Download Antimicrobial properties of magnesium chloride at low pH in the

Magnesium Research 2014; 27 (2): 57-68
ORIGINAL ARTICLE
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Antimicrobial properties of
magnesium chloride at low pH in the
presence of anionic bases
Pía Oyarzúa Alarcón1 , Katherine Sossa1,2 , David Contreras3 , Homero Urrutia1 ,
Andreas Nocker4
1 Laboratorio de Biopelículas y Microbiología Ambiental, Centro de Biotecnología, Universidad de Concepción, PO Box 160-C, Concepción, Chile; 2 Facultad de Ciencias Forestales,
Universidad de Concepción, PO Box 160-C, Concepción, Chile; 3 Facultad de Ciencias Químicas, Centro de Biotecnología, Universidad de Concepción, PO Box 160-C, Concepción,
Chile; 4 Cranfield Water Science Institute, School of Applied Sciences, Cranfield University,
Cranfield, Bedfordshire, MK43 0AL, United Kingdom
Correspondence: Andreas Nocker, Cranfield Water Science Institute, School of Applied Sciences, Cranfield University, Cranfield, Bedfordshire, MK43 0AL, United Kingdom
<[email protected]>
Abstract. Magnesium is an element essential for life and is found ubiquitously in all organisms. The different cations play important roles as enzymatic
co-factors, as signaling molecules, and in stabilizing cellular components. It is
not surprising that magnesium salts in microbiological experiments are typically associated with positive effects. In this study with Listeria monocytogenes
as a model organism, we focus however on the usefulness of magnesium (in
form of MgCl2 ) as a stress enhancer. Whereas MgCl2 does not affect bacterial
viability at near-neutral pHs, it was found to strongly compromise culturability and redox activity when cell suspensions were exposed to the salt at acidic
pH. The principle was confirmed with a number of gram-negative and grampositive species. The magnesium salt dramatically increased the acidity to a
level that was antimicrobial in the presence of anionic bases such as phosphate,
lactate, or acetate, but not TRIS. The antimicrobial activity of MgCl2 was much
stronger than that of NaCl, KCl, or CaCl2 . No effect was observed with MgSO4
or when cells were exposed to MgCl2 in phosphate buffer with a pH≥5. Acid
stress was reinforced by an additional, salt-specific effect of MgCl2 on microbial
viability that needs further examination. Apart from its implications for surface
disinfection, this observation might support the commonly stated therapeutic
properties of MgCl2 for the treatment of skin diseases (with healthy skin being
an acidic environment), and could contribute to understanding why salt from
the Dead Sea, where Mg2+ and Cl- are the most abundant cation/anion, has
healing properties in a microbiological context.
doi:10.1684/mrh.2014.0362
Key words: bacteria, magnesium chloride, sodium chloride, pH, antimicrobial, acid
stress
Many important findings about the effects of different salts and cations on bacterial viability were
made in the first three decades of the 20th century which many considered to be the ‘golden age’
of this type of research. Cations were reported to
exert a highly characteristic effect upon bacteria:
low concentrations of given salts were reported
to favor viability, whereas higher concentrations
were associated with growth inhibition [1-3]. This
effect was visualized by an optimum curve that
generally held true for all cations ; however, the
concentrations at which the transition between
57
To cite this article: Oyarzúa Alarcón P, Sossa K, Contreras D, Urrutia H, Nocker A. Antimicrobial properties of magnesium
chloride at low pH in the presence of anionic bases. Magnes Res 2014; 27(2): 57-68 doi:10.1684/mrh.2014.0362
P. OYARZÚA ALARCÓN, ET AL.
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beneficial and toxic occurred seemed to vary
greatly among the different cations. Salts such
as NaCl or KCl, which are typically ‘favorable’
(for example, in growth media or physiological
saline solution), were reported to be inhibitory
in sufficiently high concentrations. Whereas the
latter correlates with common scientific ‘gut feeling’ and the concept of osmotic stress, the same
principle could be implied to suggest that even
highly toxic substances such as HgCl2 , PbCl2
and other heavy metals might have a stimulating effect on bacterial growth in sufficiently low
concentrations. Although there was no clear explanation of this empiric observation [3], the effects
of different cations and their specific efficiencies
(both in regard to stimulation and inhibition)
resulted in the quantitative assignment of ‘specific potency’ factors. Na+ served as a reference
and was assigned a potency factor of 1. Examples
of potencies that were reported include: K+ = 1.2,
Mg2+ = 9.4, Ca2+ = 12, Mn2+ = 400, Zn2+ = 700, and
Cd2+ = 3000 [4].
Whereas these studies focused on the direct
effect of the different ions on bacterial viability,
this study addresses their impact under different pH conditions and buffer systems. The project
was motivated by previous findings that bacterial
viability was much more strongly affected by desiccation in the presence of MgCl2 compared with
other salts [5], that magnesium salts have antiseptic properties in treatments involving Dead
Sea salts, and that the effects of different salts
on bacterial viability depended greatly upon pH.
Whereas the presence of salts typically had no
effect at a neutral pH (compared to a sample without salt), a slightly stronger effect was observed in
the acidic range.
Listeria monocytogenes served as a model organism because of our laboratory’s interest in this
food-borne pathogen, although Escherichia coli,
Salmonella typhimurium, Enterococcus faecium,
and Staphylococcus aureus were also used. Plating onto nutrient agar was further supplemented
by cultivation-independent measurement of redox
activity. Over the course of the study, we developed the hypothesis that the impact on bacterial
viability is, in part, due to the enhancement of
acid stress. Whereas it is known that an aqueous
solution of MgCl2 is slightly acidic as a result of
hydrolysis [6], the effect is greatly potentiated in
the presence of naturally occurring, strong inorganic and organic bases. The idea was addressed
in more detail because of the lack of understanding
58
of how salts and different acid-base combinations
can affect microbial viability. The research has
potential implications for enhancing antimicrobial action and for medical treatments. Low pH
and the presence of inorganic and organic bases
is a situation typically encountered on the skin
where MgCl2 has been attributed healing and
antiseptic properties. Whereas detailed information about the effect of the salt on skin barrier
function and its anti-inflammatory and biochemical properties in eukaryotic cells has accumulated
[7-9], there is little knowledge with regard to its
effect on microbial viability.
Materials and methods
Bacterial strains and growth conditions
The bacterial strains used for this study included
Listeria monocytogenes (ATCC 19115), Staphylococcus aureus (ATCC 2913), Escherichia coli
(K-12), Salmonella enterica serovar Typhimurium
(isolated from human feces), Enterococcus faecium
(isolated from vaginal excretions); where not indicated otherwise, strains were from the Facultad
de Ciencias Biológicas, University of Concepción.
All bacteria were grown on tryptic soy agar (TSA;
Becton, Dickinson and Company, Le Pont de Claix,
France) at 30 ◦ C. Liquid cultures were obtained by
inoculating 15 mL of tryptic soy broth (TSB) into
a 50 mL Falcon tube, shaken at a 45◦ angle for
18 hours, at 120 rpm and at 30◦ C. Cell density
was measured in a spectrophotometer (TU-1810
Split Beam UV-VIS, Electronic Co Ltd, Shanghai, China) at 600 nm (OD600 ) and adjusted to an
OD600 = 1.0 by addition of fresh medium. Aliquots
of 1 mL were transferred into 1.5 mL microcentrifuge tubes, and centrifuged (5,000 rpm, 5 min),
followed by careful removal of the supernatant.
Sample preparation and pH exposure
The bacterial cell pellet was resuspended in 500
␮L of phosphate buffer (100 mM) followed by addition of 500 ␮L of either water or solutions of
different salts (final salt concentrations of 10, 50,
150, 400, or 1,000 mM; final buffer concentration
of 50 mM). Phosphate buffer was adjusted to pH
values between 2 and 11. Alternatively, cells were
resuspended in TRIS, acetate or lactate solutions
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Antimicrobial properties of MgCl2
(final concentrations of 50 mM each) adjusted to a
pH of 4 by the addition of NaOH or HCl. Cells were
exposed to the different solutions without salt or
supplemented with different salts for 20 min each.
The salts used in this study were: MgCl2 (cat. nr.
BM-0970; Winkler Ltda., Santiago, Chile), NaCl
(cat. nr. SO-1455; Winkler Ltda., Santiago, Chile),
KCl (cat. nr. 1.04936.1000; Merck), MgSO4 (cat.
nr. MA-0980; Winkler Ltda.), and CaCl2 (cat. nr.
CA-0520; Winkler Ltda). After salt exposure, cells
were harvested by centrifugation (5,000 g, 5 min)
and resuspended in phosphate buffer (50 mM;
pH7).
Cultivation on plates
Aliquots of undiluted cell suspensions were
transferred into the top row of sterile, 96-well
NunclonTM plates (Nunc, Roskilde, Denmark).
Dilutions were made by stepwise mixing of 10 ␮L
of cell suspension with 90 ␮L TSB pre-aliquoted
in the lower rows. All dilutions and transfers
were made using multichannel pipettes to allow
for a rapid sample processing. Volumes of 3 ␮L
of the undiluted cell suspension and the different dilutions were spotted onto TSA Petri dishes,
with the highest cell concentration in the top row
of the grid. After brief drying, plates were incubated at 30◦ C for approximately 20 h. Images of
growth patterns were taken with a digital camera
(Scion Corporation, Japan), and visualized using
the Gel-Proanalyzer program (Media Cybernetics,
USA).
Redox activity
For the measurement of redox activity, WST8 (GenScript, Piscataway, USA) and menadione
(2-methyl-1,4-naphthoquinone; ACROS Organics,
Geel, Belgium) were dissolved in water and DMSO
respectively, to obtain stocks of 10 mM and 8 mM
that were stored at -20 ◦ C. WST-8, menadione, and
water were mixed in ratios of 9:1:10 to obtain a
detection reagent that was pre-aliquoted in 20 ␮L
volumes in black, flat-bottom, 96-well microtiter
plates (cat. nr. 5530100; Orange Scientific; Brainel’Alleud, Belgium). The reaction was started by
addition of 180 ␮L of 10-fold-diluted cell suspensions (prepared previously for the cultivation
analysis) using a multichannel pipettor. Diluted
cell suspensions were used as controls; without
dilution the signals from untreated control sam-
ples were obtained too quickly. After addition of
cells and mixing by pipetting up and down several times, plates were immediately transferred
to a TECAN F200-Pro plate reader (TECAN, Austria). Signals were measured at 450 nm every two
min for a total of three hours. Before every measurement, the plate was shaken for five seconds
(linear shaking, amplitude of 3).
Fluorescence microscopy
Cells were stained using the LIVE/DEAD®
BacLightTM bacterial viability kit (L13152; Invitrogen, Carlsbad, California). Following the manufacturer’s instructions, SYTO9 and propidium
iodide were each dissolved in 2.5 mL of sterile
water and subsequently blended to obtain a 2×
staining solution. Cell suspensions were stained
for 15 min in the dark followed by filtration on
black, 0.22 ␮m, Isopore polycarbonate filters (cat.
nr. GTBP02500, Millipore, USA). Filters were
placed on a slide using the mounting oil provided
with the kit. Images were acquired from a fluorescence Olympus BX51 microscope using a 100×
(UPlanFI, Olympus, USA) objective, FITC and PI
fluorescence filter sets (ex485/20, em535/25 and
ex540/20, em635/350, respectively), and a Cool
SNAP-Pro Digital Kit camera (Media Cybernetics Inc., USA). The software used for visualization
was Image-Pro Plus 5.1 (Media Cybernetics Inc.,
USA).
Chemical speciation and statistical
analysis
Calculation of the chemical speciation of salts
at different pH values was performed with the
software “CHEAQS pro V. 2004.1” [10]. Statistical ANOVA and Tukey analyses were performed
using statistical software GraphPad Prism 5.0
(GraphPad Software Inc., San Diego, California,
USA).
Results
Comparison of different salts
To compare the effects of different common salts
on bacterial viability, Listeria monocytogenes was
resuspended in phosphate buffer at pH7 and pH3
59
P. OYARZÚA ALARCÓN, ET AL.
buffer pH7
Comparison of effect at different pHs
buffer pH3
no
salt NaCl KCl
Mg- CaCl2 Cl2
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Serial dilution
no
Mg- Casalt NaCl KCl
Cl2 Cl2
Buffersalt pH
7.0
6.8
6.9 5.6
4.7
3.0 2.9
3.0 2.5
2.6
Figure 1. Effect of different salts on culturability
of L. monocytogenes at neutral or acidic pH. Cells
were suspended in phosphate buffers of pH7 and
pH3 (initial pH without salt) before adding salts
to a final concentration of 400 mM (or an equivalent volume of water). Samples were incubated
for 20 min, followed by resuspension, serial dilution and spotting aliquots on TSA. Measured pH
values of mixed buffer-salt solutions are indicated.
Pictures of representative plates are shown.
without salt, or supplemented with 400 mM of
NaCl, KCl, MgCl2 , or CaCl2 (figure 1). As the
addition of salt was found to change the pH of
the phosphate buffer, pH values of the buffer-salt
mixtures (that were used to resuspend cells) are
shown, in addition to initial buffer pH values without salt. None of the salts affected culturability
at pH7 (initial buffer pH). Neither was survival
compromised in the sample buffered at pH3 in
the absence of salt, suggesting that exposure to
pH3 for 20 min did not affect the culturability
of L. monocytogenes. The presence of the salts at
acidic pH on the other hand, resulted in a general
decrease in growth, with reductions of approximately 2 log units (NaCl and KCl), 4 log units
(CaCl2 ), and 5-6 log units for MgCl2 . A substantial
drop in pH when mixing buffer and salt solutions was only observed for the divalent cations,
offering a potential explanation for the effects
of MgCl2 and CaCl2 , but not for the monovalent
ions.
60
In a next step L. monocytogenes aliquots were
resuspended in phosphate buffer (initial pH
between 2 and 11) in the presence or absence of
MgCl2 (figure 2). In the absence of salt, the only
sample where viability was compromised was at
pH2, whereas culturability was comparable for all
other pHs (figure 2A). In the presence of salt, no
effect on culturability was seen for the samples
with an initial buffer pH of 5 or higher. Of the
samples with an initial buffer pH of 4 or lower
on the other hand, MgCl2 was associated with a
marked reduction of survival. The effect can, in
part, be explained by the salt-induced decrease in
pH (values of buffer-salt mixtures are indicated
in figure 2). On the other hand, culturability in
the pH3 sample (without salt) was higher than
in the pH4 sample with salt (pH of buffer-salt
mixture = 3.3), suggesting a pH-independent salt
effect of unknown nature. The same observation
applies when comparing culturability of the pH2
sample (without salt) with the one of the pH3 sample with salt (pH of buffer-salt mixture = 2.5). A
more detailed insight was obtained by measuring
redox activity (figure 2B): whereas the presence
of salt did not affect activities in the pH range 6
and 9, a positive effect (more activity) was seen
for pH11, and a negative effect (less activity) for
pH values ≤5. The positive effect of the salt in
the basic pH range is probably due to the acidifying effect of MgCl2 mitigating the stress at
high pH. Absolute differences in results between
figures 2A and B are due to differences in the
sensitivity of the two diagnostic methods with
plate reader assays being inherently less sensitive
than culture. Statistical analysis of redox activities revealed significant differences between the
mean increases in activity signals obtained for
different treatments. Significance (p) was less or
equal than 0.05.
Effect of salt concentration
To address the dependence of the effect on the salt
concentration, L. monocytogenes was exposed to
phosphate buffer at pH3 (initial pH without salt)
supplemented with increasing concentrations of
MgCl2 or NaCl (figure 3). Both salts affected viability, but at different concentrations. Compared
with a control sample without salt, the presence of
MgCl2 and NaCl reduced growth in concentrations
≥150 mM and ≥400 mM, respectively. This phe-
Antimicrobial properties of MgCl2
Initial pH of buffers (without salt)
A
pH2
-
+
pH4
-
+
pH5
-
+
pH6
-
+
pH7
-
+
pH8
-
+
pH9
-
+
+
pH10
pH11
-
-
+
+
Serial dilution
Buffer-salt
pH
2.0 1.7
3.0 2.5
4.0 3.3
-
-
-
6.0 4.7
5.0 3.7
7.0 5.6
8.0 6.5
9.0 7.0 10.0 7.0
11.0 7.0
0.8
B
WST-8 signal increase in 3h
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MgCl2 -
pH3
0.6
0.4
0.2
0
MgCl2
pH2
+
+
pH3
+
pH4
-
+
pH5
-
+
pH6
-
+
pH7
-
+
pH8
-
+
pH9
-
+
pH10
-
+
pH11
Figure 2. Effect of pH in the absence (-) and presence (+) of 400 mM MgCl2 on culturability (A) and
redox activity (B) of L. monocytogenes. Cells were exposed to different pHs for 20 min (measured pH
values of mixed buffer-salt solutions are indicated) followed by resuspension in neutral buffer. A) Serial
dilutions of cells spotted on TSA. Pictures of representative plates are shown. B) Effect of salts and pH
on redox activity. Values show the increase of WST-8 signals within 3 h. Error bars represent standard
deviations from three independent experiments.
61
P. OYARZÚA ALARCÓN, ET AL.
[MgCl2 ] (in mM)
10
50
150 400 1000
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Buffersalt pH
3.0
3.0 2.9 2.6
2.5 2.3
buffer pH7
[NaCl] (in mM)
10
50
150 400 1000
Serial dilution
no
salt
3.0 3.0
buffer pH3
no salt
no salt
+ NaCl
+ NaCl
+ MgCl2
+ MgCl2
3.0 2.9 2.7
Figure 3. Effect of increasing concentrations of
MgCl2 and NaCl on L. monocytogenes at low
pH. Cells were exposed for 20 min to different
concentrations of MgCl2 and NaCl at pH3 (initial pH), followed by assessment of culturability
(after resuspension of cells in neutral buffer, serial
dilution, and spotting of aliquots on TSA) in comparison with a control without salt. pH values of
mixed buffer-salt solutions are indicated. Pictures
of representative plates are shown.
nomenon might have been caused synergistically
by increasing osmolarity and the aforementioned
pH.
Figure 4. Microscopic images of BacLightTM stained L. monocytogenes exposed to pH7 or pH3
in the presence of MgCl2 or NaCl in comparison with controls without salt. Salt concentrations
were 400 mM each. Green color indicates that cells
have intact cell membranes, red color indicates
membrane damage. A 40 × magnification was chosen. Representative pictures are shown.
In addition to assessing culturability and redox
activity, the impact of the two salts was studied
using fluorescence microscopy by staining treated
cells with SYTO9 and propidium iodide (as part
of the LIVE/DEAD® BacLightTM kit). A green
color indicates an intact membrane, a red color
indicates membrane damage. Whereas all cells
appeared green at pH7 (initial buffer pH), independent of the presence of salt, a few red cells
were observed at pH3 without salt and in the sample containing NaCl (figure 4). In the sample with
an initial pH of 3 supplemented with MgCl2 , more
than half of the cells stained red.
of E. coli, S. typhimurium, E. faecium, and S.
aureus were exposed to pH7 and acidic pHs in
the absence of salt or in the presence of MgCl2
and NaCl (figure 5). Due to the different tolerances of the different species towards acid stress,
pH3 was chosen for the two gram-negative species
and pH2 for the two gram-positive species. No salt
effect was observed for the initial buffer pH of 7
for any of the bacteria. At acidic pHs the presence
of MgCl2 almost completely abolished growth of
all of the species, whereas the effect of NaCl varied between species. Whereas it had no effect on
the survival of gram-negative species, the impact
on gram-positive species was comparable to that
seen with MgCl2 .
Testing other bacterial species
Comparison of different anionic bases
To test whether our observations held true for
a wider range of bacterial species, pure cultures
To assess whether the effect is limited to the
presence of phosphate, L. monocytogenes was
Membrane integrity
62
Antimicrobial properties of MgCl2
E. coli
S. typhimurium
pH3
no
no
salt MgCl2 NaCl salt MgCl2 NaCl
pH7
pH3
no
no
salt MgCl2 NaCl salt MgCl2 NaCl
E. faecium
pH7
pH2
no
no
salt MgCl2 NaCl salt MgCl2 NaCl
S. aureus
pH7
pH2
no
no
salt MgCl2 NaCl salt MgCl2 NaCl
Serial dilution
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pH w/o
salt
pH7
Buffer7.0 5.6 6.8 3.0 2.5 2.9
salt pH
7.0 5.6 6.8
3.0 2.5 2.9
7.0 5.6 6.8
2.0 1.3 2.0
7.0 5.6 6.8
2.0 1.3 2.0
Figure 5. Effect of exposure of E. coli, S. typhimurium, E. faecium and S. aureus to MgCl2 and NaCl
at pH7, pH 3, or pH2. Cells were exposed to 400 mM salt for 20 min each. A cell suspension without
salt served as a control. pH values of mixed buffer-salt solutions are indicated. Following exposure,
cells were resuspended in neutral buffer, serially diluted, and aliquots spotted on TSA. Pictures of
representative plates are shown.
resuspended in solutions of other acid-base systems (figure 6). All solutions were adjusted to
pH4 prior to adding salt and to resuspending
cells. No salt effect was observed when cells were
suspended in TRIS-HCl buffer, on the level of cultivation, or on the level of redox activity. An effect of
MgCl2 on viability was again seen for both tests in
the presence of acetic acid/acetate, and especially
of lactic acid/lactate. The presence of salt resulted
in both cases in a substantial drop in pH.
Effect of the salt anion
To study the effect of the salt anion, experiments at neutral and acidic pH were performed
in the presence of MgSO4 . Samples without
salt and supplemented with MgCl2 and NaCl
served as controls (figure 7). The NaCl concentration (800 mM) chosen was twice as high
as the MgCl2 concentration (400 mM) in order
to obtain the same chloride concentration. As
expected, no effect of either salt was observed
at neutral pH on viability (cultivation and redox
activity) of L. monocytogenes. At acidic pH both
chloride salts had an impact on the two viability parameters (with MgCl2 showing more
effect than NaCl), whereas the sample containing
MgSO4 appeared comparable with the one without
salt.
Discussion
Results presented in this study indicate that
at low pH and in the presence of inorganic or
organic bases, extracellular MgCl2 at concentrations above 150 mM can result in an antimicrobial
effect. As the salt does not affect bacterial viability
under ‘normal’ conditions (i.e. near-neutral pH),
the action qualifies as synergistic with acidity. The
effect of MgCl2 was substantially stronger than
that exerted by other chloride salts and might, in
part, be explained by enhanced acidification. The
latter can be attributed to the special properties of
the Mg2+ cation which has, among the biologically
relevant cations, the smallest ionic radius and the
highest charge density [11]. This in turn results
in a strong interaction with the water molecules
that surround the cation in two shells [12]. The
polarity induced by the high charge density of the
central Mg2+ cation renders water molecules more
63
P. OYARZÚA ALARCÓN, ET AL.
A
Inner hydration
shell
Outer hydration
shell
Aqueous MgCl2-:
Weakly acidic
• PO43• Organic bases:
lactate, glycolate,
maleate, citrate,
etc.
Aqueous MgCl2 in
presence of
inorganic or
organic bases:
-
2+
Strongly acidic
B
MgCI2
MgSO4
[MgSO4]
0.25
0.25
Free Mg+2
Concentration (mol L-1)
Concentration (mol L-1)
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2+
0.20
[MgCI]+
0.15
0.10
[MgHPO4]
0.05
0.20
0.15
Free Mg+2
0.10
0.05
0.00
[MgHPO4]
0.00
1
2
3
4
pH
5
6
7
1
2
3
4
5
6
7
pH
Figure 6. Simplified models to explain the roles of the cation and anion. A) Schematic diagram of how
inorganic and organic bases increase the acidity of dissolved MgCl2 . Mg2+ cations are surrounded by 18
water molecules forming two hydration shells. Whereas aqueous MgCl2 is a weak acid with polarized
water molecules releasing protons, the presence of inorganic and organic bases enhances the release of
protons resulting in increased acidification. B) Speciation analysis of MgCl2 and MgSO4 in the pH range
between pH1 to pH7. The diagram shows the molar concentrations of free Mg2+ and the corresponding
ionic couple.
64
Antimicrobial properties of MgCl2
TRIS-HCl
-
+
Lactate
-
+
4.0
3.2
+
Serial dilution
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MgCl2
Acetate
Buffersalt pH
4.0
4.0
4.0
2.9
Figure 7. Effect of different buffers/bases at pH4
on culturability of L. monocytogenes in absence
or presence of MgCl2 (400 mM). pH values of
mixed buffer-salt solutions are indicated. Cells
were exposed to different buffer-salt mixtures for
20 min followed by resuspension in neutral buffer,
serial dilution and spotting of aliquots on TSA.
Pictures of representative plates are shown.
likely to donate a proton than in the absence of
salt. Depending on the point of view, Mg2+ ions
therefore qualify as a Lewis acid (accepting electrons, based on the Lewis acid-base theory), or
hydrated Mg2+ can be seen as an acid (donating
protons to a base, in agreement with the BrønstedLowry acid-base theory). Although there might be
other possible explanations, the strong polarity of
Mg2+ is likely to cause a slightly acidic pH when
dissolving MgCl2 in water.
Whereas the polarity of Mg2+ explains why an
aqueous MgCl2 solution is slightly acidic, the
acidification is greatly enhanced in the presence
of strong bases resulting in stronger hydrolysis
(as schematically summarized in figure 6A). The
acetate or lactate used in this study serve as
examples of molecules that can increase acidity in
the presence of MgCl2 , whereas no effect is seen
with TRIS (figure 7). The reason for the latter
can be seen in the fact that at a pH substantially lower than its pKa (8.3), the protonated and
thus positively charged TRIS is not a base. In
the cases of acetic acid (pKa = 4.79) and lactic
acid (pKa = 3.86) on the other hand, the acid-base
ratio at the experimental pH of 4 produces sufficient base molecules to result in acidification in
the presence of MgCl2 . Acidification, in turn, shifts
the acid-base equilibrium towards acidic. Only the
protonated, uncharged forms of these molecules
exert an antimicrobial effect, a mechanism shared
among weak-acid preservatives [13]. The difference in the pKa values of the two acids is probably
reason why the antimicrobial effect of the salt-acid
mixture was substantially stronger in the presence of lactate than with acetate. The acidification
of MgCl2 in the presence of phosphate buffer on
the other hand, might be better explained by the
low solubility of magnesium phosphate salts. The
protons left behind after precipitation result in
acidification. The acidification caused by different
concentrations of MgCl2 in the presence of phosphate buffer is shown in table 1.
Apart from the role of the Mg2+ cation, the
comparison between MgCl2 and MgSO4 (figure 8)
raises the question how much of the effect can be
attributed to the anion. Both salts were compared
in the presence of phosphate buffer; when added
Table 1. Effect of different concentrations of
MgCl2 on pH when added to phosphate buffer of
defined initial pH. Numbers show final pH values
after addition of aqueous salt solution. The final
concentration of phosphate buffer after MgCl2
addition was 50 mM.
MgCl2
concentration
(in mM)
initial pH
pH 11
pH 10
pH 9
pH 8
pH 7
pH 6
pH 5
pH 4
pH 3
pH 2
10
9.78
9.10
8.60
7.97
6.94
5.96
4.93
3.98
3.00
2.01
50
8.00
8.03
7.95
7.62
6.54
5.52
4.48
3.82
2.89
1.99
150
7.31
7.37
7.35
7.14
6.09
5.11
4.11
3.59
2.60
1.87
400
6.98
6.67
7.01
6.48
5.62
4.66
3.73
3.31
2.50
1.68
1000
6.63
6.66
6.43
6.14
5.05
4.07
3.20
2.90
2.26
1.32
65
P. OYARZÚA ALARCÓN, ET AL.
buffer pH3
no
salt
MgCl2
MgSO2
NaCl
7.0
5.6
6.0
6.8
no
salt
MgCl2
MgSO2
NaCl
2.5
3.1
2.9
Serial dilution
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 14/06/2017.
buffer pH7
Buffersalt pH
3.0
Figure 8. Comparison of effects of MgCl2 ,
MgSO4, and NaCl at neutral and acidic pH on culturability of L. monocytogenes. Cells were exposed
for 20 min to 400 mM MgCl2 and MgSO4 or
800 mM NaCl at pH7 and pH3 (initial buffer pH
without salt). An aliquot without salt served as
a control. pH values of mixed buffer-salt solutions are indicated. Following exposure, cells were
resuspended in neutral buffer, serially diluted,
and aliquots spotted on TSA. Pictures of representative plates are shown.
to neutral phosphate buffer, acidification resulted.
When added to phosphate buffer at pH3 on the
other hand, acidification was only observed with
MgCl2 . Differences in speciation of the two salts
at low pH might be a potential explanation of this
phenomenon. A computer-based analysis revealed
a slightly higher concentration of free Mg2+ with
MgCl2 compared to MgSO4 (figure 6B), suggesting
that free Mg2+ might play a role. A full speciation
analysis for MgCl2 when dissolved in phosphate
buffer can be found in table 2 (Appendix). Further
research will, however, be necessary to investigate
the differences between these salts in relation to
their different biological effects.
66
Although the conditions applied in this study
are ‘artificial’, they demonstrate that different
salts have very different effects on bacterial viability and that the effects are pH-dependent. At
pH conditions that are critical but sub-lethal, the
additional presence of the salt can render cells
more susceptible, exceeding the tolerable stress
intensity. Acidic pH is one of nature’s most efficient strategies to control microbial growth. Low
pH is, for example, an essential requirement for
healthy skin where the pH has been reported to
be, on average, around 4.7 as a result of acids
that are either secreted by the human body or produced by bacteria that are part of normal skin flora
[14]. Disturbance of this protective mantle is common in skin disorders such as atopic dermatitis
and eczema [15]. An increase in skin pH can be
associated with a general increase in skin colonization, a higher abundance of pathogens [16],
and modulated virulence of pathogens [17] and
their adhesion [18]. In the context of this study,
it is noteworthy that MgCl2 is the dominant salt
in the Dead Sea, with a Mg2+ concentration of
1.89 M and Cl- representing 99% of all anions
[19]. The Dead Sea has been credited with healing properties for skin diseases since historic
times. Scientific studies on its effect on microbes
are extremely rare, although antimicrobial properties have been described for Dead Sea mud
[20]. Interestingly, we could show in this study
that Staphylococcus aureus (which is a common
skin pathogen) was susceptible to the presence
of MgCl2 at an acidic pH. Microbiological studies in relation to skin seem appropriate for future
research. Anionic bases can be expected to be
present on skin in the form of skin excretion
products, bacterial metabolites and cellular debris
from dead keratinocytes accumulating on the surface. In contrast to the harsh acidic pH conditions
(typically pH 3) chosen in this study to look for an
antimicrobial effect within a short exposure time
(20 min), less severe (and thus more physiologically relevant) pH conditions might be effective
when applying longer exposure times. It is tempting to speculate that it might be beneficial to apply
alpha hydroxy acids (AHAs) in combination with
MgCl2 for skin treatment. AHAs comprise a group
of organic carboxylic compounds (lactic acid, glycolic acid, malic acid, citric acid, etc.) commonly
used in cosmetics and dermatological applications
[21]. Our findings might also shed new light on
AlCl3 which is a common ingredient of deodorants, its effect being attributed to the blocking
Antimicrobial properties of MgCl2
Table 2. Molar concentrations (M) of chemical magnesium and phosphate species at different pH
values. The speciation analysis is based on 400 mM of MgCl2 dissolved in 50 mM phosphate buffer.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 14/06/2017.
Chemical species
free Mg2+
PO4 3HPO4 2H2 PO4 H3 PO4
MgOH+
MgPO4 MgHPO4
MgH2 PO4+
MgH3 PO4 2+
MgH4 PO4 3+
MgCl+
Mg(OH)2 (s)
MgH(PO4 )(H2 O)3 (s)
Mg3 (PO4 )2 (s)
7
6
0.19
1.48E-09
7.03E-05
3.77E-05
3.07E-10
4.32E-06
8.18E-07
1.00E-03
1.03E-06
1.21E-08
3.69E-11
0.16
0
4.88E-02
0
0.19
1.46E-10
7.01E-05
3.80E-04
3.12E-08
4.29E-07
8.08E-08
9.99E-04
1.04E-05
1.22E-07
3.75E-09
0.16
0
4.84E-02
0
of sweat glands [22] and a direct toxicity of Al3+
[23]. Extrapolation of the findings of this study
might suggest that, in common with MgCl2 , some
of the antimicrobial action might also be explained
by the decrease in pH (Al3+ is a stronger Lewis
acid than Mg2+ ) [24]. Although a stronger antimicrobial effect can be obtained with such metals,
an obvious advantage of MgCl2 over more toxic
salts consists in the harmless nature of the salt
as regards to human health and environmental
impact.
Despite the focus on the enhancement of pH
stress (which we currently see as the dominant
factor for the explanation of the results obtained),
other potential mechanisms of action of MgCl2
cannot be excluded. Figure 2 suggests that acidification might not be the only factor responsible
for the impact on bacterial viability. Comparing
samples with and without MgCl2 , viability was
affected more strongly at a final pH of 3.3 in the
presence of salt than at pH 3.0 in the absence
of salt. Similarly, survival was lower at pH 2.5
with salt than at pH 2 without salt. Additionally,
results suggest distinct pH-independent effects
of different salts as demonstrated in the direct
comparison of MgCl2 and NaCl (figure 3), where
the two salts exert different effects on bacterial
viability at comparable pH values. These specific
salt effects which seem to add to the pH effect
will need confirmation and further investigation.
Factors such as osmotic pressure, transport mechanisms, and interaction of the ions with proteins
pH of solution
5
4
0.19
1.48E-11
6.89E-05
3.62E-03
2.87E-06
4.50E-08
8.37E-09
1.00E-03
1.00E-04
1.16E-06
3.45E-07
0.16
0
4.46E-02
0
0.21
1.49E-12
6.06E-05
2.75E-02
1.86E-04
5.84E-09
9.83E-10
1.00E-03
8.40E-04
8.84E-06
2.23E-05
0.18
0
1.63E-02
0
3
2
0.21
1.39E-14
7.03E-06
3.94E-02
3.27E-03
5.05E-10
1.00E-11
1.25E-04
1.28E-03
1.59E-06
4.92E-05
0.18
0
0
0
0.22
8.50E-17
4.35E-07
2.46E-02
2.06E-02
5.01E-11
6.16E-14
7.76E-06
7.99E-04
6.15E-08
1.92E-05
0.18
0
0
0
and lipids have to be considered. Apart from
affecting membrane permeability and membrane
potential, MgCl2 has been shown (for eukaryotic cells) to interact with a large number of
exchangers and channels found in cellular membranes [7]. As regards to osmotic stress, Listeria
has been reported to be extremely resistant. Liu
et al. (2005), when examining the salt tolerance
of different virulent and avirulent L. monocytogenes strains, found that all strains tested were
resistant to saturated NaCl (corresponding to
approximately 6.1 M) for at least 20 h and possibly
longer, as tested by enumeration of colony-forming
units [25]. This finding is in line with a later
study showing that no decrease in viability was
obtained when exposing Listeria to a highly concentrated NaCl solution (4.8 M) at neutral pH for
three hours [5]. Osmotic stress should therefore
not contribute greatly to the observations reported
here.
In summary, this study demonstrates the effect
that the presence of a ‘harmless’ salt can have
on microbial viability. Whereas MgCl2 does not
visibly affect cells under ‘normal’ conditions, its
presence can have a severe impact under critical (but yet sublethal) conditions. Although we
hypothesize that the effect is largely due to a
drop in pH, other factors might be involved in this
antimicrobial activity. Future studies will greatly
benefit from the incorporation of a skin model,
given its potential implications for dermatological
applications.
67
P. OYARZÚA ALARCÓN, ET AL.
Disclosure
Financial support: this work was, in part, supported by the Chilean Council for Science and
Technology (Project FONDECYT 1101009). Conflict of interest: none.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 14/06/2017.
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