Download OF AEROMONAS HYDROPHILA (HG-1), A. BESTIARUM (HG

Bull Vet Inst Pulawy 52, 45-52, 2008
INFLUENCE OF TEMPERATURE ON THE GROWTH,
PROTEASE PRODUCTION, AND HEAT RESISTANCE
OF AEROMONAS HYDROPHILA (HG-1),
A. BESTIARUM (HG-2), AND A. SALMONICIDA (HG-3)
LESZEK GUZ, AND ANTONINA SOPIŃSKA
Sub-department of Fish Diseases and Biology, Faculty of Veterinary Medicine,
Agricultural University in Lublin, 20-950 Lublin, Poland
[email protected]
Received for publication September 03, 2007
Abstract
The aim of the study was to evaluate the effect of
different temperatures on caseinase and elastase production
and growth of Aeromonas hydrophila K-101, A. bestiarum
15s, and A. salmonicida A-11 strains, isolated from diseased
carp. In order to study the influence of the temperature on
proteolytic yield and growth of the strains, standard
spectrophotometric methods were used. For the determination
of caseinase and elastase activity, the azocasein and elastinCongo red as substrates were used. It was shown that K-101,
15s, and A-11 strains isolated from motile Aeromonas
septicaemia (MAS) affected carp grew better at 28ºC than at
18ºC and 38ºC. The highest proteolytic activity of all studied
strains was obtained when the bacteria were grown at 28ºC. At
60ºC the D-value for K-101, 15s, and A-11 were 7, 4, and 3,
respectively. In summary, the temperature influenced the
growth of the strains isolated from MAS affected carp. The
adaptation of these strains to environmental factors imply their
possible long survival in the water, which is a potential threat
to public and animal health. From these results, it can be
concluded that the potential virulent ability of the ECP might
vary due to culturing at different incubation temperatures.
Key words: fish, Aeromonas, temperature.
Aeromonas species are ubiquitous inhabitants
of the aquatic environment and are also an opportunistic
and primary pathogen of fish, predisposing to the
infection as a result of stress (4, 30, 31). As part of the
normal microbiota, aeromonads usually do not cause
disease in healthy fish. Some of the aeromonads produce
a number of toxic extracellular products such as
haemolysins, cytotoxins, enterotoxins, and different
proteolytic enzymes (11, 24, 28). These properties have
been suggested to be associated with the virulence of
these pathogens (2). Widely distributed in aqueous
environments, aeromonads have been isolated from
rivers, drinking water, swimming pools, estuaries, and
lakes (4, 16). Aeromonas strains could be of public
health significance in food products that have an
extended shelf-life at refrigeration temperatures (3, 6).
The classification of the genus Aeromonas has
been dogged by confusion and controversy. According
to Joseph and Carnahan (13), this genus is now
classified within the family Aeromonadaceae and
consists of 14 different confirmed species. Species
Aeromonas hydrophila (HG-1), A. bestiarum (HG-2),
and A. salmonicida (HG-3) are included in the so-called
“A. hydrophila” complex (12).
The effect of temperature on the growth
kinetics of strains of A. hydrophila was evaluated by
Knochel (18), Stecchini et al. (37), Santos et al. (32),
Sautour et al. (33), and Wang and Gu (39). Many studies
are done to assess the influence of different factors on
the survival of A. hydrophila (15, 22, 39). Palumbo et al.
(26) studied the combined effects of temperature, NaCl,
pH, and NaNO2 on the aerobic growth of A. hydrophila.
It is well known that the temperature is an important
factor controlling the rate of development of microbial
populations. A modulation of enzyme synthesis by the
growth temperature has been observed in several
microorganisms (8, 22). There are no data concerning
the effect of temperature, protease yields and growth of
bacteria belonging to the different hybridysation groups
(HG) of A. hydrophila “complex” species.
The aim of the study was to evaluate the effect
of different temperatures on caseinase and elastase
yields and growth of A. hydrophila K-101, A. bestiarum
15s and A. salmonicida A-11 strains, isolated from
diseased carp.
Material and Methods
Bacteria and growth conditions. A.
hydrophila K-101 (HG-1), A. bestiarum 15s (HG-2),
and A. salmonicida A 11 (HG-3) strains, isolated from
motile aeromonas septicaemia (MAS) affected carp
(Cyprinus carpio L.), were kindly provided by Dr.
46
Kozińska (Department of Fish Diseases, National
Veterinary Research Institute, Poland). The bacteria
were cultured in tryptic soy agar (TSA). The agar plates
were incubated for 24 h at 28ºC. For the production of
extracellular proteases, the bacteria were grown in
tryptic soy broth (TSB) at 28ºC for 24 h. The culture
from slants was inoculated into 250 ml of TSB in 500 ml
Erlenmeyer flasks, and then incubated at three different
temperatures (18°C, 28°C, and 38ºC). The samples were
removed from the incubator at specified time intervals
(0, 6, 12, 24, 48, 72 and 96 h) and examined for bacterial
growth by determining in the spectrophotometer their
optical density at 620 nm.
The samples for the measurement of proteolytic
activity were centrifuged for 30 min at 10 000 g at 4ºC,
filtered through 0.22 µm membrane (Millipore), and
frozen at -80ºC for later analysis.
Measurement of proteolytic activity of ECPs.
Protein levels of ECP solutions were determined using
the Sigma protein assay kit with bovine albumin as a
standard.
Caseinase activity. The caseinase activity was
determined by the azocasein procedure described by
Leung and Stevenson (20), with slight Mateos et al. (22)
and own modifications. Briefly, the reaction mixture
consisting of 0.1 ml of a 10% (w/v) azocasein solution
(Sigma), 0.1 ml of supernatant fluid sample, and 2.3 ml
of 0.1 mol l-1 sodium phosphate buffer, pH 7.2, was
incubated at 28ºC for 30 min. The reaction was stopped
with 2.5 ml of 10% (w/v) trichloroacetic acid (TCA),
and after 30 min at room temperature, the precipitate
was removed by centrifugation. Equal volumes of
supernatant fluid and NaOH 1 mol l-1 were mixed and
absorbance was read at 450 nm. TCA was added to the
blank before incubation.
Elastase activity. The elastase activity was
determined by the elastin-Congo red procedure
described by Bjorn et al. (1), with slight Mateos et al.
(22) and own modifications. Briefly, 1 ml of culture
supernatant fluids was added to 2 ml of Tris-maleate
buffer (0.1 mol l-1, pH 7.0) supplemented with CaCl2
(0.001 mol l-1) containing 10 mg of elastin-Congo red.
The mixture was incubated at 28ºC for 30 min and the
reaction was stopped by the addition of 2 ml of sodium
phosphate buffer (0.7 mol l-1, pH 6.0). The precipitate
was removed by centrifugation. The blank consisted of 3
ml of the buffer containing 10 mg of elastin-Congo red.
Elastase activity was determined by reading absorbance
of the supernatant fluid at 495 nm.
Heat stability. Heat stability of the bacteria
was measured as described by Spinks et al. (35) with
own modifications. Briefly, 10 ml portions of the final
stationary phase cultures were centrifuged (35 000xg, 10
min) at 4ºC, and pellets were resuspended in sterile
distilled water to give approximate concentrations of
1010 cells ml-1. The inocula were determined by serial
dilutions and plated on TSA. A fixed volume of sterile
distilled water was placed into an Erlenmeyer flask held
in water bath at the appropriate lethal temperature
(55°C, 60°C, and 65ºC) prior to inoculation. After
temperature stabilisation, 1 ml of resuspended culture
was injected into the water medium and timing was
immediately initiated. Surviving bacteria were
enumerated by serial dilutions, plated on TSA, and then
incubated at 28ºC for 48 h. The “decimal reduction
time” (D-value) was defined as the time required to
reduce a bacterial population by 90% or 1 log reduction,
and was derived from the formula:
Dx = (T2 – T1)/(logC1 – logC2).
where Dx is the D-value in seconds for temperature x, T2
is the number of elapsed seconds at the final sample
point since time zero, T1 is the number of elapsed
seconds at the initial sample point since time zero, C1 is
the concentration of bacteria at T1, and C2 is the
concentration of bacteria at T2.
The stability of proteases was measured as
described by Khalil and Mansour (15) by subjecting the
samples to heat treatment ranging from 30 to 100ºC for
15 min. After the heat treatment, the residual proteolytic
activity was measured as described above.
Results
Three bacterial strains isolated from MAS
affected carp for the current investigations were
identified as hybridisation groups HG-1, HG-2, and HG3 (19).
After 96 h cultivation, the optical density,
expressing the growth rate at 3 different temperatures,
for A. hydrophila K-101 (HG-1), A. bestiarum 15s (HG2), and A. salmonicida A-11 (HG-3) strains were 3.2,
2.4, 1.6 at 18ºC, 4.6, 3.7, 2.8 at 28ºC, and 4.1, 2.8, 1.8 at
38ºC, respectively (Fig. 1).
The highest proteolytic activity of ECPs from
K-101, 15s, and A-11 cultures grown at 18°C, 28°C, and
38ºC was obtained when the bacteria were grown at
28ºC (Fig. 2). The lowest proteolytic activity was
obtained when the strains were grown at 18ºC, while the
cultures grown at 38ºC showed moderate proteolytic
activity (Fig. 2).
The heat resistance of the strains was studied at
50°C, 55°C, and 60ºC. D-values expressed as the time
required to achieve 90% reduction in the concentration
of bacteria from three replicate experiments were
calculated (Table 1). The reductions in bacterial count
were observed at all temperatures used (Fig. 3). The
capacity for heat resistance was greatly diminished at
60ºC with several log reductions occurring below 30 s.
The caseinase activity of K-101 and 15s ECPs
was relatively stable when heated for 15 min at 60ºC
(95% and 72.5%, respectively), although caseinases of
A-11 were more labile (30.5% activity). Complete
inactivation of the caseinolytic enzymes was observed
after heating the ECPs at 100ºC, 90ºC and 80ºC,
respectively (Fig. 4). The elastase activity of K-101, 15s,
and A-11 ECPs was stable when heated for 15 min at
50ºC (98%, 92%, and 93%, respectively). Complete
inactivation of the elastolytic enzymes was observed
after heating the ECPs at 90ºC, 90ºC, and 80ºC,
respectively (Fig.4).
47
(A)
3.5
A. hydrophila K-101
O.D. 620 nm
3
A. bestiarum 15s
A. salmonicida A 11
2.5
2
1.5
1
0.5
0
0h
6h
12 h
24 h
48 h
72 h
96 h
48 h
72 h
96 h
48 h
72 h
96 h
Time (h)
(B)
O.D. 620 nm
5
4.5
4
A. hydrophila K-101
A. bestiarum 15s
A. salmonicida A 11
3.5
3
2.5
2
1.5
1
0.5
0
0h
6h
12 h
24 h
O.D. 620 nm
Time (h)
(C)
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
A. hydrophila K-101
A. bestiarum 15s
A. salmonicida A 11
0h
6h
12 h
24 h
Time (h)
Fig. 1. Effect of incubation time and temperature on A. hydrophila, A. bestiarum, and A. salmonicida growth at 18ºC
(A), 28ºC (B), and 38ºC (C).
48
(A)
Proteolytic activity (U)
40
K-101
15s
A 11
35
30
38°C
28°C
25
20
18°C
15
10
5
0
0h
6h
12 h
24 h
48 h
72 h
96 h
Incubation time (h)
(B)
Proteolytic activity (U)
25
K-101
15s
A 11
20
38°C
28°C
15
10
5
18°C
0
0h
6h
12 h
24 h
48 h
72 h
96 h
Incubation time (h)
Fig. 2. Effect of incubation time and temperature on A. hydrophila, A. bestiarum, and A. salmonicida caseinase (A) and
elastase (B) activity. A unit of caseinolytic activity was defined as the enzyme activity in a 0.1 ml volume of sample
that produced an increase in absorbance of 0.1 at 450 nm. Elastase activity unit is expressed as the activity contained in
1 ml of supernatant fluid that increased the absorbance by 0.1 at 495 nm.
49
(A)
Survivors (%)
100
50ºC
10
55ºC
1
60ºC
0.1
0.01
0.001
0.0001
0.00001
0
10
20
30
60
90
120
Time (secs)
(B)
100
50ºC
10
55ºC
Survivors (%)
1
60ºC
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001
0
10
20
30
60
90
120
240
Time (secs)
(C)
100
50ºC
10
55ºC
Survivors (%)
1
60ºC
0.1
0.01
0.001
0.0001
0.00001
0.000001
0.0000001
0
10
20
30
60
90
120
Time (secs)
Fig. 3. The reduction of cells following exposure to heat for A. hydrophila (A), A. bestiarum (B) and A. salmonicida
(C).
50
(A)
Proteolytic activity (%)
120
A. hydrophila K-101
A. bestiarum 15s
100
A. salmonicida A 11
80
60
40
20
0
30
40
50
60
70
80
90
100
Heating temperatureo (OC)
(B)
Proteolytic activity (%)
120
A. hydrophila K-101
A. bestiarum 15s
100
A. salmonicida A 11
80
60
40
20
0
30
40
50
60
70
80
90
100
Heating temperatureo ( C)
O
Fig. 4. Heating stability of extracellular caseinase (A) and elastase (B) treated at different temperatures for 15 min.
Table 1
D-values expressed as the time required to achieve 90% reduction in the concentration of bacteria.
Means (± standard error) from three replicate experiments
Temperature (ºC)
Bacteria
A. hydrophila K-101
A. bestiarum 15s
A. salmonicida A 11
50
55
60
27 (±2)
33 (±3)
10 (±0.8)
13 (±0.9)
21 (±2)
9 (±0.4)
7 (±0.6)
4 (±0.5)
3 (±0.2)
51
Discussion
Temperature is considered as the major
controlling factor in the distribution of the bacteria in
natural environment. Temperature dependent seasonal
variations have been observed for Aeromonas sp. with
the highest population in summer and the lowest one in
winter (14). The growth temperature range for
aeromonads is from 4 to 44ºC, but individual strains
typically have a restricted growth range according to
their ecological niche, and growth of strains at both
extremes of the range are rare (4, 17). Our investigations
have shown that A. hydrophila K-101 (HG-1), A.
bestiarum 15s (HG-2) and A. salmonicida A 11 (HG-3)
strains, isolated from MAS diseased carp, grew better at
28ºC than 18ºC and 38ºC. These results are consistent
with the findings of Khalil and Mansour (15), who
found that the optimum temperature for A. hydrophila
growth in TSB medium was 30ºC, but in contrast to our
study, at this temperature the bacteria growth reached its
maximum after 24 h of incubation time. Palumbo et al.
(27) observed the same lag time at 28ºC and 37ºC with
the shortest generation time at 28ºC for one strain.
Although the optimum growth temperature is considered
to be 28ºC (29), Statner et al. (36) found that in some
cases better growth of bacteria could occur at 37ºC. The
maximum growth temperature for most strains of A.
hydrophila appears to be at least 42ºC with most
enterotoxigenic strains capable of growth at 43ºC (25,
26). Hänninen et al. (7) observed that the determination
of the tmax can be applied for differentiation of HG-1
from HG-2 and HG-3 (A. hydrophila phenospecies).
Hybridisation group of 2 and 3 strains, which in most
cases originated from water or food, had tmax about 3639ºC (7). Some species, including most A. salmonicida
strains, do not grow at 35ºC (7, 21). Merino et al. (23)
found that A. hydrophila strains grown at 20ºC
contained, relative to those cultured at 37ºC, increased
levels of the phospholipid fatty acids; hexadecanoate
and octadecanoate and reduced levels of the
corresponding saturated fatty acids. Furthermore, the
strains were more virulent for fish and mice when they
were grown at 20ºC than when they were grown at 37ºC.
They also showed increased different extracellular
activities when they were grown at 20ºC (23). Ishiguro
et al. (9) also found that virulent strains that grew at a
higher than optimal temperatures (26°C to 27ºC for the
three A. salmonicida strains studied) resulted in the
selection of spontaneous attenuated derivatives in the
initial bacterial population.
Our results indicated that the highest proteolytic
activities of all studied strains ECP were obtained when
the bacterium were grown at 28ºC. These results are
consistent with the findings of Khalil and Mansour (15),
who showed the highest proteolytic activity of ECPs at
30ºC. Mateos et al. (22) found that production of
caseinases, elastases, and growth yields of
environmental strains decreased sharply during
cultivation at 37ºC. Moreover, the human strains
differed from the environmental strains in response to
growth temperatures, their protease activity decreased at
37ºC, although growth yield was not affected.
Tsai et al. (38) found that the maximal toxin
titres were the same at both 28ºC and 37ºC, but that
toxins were produced slightly sooner at the lower
temperature.
Heat stability of three strains studied in our
experiment has shown that K-101, 15s, and A-11 were
resistant to heat with critical temperature 60ºC. Similar
results were reported by Spinks et al. (35) who studied
A. hydrophila (wild type) and found that temperature
range from 55°C to 65ºC was critical for effective
elimination of pathogenic bacterial components and
supported the thesis that hot water systems should
operate at a minimum of 60ºC.
The thermal resistance at any given temperature
may conveniently be expressed as the “decimal
reduction time” (D), which is defined as the time for the
survivors to be destroyed by one log cycle, which
represents 90% of the initial population. According to
the reported data, the heat resistance varies with species
(35). Large variations in thermal inactivation rates were
observed between the tested bacterial species as well as
between the tested temperatures for each species. The
influence of growth temperature on the heat resistance
of A. hydrophila has been reported by Spinks et al. (35).
The capacity for heat resistance of A. hydrophila was
greatly diminished at 60ºC with several log reduction
occurring within 1 min (35). Sheldon and Schuman (34)
determined D-values (1.5, 0.10, and 0.03) at 51°C,
57°C, and 60ºC, indicating that such thermal processes
can provide a large safety factor with regard to the
inactivation of A. hydrophila in liquid egg. Isonhood et
al. (10) found that A. hydrophila is not heat or
freeze/thaw resistant and does not appear to have a
measurable phenotypic cross-protective stress response
to starvation or cold storage that enhances heat or freeze
thaw tolerance. In our study, at 60ºC the D-value for K101, 15s, and A-11 were, 7, 4, and 3, respectively. For
all the strains studied, the inactivation curves were linear
at 60ºC, while survival curves at 50ºC and 55ºC were
characterised by a slower initial phase of inactivation
followed by a faster phase.
In summary, the temperature influenced the
bacterial growth of three isolates from MAS diseased
carp. Adaptations to environmental parameters by these
strains imply their possible long survival in water, which
is a potential threat to public and animal health. From
these results, it can be concluded that the potential
virulent ability of the ECP might vary due to culturing at
different incubation temperatures.
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