (Fig. 3).
6.5
46
kDa
1
2
3.4
3
30
21.5
14.5
Figure 3. Separation of peptide AS-48 by tricine-SDSPAGE. Lane 1 , Rainbow protein size markers; lane 2,
peptide AS-48 stained with Coomassie brilliant blue
R250; lane 3, peptide AS-48 overlaid with a strain of S.
aureus isolated from mastitic milk and embedded in BHI
Agar (0.75% agar, wt/vol). The active peptide band is
indicated by an arrow.
The titer of the liposome-encapsulated peptide AS-48 varied
between 400 AU/ml after proteinase K treatment, compared to
3200 AU/ml for the control sample (not treated with proteinase K),
to 1600 AU/ml after proteinase K treatment compared to 6 400
AU/ml for the control sample. This compares to an encapsulation
efficiency (E%) of between 12.5% and 25%. In general,
encapsulation efficiencies varied between 10 and 25%.
Addition of encapsulated peptide AS-48 (1000 AU/ml) to S. aureus
in lag-phase resulted in a decrease in viable cell numbers from 3 x
108 cfu/ml to 1 x 104 cfu/ml within 30 min, followed by a further
decrease over the next 210 min (3.5h) to below the detection limit
13
of 10 cfu/ml (Fig. 4). The cell numbers of the control, i.e. without
peptide AS-48 added, increased from 3 x 108 cfu/ml to 1 x 1011
cfu/ml over 300 min (5h). The optical density readings of cells that
received peptide AS-48 in lag-phase remained constant at
approximately 0.2, whereas the density of the culture increased to
approx. 6.0 in the absence of peptide AS-48 (Fig. 4). Encapsulated
peptide AS-48 (1000 AU/ml) added to a mid-exponential growth
phase culture of S. aureus (2.5 x 1010 cfu/ml) resulted in no growth
inhibition, as evident from the steady increase in viable cell counts
and optical density readings, respectively (Fig. 4).
6.5
13
6
12
Addition of peptide AS-48
5.5
11
10
9
4
8
3.5
7
3
6
2.5
5
2
4
Addition of peptide AS-48
1.5
3
1
2
0.5
1
0
0
0
30
60
90
120
150
180
211
240
270
300
Time ( minutes)
Figure 4. Effect of encapsulated peptide AS-48 (1000
AU/ml) on the growth of S. aureus. Cell counts in the
absence of peptide AS-48 (), in the presence of
peptide AS-48 added at the beginning of the lag phase
(), and added during mid-exponential growth ().
The optical density (at 600 nm) was determined for
cells growing in the absence of peptide AS-48 (), in
the presence of peptide AS-48 added at the beginning
of the lag phase (), and added during mid-exponential
growth ().
Treatment of healthy, uninfected udders with liposomeencapsulated peptide AS-48 (400 AU/ml), followed by infection with
S. aureus, resulted in variable SCC and S. aureus cell counts (Figs.
5a and b). The SCC on day 1, directly after milking, was 6.3 x
330
360
log10 cfu/ml
O.D. (600 nm)
5
4.5
14
104/ml and 4.9 x 104/ml for udders not treated with peptide AS-48
and udders treated with the peptide, respectively (Fig. 5a). Twentyfour hours later (day 2), the SCC in milk from untreated and
treated udders increased to 1.3 x 106/ml and 1 x 106/ml,
respectively (Fig. 5a). During the next 5 days, the SCC in milk from
untreated udders decreased slightly to 1.2 x 106, whereas the SCC
in milk from treated udders decreased from 1 x 106/ml to 4 x
105/ml, representing a 60% decrease in SCC.
Log10 SCC/ml
7
6
Not treated
Treated
5
4
1
2
3
4
5
6
7
Time (days)
.
Figure 5(a). The effect of liposome-encapsulated peptide AS48 on the somatic cell count (SCC) in milk sampled from
mastitis-free udders that were infected with S. aureus relative
to an untreated control (i.e. no peptide AS-48 injected into
the udders). Each mean is based on five replicates. The
experimental design involved five cows with one teat
randomly assigned to the treated and control groups
respectively. Vertical bars denote standard errors.
15
4
Log10 cfu/ml
3.5
3
Not treated
Treated
2.5
2
1.5
1
2
3
4
5
6
7
Time ( Days)
Figure 5(b). The effect of liposome-encapsulated peptide AS48 on the cell numbers of S. aureus in milk sampled from
mastitis-free udders that were infected with S. aureus relative
to an untreated control. Each mean is based on five
replicates. The experimental design involved five cows with
one teat randomly assigned to the treated and control groups
respectively. Vertical bars denote standard errors
The variation accounted for by the repeated sampling from different
animals was significant in the analysis involving SCC (P < 0.001)
and S. aureus counts (P < 0.05). Repeatability estimates (SE)
derived from these analyses were 0.26  0.19 and 0.13  0.13,
respectively. Although these estimates could not be proven as
significant (P < 0.05) from zero, the variation between animals
controlled significant (P < 0.05) portions of the overall variation in
both analyses. It was thus decided to retain this effect in the
models used.
The interaction between treatment and days post treatment was
not significant (P = 0.79) as far as SCC is concerned. A sharp
increase (P < 0.05) in SCC was recorded on day 2 (Fig. 5a). In
quarters treated with peptide AS-48 the SCC was generally lower
than in the control (untreated) quarters, with a significant (P <
0.05) difference on day 6. When the overall mean values for treated
and untreated quarters were compared, a significant difference (P <
0.01) was recorded in favour of the treated quarters.
The viable cell numbers recorded for S. aureus in milk sampled
from untreated udders increased from 1 x 102 cfu/ml (day 1) to 2.6
x 103 cfu/ml (day 4), followed by a decrease to 8 x 102 cfu/ml on
16
day 7 (Fig. 5b). The S. aureus count in milk collected from udders
pre-treated with peptide AS-48 increased from 1 x 102 cfu/ml (day
1) to 2.4 x 102 cfu/ml (day 4), but decreased to 1 x 102 cfu/ml
towards the end of the experiment, i.e. day 7 (Fig. 5b).
Since no significant interaction was recorded between treatment
and days post treatment (P < 0.05), it is appropriate to give overall
mean values for treated and control quarters. These were (on the
log10 scale) 2.79  0.18 for control quarters and 2.18  0.18 for
treated quarters. Transformed back to the normal scale (with 100
subtracted) these mean values correspond to respective S. aureus
counts of 509 and 51, i.e. a near to 90% reduction in treated
quarters.
The SCC in milk sampled from untreated S. aureus-infected
quarters remained more-or-less the same and varied from 2.9 x
106/ml to 7.4 x 106/ml over a period of 7 d (Fig. 6a). When injected
with liposome-encapsulated peptide AS-48 (1 600 AU/ml), the SCC
in milk from S. aureus-infected quarters decreased from approx. 7
x 106/ml to 1.0 x 106/ml over 7 d (Fig. 6a). The viable cell count of
S. aureus in infected milk from untreated udders increased from 1 x
104 cfu/ml to 3.9 x 104 cfu/ml over 7 d, whereas in milk from
udders that have been treated with encapsulated peptide AS-48,
the cell counts decreased from 3.2 x 104 cfu/ml to 2.3 x 102 cfu/ml
over the same period (Fig. 6b).
The between quarter variance was significant (P < 0.01) in analyses
on SCC and S. aureus counts. The respective repeatability
estimates derived from the variance components were 0.40  0.25
and 0.41  0.25.
For SCC the interaction between treatment and days post treatment
did not reach significance in this case (P = 0.06). Overall, the SCC
was reduced (P < 0.05) in treated quarters relative to control
quarters (Fig. 6a). Overall mean values (SE) for SCC were 6.70 
0.12 for control quarters, compared to 6.01  0.12 for treated
quarters. Expressed relative to the control treatment, the reduction
in treated quarters amounted to approximately 99%.
.
17
Log10 SCC/ml
8
7
6
5
4
1
2
3
4
5
6
7
Time (days)
Figure 6(a). The effect of liposome-encapsulated peptide AS48 on mastitis-infected udders. SCC counts were determined
in milk sampled from peptide AS-48-treated and not treated
udders, respectively. Each mean is based on three replicates.
The experimental design involved three cows with on teat
randomly assigned to the treated and control groups
respectively. Vertical bars denote standard errors
Log10 cfu/ml
5.5
4.5
Not treated
Treated
3.5
2.5
1.5
1
2
3
4
5
Time ( days)
6
7
18
Figure 6(b). The effect of liposome-encapsulated peptide AS48 on mastitis-infected udders. Cell counts of S. aureus were
determined in milk sampled from peptide AS-48-treated and
not treated udders, respectively. Each mean is based on three
replicates. The experimental design involved three cows with
on teat randomly assigned to the treated and control groups
respectively. Vertical bars denote standard errors.
In the case of S. aureus, treatment interacted (P < 0.01) with the
days of post treatment. In the case of untreated quarters, S. aureus
counts basically remained on the same level throughout the
monitoring period (Fig. 6b). In treated quarters there was a
significant (P < 0.01) decline in S. aureus counts, to reach levels
not significantly different (P < 0.05) from two, which corresponds to
zero for back transformed values. This decline was observed from
day 4. Differences between mean values for treated and control
quarters were significant (P < 0.05) from day 2 onwards. Overall
means (SE) were 4.1  0.3 for control quarters, compared to 2.91
 0.26 for treated quarters. Transformed back to the arithmetic
scale (with 100 subtracted) these means correspond to respective
S. aureus counts of 12 530 and 715, i.e. a reduction of up to 94%
in treated quarters.
DISCUSSION
Peptide AS-48 exerts its antimicrobial action by incorporating into
the cytoplasmic membrane of sensitive bacteria. This is followed by
pore formation, and leads to the leakage of potassium ions and
inorganic phosphate (Gálvez et al 1986, 1989; Samyn et al 1994).
In contrast to previous reports on the antimicrobial activity
spectrum of peptide AS-48 (Gálvez et al. 1986, 1989), we did not
observe inhibition of E. coli (Table 1). It may very well be that
higher concentrations of the peptide have to be used to effectively
penetrate the outer membrane of Gram-negative bacteria, as
suggested by other authors (Gálvez et al 1986, 1989; Joosten et al
1996). Peptide AS-48 did, however, inhibit the growth of S. aureus,
S. agalactiae and S. dysgalactiae which are considered to be major
pathogens involved in mastitis infection, especially in the Western
Cape region of South Africa.
Production of peptide AS-48 during late exponential growth is
typical of most bacteriocins described for lactic acid bacteria (De
Vuyst and Vandamme 1994). The molecular mass of peptide AS-48
(7.150 kDa; Fig. 2) is as previously reported for the peptide
(Martínez-Bueno et al. 1994). A clear inhibition zone which
surrounded a well-separated single peptide band of approx. 6.5 kDa
19
on a tricine-SDS gel (Fig. 3), confirmed that strain FAIRE 92
produces peptide AS-48.
Peptide AS-48 was successfully encapsulated into liposomes.
Although the antimicrobial activity recorded for the encapsulated
peptide is much lower than for the non-encapsulated peptide, the
encapsulation efficiency obtained (12 – 25% E) compared well with
encapsulation results previously reported for this specific method
(Degnan & Luchansky 1992). When encapsulated in liposomes, the
antimicrobial peptides are delivered to phagocytic cells to
accumulate intra-cellularly (Francis 1989; Onyeji et al 1994). Upon
phagocytosis of the liposomes, the antimicrobial compound is
released into the phagolysosome (Francis 1989). Virulent cells of S.
aureus may be situated in phagocytic cells and survive within
leukocytes (Bramley & Dodd 1984; Watson 1992; Onyeji et al
1994, Hébert et al 2000). Although non-encapsulated peptide AS48 would be more active in eliminating extra-cellularly located S.
aureus, encapsulation of the peptide would ensure a slow release of
antimicrobial activity and the deposition of the peptide to localised
areas within the udder.
The release of peptide AS-48 from encapsulated liposomes was
clearly demonstrated when added to lag-phase cells of S. aureus
(Fig. 4). The lack of inhibition recorded when encapsulated peptide
AS-48 was added to a mid-exponential phase culture of S. aureus
suggests that older cells are either less sensitive to the peptide, or
that the peptide was present at sub-lethal concentrations, as
previously reported for lacticin 3147 in similar experiments
(Twomey et al 2000).
Based on results obtained in the prevention and treatment
experiments with encapsulated peptide AS-48 injected into the
udders of healthy and mastitis-infected cows, respectively, better
results were obtained when the peptide was used to treat S. aureus
infection than when the peptide was used to prevent S. aureus
infection. However, in none of the two experiments, S. aureus was
completely eliminated. Nevertheless, this study clearly indicated the
potential use of peptide AS-48 in the prevention and treatment of
S. aureus mastitis. Further research is needed to optimize the
encapsulation efficiency of peptide AS-48 to yield higher levels of
antimicrobial activity before it can be used in the treatment or
prevention of mastitis in dairy cows.
Acknowledgements:
20
We are grateful to Dr. J. Kitching, Veterinary laboratory,
Stellenbosch, South Africa, for the strains of S. aureus, S.
agalactiae, S. dysgalactiae and E. coli and Ms R. Bauer for technical
advice.
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