Visible light mediated killing of multidrug-resistant bacteria using photoacids

Article abstract of DOI:10.1039/c2tb00317a

Increasing acidity is a promising method for bacterial inactivation by inhibiting the synthesis of intracellular proteins at low pH. However, conventional ways of pH control are not reversible, which can cause continuous changes in cellular and biological

Full text of DOI:10.1039/c2tb00317a

Journal of
Materials Chemistry B
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PAPER
Visible light mediated killing of multidrug-resistant
bacteria using photoacids
Cite this: J. Mater. Chem. B, 2013, 1,
97
9
ab
b
c
b
d
Yang Luo, Chaoming Wang, Ping Peng, Mainul Hossain, Tianlun Jiang,
a
c
b
Weiling Fu, Yi Liao and Ming Su*
Increasing acidity is a promising method for bacterial inactivation by inhibiting the synthesis of intracellular
proteins at low pH. However, conventionalways of pH control are not reversible, which can cause continuous
changes in cellular and biological behaviours and are harmful to the host. Utilizing a photoacid that can
reversibly alter pH over two units, we demonstrated a strong bacterial inhibition assisted by visible light.
The pH value of the solution reverts back to the original level immediately after the irradiation is stopped.
If a photoacid is combined with colistin, the minimum inhibitory concentration (MIC) of colistin on
Received 28th October 2012
Accepted 27th November 2012
ꢁ1
multidrug-resistant (MDR) Pseudomonas aeruginosa can be improved ꢀ32 times (from 8 to 0.25 mg mL ),
which significantly decreases the toxicity of colistin in clinics. Evidenced by the extremely low toxicity of
the photoacid, this strategy is promising in MDR bacteria killing.
DOI: 10.1039/c2tb00317a
www.rsc.org/MaterialsB
1
Introduction
Global morbidity and mortality induced by infectious diseases
are exaggerated with the prevalence of multidrug-resistant
(MDR) bacterial pathogens, primarily due to over or misuse of
antibiotics. Most of the existing antibiotics fail to deal with
MDR bacteria such as methicillin-resistant Staphylococcus
aureus, extended-spectrum b-lactamase-producing enteric
bacteria, and Mycobacterium tuberculosis. Thus, improving effi-
cacy of antibiotics and reducing resistance, together with
Fig. 1 Scheme of the visible light enabled antibacterial strategy.
nephrotoxicity and neurotoxicity, are of utmost importance in
14,15
1
proteins at low pH.
But the conventional ways of pH control

ghting against MDR bacteria.
are not reversible, which can cause continuous changes in
cellular and biological behaviors. Ideally, the pH of a bacterial
suspension can be reduced to kill bacteria, and recover back to
normal aer that. Photoswitchable chemicals can be used to
tune pH based on illumination with light of different wave-
Pseudomonas aeruginosa (PA), a leading cause of hospital
infections, is difficult to treat because of the wide spread MDR
strain. Colistin (polymyxin E) is highly effective in ghting
against MDR PA by destroying the bacterial outer membrane
2
–5
and allowing small molecules to ow out of the cytoplasm.
16–19
lengths, mostly in the ultraviolet range.
Recently, we developed a type of photoacid that can revers-
However, colistin can only be considered as the last-line treat-
ment option for MDR organisms due to its serious nephrotox-
2
0
6
–10
ibly alter the pH by ꢀ2 pH units using visible light irradiation.
icity and neurotoxicity,
together with the appearance of
11
Here we report a novel photoswitchable antibacterial strategy
via controllable adjustment of bacterial intercellular pH, by
irradiating the photoacid using a visible light, to increase the
antibacterial activity of colistin (Fig. 1).
resistance to colistin among these organisms. To reduce the
colistin induced damage to the human body, a combination of
therapies is increasingly being considered.
12,13
Among all
possibilities, increasing acidity is an effective method for
bacterial inactivation by inhibiting the synthesis of intracellular
2
Experimental procedure
a
Materials
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical
University, Chongqing 400038, China
Sodium chloride (NaCl), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), RPMI 1640 culture media,
penicillin, streptomycin, fetal bovine serum (FBS), and Dul-
becco's phosphate-buffered saline (D-PBS) were from Sigma-
Aldrich (St Louis, MO). Yeast extract, bacteriological agar, and
b
NanoScience Technology Center, University of Central Florida, Orlando, Florida
3
2826, USA. E-mail: Ming.Su@ucf.edu
c
Department of Chemistry, University of Central Florida, Orlando, Florida 32826, USA
d
Department of Transfusion Medicine, Southwest Hospital, Third Military Medical
University, Chongqing 400038, China
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BD falcon multi-well at-bottom plate were from VWR (West was grown in deionized (DI) water (pH 7.4) until a density
6
ꢁ1
Chester, PA). The LIVE/DEAD BacLight bacterial viability kit of ꢀ10 mL was reached. Then the protonated merocyanine
was from Invitrogen (Carlsbad, CA). Trypton powder was (MEH) was introduced into the bacterial suspension at the nal
obtained from MO BIO (Carlsbad, CA). Ultrapure water concentration of 500 mM. Aer different times (1, 2, 3, 4 day
ꢁ1
(
18.2 MU cm ) from Nanopure System (Barnstead, Kirkland, respectively), 100 mL of the bacterial suspension was inoculated
WA) was used throughout. The photoreactions were performed and cultured on a LB agar plate for 24 h, followed by counting
on a 120 blue LED high-output array with an emission wave- the colony-forming units (CFUs) of PA in the plate.
length of 470 nm. Products of the esterication reaction were
analyzed by PerkinElmer series 200 High-performance liquid
chromatography (HPLC). A Fisher Scientic Accument AR15
Statistical analysis
Six independent duplicates were performed for each test and
the data were shown as mean ꢃ standard error. All the gures
were plotted using the Origin 8.5 soware (OriginLab, North-
ampton, MA) and statistical analyses were performed using
SPSS 16.0 (SPSS Inc, Chicago, Illinois). One-way ANOVA and LSD
tests were applied to compare the difference between the
numbers of bacterial colony aer different treatments. p < 0.05
was considered statistically signicant.
benchtop meter and pH combination electrodes were used to
measure the pH change. A Varian Cary 50 Scan UV-Vis spec-
trophotometer was used to measure the UV absorption spectra.
A PA reference strain (ATCC 27853) and multi-drug resistant
PA (ATCC 15442), a HeLa (CLL-2) cell line, and a HEK-293 (CRL-
1573) cell line were obtained from American Type Culture
Collection (ATCC, Manassas, VA). All chemicals used in this
study were of analytical grade and used without further
purication.
3
Results and discussion
Synthesis of photoacids
2
0
Fig. 2 shows that the synthesized photoacid is a visible-light
activatable merocyanine derivative that has a long proton-
dissociation state lifetime (70 s) due to a sequential intra-
molecular reaction. Fig. 2A illustrates the structural changes of
the photoacid with or without irradiation. In the dark, the
photoacid exists mainly as a MEH with a propyl sulfonate group
on the nitrogen of the indoline moiety. The absorption of
deprotonated merocyanine (ME) can also be observed, indi-
cating that MEH is weakly acidic. A 600 mM MEH solution has a
A photoacid was synthesized following a literature method.
Mainly, starting material 2,3,3-trimethyl-1-(3-sulfonatepropyl)-
H-indolium was synthesized at the beginning. Then 2,3,3-tri-
3
methyl-1-(3-sulfonatepropyl)-3H indolium (100 mg, 0.36 mmol)
and 2-hydroxybenzaldehyde (48 mg, 0.39 mmol) were added
into anhydrous ethanol (2 mL), followed by reuxing overnight.
The photoacid was obtained by ltration (110 mg, 80% yield).
Cytotoxicity assay
a
pH value of 5.8, from which the pK of MEH can be calculated to
The cytotoxicity of photoacid on bacteria was performed using a
bacterial viability assay. For dual staining the bacteria, the
be 7.8. Aer irradiation by a visible light with a wavelength
within its charge transfer band (Fig. 2B), the pH drops by ꢀ2
units to 3.3 (close to the theoretical value of the complete proton
dissociation of 3.2). When the light was turned off, the pH value
increases quickly to ꢀ4.7 in 70 s, and gradually returns to its
original level of 5.8 in ꢀ5 min. The characteristics of visible
5
ꢁ1
bacteria in the water were diluted to a density of 10 CFU mL
according to the absorbance at 600 nm. 3 mL of the dye mixture
containing an equal volume of green-uorescent SYTOꢀ 9
(
stain and red-uorescent propidium iodide stain) was mixed
with 1 mL bacterial suspension and incubated at 37 C in the
ꢂ
dark for 15 min, followed by observation under a uorescence
microscope. For live or dead cell calibration, 0.6 mL of the dye
mixture and 200 mL of the bacterial suspension were added into
each well of the 96-well microplate. Aer incubation for 15 min,
the uorescent signals were measured at 530 nm/630 nm (dual

uorescence). A cell-free control was used to remove the
orescent backgrounds.

The cytotoxicities of photoacid on mammal cells were tested
using MTT assay aer incubating with various concentrations
of photoacid (5, 50, and 500 mM). Firstly, the HeLa cells or HEK-
2
8
93 cells were grown in the microwell of the 96-well plate till
0% conuence. Then the photoacid (combined or not
combined with colistin) was incubated with the cells for 3 days,
followed by absorbance measurement.
Bactericidal activity
The bactericidal activity of the photoacid (with/without
combined with colistin) was determined by monitoring the
Fig. 2 Chemical characteristics of photoacid. (A) pH change with or without
visible light illumination. (B) Absorbance of MEH after irradiation. (C) Illustration
growth of PA in the presence of the photoacid. Briey, PA of the reaction between MEH and SP.
9
98 | J. Mater. Chem. B, 2013, 1, 997–1001
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light excitation and reversibility make it more attractive in
Firstly, the bactericidal activity of visible light is tested by
biological applications than conventional photoacids, because measuring the bacterial population aer exposure at 470 nm for
no UV light is involved. a long time. Results show that there is no signicant difference
The photochemical reaction aer irradiation is monitored by between bacteria numbers in the illuminated sample and the
UV-Vis absorption spectroscopy. Fig. 2C shows that the control sample (non-illuminated) even aer 24 h of incubation
absorption of MEH (lmax ¼ 424 nm) decreases upon irradiation (p > 0.05), indicating that the exposure to visible light (470 nm)
and an absorption peak of SP (lmax ¼ 325 nm) increases has no direct bactericidal effect. Meanwhile, the cytotoxicities of
accordingly.
the photoacid on bacteria before and aer illumination are
To assess the antibacterial activity of the photoacid, two tested with MTT assay. Results show that 500 mM MEH kills only
strains of PA (reference strain and MDR strain) are tested. 25.7% of MDR PA cells in 24 h of incubation without illumi-
Bacteria are inoculated in DI water until they reach a value of nation, revealing its low cytotoxicity on bacterial cells. Consid-
6
ꢁ1
ꢁ1
1
0 CFU mL . DI water is taken as solvent to eliminate ering that the illuminated photoacid kills 5 log10 CFU mL
the buffering ability against the pH change. Fig. 3A shows the MDR PA within 2 h, we consider the killing effect is mainly due
illuminated photoacid or colistin alone can only inhibit the to the pH change of the solution.
growth of MDR PA, but the combination of illuminated photo-
To verify this hypothesis, the growth rate of MDR PA at
acid and colistin kills most of the MDR PA. Fig. 3B shows that in different pHs (ranging from 2 to 7.4) is determined. Fig. 3C
the presence of MEH, illumination with visible light (470 nm) shows that the bacterial population increases ꢀ18% within 24 h
ꢁ
1
for 1 h kills ꢀ99% (ꢀ2 log CFU mL decrease) of MDR PA, at pH 7.4 due to natural growth. No signicant difference is
1
0
whereas MDR PA population increases ꢀ20% in the absence of observed in the bacterial population between pH 6 and 7.4
MEH. The 20% increase of the bacterial population is mainly ( p > 0.05), showing that MDR PA can grow well under a pH
due to the natural growth of bacteria in DI water. Meanwhile, range of 6–7.4. But the bacterial population begins to decrease
ꢁ1
ꢁ1
1
mg mL colistin alone kills ꢀ3.5 log10 CFU mL MDR PA signicantly when the pH is lower than 5. The bacterial pop-
ꢁ1
within 1 h. Interestingly, in the presence of MEH and colistin, ulation decreases to 1.5 and 3 log10 CFU mL at pH 4 and 3
the antibacterial activity of colistin is signicantly improved by within 1 h, respectively. At pH 2, all bacteria are killed (5.5 log10
ꢁ
1
ꢁ1
decreasing ꢀ5.5 log10 CFU mL bacteria number aer 30 min CFU mL decrease) within 20 min, indicating that the pH value
illumination. The results clear show that the combination of has a signicant inuence on the bacterial growth, which is
photoacid and colistin can signicantly improve the antibac- consistent with previous reports that the optimal pH range for
21
terial activity. The following experiments are carried out to the growth of PA is 5.6–8.5. The antibacterial activity of the
determine whether this enhanced capability is due to visible illuminated photoacid (2 log10 CFU mL decrease) is within the
light illumination or from the cytotoxicity of the photoacid.
ꢁ
1
ꢁ
1
range of 1.5 and 3 log10 CFU mL at pH 4 and 3. Considering
that the illuminated photoacid (pH 3.3) is also within the range
mentioned above, it can be concluded that the antibacterial
activity of the illuminated photoacid is mainly due to the pH
decrease aer illumination. These results show that visible light
illuminated MEH can induce a pH drop of 4 to 3 in solution,
which can signicantly inhibit the growth of MDR PA. Regrowth
of bacteria is observed aer a few hours when the pH returns to
7.4, indicating that inactivated bacteria are still alive and can
grow into colonies. The bacterial regrowth aer acid treatment
is likely due to the adaption of MDR PA on environmental
stress, which means that lowering the pH alone is not a
powerful antibacterial strategy (Fig. 4A and B).
Colistin demonstrates a concentration dependent killing of
MDR PA at pH 7.4. Our results show that the minimal inhibition
ꢁ
1
concentration (MIC) of colistin on MDR PA is 8 mg mL
,
ꢁ
1
meaning that 8.0 mg mL colistin can completely inhibit the
ꢁ1
growth (ꢀ6 log10 CFU mL
decrease) of MDR PA in 1 h
(
Fig. 4A). Lower colistin concentrations can only partially
ꢁ
1
inhibit the growth of MDR PA. Colistin at 4.0, 2.0, 1.0 mg mL
ꢁ1
can decrease 4.5, 3.0, 1.5 log10 CFU mL of MDR PA, respec-
tively. No signicant antibacterial activity is observed for 0.5 mg
ꢁ1
mL colistin when compared with the negative control ( p >
0
.05). Meanwhile, MDR PA regrows 2–3 days later aer being
Fig. 3 Antibacterial activity of the illuminated photoacid. (A) Cultured MDR PA
after treating with PBS (A1), colistin (A2), illuminated photoacid (A3), and colistin
together with the illuminated photoacid (A4); (B) effect of irradiation time on the
bacterial population; (C) effect of different pH values on the bacterial population.
The error bars represent standard errors of 6 independent tests.
ꢁ1
treated by colistin (<8.0 mg mL ), which is consistent with our
previous reports that high dose of colistin is required to kill
MDR PA due to increased resistance by accelerating the
synthesis of intracellular proteins to reduce the effect of
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lipopolysaccharides (LPS) in the outer membrane of bacteria.
The disruptions of outer and cytoplasmic membranes are fol-
lowed by the leakage of small intracellular molecules, and
bacterial killing occurs within few minutes. Generally, a rela-
tively high concentration of colistin is needed to trigger
disruption of the membrane, because bacteria can repair a
2
+
2+
slight displacement of Mg and Ca ions in the membrane at
the optimal intracellular pH (pH ). But if the cytoplasmic
i
+
membrane is disrupted seriously, extracellular H (high
concentration) can quickly diffuse into the cytoplasm and a
considerable number of small intracellular molecules ow out
of the membrane, which overwhelmed the ability of bacteria to
maintain optimal pH
In addition, the change of pH
the displacement of Mg and Ca in the membrane, acceler-
i
and induced irreversible bacterial death.
hinders the ability to compensate
i
2
+
2+
Fig. 4 Synergic antibacterial activity of colistin with photoacid. (A) Antibacterial ating bacterial killing. Thus, the antibacterial activity of colistin
activity of different colistin concentrations on MDR PA; (B) synergic antibacterial is signicantly enhanced by decreasing the MIC in the case of
activity of different colistin concentrations with 500 mM MEH on MDR PA; (C)
combination.
antibacterial activity of different colistin concentrations on reference PA (27853);
The cytotoxicity of MEH on mammalian cells is also studied
(
D) synergic antibacterial activity of different colistin concentrations with 500 mM
either with or without colistin. HeLa (cervical cancer cell) and
HEK-293 (human renal cell) are used to assess the cytotoxicity of
photoacid because the kidney toxicity is frequently reported.
We are Fig. 5A shows that cytotoxicities of the photoacid depend on its
MEH on reference PA strain (27853); n ¼ 6.
22–24
extracellular pH change on inner organelles.
wondering whether the illuminated photoacid can decrease the concentration: no signicant cytotoxicity is observed for 5 and
antibacterial resistance by inhibiting the protein synthesis. 50 mM photoacid up to 72 h when compared with the control
Fig. 4B shows that the combination of colistin and photoacid ( p > 0.05). And for 500 mM photoacid, only 10% HeLa cells and
provides higher antibacterial activity than colistin or photoacid 16% HEK-293 cells are killed aer 72 h treatment, respectively.
alone. Aer illuminating (470 nm) the bacterial solution con- These results reveal that the photoacid has low toxicity to
taining photoacid and colistin, the MIC of colistin on MDR PA mammalian cells even at high concentration. Meanwhile,
ꢁ
1
dropped from 8.0 to 0.25 mg mL . In addition, no regrowth is Fig. 5B shows that the cytotoxicity of colistin is highly concen-
ꢁ
1
observed aer 4 days culturing, in the presence of 0.25 mg mL
tration dependent: an extremely low concentration of colistin
ꢁ1
colistin and 500 mM MEH. These results clearly show that low (0.25 mg mL ) imposes nearly no toxicity on both HeLa and
pH can signicantly enhance the antibacterial activity of HEK-293 cells, 1 mg mL of colistin kills 19% HeLa cells and
colistin and prevent the regrowth of MDR PA. To verify the 28% HEK-293 cells, while 8 mg mL colistin kills 50% HeLa
ꢁ1
ꢁ
1
feasibility of this combination in killing bacteria, the killing of a cells and 84% HEK-293 cells within 72 h.
reference PA strain (27538) is also studied (Fig. 4C and D). A
This high cytotoxicity of colistin can be adopted to explain
MIC of 1 mg mL colistin on PA 27538 is observed without the the serious nephrotoxicity during MDR PA treatment in
ꢁ
1
7
,8,24,25
presence of photoacid, and the regrowth can be observed 2 days clinics.
aer colistin treatment. However, in the presence of 500 mM reduced by decreasing its dosage. Fig. 5B shows that the
The side effect of colistin can be signicantly
ꢁ
1
MEH assisted with illumination, the MIC of colistin decreases combination of 500 mM photoacid and 0.25 mg mL colistin
to 0.25 mg mL (Table 1). Thus the synergic effect of visible kills 13% HeLa cells and 21% of HEK-293 cells aer 72 h
light assisted MEH and an extremely low concentration of treatment, which are much lower than that of 8 mg mL colistin
colistin (0.25 mg mL ) can effectively kill PA (both laboratory on HeLa cells (74%) and HEK-293 cells (81%) alone (p < 0.05).
and MDR strain), and no regrowth has been observed within 5
days of culture.
ꢁ1
ꢁ1
ꢁ
1
The synergic antibacterial activity of photoacid and colistin
can be understood as below. Colistin kills bacteria by destroying
the outer membrane of bacterial cells. The polycationic colistin
2
+
2+
molecules can displace Mg and Ca ions that cross-bridge
between adjacent negatively charged phosphate groups of
Table 1 MICs of colistin on PA
Reference PA (27853)
MDR PA
8 mg mL
Fig. 5 Cytotoxicity of photoacid and colistin. (A) Concentration dependent
cytotoxicity of MEH on HeLa cells and HEK-293 cells. (B) Cytotoxicity of the anti-
bacterial combination including MEH and different concentrations of colistin.
Error bars represent the standard errors of 6 independent tests.
ꢁ
1
ꢁ1
No photoacid
1 mg mL
0.25 mg mL
ꢁ1
ꢁ1
5
00 mM photoacid
0.25 mg mL
1
000 | J. Mater. Chem. B, 2013, 1, 997–1001
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The cytotoxicity of the illuminated photoacid (SP) is not
assessed directly, because the entire monolayer cultured cells
die within 30 min in such a low pH (3.3) condition. It is difficult
to differentiate whether the cell death is caused by low pH or the
cytotoxicity of SP. But, previous studies have shown that 8-
methoxy-6-nitro-BIPS (SP derivative) did not affect the cellular
survival of THP-1, AGS, and A549 cells for 24 h at concentration
6 C. Y. Cheng, W. H. Sheng, J. T. Wang, Y. C. Chen and
S. C. Chang, Int. J. Antimicrob. Agents, 2010, 35, 297–300.
7 S. J. Wallace, J. Li, R. L. Nation, C. R. Rayner, D. Taylor,
D. Middleton, R. W. Milne, K. Coulthard and
J. D. Turnidge, Antimicrob. Agents Chemother., 2008, 52,
1159–1161.
8 M. E. Falagas, P. L. Rafailidis, S. K. Kasiakou, P. Hatzopoulou
and A. Michalopoulos, Clin. Microbiol. Infect., 2006, 12, 1227–
1230.
ꢁ
4
ꢁ9
26
ranging from 10 to 10 M. Photochromic molecules from
the SP family also have no effect on the cellular survival of HEK-
27
2
93 cells. Compared to these SP derivatives reported before,
9 B. Lin, C. Zhang and X. Xiao, J. Vet. Pharmacol. Ther., 2005,
28, 349–354.
our SP molecule has a similar structure except for lacking a
+
NH3 group, indicating a much lower cytotoxicity than those 10 M. E. Falagas, M. Rizos, I. A. Bliziotis, K. Rellos,
reported SP derivatives. S. K. Kasiakou and A. Michalopoulos, BMC Infect. Dis.,
005, 5, 1.
2
4
Conclusions
11 K. R. Peck, M. J. Kim, J. Y. Choi, H. S. Kim, C. I. Kang,
Y. K. Cho, D. W. Park, H. J. Lee, M. S. Lee and K. S. Ko,
J. Med. Microbiol., 2012, 61, 353–360.
A strong bactericidal activity is observed by combining the
visible light illumination of photoacid and colistin. The neph-
rotoxicity of colistin can be drastically decreased by reducing
the MIC of colistin when combined with a photoacid, indicating
a promising approach in MDR bacteria killing. In the future,
photoacids may be incorporated into the structure of a drug
carrier. Visible light irradiation will produce a temporary and
localized pH drop that increases the bactericidal activity of
the drug.
1
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