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Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium
Journal Pre-proof Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium Indu Pant, Rabindranath Shashidhar PII:
S0969-8043(19)30413-0
DOI:
https://doi.org/10.1016/j.apradiso.2019.108990
Reference:
ARI 108990
To appear in:
Applied Radiation and Isotopes
Received Date: 27 March 2019 Revised Date:
31 October 2019
Accepted Date: 17 November 2019
Please cite this article as: Pant, I., Shashidhar, R., Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium, Applied Radiation and Isotopes (2020), doi: https:// doi.org/10.1016/j.apradiso.2019.108990. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical Abstract
RADIATION
ACID & ALKALINE STRESS
SALT STRESS
Salmonella Typhimurium+ D2O
Reduced growth rate Sensitive to radiation without media Resistant to radiation in medium 10% reduction in cell number in salt stress in D2O Dies after 24 h in both alkaline and acidic stress
1
Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium
2 3
a Indu Pant and Rabindranath Shashidhar*
4 5
a Food Technology Division, Bhabha Atomic Research Centre, Mumbai, India
6 7
Running Title: Effect of deuterium oxide on the stress tolerance capability of Salmonella
8
typhimurium
9 10
Keywords: Heavy water, Salmonella typhimurium, growth rate, radiation stress, salt stress, D10,
11
alkaline stress, acidic stress, biofilm, heat stress.
12 13 14 15 16
*Corresponding author: Food Technology Division, Bhabha Atomic Research Centre, Trombay,
17
Mumbai, India and Life Sciences, Homi Bhabha National Institute (DAE-Deemed University),
18
Trombay, Mumbai 400094, India. Tel: +91 022-25593961; E-mail: [email protected]
19 20
Abstract
21
The heavy isotope of water is used in understanding the physiology of bacteria. Deuterium (D2O)
22
reduces chemical reaction kinetics. In the present study, the survivability of the food-borne pathogen
23
Salmonella typhimurium grown in D2O supplemented medium is studied under various stress
24
conditions. The growth of S, typhimurium was studied in rich (Luria Broth–LB) and minimal medium
25
(M9) prepared in D2O. The reduced growth rate of S. typhimurium in M9 (2.4 fold) as compared to
26
that in LB (1.6 fold) was observed. S. typhimurium grown in D2O supplemented medium was
27
significantly more tolerant to heat and gamma radiation (1.2 fold), but was sensitive to extreme pH
28
(both alkaline and acidic) and osmotic stress (10 fold). These results suggest that the change in the
29
biological reaction kinetics in the cell due to D2O may modify the stress tolerance of S. typhimurium.
30
This is the first study carried out to understand how a bacterial system (S. typhimurium) in D2O
31
responds to different stresses. This study suggests that investigations on bacterial physiology in D2O
32
supplemented medium helps in understanding the underlying mechanisms of stress tolerance.
33 34
Keywords: Heavy water, Salmonella typhimurium, growth rate, salt stress, D10, alkaline stress, acidic
35
stress, biofilm, heat stress, radiation stress.
36
Introduction
37
Water is essential for life on the Earth. For a long period, the chemical nature of water was a mystery.
38
Today, the unique properties of water are known. Pure heavy water, D2O, is the oxide of the heavy
39
stable isotope of hydrogen, deuterium, denoted by the symbols 2H or D. The proportion of heavy water
40
varies in natural water; river water contains 0.02-0.03 mol% of D2O, and the water of Antarctic ice
41
contains 0.015 mol% of D2O (Mosin and Ignatov., 2014). There is no difference in the chemical
42
structure of D2O and H2O. However, a small difference exists in the length of covalent H-O bonds and
43
the angle between them. D2O has a molecular mass 10% higher than that of H2O. The enhanced
44
strength of inter- and intra-molecular hydrogen bonds has consequences for biological macromolecules.
45
The biological effects of deuterium oxide on a wide variety of microorganisms like yeast (Richards.,
46
1934), Escherichia coli (Katz., 1960), and algae (Strain et al., 1959); plants such as tobacco (Lewis.,
47
1934), Arabidopsis (Bhatia and Smith., 1968), and various grasses (Crumley., 1950); and animals
48
including mice (Katz., 1962) and dogs (Katz., 1960) were studied. Some variants of E. coli (Giovanni.,
49
1961), Bacillus thuringiensis, Bacillus subtilis, an autotrophic nitrite-oxidizing bacterium (Nitrospira
50
moscoviensis), an autotrophic ammonia-oxidizing archaeon (Nitrosophaera gargensis), and autotrophic
51
methanogenic archaea (Methanobrevibacters mithii and Methanocorpusculum labreanum) (Berry D et
52
al., 2015) can survive in 90% (v/v) D2O. Plant cells can survive in up to 75% (v/v) D2O, and animal
53
cells survive up to 30% (v/v) D2O (Mosin and Ignatov., 2014). Excess deuterium in water reduced the
54
synthesis of proteins and nucleic acids and changed the enzymatic kinetic rates and morphology of the
55
cell (Schroeter et al., 1992; Takahashi et al.,1983; Caldwell et al.,1939). The cell division rate was
56
reduced on the subsequent increase in the heavy water concentration in the growth medium (Gross and
57
spindle.,1960). The antimitotic action of D2O was observed in all the stages of the mitotic cycle and
58
during cytokinesis in the cells of Arbacia (Gross and spindle.,1960).
59
In E. coli, slow internalisation of deuterium into substrates resulted in change in the metabolic pathway
60
and enzymatic reaction rate (Hochuli et al., 2000; Zhang et al., 2009). Lower mutation rates after
61
ultraviolet irradiation was reported in deuterated medium than in non-deuterated medium (Flaumenhaft
62
and Katz., 1960). Flaumenhaft E et al. (1960) also showed the morphological changes in the nuclei of
63
green algae grown in D2O, with proteins becoming darkly stained because of higher concentration of
64
the free basic groups.
65
Salmonella species is an important food-borne pathogen (Hendriksen et al., 2011). Salmonella is
66
prevalent in poultry, pigs, cattle, fruits, and vegetables. It can enter the food chain and survive with
67
limited nutrients. Many epidemics caused by Salmonella species have been reported. The presence of
68
Salmonella species is a severe problem in most foods, particularly dry and semi-dry products (e.g.,
69
milk powder, spices).
70
S. typhimurium causes systemic disease in mice similar to Salmonella typhi in humans (Teuber et al.,
71
1999). Therefore, S. typhimurium is used for molecular biology and stress-related studies. Salmonella
72
enterica serovar typhimurium is the most prevalent serotype in the Indian sub-continent (Gupta and
73
Verma.,1993) and hence, S. typhimurium was used as a model organism in the present study.
74
The effect of heavy water on the stress physiology of food-borne pathogens is a new area of study.
75
Better understanding of the underlying molecular physiology of stress tolerance would give an insight
76
into the applied aspects of food microbiology, such as design of novel food processing methods and
77
spoilage prevention tools.
78 79
Material and Methods
80
Chemicals
81
The 99.9% deuteriated water was obtained from Heavy Water Board (Mumbai, India). All the
82
chemicals (Sodium chloride, glutaraldehyde, sodium bicarbonate, sodium hydroxide, sterile distilled
83
water, phosphate buffer saline (pH 7.4), crystal violet solution, and potassium dihydrogen phosphate)
84
were procured from Hi-Media Laboratories Mumbai, India.
85
Microbiological media used in the studies were from Hi-Media Laboratories. Luria agar, Luria broth,
86
and tryptic soy media were used. M9 medium (M9 salt, 5X-20 mL, 20% glucose-2 mL, MgSO4-200
87
µL, CaCl2-10 µL, water-78 mL) was prepared. (Cold Spring Harbor Laboratory., 2010).
88
Instruments
89
Gamma chamber 5000 (Board of Radioisotope and Technology, Mumbai, India) at a dose rate of 0.083
90
kGy/min was used for irradiating the cells. The dose rate of radiation sources was measured using the
91
Fricke dosimeter. In experimental samples, the variations in dose absorbed were minimised by placing
92
the samples within a uniform area of the radiation field.
93
All the growth curve experiments were performed in Microtiter plates (96 well, Becton Dickinson
94
Labware, USA) and Bio-Tek microtiter plate reader (US). Water bath (Biosan, WB-4MS, Latvia,
95
Europe) was used for giving heat stress to the bacteria.
96
Microbiological methods
97
Bacterial culture and growth conditions
98
Salmonella typhimurium LT2 strain MTCC 98 procured from Microbial Type Culture Collection,
99
Chandigarh, India.
100
S. typhimurium was grown overnight in LB/M9 at 37°C at 150 rpm. Overnight culture of S.
101
9 -1 typhimurium (10 CFU mL ) was taken and washed thrice with 0.85% (w/v) saline. The washed cells
102
7 -1 were resuspended in saline and further diluted to 10 CFU mL . The end point of all the stress
103
survival assay (radiation, osmotic, pH extremes, and heat stress) was determined using the standard
104
plate count method (Willey et al., 2011). In the standard plate count method, cell dilutions were made
105
in saline and 100 µL of appropriate dilution was plated on plate count agar medium and kept for
106
-1) incubation at 37°C for different time intervals (24 and 48 h). The cell count (CFU mL was
107
calculated using the formula: Number of colonies x dilution factor/volume of dilution plated (mL)
108
(Willey et al., 2011).
109
Effect of D2O on the growth rate of S. typhimurium in different medium
110
7 -1 Salmonella typhimurium (10 CFU mL ) was grown in M9 and Luria broth (LB) in microtiter plates,
111
and their growth was monitored by measuring OD at 600 nm by using a plate reader at 37'C for 18 h.
112
The growth rate was calculated using the equation ln(N2/N1) =k(t2-t1) (Willey et al., 2011). In both LB
113
and M9 medium, after 24 h of incubation, CFU mL
114
count method as mentioned in microbiological methods.
115
Effect of D2O on radiation sensitivity of S. typhimurium
116
The D10 value of bacteria is defined as the radiation dose required to inactivate 90% of a viable
117
population. The 18-h-old S. typhimurium cells grown in LB at 37ºC were used for this study. Radiation
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7 doses of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 kGy were given to 1 ml of 10 cells dispensed in saline prepared in
119
D2O or H2O and media prepared in D2O or H2O in microfuge tubes. For calculating the number of cells
120
that survived after irradiation, the standard plate count method was used. The plates were incubated at
121
37°C for 24 h, and the slopes of the individual survivor curves were calculated by linear regression
122
with a computer program (Microsoft Excel 2007). Negative reciprocal of survival curve slope was used
123
to calculate D10 value (Saroj et al., 2006).
124
Effect of D2O on salt stress tolerance of S. typhimurium
125
S. typhimurium (107CFU mL-1) cells were resuspended in 2.5M NaCl solution made in D2O or H2O
126
and were incubated at 37°C in shaking condition for 24 h. The cell survival after osmotic stress was
127
checked at 0 and 24 h using the standard plate count method as mentioned in microbiological methods.
-1
was also calculated using the standard plate
128
Effect of D2O on alkaline stress tolerance of S. typhimurium
129
S. typhimurium cells were resuspended in pH 9, sodium bicarbonate buffer made in D2O, and H2O, and
130
incubated at 37ºC in shaking condition. Cell survival was checked at 0 and 24 h using the standard
131
plate count method as mentioned in microbiological methods.
132
Effect of D2O on acidic stress tolerance of S. typhimurium
133
Salmonella Typhimurium cells were resuspended in pH 4.5, phosphate buffer made in D2O, and H2O,
134
and incubated at 37ºC in shaking condition. The cell survival was checked at 0, 24, and 48 h using the
135
standard plate count method as mentioned in microbiological methods.
136
Effect of D2O on heat stress tolerance of S. typhimurium
137
An overnight culture of S. typhimurium was suspended in saline (0.85%) made in D2O or H2O, and heat
138
shock at a temperature of 55ºC was given for 5 min and 10 min in a water bath. The cell survival was
139
checked at 0, 5, and 10 min using the standard plate count method as mentioned in microbiological
140
methods.
141
Effect of D2O on biofilm formation of S. typhimurium
142
Biofilms are surface-attached microbial communities in which microbial cells are embedded in self-
143
produced extracellular polymeric substances. S. typhimurium was grown in Tryptic Soy Broth (TSB)
144
medium amended with D2O or H2O in a 96-well microtiter plate, and biofilm formation was checked
145
using specific biofilm formation index (Naves et al., 2008). Total cell mass was measured at OD 600
146
nm. The wells were washed thrice with sterile water to remove loosely attached or planktonic cells.
147
Microtiter plates were air dried and then oven dried at 50°C for 30 minutes. Following drying, cells
148
were fixed with 200 µL of 2.5% glutaraldehyde in PBS (pH 7.4) for 10 minutes. The wells were
149
washed thrice with distilled water and oven dried at 50°C for 30 min. Wells were stained with 0.2%
150
crystal violet solution and incubated at room temperature for 15 min after which the plates were
151
washed thrice with distilled water to remove unabsorbed stain. Microtiter plates were then air dried for
152
30 min and then 200 µL of ethanol-acetone mix (80%-20%) was added to each well to destain the
153
biofilm, and the concentration of crystal violet absorbed by the cells was measured at OD at 570 nm.
154
Biofilm formation was classified into three categories according to (Jahid et al., 2013): no biofilm
155
(SBF<0.1), weak (0.11).
156
SBF is specific biofilm formation index that represents the amount of biofilm formed in a specific
157
condition which is calculated using the formula SBF=B-NC/G (Naves et al., 2008) where B= OD at
158
570 nm of attached and stained bacteria, NC= OD at 570 nm of stained control well containing only
159
bacteria-free medium, and G= OD at 600 nm of cell growth in medium.
160
Statistical analysis
161
All the tests were carried out in triplicates with appropriate biological replicates. The mean and
162
standard deviation of the replicates are provided wherever required. Test of significance was carried out
163
at p < 0.05.
164
Results and discussion
165
S. typhimurium was grown in both LB and M9 media (Fig. 1 and 2) containing different concentrations
166
of D2O (20-100%). There was no observable change in the growth rate and pattern of growth in D2O
167
containing LB medium. This may be due to the rich medium components. The nutrients can
168
compensate and relegate the slower metabolism caused by D2O. The rich medium components are
169
more protiated. Hence, this provides a lesser chance of deuterium incorporation into the important
170
cellular machinery. Therefore, complete effect of the deuterium oxide on the growth could not be
171
observed.
172
The growth rate of S. typhimurium decreased with the increased concentration of D2O in M9 medium
173
(Fig. 3). The prominent reduction in growth was seen in M9 medium at a concentration ≥ 50% D2O (v
174
-1 /v) (Fig. 2). The growth rate of S. typhimurium in M9 medium prepared in 100% D2O was 0.15 h as
175
compared to medium prepared in 100% H2O, in which the growth rate was 0.36 h
176
the growth rate of S. typhimurium was 2.4-fold more than that observed in D2O supplemented minimal
177
-1 medium. The cell number in M9 minimal medium prepared in D2O reached 7 log CFU mL after 24
178
hours as compared to 9 log CFU mL -1 in the medium prepared in H2O. The decreased growth rate
179
may be due to overall slow kinetics of deuterated molecules, which slow down the metabolism. Also,
180
when D2O is mixed with H2O, it results in non-uniformity in water density; this further slows down the
181
metabolic process. Hence, low growth rate at higher D2O concentration was observed. These
182
observations suggest that D2O retards the growth of S. typhimurium in M9 medium. Further, in the
183
minimal medium (M9), cells synthesize all the components de novo. The de novo metabolism
184
incorporates more D2O into important cellular machinery of the cell. Therefore, the overall metabolism
185
of bacterial cells slows down in D2O supplemented minimal medium.
186
Inhibition of growth of the bacteria due to heavy water was reported in other studies. In 1987, Hakura
187
et al. showed a decrease in growth rate in variants of E. coli in D2O when compared to that in H2O.
188
However, there was no comparison in the growth rate with respect to the percentage of D2O in the
189
medium (Hakura et al., 1987). The results shown in Fig. 1 and 2 are also in corroboration with the
190
previous observation made by Lewis et al. (1934), where they had observed inhibition of sprouting of
-1
(Fig. 2). In water,
191
tobacco plant with the increased concentration of D2O. Similar to these observations, the human
192
pancreatic tumour cells showed a slow growth in D2O containing medium (Hartmann et al., 2005).
193
The growth of S. typhimurium growth in 20% D2O containing medium and 100% H2O containing
194
medium was comparable. This is an interesting observation. The chances of deuterium incorporation
195
were low in LB amended with 20% D2O as the percentage of deuterium is less. Further, LB medium is
196
more protiated and provides more H in medium than D. Therefore, the optimum natural isotopic
197
environment did not change in the cells grown in 20% D2O containing medium. As a rule, living
198
organisms “resist” changes in their isotopic environment, preferring natural isotopic abundances. This
199
preference could be due to the evolutionary optimization and an additional effect could be because
200
of “isotopic resonance” (Xueshu and Zubarev., 2015).
201
The rate of chemical and biochemical reactions is affected by isotopic composition of the reactants.
202
Progressive increase in the amount of stable isotope leads to the slow chemical reactions. The isotopic
203
resonance hypothesis suggests that the reaction rate depends on the enrichment degree and is not
204
monotonous. The isotopic resonance hypothesis also suggests that at some “resonance” isotopic
205
compositions, the kinetics increases, while at “off-resonance” compositions, the same reactions
206
progress slower (Xueshu and Zubarev., 2015).
207
Ball. E. (1933) observed that water containing 0.06 mol% of heavy isotope had reduced cell disjunction
208
in Spirogyra, which led to greater longevity. Lobyshev (1978) showed that Na, K-ATPase activity
209
increased with a lower concentration of D2O and decreased with a higher concentration. Other studies
210
also showed the anomalous behaviour in growth in different organisms. In 1935, Curry et al. showed
211
that diluted heavy water increases the mass of Aspergillus. Growth of Atropa belladonna in 30% D2O
212
was the same as that in H2O (Uphaus.,1965). Further, there was an increase in multiplication of
213
poliomyelitis virus in 20-50% D2O (Carpi.,1960). The rate of the reaction of an enzyme depends on the
214
concentration of heavy isotopes in the medium. The biological system tends to keep equilibrium with
215
natural isotope abundance.
216
The D10 value of S. typhimurium in D2O containing medium and H2O containing medium, was 263 Gy
217
and 217 Gy respectively (Fig. 4). In D2O medium, cells were 1.2-fold more resistant to radiation.
218
However, the radiation sensitivity of S. typhimurium in saline differed from the enriched medium. The
219
D10 of S. typhimurium in saline prepared with D2O and saline amended with H2O was 89 Gy and 156
220
Gy, respectively (Fig. 4). The S. typhimurium cells were sensitive to radiation in D2O amended saline
221
as compared to H2O amended saline. The reason may be due to higher damage to DNA because of
222
enhanced oxidative stress induced by the combination of D2O and the radiation (Newo et al., 2004)
223
Cells are resistant to radiation in rich medium (Urbano G.M et al., 2005). In this study, we also
224
observed that D2O along with media component synergistically provided better protection. In medium,
225
the constituents of the medium compete with the cells for radiolytic free radicals and thereby reduce the
226
effect of radiation and make the organism more resistant to radiation (Urbano G.M et al., 2005).
227
S. typhimurium grown in D2O medium was sensitive to osmotic stress (Fig. 5). The 90% reduction in
228
cell number was observed within 24 h in D2O medium. However, S. typhimurium in H2O was
229
unaffected by the osmotic stress (Fig. 5). The D2O reduces GFP’s expression in prokaryotic cell-free
230
assay (Hohlefelder et al., 2013). The D2O may inhibit gene expression at the transcription and
231
translational level. Increased gene expression is required for the survival of S. typhimurium in higher
232
salt concentration. The reduced expression of the genes involved in osmotic stress tolerances such
233
asproU, prop may be responsible for the decreased survival of Salmonella Typhimurium (Dunlap and
234
C. Sonka., 1985). Andjus and Vucelic (1990), found that D2O induces osmotic shock and cause an
235
efflux of intracellular potassium from algal cells; a similar condition may be responsible for decreased
236
S. typhimurium survival in D2O medium.
237
S. typhimurium was sensitive to extreme pH condition in D2O. In both alkaline and acidic conditions, S.
238
typhimurium did not survive up to 24 h in D2O (Fig. 6 and 7). Hydrogen is necessary for the oxidation
239
and reduction processes in the cell; therefore, changes in protium and deuterium isotope may cause
240
enhanced or reduced biochemical activity in the cell. Exchange of solvent, i.e., water by D2O, does not
241
lead to a significant change in pH as the pH of deuterated water is 7.4 and that of regular water is 7
242
(Pabbo and Bates.,1969). Death of bacteria in the alkaline and acidic environments in the heavy water
243
may also be because of enhanced isotopic enrichment of DNA which leads to instability in DNA
244
moiety and thus cell death. (Farthing et al.,2017).
245
S. typhimurium could tolerate heat better in D2O supplemented medium than in H2O supplemented
246
medium. The poor hydration of proteins in the deuterated medium results in reduced molecular motion
247
(Cioni et al., 2002). Thus, the stability of the deuterated macromolecule may be responsible for the heat
248
resistance. In Drosophila, a rapid enhancement in temperature tolerance was observed in D2O
249
(Pittendrigh and Cosbey., 1974). Deuterium bonds are more stable than protium bonds. Our results are
250
in correlation with previous observations which showed that D2O enhances the thermostability of
251
various vaccines (Wu R et al., 1995).
252
Biofilm formation is an essential physiological parameter concerning survival in extreme stress
253
conditions (Yin et al., 2019). D2O did not support biofilm formation as it delays the growth of bacteria
254
and slow growth is not suitable for biofilm formation. The biofilm formation capability of S.
255
typhimurium in H2O and D2O was compared and the SBF value was 0.071 in D2O and 0.141 in H2O.
256
This proves that heavy water does not support biofilm formation by S. typhimurium.
257
Conclusions
258
In bacterial physiological studies, we can replace H2O with D2O. D2O provides a unique advantage to
259
understand the bacterial physiology in a slowed metabolic environment inside the cell which mimics
260
nutrient limited conditions of the environment. Therefore, these results could be very interesting to
261
study and understand the metabolic dynamics inside the cells. S. typhimurium, a food-borne pathogen,
262
exhibited varied abiotic stress response in D2O. S. typhimurium was resistant to radiation and heat
263
stress and sensitive to osmotic and pH stress. These observations suggest that D2O modulates cellular
264
homeostasis that renders cells sensitive or resistant to various abiotic stress. This is the first study
265
where influence of heavy water stress on bacterial physiology along with other stress was studied.
266
Future studies will help us in determining how heavy water stress changes the regulation of different
267
regulons leading to varied responses of S. typhimurium to abiotic stress.
268 269
Conflicts of interest
270
The authors declare no conflict of interest.
271 272
Acknowledgement
273
The authors are very grateful to the Heavy Water Board, which aided access to heavy water to complete
274
this study.
275 276
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Fig. 1: Effect of heavy water on the growth rate of Salmonella typhimurium at 37°C in nutrient-rich LB medium. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 2: Effect of heavy water on the growth rate of Salmonella typhimurium in minimal medium, M9 at 37°C. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 3: Effect on the growth rate of Salmonella typhimurium in different concentrations of heavy water in M9, minimal media. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig. 4: Effect of heavy water on the radiation sensitivity of Salmonella typhimurium, both in presence of medium component and without any medium component. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig.5: Effect of heavy water on Salmonella typhimurium in 2.5M salt stress. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows that significant difference was observed in salt stress condition after 24 hs in H2O according to ANOVA test (p < 0.05).
Fig.6: Effect of heavy water on Salmonella typhimurium in alkaline stress (pH 9). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in the survival of S. typhimurium in 100% D2O and 100% H2O in alkaline stress (pH 9) according to ANOVA test (p < 0.05).
Fig.7: Effect of heavy water on S. typhimurium in acidic stress (pH 4.5). Experiments were performed in triplicates. Error bars are standard deviation of the mean from three replicates. * shows significant difference in survival of S. typhimurium in 100% D2O and 100% H2O in acidic stress (pH 4.5) according to ANOVA test (p < 0.05).
Fig. 1: Effect of heavy water on growth rate of Salmonella Typhimurium at 37°C in nutrient rich, LB medium. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig.6: Effect of heavy water on Salmonella Typhimurium in alkaline stress (pH 9). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in survival of Salmonella Typhimurium in 100% D2O and 100 H2O in alkaline stress (pH 9) according to ANOVA test (p < 0.05).
Fig.7: Effect of heavy water on Salmonella Typhimurium in acidic stress (pH 4.5). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in survival of Salmonella Typhimurium in 100% D2O and 100 H2O in acidic stress (pH 4.5) according to ANOVA Test (p < 0.05).
Fig. 2: Effect of heavy water on growth rate of Salmonella Typhimurium in minimal medium, M9 medium at 37°C. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 3: Effect on growth rate of Salmonella Typhimurium in different concentration of heavy water in M9, minimal media. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig. 4: Effect of heavy water on radiation sensitivity of Salmonella Typhimurium, both in presence of medium component and without any medium component. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig.5: Effect of heavy water on Salmonella Typhimurium in 2.5M salt stress. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows that significant difference was observed in salt stress condition after 24 hours in H2O according to ANOVA test (p < 0.05).
Highlights 1. There was no significant change observed in the growth rate of Salmonella
typhimurium in D2O amended LB medium, whereas in minimal media a significant difference in growth rate was observed. 2. S. typhimurium was sensitive to radiation in saline amended with D2O, whereas it was
resistant to radiation in LB amended with D2O. 3. In 2.5M salt stress, there was a 90% decrease in the number of S. typhimurium in
D2O. 4. S. typhimurium was sensitive to alkaline and acidic stress in the presence of D2O. 5. S. typhimurium was resistant to heat stress in the presence of D2O.
This is the first study where the influence of heavy water stress on bacterial physiology along with other stress was studied.
T
Conflicts of interest The authors declare no conflict of interest.
S0969-8043(19)30413-0
DOI:
https://doi.org/10.1016/j.apradiso.2019.108990
Reference:
ARI 108990
To appear in:
Applied Radiation and Isotopes
Received Date: 27 March 2019 Revised Date:
31 October 2019
Accepted Date: 17 November 2019
Please cite this article as: Pant, I., Shashidhar, R., Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium, Applied Radiation and Isotopes (2020), doi: https:// doi.org/10.1016/j.apradiso.2019.108990. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Graphical Abstract
RADIATION
ACID & ALKALINE STRESS
SALT STRESS
Salmonella Typhimurium+ D2O
Reduced growth rate Sensitive to radiation without media Resistant to radiation in medium 10% reduction in cell number in salt stress in D2O Dies after 24 h in both alkaline and acidic stress
1
Understanding the influence of heavy water stress on the physiology of Salmonella typhimurium
2 3
a Indu Pant and Rabindranath Shashidhar*
4 5
a Food Technology Division, Bhabha Atomic Research Centre, Mumbai, India
6 7
Running Title: Effect of deuterium oxide on the stress tolerance capability of Salmonella
8
typhimurium
9 10
Keywords: Heavy water, Salmonella typhimurium, growth rate, radiation stress, salt stress, D10,
11
alkaline stress, acidic stress, biofilm, heat stress.
12 13 14 15 16
*Corresponding author: Food Technology Division, Bhabha Atomic Research Centre, Trombay,
17
Mumbai, India and Life Sciences, Homi Bhabha National Institute (DAE-Deemed University),
18
Trombay, Mumbai 400094, India. Tel: +91 022-25593961; E-mail: [email protected]
19 20
Abstract
21
The heavy isotope of water is used in understanding the physiology of bacteria. Deuterium (D2O)
22
reduces chemical reaction kinetics. In the present study, the survivability of the food-borne pathogen
23
Salmonella typhimurium grown in D2O supplemented medium is studied under various stress
24
conditions. The growth of S, typhimurium was studied in rich (Luria Broth–LB) and minimal medium
25
(M9) prepared in D2O. The reduced growth rate of S. typhimurium in M9 (2.4 fold) as compared to
26
that in LB (1.6 fold) was observed. S. typhimurium grown in D2O supplemented medium was
27
significantly more tolerant to heat and gamma radiation (1.2 fold), but was sensitive to extreme pH
28
(both alkaline and acidic) and osmotic stress (10 fold). These results suggest that the change in the
29
biological reaction kinetics in the cell due to D2O may modify the stress tolerance of S. typhimurium.
30
This is the first study carried out to understand how a bacterial system (S. typhimurium) in D2O
31
responds to different stresses. This study suggests that investigations on bacterial physiology in D2O
32
supplemented medium helps in understanding the underlying mechanisms of stress tolerance.
33 34
Keywords: Heavy water, Salmonella typhimurium, growth rate, salt stress, D10, alkaline stress, acidic
35
stress, biofilm, heat stress, radiation stress.
36
Introduction
37
Water is essential for life on the Earth. For a long period, the chemical nature of water was a mystery.
38
Today, the unique properties of water are known. Pure heavy water, D2O, is the oxide of the heavy
39
stable isotope of hydrogen, deuterium, denoted by the symbols 2H or D. The proportion of heavy water
40
varies in natural water; river water contains 0.02-0.03 mol% of D2O, and the water of Antarctic ice
41
contains 0.015 mol% of D2O (Mosin and Ignatov., 2014). There is no difference in the chemical
42
structure of D2O and H2O. However, a small difference exists in the length of covalent H-O bonds and
43
the angle between them. D2O has a molecular mass 10% higher than that of H2O. The enhanced
44
strength of inter- and intra-molecular hydrogen bonds has consequences for biological macromolecules.
45
The biological effects of deuterium oxide on a wide variety of microorganisms like yeast (Richards.,
46
1934), Escherichia coli (Katz., 1960), and algae (Strain et al., 1959); plants such as tobacco (Lewis.,
47
1934), Arabidopsis (Bhatia and Smith., 1968), and various grasses (Crumley., 1950); and animals
48
including mice (Katz., 1962) and dogs (Katz., 1960) were studied. Some variants of E. coli (Giovanni.,
49
1961), Bacillus thuringiensis, Bacillus subtilis, an autotrophic nitrite-oxidizing bacterium (Nitrospira
50
moscoviensis), an autotrophic ammonia-oxidizing archaeon (Nitrosophaera gargensis), and autotrophic
51
methanogenic archaea (Methanobrevibacters mithii and Methanocorpusculum labreanum) (Berry D et
52
al., 2015) can survive in 90% (v/v) D2O. Plant cells can survive in up to 75% (v/v) D2O, and animal
53
cells survive up to 30% (v/v) D2O (Mosin and Ignatov., 2014). Excess deuterium in water reduced the
54
synthesis of proteins and nucleic acids and changed the enzymatic kinetic rates and morphology of the
55
cell (Schroeter et al., 1992; Takahashi et al.,1983; Caldwell et al.,1939). The cell division rate was
56
reduced on the subsequent increase in the heavy water concentration in the growth medium (Gross and
57
spindle.,1960). The antimitotic action of D2O was observed in all the stages of the mitotic cycle and
58
during cytokinesis in the cells of Arbacia (Gross and spindle.,1960).
59
In E. coli, slow internalisation of deuterium into substrates resulted in change in the metabolic pathway
60
and enzymatic reaction rate (Hochuli et al., 2000; Zhang et al., 2009). Lower mutation rates after
61
ultraviolet irradiation was reported in deuterated medium than in non-deuterated medium (Flaumenhaft
62
and Katz., 1960). Flaumenhaft E et al. (1960) also showed the morphological changes in the nuclei of
63
green algae grown in D2O, with proteins becoming darkly stained because of higher concentration of
64
the free basic groups.
65
Salmonella species is an important food-borne pathogen (Hendriksen et al., 2011). Salmonella is
66
prevalent in poultry, pigs, cattle, fruits, and vegetables. It can enter the food chain and survive with
67
limited nutrients. Many epidemics caused by Salmonella species have been reported. The presence of
68
Salmonella species is a severe problem in most foods, particularly dry and semi-dry products (e.g.,
69
milk powder, spices).
70
S. typhimurium causes systemic disease in mice similar to Salmonella typhi in humans (Teuber et al.,
71
1999). Therefore, S. typhimurium is used for molecular biology and stress-related studies. Salmonella
72
enterica serovar typhimurium is the most prevalent serotype in the Indian sub-continent (Gupta and
73
Verma.,1993) and hence, S. typhimurium was used as a model organism in the present study.
74
The effect of heavy water on the stress physiology of food-borne pathogens is a new area of study.
75
Better understanding of the underlying molecular physiology of stress tolerance would give an insight
76
into the applied aspects of food microbiology, such as design of novel food processing methods and
77
spoilage prevention tools.
78 79
Material and Methods
80
Chemicals
81
The 99.9% deuteriated water was obtained from Heavy Water Board (Mumbai, India). All the
82
chemicals (Sodium chloride, glutaraldehyde, sodium bicarbonate, sodium hydroxide, sterile distilled
83
water, phosphate buffer saline (pH 7.4), crystal violet solution, and potassium dihydrogen phosphate)
84
were procured from Hi-Media Laboratories Mumbai, India.
85
Microbiological media used in the studies were from Hi-Media Laboratories. Luria agar, Luria broth,
86
and tryptic soy media were used. M9 medium (M9 salt, 5X-20 mL, 20% glucose-2 mL, MgSO4-200
87
µL, CaCl2-10 µL, water-78 mL) was prepared. (Cold Spring Harbor Laboratory., 2010).
88
Instruments
89
Gamma chamber 5000 (Board of Radioisotope and Technology, Mumbai, India) at a dose rate of 0.083
90
kGy/min was used for irradiating the cells. The dose rate of radiation sources was measured using the
91
Fricke dosimeter. In experimental samples, the variations in dose absorbed were minimised by placing
92
the samples within a uniform area of the radiation field.
93
All the growth curve experiments were performed in Microtiter plates (96 well, Becton Dickinson
94
Labware, USA) and Bio-Tek microtiter plate reader (US). Water bath (Biosan, WB-4MS, Latvia,
95
Europe) was used for giving heat stress to the bacteria.
96
Microbiological methods
97
Bacterial culture and growth conditions
98
Salmonella typhimurium LT2 strain MTCC 98 procured from Microbial Type Culture Collection,
99
Chandigarh, India.
100
S. typhimurium was grown overnight in LB/M9 at 37°C at 150 rpm. Overnight culture of S.
101
9 -1 typhimurium (10 CFU mL ) was taken and washed thrice with 0.85% (w/v) saline. The washed cells
102
7 -1 were resuspended in saline and further diluted to 10 CFU mL . The end point of all the stress
103
survival assay (radiation, osmotic, pH extremes, and heat stress) was determined using the standard
104
plate count method (Willey et al., 2011). In the standard plate count method, cell dilutions were made
105
in saline and 100 µL of appropriate dilution was plated on plate count agar medium and kept for
106
-1) incubation at 37°C for different time intervals (24 and 48 h). The cell count (CFU mL was
107
calculated using the formula: Number of colonies x dilution factor/volume of dilution plated (mL)
108
(Willey et al., 2011).
109
Effect of D2O on the growth rate of S. typhimurium in different medium
110
7 -1 Salmonella typhimurium (10 CFU mL ) was grown in M9 and Luria broth (LB) in microtiter plates,
111
and their growth was monitored by measuring OD at 600 nm by using a plate reader at 37'C for 18 h.
112
The growth rate was calculated using the equation ln(N2/N1) =k(t2-t1) (Willey et al., 2011). In both LB
113
and M9 medium, after 24 h of incubation, CFU mL
114
count method as mentioned in microbiological methods.
115
Effect of D2O on radiation sensitivity of S. typhimurium
116
The D10 value of bacteria is defined as the radiation dose required to inactivate 90% of a viable
117
population. The 18-h-old S. typhimurium cells grown in LB at 37ºC were used for this study. Radiation
118
7 doses of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 kGy were given to 1 ml of 10 cells dispensed in saline prepared in
119
D2O or H2O and media prepared in D2O or H2O in microfuge tubes. For calculating the number of cells
120
that survived after irradiation, the standard plate count method was used. The plates were incubated at
121
37°C for 24 h, and the slopes of the individual survivor curves were calculated by linear regression
122
with a computer program (Microsoft Excel 2007). Negative reciprocal of survival curve slope was used
123
to calculate D10 value (Saroj et al., 2006).
124
Effect of D2O on salt stress tolerance of S. typhimurium
125
S. typhimurium (107CFU mL-1) cells were resuspended in 2.5M NaCl solution made in D2O or H2O
126
and were incubated at 37°C in shaking condition for 24 h. The cell survival after osmotic stress was
127
checked at 0 and 24 h using the standard plate count method as mentioned in microbiological methods.
-1
was also calculated using the standard plate
128
Effect of D2O on alkaline stress tolerance of S. typhimurium
129
S. typhimurium cells were resuspended in pH 9, sodium bicarbonate buffer made in D2O, and H2O, and
130
incubated at 37ºC in shaking condition. Cell survival was checked at 0 and 24 h using the standard
131
plate count method as mentioned in microbiological methods.
132
Effect of D2O on acidic stress tolerance of S. typhimurium
133
Salmonella Typhimurium cells were resuspended in pH 4.5, phosphate buffer made in D2O, and H2O,
134
and incubated at 37ºC in shaking condition. The cell survival was checked at 0, 24, and 48 h using the
135
standard plate count method as mentioned in microbiological methods.
136
Effect of D2O on heat stress tolerance of S. typhimurium
137
An overnight culture of S. typhimurium was suspended in saline (0.85%) made in D2O or H2O, and heat
138
shock at a temperature of 55ºC was given for 5 min and 10 min in a water bath. The cell survival was
139
checked at 0, 5, and 10 min using the standard plate count method as mentioned in microbiological
140
methods.
141
Effect of D2O on biofilm formation of S. typhimurium
142
Biofilms are surface-attached microbial communities in which microbial cells are embedded in self-
143
produced extracellular polymeric substances. S. typhimurium was grown in Tryptic Soy Broth (TSB)
144
medium amended with D2O or H2O in a 96-well microtiter plate, and biofilm formation was checked
145
using specific biofilm formation index (Naves et al., 2008). Total cell mass was measured at OD 600
146
nm. The wells were washed thrice with sterile water to remove loosely attached or planktonic cells.
147
Microtiter plates were air dried and then oven dried at 50°C for 30 minutes. Following drying, cells
148
were fixed with 200 µL of 2.5% glutaraldehyde in PBS (pH 7.4) for 10 minutes. The wells were
149
washed thrice with distilled water and oven dried at 50°C for 30 min. Wells were stained with 0.2%
150
crystal violet solution and incubated at room temperature for 15 min after which the plates were
151
washed thrice with distilled water to remove unabsorbed stain. Microtiter plates were then air dried for
152
30 min and then 200 µL of ethanol-acetone mix (80%-20%) was added to each well to destain the
153
biofilm, and the concentration of crystal violet absorbed by the cells was measured at OD at 570 nm.
154
Biofilm formation was classified into three categories according to (Jahid et al., 2013): no biofilm
155
(SBF<0.1), weak (0.1
156
SBF is specific biofilm formation index that represents the amount of biofilm formed in a specific
157
condition which is calculated using the formula SBF=B-NC/G (Naves et al., 2008) where B= OD at
158
570 nm of attached and stained bacteria, NC= OD at 570 nm of stained control well containing only
159
bacteria-free medium, and G= OD at 600 nm of cell growth in medium.
160
Statistical analysis
161
All the tests were carried out in triplicates with appropriate biological replicates. The mean and
162
standard deviation of the replicates are provided wherever required. Test of significance was carried out
163
at p < 0.05.
164
Results and discussion
165
S. typhimurium was grown in both LB and M9 media (Fig. 1 and 2) containing different concentrations
166
of D2O (20-100%). There was no observable change in the growth rate and pattern of growth in D2O
167
containing LB medium. This may be due to the rich medium components. The nutrients can
168
compensate and relegate the slower metabolism caused by D2O. The rich medium components are
169
more protiated. Hence, this provides a lesser chance of deuterium incorporation into the important
170
cellular machinery. Therefore, complete effect of the deuterium oxide on the growth could not be
171
observed.
172
The growth rate of S. typhimurium decreased with the increased concentration of D2O in M9 medium
173
(Fig. 3). The prominent reduction in growth was seen in M9 medium at a concentration ≥ 50% D2O (v
174
-1 /v) (Fig. 2). The growth rate of S. typhimurium in M9 medium prepared in 100% D2O was 0.15 h as
175
compared to medium prepared in 100% H2O, in which the growth rate was 0.36 h
176
the growth rate of S. typhimurium was 2.4-fold more than that observed in D2O supplemented minimal
177
-1 medium. The cell number in M9 minimal medium prepared in D2O reached 7 log CFU mL after 24
178
hours as compared to 9 log CFU mL -1 in the medium prepared in H2O. The decreased growth rate
179
may be due to overall slow kinetics of deuterated molecules, which slow down the metabolism. Also,
180
when D2O is mixed with H2O, it results in non-uniformity in water density; this further slows down the
181
metabolic process. Hence, low growth rate at higher D2O concentration was observed. These
182
observations suggest that D2O retards the growth of S. typhimurium in M9 medium. Further, in the
183
minimal medium (M9), cells synthesize all the components de novo. The de novo metabolism
184
incorporates more D2O into important cellular machinery of the cell. Therefore, the overall metabolism
185
of bacterial cells slows down in D2O supplemented minimal medium.
186
Inhibition of growth of the bacteria due to heavy water was reported in other studies. In 1987, Hakura
187
et al. showed a decrease in growth rate in variants of E. coli in D2O when compared to that in H2O.
188
However, there was no comparison in the growth rate with respect to the percentage of D2O in the
189
medium (Hakura et al., 1987). The results shown in Fig. 1 and 2 are also in corroboration with the
190
previous observation made by Lewis et al. (1934), where they had observed inhibition of sprouting of
-1
(Fig. 2). In water,
191
tobacco plant with the increased concentration of D2O. Similar to these observations, the human
192
pancreatic tumour cells showed a slow growth in D2O containing medium (Hartmann et al., 2005).
193
The growth of S. typhimurium growth in 20% D2O containing medium and 100% H2O containing
194
medium was comparable. This is an interesting observation. The chances of deuterium incorporation
195
were low in LB amended with 20% D2O as the percentage of deuterium is less. Further, LB medium is
196
more protiated and provides more H in medium than D. Therefore, the optimum natural isotopic
197
environment did not change in the cells grown in 20% D2O containing medium. As a rule, living
198
organisms “resist” changes in their isotopic environment, preferring natural isotopic abundances. This
199
preference could be due to the evolutionary optimization and an additional effect could be because
200
of “isotopic resonance” (Xueshu and Zubarev., 2015).
201
The rate of chemical and biochemical reactions is affected by isotopic composition of the reactants.
202
Progressive increase in the amount of stable isotope leads to the slow chemical reactions. The isotopic
203
resonance hypothesis suggests that the reaction rate depends on the enrichment degree and is not
204
monotonous. The isotopic resonance hypothesis also suggests that at some “resonance” isotopic
205
compositions, the kinetics increases, while at “off-resonance” compositions, the same reactions
206
progress slower (Xueshu and Zubarev., 2015).
207
Ball. E. (1933) observed that water containing 0.06 mol% of heavy isotope had reduced cell disjunction
208
in Spirogyra, which led to greater longevity. Lobyshev (1978) showed that Na, K-ATPase activity
209
increased with a lower concentration of D2O and decreased with a higher concentration. Other studies
210
also showed the anomalous behaviour in growth in different organisms. In 1935, Curry et al. showed
211
that diluted heavy water increases the mass of Aspergillus. Growth of Atropa belladonna in 30% D2O
212
was the same as that in H2O (Uphaus.,1965). Further, there was an increase in multiplication of
213
poliomyelitis virus in 20-50% D2O (Carpi.,1960). The rate of the reaction of an enzyme depends on the
214
concentration of heavy isotopes in the medium. The biological system tends to keep equilibrium with
215
natural isotope abundance.
216
The D10 value of S. typhimurium in D2O containing medium and H2O containing medium, was 263 Gy
217
and 217 Gy respectively (Fig. 4). In D2O medium, cells were 1.2-fold more resistant to radiation.
218
However, the radiation sensitivity of S. typhimurium in saline differed from the enriched medium. The
219
D10 of S. typhimurium in saline prepared with D2O and saline amended with H2O was 89 Gy and 156
220
Gy, respectively (Fig. 4). The S. typhimurium cells were sensitive to radiation in D2O amended saline
221
as compared to H2O amended saline. The reason may be due to higher damage to DNA because of
222
enhanced oxidative stress induced by the combination of D2O and the radiation (Newo et al., 2004)
223
Cells are resistant to radiation in rich medium (Urbano G.M et al., 2005). In this study, we also
224
observed that D2O along with media component synergistically provided better protection. In medium,
225
the constituents of the medium compete with the cells for radiolytic free radicals and thereby reduce the
226
effect of radiation and make the organism more resistant to radiation (Urbano G.M et al., 2005).
227
S. typhimurium grown in D2O medium was sensitive to osmotic stress (Fig. 5). The 90% reduction in
228
cell number was observed within 24 h in D2O medium. However, S. typhimurium in H2O was
229
unaffected by the osmotic stress (Fig. 5). The D2O reduces GFP’s expression in prokaryotic cell-free
230
assay (Hohlefelder et al., 2013). The D2O may inhibit gene expression at the transcription and
231
translational level. Increased gene expression is required for the survival of S. typhimurium in higher
232
salt concentration. The reduced expression of the genes involved in osmotic stress tolerances such
233
asproU, prop may be responsible for the decreased survival of Salmonella Typhimurium (Dunlap and
234
C. Sonka., 1985). Andjus and Vucelic (1990), found that D2O induces osmotic shock and cause an
235
efflux of intracellular potassium from algal cells; a similar condition may be responsible for decreased
236
S. typhimurium survival in D2O medium.
237
S. typhimurium was sensitive to extreme pH condition in D2O. In both alkaline and acidic conditions, S.
238
typhimurium did not survive up to 24 h in D2O (Fig. 6 and 7). Hydrogen is necessary for the oxidation
239
and reduction processes in the cell; therefore, changes in protium and deuterium isotope may cause
240
enhanced or reduced biochemical activity in the cell. Exchange of solvent, i.e., water by D2O, does not
241
lead to a significant change in pH as the pH of deuterated water is 7.4 and that of regular water is 7
242
(Pabbo and Bates.,1969). Death of bacteria in the alkaline and acidic environments in the heavy water
243
may also be because of enhanced isotopic enrichment of DNA which leads to instability in DNA
244
moiety and thus cell death. (Farthing et al.,2017).
245
S. typhimurium could tolerate heat better in D2O supplemented medium than in H2O supplemented
246
medium. The poor hydration of proteins in the deuterated medium results in reduced molecular motion
247
(Cioni et al., 2002). Thus, the stability of the deuterated macromolecule may be responsible for the heat
248
resistance. In Drosophila, a rapid enhancement in temperature tolerance was observed in D2O
249
(Pittendrigh and Cosbey., 1974). Deuterium bonds are more stable than protium bonds. Our results are
250
in correlation with previous observations which showed that D2O enhances the thermostability of
251
various vaccines (Wu R et al., 1995).
252
Biofilm formation is an essential physiological parameter concerning survival in extreme stress
253
conditions (Yin et al., 2019). D2O did not support biofilm formation as it delays the growth of bacteria
254
and slow growth is not suitable for biofilm formation. The biofilm formation capability of S.
255
typhimurium in H2O and D2O was compared and the SBF value was 0.071 in D2O and 0.141 in H2O.
256
This proves that heavy water does not support biofilm formation by S. typhimurium.
257
Conclusions
258
In bacterial physiological studies, we can replace H2O with D2O. D2O provides a unique advantage to
259
understand the bacterial physiology in a slowed metabolic environment inside the cell which mimics
260
nutrient limited conditions of the environment. Therefore, these results could be very interesting to
261
study and understand the metabolic dynamics inside the cells. S. typhimurium, a food-borne pathogen,
262
exhibited varied abiotic stress response in D2O. S. typhimurium was resistant to radiation and heat
263
stress and sensitive to osmotic and pH stress. These observations suggest that D2O modulates cellular
264
homeostasis that renders cells sensitive or resistant to various abiotic stress. This is the first study
265
where influence of heavy water stress on bacterial physiology along with other stress was studied.
266
Future studies will help us in determining how heavy water stress changes the regulation of different
267
regulons leading to varied responses of S. typhimurium to abiotic stress.
268 269
Conflicts of interest
270
The authors declare no conflict of interest.
271 272
Acknowledgement
273
The authors are very grateful to the Heavy Water Board, which aided access to heavy water to complete
274
this study.
275 276
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Fig. 1: Effect of heavy water on the growth rate of Salmonella typhimurium at 37°C in nutrient-rich LB medium. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 2: Effect of heavy water on the growth rate of Salmonella typhimurium in minimal medium, M9 at 37°C. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 3: Effect on the growth rate of Salmonella typhimurium in different concentrations of heavy water in M9, minimal media. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig. 4: Effect of heavy water on the radiation sensitivity of Salmonella typhimurium, both in presence of medium component and without any medium component. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig.5: Effect of heavy water on Salmonella typhimurium in 2.5M salt stress. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows that significant difference was observed in salt stress condition after 24 hs in H2O according to ANOVA test (p < 0.05).
Fig.6: Effect of heavy water on Salmonella typhimurium in alkaline stress (pH 9). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in the survival of S. typhimurium in 100% D2O and 100% H2O in alkaline stress (pH 9) according to ANOVA test (p < 0.05).
Fig.7: Effect of heavy water on S. typhimurium in acidic stress (pH 4.5). Experiments were performed in triplicates. Error bars are standard deviation of the mean from three replicates. * shows significant difference in survival of S. typhimurium in 100% D2O and 100% H2O in acidic stress (pH 4.5) according to ANOVA test (p < 0.05).
Fig. 1: Effect of heavy water on growth rate of Salmonella Typhimurium at 37°C in nutrient rich, LB medium. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig.6: Effect of heavy water on Salmonella Typhimurium in alkaline stress (pH 9). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in survival of Salmonella Typhimurium in 100% D2O and 100 H2O in alkaline stress (pH 9) according to ANOVA test (p < 0.05).
Fig.7: Effect of heavy water on Salmonella Typhimurium in acidic stress (pH 4.5). Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows significant difference in survival of Salmonella Typhimurium in 100% D2O and 100 H2O in acidic stress (pH 4.5) according to ANOVA Test (p < 0.05).
Fig. 2: Effect of heavy water on growth rate of Salmonella Typhimurium in minimal medium, M9 medium at 37°C. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates.
Fig. 3: Effect on growth rate of Salmonella Typhimurium in different concentration of heavy water in M9, minimal media. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig. 4: Effect of heavy water on radiation sensitivity of Salmonella Typhimurium, both in presence of medium component and without any medium component. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. Values having different letters are significantly different from each other according to ANOVA test (p < 0.05).
Fig.5: Effect of heavy water on Salmonella Typhimurium in 2.5M salt stress. Experiments were performed in triplicates; Error bars are standard deviation of the mean from triplicates. * shows that significant difference was observed in salt stress condition after 24 hours in H2O according to ANOVA test (p < 0.05).
Highlights 1. There was no significant change observed in the growth rate of Salmonella
typhimurium in D2O amended LB medium, whereas in minimal media a significant difference in growth rate was observed. 2. S. typhimurium was sensitive to radiation in saline amended with D2O, whereas it was
resistant to radiation in LB amended with D2O. 3. In 2.5M salt stress, there was a 90% decrease in the number of S. typhimurium in
D2O. 4. S. typhimurium was sensitive to alkaline and acidic stress in the presence of D2O. 5. S. typhimurium was resistant to heat stress in the presence of D2O.
This is the first study where the influence of heavy water stress on bacterial physiology along with other stress was studied.
T
Conflicts of interest The authors declare no conflict of interest.