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OJHAS Vol. 6, Issue
2: (2007 Apr-Jun) |
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Survivability
of Salmonella typhimurium L1388 and
Salmonella enteritidis L1225
under stressful growth conditions. |
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Ngwai YB, Department
of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy,
Niger Delta University, Wilberforce Island, PMB 071, Yenagoa, Bayelsa
State, Nigeria Wambebe
C, International
Biomedical Research in Africa (IBRIA),
P.M.B. Garki, Abuja, Nigeria Adachi Y, Animal Health
Laboratory, School of Agriculture, Ibaraki University, 3-21-1 Ami, Ami-machi,
Ibaraki, 300-0393, Japan |
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Address For Correspondence |
Dr.
Yakubu B. Ngwai, Department
of Pharmaceutical Microbiology and Biotechnology, Faculty
of Pharmacy, Niger Delta
University, Wilberforce Island, P.M.B. 071
Yenagoa, Bayelsa
State, Nigeria
E-mail:
ybngwai@yahoo.com
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Ngwai YB, Wambebe
C, Adachi Y. Survivability
of Salmonella typhimurium L1388 and Salmonella enteritidis L1225
under stressful growth conditions
Online J Health Allied Scs. 2007;2:4 |
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Submitted Feb 24, 2007;
Suggested revision: Jun 17, 2007; Resubmitted: Jul 18, 2007; Accepted:
Nov 7, 2007; Published: Nov 10, 2007 |
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Abstract: |
In an earlier
study with Salmonella typhimurium L1388 (ST) and Salmonella
enteritidis L1225 (SE) isolated from diseased chickens, we found that
SE formed more biofilm than ST on abiotic surfaces in a time-dependent
manner. Since the ability of salmonellae to survive extreme environment
is related to their virulence, the present study examined the survival
of Salmonella typhimurium L1388 and Salmonella
enteritidis
L1225 under the usual stresses that salmonellae encounter during their
life cycle. This is with a view to understanding the strains’ stress
tolerance that could be used to explain their virulence. Incubation
at 37oC for various time periods was done for: i) stationary
phase (SP) cells at pH 2.6; ii) log-phase (LP) cells at pH 4.0; log-phase
or stationary phase cells in broth containing iii) hydrogen peroxide,
iv) sodium chloride and v) ethanol; vi) stationary phase cells in Hank’s
balanced salt solution (single strength) containing 10% human serum;
and vii) prolong stationary phase cells. Stationary phase cells were
also incubated at 52oC for 15 min. Surviving cells at the
various incubation times were counted on trypticase soy agar (TSA) after
appropriate dilution in saline and overnight incubation at 37oC.
Growth iron-poor medium was determined by growing a single bacterial
colony in Medium A with shaking at 37oC or 40oC
for 24 h. Statistics was done by one-way analysis-of-variance (ANOVA)
at P = 0.05. Differences in the survival of ST and SE were insignificant
(p>0.05) in acid pH at both pH 4.0 (p = 0.3783) and pH 2.6
(p = 0.4711); at high salinity for log-phase (p = 0.1416) and stationary
phase (p = 0.1816) cells; in ethanol (p = 0.5984), human serum (p =
0.8139), prolonged stationary phase (p = 0.3506); and under heat (p
= 0.5766). SE was significantly (p<0.05; p = 0.0031) more
tolerant to oxidative-killing by hydrogen peroxide. Culturable growth
of the ST and SE in an iron-poor medium A revealed insignificant differences
at 37oC (p = 0.8381) but marginally significant at 40oC
(p = 0.0508). Thus, with the exception of survival in hydrogen peroxide,
SE had similar response pattern with ST to the usual stresses that salmonellae
encounter during their life cycle, despite the former’s preferential
ability to form biofilm on abiotic surfaces. The relationship between
the observed enhanced ability of SE to survive in hydrogen peroxide
and virulence need to be investigated in subsequent study.
Key Words:
Salmonella typhimurium; Salmonella enteritidis; Survival; Stress |
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Members of
the genus Salmonella (Family: Enterobacteriaceae) have capacity
for survival in diverse environments as well as the capability of infecting
a wide range of vertebrate hosts.(1) During their life cycles, salmonellae
encounter diverse environmental stresses, such as nutrient deprivation,
pH extremes, oxidative stress, osmotic shock, DNA damage and heat shock,
(2) which may significantly influence their survival and virulence.(3) These stresses, depending on severity and duration of exposure,
usually inhibit either the growth or survival, or cause a loss of viability
of the cells.(4) Consequently, the ability of salmonellae to mount
up an effective stress response is also critical to their virulence.
Over
2,400 serotypes of the genus Salmonella are believed to exist;
and of this number, the serotypes Typhimurium and Enteritidis are more
frequently isolated in human and animal salmonellosis.(5, 6) The consumption
of infected poultry meat and eggs is a major source of human cases of
infections caused by Typhimurium and Enteritidis.(7) Therefore, these
serotypes remain a Public health problem worldwide, particularly with
respect to food safety.(8)
Salmonella
typhimurium L1388 and Salmonella enteritidis L1225 strains were
isolated from diseased chicken at the Japanese National Institute of
Infectious Diseases. We have found that SE produces more biofilm on
abiotic surfaces than ST in vitro.(9) In this study, we examined the
survival of Salmonella typhimurium L1388 and Salmonella
enteritidis L1225 under the usual stresses that salmonellae encounter
during their life cycle. This is with a view to understanding the strains’
stress tolerance. Studies of this nature have application in explaining
virulence of bacteria.
Bacterial
isolates and culture media
Salmonella
typhimurium L1388 and Salmonella enteritidis L1225, both of which
were isolated from chicken in Japan, were used in this study. Both strains
were propagated in either trypticase soy broth (TSB; BBL, U.S.A.) or
Luria-Bertani broth (LB; Daigo, Inc., Japan) as indicated in the text.
Solid media culture was grown on trypticase soy agar (TSA; BBL, U.S.A.)
or LB agar (Daigo, Inc., Japan). Unless otherwise indicated, all chemicals
used were from Wako Chemical Company, Japan.
Survival
in acidic medium
Survival of
log-phase (LP: 4-h LB culture) and stationary phase (SP: overnight LB
culture) cells in pH-adjusted LB broth was assayed based on Fang et
al. (10) with modifications. 2 ml of LB (pH 4.0 or 2.6; adjusted with
2 N HCl) was inoculated with 100 µl of overnight LB broth culture of
bacteria and incubated with shaking at 37oC. Samples were
drawn at times T = 0 h (before incubation) and T = 2 h of incubation
(pH 4.0) or T = 5 min (pH 2.6) of incubation, diluted in normal saline
and viable cells were counted after 18-h incubation on TSA at 37oC.
Survival (%) was calculated from ([Bacterial CFU per ml at T = 2 h or
5 min / Bacterial CFU per ml at T = 0 h] x 100). Results are means of
six independent experiments.
Survival
in hydrogen peroxide
The ability
of isolates to tolerate reactive oxygen intermediates produced by hydrogen
peroxide (H2O2) was evaluated by their survival
in LB broth containing 15 mM H2O2. Briefly, 2
ml of LB-15 mM H2O2
broth was inoculated with 100 µl of overnight LB broth culture of bacteria
and incubated with shaking at 37oC. Samples were drawn at
times T = 0 h (before incubation) and T = 1 h of incubation, diluted
in normal saline and viable cells were counted after overnight incubation
on TSA at 37oC. Survival (%) was calculated from ([Bacterial
CFU per ml at T = 1 h / Bacterial CFU per ml at T = 0 h] x 100). Results
are means of six independent experiments.
Survival
at high salinity
Survival of
log-phase (LP: 4-h TSB culture) and stationary phase (SP: overnight
TSB culture) cells in NaCl-adjusted TSB broth as described previously,
(11) with modifications. Briefly, 2 ml of TSB supplemented with NaCl
to a final concentration of 10% (equivalent to water activity [aw]
of < 0.94) (12) was inoculated with overnight broth culture of bacteria,
and incubated at 37oC with shaking for 2 h. Samples were
taken at times T = 0 h (before incubation) and T = 2 h of incubation,
and viable cells determined as before. Survival (%) of bacteria was
calculated by comparing the bacterial CFU at T = 2 h and T = 0 h as
in other survival assays described earlier. Results are means of three
experiments.
Survival
in ethanol
Ethanol is
an acceptable food additive. Hence, the need to investigate the ability
of ethanol to kill ST and SE. Survival in ethanol was investigated in
accordance with St. John et al. (13) with minor modifications. 2 ml
of LB broth containing 10 % ethanol (LBEtOH) was inoculated
with 0.1 ml of overnight broth culture of bacteria, and incubated with
shaking at 37oC for 3 h. Samples were taken at times, T =
0 h (before incubation) and T = 3 h of incubation, and viable cells
determined as described above. Results are means of three experiments.
Survival
at elevated temperature
The method
described by Jørgensen et al. (14) was used. Briefly, 2 ml of TSB was
inoculated with 0.1 ml of overnight broth culture of bacteria, and incubated
at 52oC with shaking. Samples were taken at times T = 0 min
(before incubation) and T = 15 min, diluted in saline, and the number
of viable cells were counted after overnight incubation on TSA at 37oC.
Survival (%) was determined from ([Bacterial CFU per ml at T = 15 min
/ Bacterial CFU per ml at T = 0 h] x 100); Results are averages of three
separate determinations.
Survival
in prolonged stationary phase
Survival in
prolonged stationary phase was examined as previously described, (10)
with slight modifications. Briefly, one colony of a strain was grown
in 2 ml of LB broth for 24 h at 37oC; 100 µl samples were
taken, diluted appropriately in normal saline, spread on TSA, and viable
cells counted after overnight incubation at 37oC. The 24-h
broth cultures were incubated further for another 144 h (i.e. a total
168 h), and the number of viable cells was determined after appropriate
dilution and overnight incubation on TSA as before. Survival (%) after
168 h of incubation was calculated from ([Bacterial CFU per ml after
168 h / bacteria CFU per ml after 24 h incubation] x 100). Experiments
were done twice using duplicate samples (i.e., n = 4), and error bars
are standard deviations of individual results from their respective
means.
Survival
in human serum
Bacterial survival
in human serum was assayed as previously described. (15) Counts were
determined at times T = 0 h and T = 2 h following addition of the inoculum
to 10% normal human serum in single strength Hank’s balanced salt
solution (HBSS: 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4,
0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM
MgSO4, 4.2 mM NaHCO3, 5.6 mM D-glucose, 0.02%
phenol red, distilled water to 1000 ml; membrane filter-sterilized (pore
size: 0.45 µm). Survival, expressed as Serum Resistance Factor, was
determined from the relation: CFU at T = 2 h / CFU at T = 0 h. Results
are means of three independent determinations.
Growth
in iron-poor Medium A
Growth of bacteria
under iron-limited condition was monitored in an iron-poor medium. (16)
Single colony of bacteria reactivated from a -80oC stock
culture was inoculated into 2 ml of a membrane filter-sterilized (pore
size: 0.4 µm) iron-poor medium (Medium A: 0.7 g K2HPO4,
0.4 g KH2PO4, 0.2 g (NH4) 2
SO4, 0.02 g MgSO4.7H2O, 0.5% D-glucose,
Distilled water to 100 ml; pH 6.95). The inoculated medium was then
incubated (with shaking) at 37oC for 24 h; and number of
colony forming units was counted on TSA, after appropriate dilution
with normal saline and overnight incubation at 37oC. Growth
was expressed as CFU of bacteria per milliliter of culture. Results
are means of four independent experiments.
Statistical
analysis
Data were analyzed
by the one-way analysis of variance (ANOVA) using Smith Statistical
Package, version 2.5 and significance of results determined at the 5
% probability level (that is, at P = 0.05).
Survival
in acidic medium
For both isolates,
survival at pH 2.6 was less than at pH 4.0 (Fig. 1). However, differences
in the survival of ST and SE were insignificant (p>0.05) at both pH
4.0 (p = 0.3783) and pH 2.6 (p = 0.4711).
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Figure 1: Survival of Salmonella
typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in acid pH. Survival of bacteria was determined
at 37oC for 5 min in LB broth (pH 2.6) or for 2 h in LB broth
(pH 4.0). Viable cells were counted on TSA after dilution in saline
and overnight incubation at 37oC; Survival (%) survival was
determined from the relation: (Bacterial CFU ml-1 at stated incubation
time / Bacterial CFU ml-1 at time, T = 0 h) x 100. Experiments were
done three times, and error bars are standard deviations from mean values.
LP-pH 4.0, log-phase cells exposed to pH 4.0 and SP-pH 2.6, stationary
phase cells exposed to pH 2.6. |
Survival
in hydrogen peroxide
SE was significantly
(p<0.05; p = 0.0031) more tolerant than ST to oxidative-killing by
hydrogen peroxide (Fig. 2).
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Figure 2:
Survival of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in hydrogen peroxide. Bacterial survival
was determined at 37oC in LB-15 mM H2O2
for 2 h, and viable cells were counted on TSA after appropriate dilution
in saline and overnight incubation at 37oC. Survival (%)
was calculated from the relation: (Bacterial CFU ml-1 T = 2 h / Bacterial
CFU ml-1 at time, T = 0 h) x 100. Experiments were done three times,
and error bars are standard deviations of individual results from their
respective means. |
Survival
at high salinity
Survival at
high salinity is as shown in Figure 3. The results indicate that differences
in the tolerance of ST and SE to the osmotic up-shift generated by high
salinity were insignificant (p>0.05) for log-phase (p = 0.1416) and
stationary phase (p = 0.1816) cells.
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Figure 3:
Survival of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in high salinity. Bacterial survival was
determined at 37oC in TSB-10% NaCl for 2 h, and viable cells
were counted on TSA after appropriate dilution in saline and overnight
incubation at 37oC. Survival (%) was calculated from the
relation: (Bacterial CFU ml-1 T = 2 h / Bacterial CFU ml-1 at time,
T = 0 h) x 100. Experiments were done three times, and error bars are
standard deviations of individual results from their respective means.
LP, log-phase cells; SP, stationary phase cells. |
Survival
in ethanol
At 10% ethanol
that is a common level in many beverages such as wine, survival of both
ST and SE was dramatically reduced within 3 h (Figure 4). However, differences
in the survival of the two isolates were insignificant (p > 0.05; p =
0.5984).
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Figure 4:
Survival of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in ethanol. Bacterial survival was determined
at 37oC in LB-10% Ethanol for 3 h, and viable cells were
counted on TSA after appropriate dilution in saline and overnight incubation
at 37oC. Survival (%) was calculated from the relation: (Bacterial
CFU ml-1 T = 3 h / Bacterial CFU ml-1 at time, T = 0 h) x 100. Experiments
were done three times, and error bars are standard deviations of individual
results from their respective means. |
Survival
at elevated temperature
Survival of
both ST and SE were significantly reduced on exposure to 52oC
(Figure 5). However, differences in survival between the isolates were
insignificant (p>0.05; p = 0.5766).
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Figure 5:
Survival of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) at elevated temperature. Bacterial cells
were incubated (with shaking) for 15 min at 52oC in TSB,
and viable cells were counted on TSA after appropriate dilution in saline
and overnight incubation at 37oC. Survival (%) was calculated
from the relation: (Bacterial CFU ml-1 T = 15 min / Bacterial CFU ml-1
at time, T = 0 h) x 100. Experiments were done three times, and error
bars are standard deviations of individual results from their respective
means. |
Survival
in prolonged stationary phase
Our findings
on prolonged stationary phase survival (shown in Figure 6) indicated
that differences in the survival of ST and SE were insignificant (p>0.05;
p = 0.3506).
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Figure
6: Survival of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in prolonged (7 days) stationary phase.
Bacteria were grown in TSB at 37oC for seven days. Viable
cells were counted (both on Day 1 and Day 7) on TSA after appropriate
dilution in saline and overnight incubation at 37oC. Survival
(%) was determined from the relation: (Bacterial CFU ml-1
on Day 7 / Bacterial CFU ml-1 on Day 1) x 100. Experiments
were done twice using duplicate samples (i.e., n = 4), and error bars
are standard deviations of individual results from their respective
means. |
Survival
in human serum
The resistances
of ST and SE to the complement-killing action of human serum were generally
similar (Fig. 7) as the relative differences observed were insignificant
(p>0.05; p = 0.8139).
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Figure
7: Serum resistance of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE). Counts were determined at times T = 0
h and T = 2 h following addition of the inoculum to 10% normal human
serum in single strength Hank’s Balanced Salt Solution (HBSS) at 37oC.
Resistance factor was calculated from the relation: CFU at T = 2 h /
CFU at T = 0 h. Error bars are standard deviations from means of three
independent experiments. |
Growth
in iron-poor Medium A
Culturable
growth of the ST and SE in an iron-poor medium A is as shown in Figure
8. Although for both isolates, growth was uninhibited at 37oC
and 40oC, growth at 37oC was siginificantly (p<0.05)
more than at 40oC. The differences between the isolates in
growth were insignificant at 37oC (p = 0.8381) and marginally
significant at 40oC (p = 0.0508).
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Figure
8: Growth of Salmonella typhimurium
L1388 (ST) and Salmonella enteritidis
L1225 (SE) in iron-poor medium. Bacteria were grown
in low-iron Medium A (Pollack et al., 1970), then incubated (with shaking)
at 37oC or 40oC for 24 h; and the number of colony
forming units was counted on TSA, after appropriate dilution with normal
saline and overnight incubation at 37oC. Growth was expressed
as CFU of bacteria per milliliter of culture. Results are means of four
independent experiments. |
The first major
stress that Salmonella encounters after an oral infection is
exposure to acidic gastric contents; but the most clinically relevant
acid exposure occurs after invasion of the intestinal mucosa, within
the phagolysosome, where the internal pH is 3 or 4.(17) Therefore,
the similar acid tolerance of ST and SE is probable indication that
they may survive within the phagolysosome.
The
greater tolerance of SE to hydrogen peroxide than ST suggests possible
differences in the response of ST and SE to the lethal effect of reactive
oxygen species formed as by products of respiratory burst that occurs
in the phagolysosome after invasion of the intestinal mucosa.(2) It
has been reported that resistance to killing by reactive oxygen (RO)
or nitrogen (RN) intermediates is associated with increased virulence
of Salmonella Typhimurium.(18)
Salmonella
grow optimally at a water activity (aw) value of 0.99. (19)
An osmotic up-shift usually lowers aw,
and consequently impairs growth. The insignificant differences in the
tolerance of ST and SE to the osmotic up-shift generated by high salinity
indicates a similar capacity for persistence in low aw environments.
The insignificance of the differences observed in the survival of the
two isolates in ethanol is relevant in the food industry, which strongly
relies on the acid or alcoholic conditions to inactivate pathogens.
High-temperature
heating is known to induce protein denaturation through vibration of
water molecules to break disulfide and hydrogen bonds of intracellular
proteins.(20) The similarity in heat tolerance of ST and SE suggests
possible similarity in their protein composition.
The
similarity in serum sensitivity could be accounted for by the smooth
lipopolysaccharide (LPS) structure of the isolates (Data not shown).
The ability of Salmonella to withstood host defense mechanisms
is determined by certain structural and physiological attributes that
act together or independently to promote the survival and growth of
Salmonella in host cells. One of such attributes is serum resistance,
(7) which is complement-mediated, LPS structure-dependent and plasmid-encoded.(21) Human serum was used for the test because it is known to function
more efficiently in bacterial systems than either rabbit or guinea-pig
serum.(22)
Since
both strains survived to a similar extent under conditions of nutrient
limitation, such as that observed in prolonged stationary phase, they
may have similar persistence within the phagosome. (23) It is known
that nutrient limitation is a key regulatory signal of some virulence
genes in salmonellae.(24)
When
deprived of iron, Salmonella typhimurium synthesizes the high-affinity
iron chelator enterochelin (enterobactin), as well as produce a specific
transport system for this siderophore.(25) Like in many Gram-negative
bacteria, Salmonella typhimurium do not grow well at elevated
temperatures unless the growth medium is supplemented with iron.(26)
In S. typhimurium, the requirement for additional iron
is due, at least in part, to decreased biosynthesis of the phenolate
siderophore enterochelin at the elevated temperature.(26)
In
conclusion, but for the significant variation in response to hydrogen
peroxide, little insignificant variations were observed in the stress
tolerance response of ST and SE. These observations will need to be
correlated with virulence of the strains investigated.
We are grateful
to Rikkyo University, Japan for The Bishop William’s Fellowship support
to Yakubu B. Ngwai.
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