the role of the methylene blue and toluidine blue monomers and dimers in the photoinactivation of...
TRANSCRIPT
Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
www.elsevier.com/locate/jphotobiol
The role of the methylene blue and toluidine blue monomersand dimers in the photoinactivation of bacteria
Marina N. Usacheva, Matthew C. Teichert, Merrill A. Biel *
Advanced Photodynamic Technologies, Inc., Minneapolis, MN 55414, USA
Received 14 March 2002; received in revised form 11 April 2003; accepted 17 June 2003
Abstract
The interactions between the phenothiazine dyes, methylene blue (MB) and toluidine blue (TB), and bacteria (Staphylococcus
aureus, Streptococcus pneumoniae, Enterococcus faecalis, Hemophilus influenzae, Escherichia coli and Pseudomonas aeruginosa) were
studied spectrophotometrically. This demonstrated that a metachromatic reaction took place between the dyes and bacteria.
Furthermore, bacteria induced additional dimerization of MB and TB. The effective dimerization constants of MB and TB were
evaluated in the presence of each bacterial strain at a concentration of 108 CFU/ml. The analysis of the effective dimerization
constants for MB and TB in the presence of bacteria indicated that the ability to form dimers was greater for TB than for MB.
Gram-negative bacteria induced the dye dimerization more intensely than gram-positive bacteria. There was a correlation between
the ability of each dye to form dimers in the presence of bacteria and the relative photobactericidal efficacy of each dye against these
bacteria. These results provide evidence confirming the essential role of the dye dimers in bacterial photodamage.
� 2003 Elsevier B.V. All rights reserved.
Keywords: Photoinactivation of bacteria; Photosensitizers; Phenothiazine dyes; Dye dimerization; Metachromatic effect
1. Introduction
Our previous comparative study of the photobacte-
ricidal activity of the phenothiazine dyes methylene blue
(MB) and toluidine blue (TB) demonstrated that TB was
far more active in killing gram-positive (Staphylococcus
aureus, Streptococcus pneumoniae, Enterococcus faecalis)and gram-negative bacteria (Hemophilus influenzae,
Escherichia coli and Pseudomonas aeruginosa) under red
laser light conditions when compared to MB [1]. These
conclusions were consistent with those described by
Wainwright et al. [2] and Wilson [3].
It is common knowledge that both TB and MB have
a similar chemical structure and photochemical prop-
erties. The only distinguishing characteristic between thedyes is the partition coefficient (P), which is almost 3-
fold higher for TB than for MB [1]. This should be an
indication of the greater ability of TB molecules to
* Corresponding author.
E-mail address: [email protected] (M.A. Biel).
1011-1344/$ - see front matter � 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2003.06.002
permeate and accumulate in the hydrophobic region of
the cellular membrane and therefore have a greater
photobactericidal activity. However, some of our results
and those found in the literature are not always con-
sistent with this conclusion. We observed that MB had a
higher photobactericidal activity than TB against S.
aureus 6538 [1]. Wainwright et al. [2] demonstrated thatMB and TB had the same photobactericidal activity
against S. aureus NCTC 6571. Wilson et al. [4] observed
that MB was more effective than TB against Fusobac-
terium nucleatum. Millson et al. [5] revealed that TB and
MB have the same photobactericidal efficacy against
Helicobacter pylori in vivo.
It is difficult to predict the efficacy of photosensitizers
as many factors can contribute to their cellular photo-activity. Generally, the photosensitizing efficacy of dyes
is evaluated on the basis of the photophysical and
photochemical properties of their monomer species.
Although, in accordance with the ‘‘Kautsky effect’’ of
photosensitization, dye dimers or aggregates are the
principal species that participate in photooxidation [6].
This is supported by the investigation of Bartlett and
88 M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
Indig [7] that demonstrated the participation of the dye
dimers (including MB dimers) bound to proteins in the
photosensitization and their effect on the contribution of
Type I and Type II mechanisms to the total photosen-
sitization. Therefore, the differences in the excitationenergetics and the geometrical structure of the dye
monomer and dimer species must be considered in
evaluating the dye-photosensitizing efficacy.
From our previous results [1] and the reports of
Wilson et al. [2,3], the lethal concentrations of MB and
TB necessary to eradicate gram-negative organisms are
in the range where both dyes form dimers in aqueous
solutions. As suggested by Kasha [8] and in the discus-sion with Ito [9], the association tendency of both dyes
to form dimers should be enhanced on the surface of
biological substrates and affect their photosensitizing
efficacy.
The aim of the present study was to determine the
predominant type of TB and MB species responsible for
bacterial killing in order to account for the differences in
the photosensitizing efficacy between the dyes. For thispurpose, we examined the spectral changes of both dyes
in the presence of different bacteria, estimated the values
of the effective dimerization constant (eff :KD) induced by
these microorganisms, compared the eff :KD values and
the photobactericidal efficacy of MB and TB for
Staphylococcus aureus, Streptococcus pneumoniae, Van-
comycin resistant Enterococcus faecalis, Hemophilus in-
fluenzae, Escherichia coli ATCC 25922, Escherichia coli
ATCC 35812, and Pseudomonas aeruginosa, and esti-
mated the phototoxicity produced with the dyes and
laser light at the wavelengths corresponding to the dimer
and monomer peak of each dye against Staphylococcus
aureus ATCC 25923 (S. aureus), Escherichia coli ATCC
25922 (E. coli) and Pseudomonas aeruginosa ATCC
27853 (P. aeruginosa).
2. Materials and methods
2.1. Dyes
TB O (90%TB, Sigma–Aldrich, St. Louis, Missouri,
USA) and MB (87% MB, Sigma–Aldrich, St. Louis,
Missouri, USA) were used after three recrystallizationsfrom ethanol–distilled water. Additionally, MB was
dried at 106 �C in an incubator. The purity of dyes was
checked from their molar extinction coefficient [10,11].
All experiments were performed in parallel with purified
and commercial (non-purified) dyes. The commercial
dyes were used in order to approximate clinical trial
conditions. 0.45% saline was used as a solvent in all
experiments. Stock solutions of MB and TB at theconcentration of 1 mM were prepared by weight with
0.45% saline.
2.2. Bacteria strains
The following gram-positive and gram-negative or-
ganisms were used in this study: Staphylococcus aureus
ATCC 25923, Streptococcus pneumoniae ATCC 49619(S. pneumoniae), Vancomycin resistant Enterococcus
faecalis ATCC 51299 (E. faecalis), Hemophilus influen-
zae ATCC 49247 (H. influenzae), Escherichia coli ATCC
25922, Escherichia coli ATCC 35812 (E. coli), and
Pseudomonas aeruginosa ATCC 27853. S. aureus, E.
faecalis, E. coli and P. aeruginosa were aerobically
grown on blood agar at 37 �C for 24 h. S. pneumoniae
was grown on blood agar at 37 �C for 24 h in a CO2-incubator. H. influenzae was grown on chocolate agar in
a CO2-incubator at 37 �C for 24 h. The bacteria were
then harvested by centrifugation and suspended in
0.45% saline at a stock concentration of (1–2)� 109
CFU/ml.
Spectrophotometric study was carried out with a
Beckman DU-7 spectrophotometer using a 1-cm or 0.1-
cm path length cell. Samples for spectrophotometricmeasurements were prepared by dilutions of the dye
stock solutions. The dye solution concentration ranged
from 1 to 300 lM. Shortly before the measurements
were taken, 0.5 ml of the appropriate fresh bacterial
suspension was added to each dye solution. The total
volume of the solutions was 1 ml. The absorption
spectra of the dyes were obtained in the visible region
from 500 to 700 nm where the monomer and dimerabsorption bands of these dyes were distinct. The ref-
erences were 0.45% saline or a bacterial suspension in
0.45% saline at the same bacterial concentration. The
absorption spectra of the solutions were recorded im-
mediately after their preparation. Two different series of
the dye solutions were examined, in which: (1) a dye
concentration was fixed but the concentration of mi-
croorganisms progressively increased; (2) the bacterialconcentration was fixed but the concentration of dye
progressively increased.
2.3. Estimation of the dimerization constants
The estimation of the effective dimerization constants
of dyes in the presence of bacteria was performed using
the graphical method of Khachaturyan et al. [12] for theMB dimerization in the presence of salts. This method
assumed that the Lambert–Beer�s law deviations ac-
counting for the changes of a dye absorbance, A, with
the dye concentration are caused by dimer formation in
accordance with the process
MþM ¼ D;
where M and D are monomer and dimer species, re-
spectively. The dimerization constant of this process,
KD, is
M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98 89
KD ¼ ½D�½M�2
; ð1Þ
where [D] and [M] are the concentrations of dimers and
monomers, respectively.
It has been shown that KD is related to the deviation
value of the absorbance from the absorbance value
provided by the Lambert– Beer�s law, DA, as follows
KD ¼ DADe
2 A� c0eD2
� �2 ; ð2Þ
where DA ¼ ðc0eM � AÞ, c0 is the analytical molar con-
centration of the dye, eM and eD are the molar extinction
coefficients of the monomer and dimer of dye, respec-tively, and
De ¼ eM�
� eD2
�:
After some transformations of Eq. (2), we obtain thefinal Eq. (3), which is suitable for plotting
1ffiffiffiffiffiffiffiDA
p ¼ �bþ c0DA
� �tan a; ð3Þ
where b �ffiffiffiffiffiffiffiffiffi2KD
p=
ffiffiffiffiffiffiDe
pand tan a ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffi2KDDe
p.
KD could be determined from the slope of the plot
described by the Eq. (3)
KD ¼ 0:5b tan a: ð4ÞFor the calculation of KD, a series of solutions at pro-
gressively increasing concentrations of dye, c0, and at a
fixed concentration of bacteria was prepared for each
experiment. The same stock solution of each bacterial
strain was used for the experiments with MB and TB.The intensity of absorption, A, at 664 and 631 nm for
MB and TB, respectively, at progressively increasing
concentrations of each dye was measured with and
without bacteria. The molar extinction coefficient of the
monomer, eM, was determined for the most dilute dye
solutions that were used (less than 4 lM).
2.4. Investigation of the phototoxicity of MB and TB
induced by laser light at the wavelengths corresponding to
the dimer and monomer peak of each dye
A series of solutions with increasing concentrations of
both dyes and a fixed concentration of bacteria was used
in this study. For this purpose, equal volumes of the
stock bacterial suspension at a concentration of
1.5� 108 CFU/ml were transferred to test tubes. Asuitable volume of the stock dye solution of MB or TB
(1 mM) was added to each tube to give final dye con-
centrations ranging from 25 to 375 lM. The appropriate
volume of 0.45% saline was added to each tube for a
total volume of 1 ml. Then 50 ll of each bacterial sus-
pension at the same dye concentration was placed into
duplicate glass vials. One of these vials was exposed to
laser light corresponding to the dimer peak of the dye (at
610 nm for MB and 585 nm for TB). Another one was
exposed to laser light corresponding to the dye mono-
mer peak (at 664 nm for MB and 630 nm for TB). The
fluence and light intensity of laser light corresponding tothe dye dimer and monomer wavelengths were kept the
same. After irradiation, the suspensions were serially
diluted in 0.45% saline and 0.5-ml aliquots were spread
over the surface of blood agar. Plates were incubated for
24 h at 37 �C and counted for Colony Forming Units
per ml (CFU).
The red light sources used were an argon pumped-dye
laser (MDS-90, California Laboratories, Inc., Califor-nia, USA) tuned to emit light at 585 nm, 610 nm or 630
nm and a diode laser emitting light at 664 nm (Photo-
Point DD4, Miravant Systems, Inc., Santa Barbara,
California, USA).
3. Results
3.1. Spectra of MB and TB with or without the presence
of bacteria
Figs. 1a and 2a illustrate the molar extinction curves
of MB and TB in 0.45% saline at concentrations ranging
from 7.5 to 375 lM without bacteria. Each of the molar
extinction curves is defined as the superposition of the
two bands responsible for monomer and dimer ab-sorption in the visible region of the spectra. All curves
passed through the isosbestic point. In diluted solutions,
the peaks of the MB monomer and dimer bands are
located at 664 and 615 nm, respectively; the corre-
sponding peaks of TB are located at 631 nm and ap-
proximately 600 nm. With increasing dye concentration,
the molar extinction coefficient at a monomer peak of
TB and MB diminished (hypochromic effect), the dimerpeak of the dyes shifted toward the shorter wavelengths
of the spectrum (hypsochromic effect) and the ratio of
the dimer to the monomer absorbance of each dye,
AD=AM, increased in a gradual manner (Figs. 1c and 2c).
Addition of any bacterial suspension to the MB and
TB solutions caused a further depression of the mono-
mer absorption band of each dye, an increase in the
AD=AM and a further shift in the dimer peaks of bothdyes. Because the metachromasy effect is similar for
both dyes in the presence of bacteria, only two illus-
trations are given below to show the above-described
behavior of the dye spectra in the presence of a gram-
positive strain and a gram-negative strain. Figs. 1b and c
demonstrate the metachromasy effect for MB in the
presence of S. pneumoniae, whereas Figs. 2b and c show
the same effect for TB in the presence of P. aeruginosa.Both bacteria were used at a concentration of 108 CFU/
ml. A comparison of Figs. 1a and 2a with Figs. 1b and
2b demonstrates that introducing bacteria resulted in a
λ (nm)λ (nm)500 550 600 650 700
ε(M
-1 cm
-1)
0
20000
40000
60000
80000
10 20 30 40 50
AD/A
M
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
(a) (b)
1
34
5
1
2
3
2
4
500 550 600 650 700
ε(M
-1 cm
-1)
0
20000
40000
60000
80000 2
3
4
5
Fig. 1. Effect of S. pneumoniae on the spectral characteristics of MB. Extinction coefficients curves of MB at the dye concentrations of: (1) 7.5 lM, (2)
17 lM, (3) 24 lM, (4) 44 lM, (5) 3750 lM without bacteria (a) and (b) in the presence of S. pneumoniae at a concentration of 108 CFU/ml. (c)
Dependence of the ratio of the intensity of the dimer maximum to the monomer maximum, AD=AM, on the MB concentrations without (d) and in the
presence of S. pneumoniae at the concentration of 108 CFU/ml (j).
90 M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
decrease of the molar extinction coefficients of both dyes
and an increase in the AD=AM. The position of the
isosbestic point for both dyes in the presence of bacteria
changed when compared to the isosbestic point without
bacteria. This new isosbestic point disappeared at rela-
tively high concentrations of each dye (Figs. 1b and 2b).At approximately the same time the dimer peak began
to shift smoothly by 3–5 nm to the red region of the
spectra.
The same trend of spectral changes for both dyes was
observed when the dye was kept at a fixed concentra-
tion, but the bacterial concentration was progressively
increased. In the present paper, we restricted our con-
sideration to the TB–H. influenzae and MB–E. coli sys-tems (Figs. 3a and b), although analogous changes were
observed in the presence of every other strain of bacteria
that was studied. Fig. 3a and b demonstrate the spectral
changes of the TB and MB solutions following the ad-
dition of increasing amounts of H. influenzae or E. coli,
respectively. As before, the dye absorbance gradually
decreased and the AD=AM ratio increased. Each time, as
the bacterial concentration was increased up to 1� 109
CFU/ml, the attainable position of the dimer band was
approximately at 585–590 nm for TB and 605–610 nm
for MB in the presence of different bacteria.
3.2. Effect of bacteria on the ratio of AD=AM and the
position of the blue maximum
Fig. 4 demonstrates how the value of AD=AM changed
for both MB and TB at a fixed dye concentration in the
presence of increasing concentrations of different bac-
teria. The degree of changes in the AD=AM for both dyes
was greater in the presence of gram-negatives than
gram-positives. The same is true for the shift of fre-quency. In the presence of gram-negatives, the short
wavelength peak of both dyes was displaced 5–10 nm
toward the blue region of the spectra, whereas the gram-
positives resulted in a 3–5-nm blue shift of the short
wavelength peak. Among the gram-positive bacteria, the
most intensive changes occurred in the interaction be-
tween the dyes and S. pneumoniae. In turn, H. influenzae
.
5 10 15 20 25 30
AD/A
M
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
(a) (b)λ (nm)550 600 650 700
ε (M
-1 c
m-1
)
ε (M
-1 c
m-1
)
0
10000
20000
30000
40000
50000
60000
1
5
4
2
1
3
5
4
λ(nm)550 600 650 700
0
10000
20000
30000
40000
50000
60000
1
23
4
Fig. 2. Effect of P. aeruginosa on the spectral characteristics of TB. Extinction coefficient curves of TB at the dye concentrations of: (1) 9 lM, (2) 15
lM, (3) 24 lM, (4) 30 lM, (5) 157 lMwithout bacteria (a) and (b) in the presence of P. aeruginosa at a concentration of 108 CFU/ml. (c) Dependence
of the ratio of the intensity of the dimer maximum to the monomer maximum, AD=AM on the TB concentrations without (d) and in the presence of
P. aeruginosa at the concentration of 108 CFU/ml (j).
M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98 91
resulted in the greatest changes in the dye spectra among
the gram-negative organisms.
3.3. Effect of bacterial concentration on the dye monomer
band absorbance
Figs. 5 and 6 demonstrate the behavior of the dye
monomer absorbance following the addition of in-
creasing amounts of bacteria. The monomer absorbance
peak of both dyes progressively decreased, reached a
minimum and then began to rise again, when high
concentrations of gram-negative bacteria (more than 109
CFU/ml) were added to the dye solutions. Thus, in the
presence of gram-negative bacteria, the plots were de-
fined as the curves with descending and ascending
branches. Furthermore, in the presence of gram-nega-
tives, both the dimer and monomer peaks of MB and TB
began to shift toward longer wavelengths and the isos-
bestic point disappeared simultaneously with the revival
of a monomer band absorbance (Fig. 3). In the presenceof the gram-positive bacteria, the corresponding plots
exhibited only a descending branch over a range of the
bacterial concentrations.
Figs. 5 and 6 demonstrate that the depth and the
position of the minimum of the plots of absorbance vs.concentration of the gram-negative bacteria depended
on the bacterial strain. The depth increased in the fol-
lowing order for MB: E. coli 35812<P. aeruginosa<E.
coli 25922<H. influenzae. A somewhat different order
was seen for TB, namely: E. coli 35812<E. coli
25922<P. aeruginosa<H. influenzae. The position of
the minimum on the plots for both dyes changed as
following: P. aeruginosa<E. coli 35812�E. coli
25922<H. influenzae.
3.4. Estimation of the effective dimerization constant of
MB and TB in the presence of bacteria
We attempted to estimate the dimerization constant
values in the dye-bacterial solution in order to determine
the quantitative differences in the ability of MB and TBto dimerize in the presence of bacteria. But in so doing,
550 600 650 700
Abs
orba
nce
0.0
0.5
1.0
1.5
1
2
3
4
5
λ (nm)500 600 700
Abs
orba
nce
0.0
0.1
0.2
0.3
0.4
1
23
45
(a)
Fig. 3. Effect of additives of bacteria on the dye absorption spectra in
0.45% saline: (a) TB (at a concentration of 10 lM) in the presence of
H. influenzae at the concentrations of: (1) 0, (2) 1.0� 108 CFU/ml, (3)
2.5� 108 CFU/ml, (4) 4.0� 108 CFU/ml, and (5) 11.0� 108 CFU/ml;
(b) MB (at a concentration of 15 lM) in 0.45% saline in the presence of
E. coli 25922 at the concentrations of: (1) 0, (2) 1.0� 108 CFU/ml, (3)
2.5� 108 CFU/ml, (4) 4.0� 108 CFU/ml, (5) 8.0� 108 CFU/ml.
[Bact.] x107 (CFU/ml)
0 50 100
AD/A
M
0.85
0.90
0.95
1.00
1.05
1.10
0 50 100
AD/A
M
0.55
0.60
0.65
0.70
0.75
0.80
0.85
(a)
Fig. 4. Dependence of the ratio of the intensity of the dimer maximum
to the monomer maximum, AD=AM, for TB (a) and for MB (b) on the
concentrations of following bacteria: (d) H. influenzae, (j) P. aeru-
ginosa, (m) E. coli 25922, (.) S. pneumoniae, (r) E. faecalis.
92 M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
we simplified our systems to consider the dye behavior
with increasing dye concentrations at a fixed bacterial
concentration at 108 CFU/ml. This was approximately
the same concentration of bacteria that had been used
for evaluating the photobactericidal efficacy of both
dyes [1]. Also, this fell into the concentration range of
bacteria corresponding to the descending branch in-cluding a bottom of the curves in Figs. 5 and 6. We
believe that under these conditions both dyes exist as
free monomers (Mfree) and dimers (Dfree) and bound to
polymer surface monomers (Mbound) and dimers
(Dbound) in bacterial suspensions. There is an equilib-
rium involving free and bound dye molecule, which is
supported by the existence of isosbestic points (Figs. 1b
and 2b). The equilibrium constant in such a system is
complex because of the participation of free and bacte-
rial cell surface bound monomer and dimer species of
the dye in the equilibrium process.
We measured the absorbance, A, at the monomer
maximum of each dye in the presence of different bac-
teria using a range of dye concentrations where theisosbestic point existed. The bacterial concentration was
the same for all experiments at approximately 108 CFU/
ml. In this case, Lambert–Beer�s law absorbance devia-
tion, DA, was presumed to be due to dye dimers induced
by bacteria. In the examined systems, the expression for
the absorbance of the bacteria–dye suspension in a 1-cm
path length cuvette is:
A ¼ eMf ½Mfree� þ eMb½Mbound� þ eDf ½Dfree� þ eDb½Dbound�;ð5Þ
where eMf , eMb, eDf and eDb are the molar extinction
coefficients of free and bound monomer and dimer
8.0 8.5 9.0 9.5 10.0
A 66
5 nm
0.6
0.7
0.8
0.9
Viable count (log10CFU/ml)8.0 8.5 9.0 9.5 10.0
A 66
5 nm
0.4
0.5
0.6
0.7
0.8
(a)
Fig. 5. Dependence of the MB absorption at 665 nm on the concen-
trations of gram-negative bacteria (a): (j) E. coli 35812, (m) P. aeru-
ginosa, (d) E. coli 25922, (.) H. influenzae, and gram-positive bacteria
(b): (j) E. faecalis, (d) S. pneumoniae.
(a)
8.0 8.5 9.0 9.5 10.0
A 63
1 nm
0.54
0.56
0.58
0.60
0.62
Viable count (log10CFU/ml)
8.0 8.5 9.0 9.5 10.0
A 63
1 nm
0.3
0.4
0.5
0.6
Fig. 6. Dependence of the TB absorption at 631 nm on the concen-
trations of gram-negative bacteria (a): (j) E. coli 35812, (m) P. aeru-
ginosa, (d) E. coli 25922, (.) H. influenzae, and gram-positive bacteria
(b): (j) E. faecalis, (d) S. pneumoniae.
M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98 93
species, correspondingly, and ½Mfree], ½Mbound], ½Dfree],
and [Dbound] are the concentrations of free and bound
monomer and dimer species of dye. Eq. (5) may be
simplified if we assume that eMf � eMb and eDf � eDb, ashad already been shown by Massari and Pascolini [13]
and Nakagaki et al. [14]. Then
A ¼ eM½Mtotal� þ eD½Dtotal�; ð6Þwhere total concentrations of monomers and dimers
are ½Mtotal� ¼ ½Mfree� þ ½Mbound� and ½Dtotal� ¼ ½Dfree�þ½Dbound� correspondingly, and eM and eD are the molar
extinction coefficients of the monomer and dimer of
each dye at the monomer band maximum.
In the following, we considered the effective dimer-ization constant, eff :KD, regardless of the final location
of dimer species (the solution or the bacterial cell sur-
face), instead of a more complex true dimerization
constant. We assumed that eff :KD would characterize the
relative ability of a dye to predominantly form dimers in
the presence of a variety of microorganisms. Using this
method, the values of eff :KD for MB and TB in 0.45%
saline without bacteria (when ½Mbound� ¼ 0, ½Dbound� ¼0) were identical to the true value of KD for these dyes in
0.45% saline.
The effective dimerization constant may be written as
eff :KD ¼ Dtotal
M2total
ð7Þ
The values of the concentration of monomer and
dimer species are related by the following equation
c0 ¼ ½Mtotal� þ 2½Dtotal�; ð8Þwhere c0 is the concentration of dye.
Using the analytical molar concentration of a dye in
the solutions, c0, and the appropriate A value in the
presence of bacteria it was possible to estimate graphi-
cally the values of eff :KD for all of the dye–bacteria
systems.
Our investigation has demonstrated that the depen-
dencies of 1=ffiffiffiffiffiffiffiDA
pvs. c0=DA described by Eq. (3), were
represented by the linear plots with the correlation co-
efficient of 0.93–0.99. Calculated from the slopes of
Table 1
Effective dimerization constants and the relative photobactericidal efficacies of MB and TB
Organisms Methylene blue Toluidine blue
Dimerization constant
(eff:KD � 103 M�1)
Relative PB efficacy
(MB lM)
Dimerization constant
(eff:KD � 103 M�1)
Relative PB efficacy
(TB lM)
No bacteria
Distilled water 4.7 N/a 10.3 N/a
0.45% Saline 7.5 N/a 24 N/a
Gram-positive
S. pneumoniae 49619 20 25 77 4
E. faecalis 51299 10.3 42 45 6
S. aureus 25923 10.2 42 35 27
Gram-negative
H. influenzae 49247 80 15 130 6
E. coli 25922 33 180 80 170
E. coli 35812 20 250 51 150
P. aeruginosa 27853 35 500 120 200
94 M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
these plots, the values of eff :KD for both dyes with and
without microorganisms are shown in Table 1. This data
was compared with the eff :KD, which were evaluated in
the commercial dye-microorganism solutions. The trend
of the dependence of the eff :KD values on both dye andbacteria was the same for both purified and commercial
dyes.
Table 1 shows that the calculated values of KD for
MB and TB in distilled water are equal to 4.7� 103 and
10.3� 104 M�1, respectively. The latter value agreed
with the value of KD for MB previously determined in
the literature [15]. The values of KD for MB and TB in
0.45% saline were 7.5� 103 and 24.0� 103 M�1, re-spectively. It can be seen that, in the absence of bacteria,
both dyes had a greater ability to form dimers in 0.45%
saline than in distilled water. These results are in ac-
cordance with reports [12,16] that have indicated the
capacity of inorganic anions, in particular chloride, to
induce the aggregation of phenothiazine dyes. In com-
parison with MB, TB had a greater ability to form di-
mers both in water and in 0.45% saline.When bacteria were added, the ability of both dyes to
form dimers sharply increased. The gram-negative or-
ganisms induced a far greater dimerization of the dyes
than the gram-positive bacteria. But in either case, the
TB effective dimerization constants were 2.5–4 times
higher than those of MB.
3.5. The effect of the wavelength of laser light on the
efficacy of bacterial killing using different concentrations
of MB and TB
In order to clarify whether or not the dye dimers are
involved in bacterial killing we studied the photobacte-
ricidal efficacy of MB and TB against several pathogens
at wavelengths corresponding to the dimer and mono-
mer peak of each dye. In these experiments, a series of
suspensions containing bacteria (P. aeruginosa, E. coli
25922 and S. aureus) at a fixed concentration and pro-
gressively increasing concentrations of the dyes were
used. We postulated that the dye dimer concentration
would rise when the total dye concentration increased.This would affect the ratio of the photobactericidal ef-
ficacy of dye induced by laser light at different wave-
lengths if the dimers were participants in the bacterial
damage. Figs. 7 and 8 demonstrate how the suscepti-
bility of the bacterial strains changed with the dye
concentration when the bacterial suspensions were ex-
posed to light at wavelengths corresponding to the di-
mer and monomer peak of each dye. It can be seen thatthe numbers of bacteria killed with each dye and light at
the dimer wavelength of excitation rose faster than those
killed at the monomer wavelength of excitation with
increasing dye concentrations. At the highest concen-
trations of MB and TB, the effect on the killing at the
dimer wavelengths even exceeded the effect produced at
the monomer wavelengths. These results demonstrated
the involvement of the dye dimers in bacterial killing.
4. Discussion
MB and TB are known metachromatic dyes [10,17].
In certain circumstances, such as increasing dye con-
centration, or the presence of anionic polymers (chro-
motropes) or inorganic salts, their spectra change due toelectrostatic and hydrophobic interactions between the
molecules of dye and the nearest partner and the inter-
action of the p-electrons between adjacent dye mole-
cules. These interactions result in dye aggregation,
which in some cases is restricted to dimerization [10].
The generally accepted criteria for these changes, known
as the metachromasy effect, are a shift in the absorption
maximum of the dye to a shorter wavelength (hypso-
0 100 200 300 400
Bac
teria
kill
ed (
log 10
CF
U/m
l)
0
1
2
3
4
5
MB (µM)MB (µM)
0 100 200 300 400
Bac
teria
kill
ed (
log 10
CF
U/m
l)
0
1
2
3
4
0 50 100 150 200
Bac
teria
kill
ed (
log 10
CF
U/m
l)
0
1
2
3
4
(a) (b)
Fig. 7. Effect of the wavelength of irradiation on the number of bacteria killed with combined action of MB at enhanced concentrations and laser
light at 610 nm (�) and 664 nm (j). Examined bacteria: (a) E. coli 25922, (b) P. aeruginosa, and (c) S. aureus 25923. Light intensity 100 mW/cm2,
light fluence 20 J/cm2 for (A) and (B). Light intensity 50 mW/cm2, light fluence 5 J/cm2 for (C).
M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98 95
chromic effect), a decrease in its molar absorbance at the
long wavelength absorption maximum (hypochromic
effect) and an increase in the AD=AM ratio. The previ-
ously described spectral behavior of increasing concen-
trations of MB and TB (Figs. 1a and 2a) demonstratesthat the metachromatic effect was caused by the increase
in the dye concentration. This is qualitatively the same
as that reported by Michaelis and Granick [10] and
Ruprecht and Baumgartel [15] for MB and TB in dis-
tilled water and results from the enhancement of the
dye dimerization when the MB or TB concentration is
increased.
Another type of metachromasy, the chromotropemetachromasy, induced mainly by anionic polymers, is
demonstrated in the solutions of both dyes in the pres-
ence of bacteria (Figs. 1–3). This is supported by the
hypsochromic and hypochromic effects observed in the
dye spectra in the presence of bacteria. We believe these
effects were promoted by the electrostatic interaction
between the cationic dyes and the negatively charged
polymers on the bacterial cell surface. This in turn in-duced the dimerization of adjacent dye molecules bound
to the anionic site of polymers and the formation of
bound dimers on the bacterial cell surface as witnessed
by an increase in the AD=AM (Fig. 5). Based on our
findings and those in the literature [18,19], it can be
hypothesized that the dye–dye interaction corresponded
to the descending branch of our graphs (Figs. 5 and 6).
The ascending branch observed in the gram-negative
bacterial systems could be associated with the formationof bound dye monomers that occupy chemically differ-
ent binding sites on biopolymers. The latter occurred
when the bacterial concentration was relatively high. In
this case, the monomer peak absorbance might occa-
sionally rise higher than the initial peak [18].
Thus, our findings demonstrate that bacteria induced
additional association of MB and TB on the surface of
the bacterial cell. The spectra of both dyes in the pres-ence of bacteria represented a superposition of mono-
mer and dimer bands with a different contribution of the
latter one. The monomer bands were located at 664 nm
for MB and 631 nm for TB. The dimer bands were lo-
cated in region of 605–610 nm for MB and 585–590 nm
for TB. A third band, which would correspond to higher
dye aggregates and is located in region of 560–580 nm
for MB [16] and of 530–560 nm for TB [20], was notobserved at bacterial concentrations used in our exper-
iment.
We believe that the metachromasy effect in the dye–
bacterial system is restricted to dimerization because of
TB (µM)
0 100 200 300
Bac
teria
kill
ed (
log 10
CF
U/m
l)
0
1
2
3
4
5
0 100 200 300
Bac
teria
kill
ed (
log 10
CF
U/m
l)
0
1
2
3
4
5
6
(a)
Fig. 8. Effect of the wavelength of irradiation on the number of killed
bacteria with combined action of TB at enhanced concentrations and
laser light at 585 nm (�) and 630 nm (j). Examined bacteria: (a)
E. coli 25922, (b) P. aeruginosa. Light intensity 100 mW/cm2, light
fluence 20 J/cm2 for (A) and (B).
96 M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98
the absence of subsidiary peaks other than the monomer
and dimer bands in the dye spectra. One might expectthat MB and TB would form higher aggregates on the
bacterial surface based on the Bradley and Wolf [19]
study of dye adsorbing on the surface of a single type of
polymer. However our results do not support this find-
ing. Probably, the difference between the dye behavior in
our bacterial systems is due to the complexity of the
bacterial cell surface vs. the relative simplicity of the
single polymer used by Bradley and Wolf [19].In the single polymer model system, the polymer has
a restricted number of dye binding sites. With a pro-
gressive increasing dye concentration the dye molecules
would occupy each of the polymer�s binding sites
forming higher aggregates [18]. Then, if the polymer
concentration were increased, the dye molecules would
redistribute and occupy new free binding sites, resulting
in a decrease in the number of bound dye molecules inan aggregate.
The surface of bacteria consists of many different
polymers that may be potentially involved in the inter-
action with dye. An increase in the polyanion concen-
tration and consequently in the numbers of various
polymers can lead to a decrease in dye aggregation [21].
The sequence of interaction between the dyes and dif-
ferent cellular polymers is probably determined by thechemistry of the vacant sites on a polymer. Initially the
dye molecules would occupy the sites of the polymer
that form the strongest complex with the dye. The
isosbestic point between the monomer and dimer peak
suggests there is equilibrium between the monomer and
dimer species bound to this polymer (Figs. 1b and 2b).
In this case the dimer peak shifted towards the blue
spectral region. When all of the binding sites on themost active polymer are occupied, the dye molecules
then can bind to polymers involved in fewer complexes.
This process is probably followed by the disappearance
of the isosbestic point. Because this complex is weaker
than previous one, the peak of the bound dimer must
shift slightly to the red spectral region relative to the first
one. The deviation of the AD=AM vs. the dye concen-
tration plot from linearity (Fig. 1c) may be associatedwith the formation of dye dimers bound to another
biopolymer rather than the formation of higher aggre-
gates. In the latter case, the further shift of the dimer
peak to the blue spectral region would be expected in the
presence of excess dye along with the depression of the
monomer band but this was not the case in our systems.
In addition, the restriction of the metachromasy effect to
dimer formation in the dye-single polymer model wasdemonstrated in the literature, in particular for MB
adsorbing on the surface of polysodium (4-vinylphe-
nylsulfate) [16] and oxidized starch [22]. From this it can
be deduced that the degree of dye aggregation depends
on the type of polymer inducing aggregation. There are
many reasons likely that may explain why dye interac-
tions with a variety of biopolymers could be restricted to
dye dimers.The extent of the metachromatic reaction between
each dye and bacteria may be characterized by the
degree of different qualitative spectral changes such as
hypochromism, hypsochromism, the AD=AM ratio
(Figs. 4–6), as well as the effective dimerization con-
stants. The depth of the lowest point of the graphs in
Figs. 5 and 6 indicates the relative efficacy of the
metachromatic reaction between the dyes and thegram-negative bacteria. The relative metachromatic
efficacy increased as follows: E. coli 35812<P. aeru-
ginosa<E. coli 25922<H. influenzae for MB and E.
coli 35812<E. coli 25922<P. aeruginosa<H. influ-
enzae for TB. Both dyes interacted the most with H.
influenzae. The analysis of the effective dimerization
constants for MB and TB in the presence of bacteria
allows us to conclude that the ability to form dimerswas greater for TB than for MB. In general, gram-
negative bacteria induced the dye dimerization more
significantly than gram-positive bacteria.
M.N. Usacheva et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 87–98 97
The relationship between the ability of MB and TB to
dimerize in the presence of bacteria and the photobac-
tericidal efficacy of dyes against pathogenic organisms
was of particular interest to us. In this study, the values
of the effective dimerization constant for both dyes inthe presence of bacteria were compared with the relative
photobactericidal efficacy of these dyes against each
previously evaluated bacteria [1]. Recall that the relative
dye photobactericidal efficacy was determined as the
minimum dye concentration corresponding to the onset
of the plot plateau, this is, the amount of the killed
bacteria vs. the dye concentration [1]. Therefore, the
minimum lethal concentration of TB that is effective ineradicating each corresponding microorganism is sig-
nificantly below that of MB but the value of eff :KD for
TB in the presence of each bacterial strain is far greater
than that for MB. This indicates a parallelism between
the dye photobactericidal efficacy and its ability to form
dimers. On the basis of our findings, TB is a more ef-
fective photobactericide and has a greater ability to di-
merize than that of MB. This pattern was consistentlyobserved in both gram-positive and gram-negative
bacteria. In addition, this trend between the photobac-
tericidal efficacy and the ability to dimerize was constant
for each dye in the presence of different gram-positive
organisms. So, the ability of MB and TB to form dimers
induced by gram-positive bacteria and the dye photo-
bactericidal efficacy increased as follows: S. aureus<E.
faecalis<S. pneumoniae. The same is true for the com-parison between the photobactericidal efficacy of the
dyes and the values of the AD=AM ratio characterizing
the extent of dimerization.
The correlation between the dye photobactericidal
efficacy and the dimerization was not consistent among
gram-negative bacteria. Gram-negative bacteria do not
always adhere to the relationship between a dye�s pho-tobactericidal efficacy and its dimerization ability. Inparticular, the combinations of dye and E. coli (two
different strains) do not adhere to this relationship.
Based on the literature [23], we believe mechanisti-
cally that the dye predominantly interacts with the li-
popolysaccharide-covered surface of gram-negative
bacteria. Among different bacterial strains, the variance
in the dye�s photobactericidal efficacy and its ability to
dimerize might be the result of the structural differencesin the lipopolysaccharides of the cell membrane in var-
ious bacteria [19]. It is known that the polymer config-
uration could affect the relative orientation of the dye
bound to an adjacent site and therefore affects sub-
sequent dye–dye interactions on the polymer surface
[24]. This could produce different concentrations of
bound dimers and bound monomers on the cell mem-
brane surface. This finding is confirmed in Figs. 5a and6a, which demonstrate that the different minimum po-
sitions on the graphs corresponding to beginning dye
dissociation on the bacterial cell surface, are different
among gram-negative bacteria. Nonetheless, alternative
possibilities involving the adsorption of the dye on other
biopolymers of the bacterial cell surface along with
lipopolysaccharides cannot be ruled out. In any case, the
general trend of a parallelism between a dye�s photo-bactericidal efficacy and its ability to form dimers is true
for gram-negative bacteria.
The clear evidence of the dye dimer participation in
the photosensitized killing of bacteria results from the
experiment estimating the effects of different wave-
lengths of irradiation in the presence of high concen-
trations of MB and TB on the photosusceptibility of
bacteria. In the present paper we do not determine themechanism of the photokilling of bacteria due to dye
dimers. Probably, the role of Type I and Type II
mechanisms changes with increasing dye concentrations
according to Bartlett and Indig [7]. In order to clarify
this, a separate investigation is required.
In summary, not only a dye�s hydrophobicity (as in-
dicated by the partition coefficient), but also whether the
dye monomers or dimers are bound to the bacterial cellmembrane surface and the ratio of their concentrations
are important characteristics in determining the photo-
bactericidal potential of a dye. Therefore, the dye dimer
species as well as the dye monomer species are respon-
sible for photodestruction of bacteria. It follows that the
knowledge of the photophysical characteristics of the
dye�s dimers along with those of the monomer species is
necessary to determine a dye�s efficacy as a photosensi-tizer.
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