Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)
Science against microbial pathogens: photodynamic therapy approaches
Constance L.L. Saw1
1
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160
Frelinghuysen Road, Piscataway, 08854 New Jersey, USA. E-mail: [email protected]
There is an emerging area of research to identify the application of photodynamic therapy (PDT) as a means to kill
microbial pathogens. In fact, the first recorded observation in more than 100 years ago of photodynamic processes was
inactivation of microorganism. In this volume of the Formatex Microbiology book titled: Science against microbial
pathogens: communicating current research and technological advances, this chapter will focus on the use of
photosensitizer and light as anti-microbial agent against various microbes in different settings. The mechanism of action of
PDT inactivating microorganisms, anti-microbial photosensitizing agents and light sources used for eliminating
microorganisms will be covered. The success and challenges of using PDT to eradicate bacteria including antibiotic
resistant bacteria will be discussed.
Keywords PDT; against microbial; antimicrobial; photosensitizers; light
1. Introduction
There is an emerging area of research to identify the application of photodynamic therapy (PDT) as a means to kill
microbial pathogens. In fact, the first recorded observation in more than 100 years ago of photodynamic processes was
inactivation of microorganism, paramecia by Oscar Raab [1]. It was an incidental finding that in the presence of
acridine and illumination from a thunderstorm, resulted in the death of paramecia. He demonstrated that the death of
paramecia was possible only when light and acridine were present. Additionally, it was found that the toxic effect was
not due to heat [2] and the term ‘photodynamic reaction’ was coined in 1904 [3]. For detailed history of PDT, please
refer to existing literature [4]. PDT is based on the dual selectivity: (i) selective localization of photosensitizer targeting
at tumor or other lesion of interest and (ii) specific delivery of light eliciting the PDT at the target sites. Although PDT
was originally developed as a cancer therapy approach and it is still being developed [5], furthermore it has already
been developed as a treatment for age-related macular degeneration [6, 7], psoriasis [8, 9], barrett’s oesophagus [10, 11]
etc. Taking PDT in cancer as a classic example, after photosensitizers have accumulated in pre-cancerous and cancerous
tissues, then appropriate light of specific wavelength will be applied, causing the tumor undergo photo-induced
chemical reactions that cause apoptosis or necrosis [12]. Similarly, the uptake of photosensitizers and upon activation
by light, PDT induced destruction to pathological or infectious tissues/regions is the common working mechanism
among various diseases, including infection.
2. Mechanism of photodynamic reaction
There are two mechanisms involved in the photodynamic reaction / PDT. Upon irradiation with an appropriate
wavelength of light, a photosensitizer will be activated from its lowest energy ground state to a higher energy triplet
stage, which will further react directly with biomolecules to produce free radicals (Type I mechanism) or react with
oxygen to form reactive singlet oxygen, 1O2 (Type II mechanism) [13]. 1O2 is very reactive and has strong oxidizing
power that can produce potent cytotoxic effects [4]. In cells, it has been reported that 1O2 has a lifetime of less than 0.05
s and a maximal diffusion distance of 0.02 m from the site of its production [14], such characteristics explain in part
the specificity of photodynamic reaction for PDT in cancers or photodynamic inactivation (PDI) for killing of antibiotic
resistant bacteria. Therefore, conventionally it is thought that the targets of PDT are places where the photosensitizer is
localized [15]. However, recent findings suggest that even if some photosensitizers do not bind to the bacteria, yet can
cause PDI of bacteria if the distance between the singlet oxygen source and bacteria is close [16]. Please see further
discussion in section 5, mechanism of PDI damage to bacteria.
3. The need to search for new antibacterial therapeutics
Due to the emergence of antibiotic resistance bacteria, particularly with Staphylococcus aureus after the introduction of
methicillin [17], there is an urgent need to find alternative antibacterial therapeutics. While hospital-associated
Methicillin-resistant Staphylococcus aureus (HA-MRSA) was once observed in immunocompromised hosts, the rapid
emergence of community-associated MRSA (CA-MRSA) has caused a concern and the global epidemiology of CAMRSA appears to be heterogenous [18]; moreover, both HA-MRSA and CA-MRSA have been found to circulate in
community due to loose used of terms [19]. Since the fist report of vancomycin-intermediate Staphylococcus aureus
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Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
(VISA) in 1996 in Japan [20], the appearance of MRSA gaining new resistance against vancomycin has been observed
repeatedly in other countries including USA. Thus, there is an urgent need to develop new anti-microbial strategies in
addition to the implementation of some precautions such as educating the health-care providers, reduction of
unnecessary use of antibiotics and local disinfectants.
4. PDT against microbial pathogens
Various studies have shown that there is a fundamental difference in susceptibility to PDT between Gram (+) and Gram
(-) bacteria. In general, anionic and neutral photosensitizers are efficiently bound to Gram (+) bacteria, and they
photodynamically inactivate these Gram (+) bacteria effectively after illuminating with appropriate light, which is not
the case for Gram (-) bacteria [21]. The higher susceptibility of Gram (+) species is explained by their physiology where
their cytoplasmic membrane is surrounded by a relatively porous layer of peptidoglycan and lipoteichoic acid that
allows photosensitizers to cross [21]. For Gram (-) bacteria, they have an additional negatively charged outer membrane
that forms a protective barrier between the cells and the environment. Several approaches have been adopted to kill
Gram (-) bacteria using PDT.
4.1 Pre-treatment with ethylenediaminetetraacetic-acid (EDTA)
Treatment with EDTA will made Gram (-) cells lose up to 50% of their lipopolysaccharide (LPS) because of the
increased electrostatic repulsion between LPS caused by removal of divalent cations upon EDTA treatment. Bertoloni
et al. tested the pre-treatment with tris-EDTA could induce photosensitivity and the analysis on irradiated cells suggest
that the cytoplasmic membrane was an important target of the photoprocess [22].
4.2. Pre-treatment with polycation
Pre-treatment of E. coli and P. aeruginosa with polycation polymyxin B nonapeptide (PMBN) would assist the uptake
of deuteroporphyrin and with the exposure of light, inhibited the cell growth, which was not seen when PMBN was
used alone [23]. The main advantage of using PMBN is that this is an agent acting on the growing bacteria in their
neutral environment in the culture medium, it is known as a membrane disorganizing agent working on the structure but
does not cause metabolic leakage from the cells [24]. Unlike EDTA, PMBN does not cause the release of LPS, but
expands the outer leaflet of the outer membrane such that the expanded membrane will become more permeable and
facilitate the partition of the hydrophobic molecules from the external medium [21]. It was also shown that there was an
interaction between PMBN and deuteroporphyrin which was thought to be assisting the penetration of deuteroporphyrin
[23]. This method has been used to kill a multi-antibiotic resistant strain of a pathogenic aerobic Gram (-) bacteria,
Acinetobacter baumannii, significantly by deuteroporphyrin after pre-treatment with PMBN [25]. Importantly, it is
worth taking note that this particular strain of A. baumannii was resistant to the following antibiotics: ampicillin,
mezlocillin, piperacillin, cefoxitin, ceftriaxone, aztreonam, tetracycline, chloramphenicol, gentamycin, tobramycin,
amikacin, cirprofloxacin and morfloxacin.
4.3. Using photosensitizers with intrinsic positive charge
Because Gram (-) bacteria are resistant to PDI that are commonly lead to PDI of Gram (+) bacteria [21], and
photosensitizers bearing cationic charge or the use of agents such as PMBN that increase the permeability of the outer
membrane of Gram (-) are reported to be effectively increase killing Gram (-) bacteria [23], therefore, photosensitizers
with positive charge are preferred to combat Gram (-) bacteria [26-28]. Studies have shown that photosensitizers that
have more cation charges are better in killing the bacteria [29], the cationic porphyrins having three and four charges
were highly effective in PDI of both Gram (+) and Gram (-) bacteria, and the mono-cationic photosensitizer was the least
effective [30]. Though some studies show the contradicting findings that some di- and tricationic porphyrins were more
effective than tetracationic photosensitizers [31], other factors to be considered are
substitution groups on the ring [30].
charge distribution and nature of
4.4 Using cell-penetrating polymer to improve delivery of photosensitizers
This approach is similar to the treatment with EDTA, however, the photosensitizers are delivered together with the
cationic and often amphipathic, cell-penetrating polymers such as Tat peptide was used to conjugate
tetrakis(phenyl)porphyrin [32]. Though the Tat peptide is a positively-charged mammalian cell-penetrating peptide with
potent antimicrobial activity, the Tat-porphyrin conjugate was found to have the bactericidal effect for both Gram (+)
and Gram (-) bacteria, and dependent on the concentration and light dose [32]. The PDI of the bacteria was attributed to
the membrane destabilization synergistic action of the Tat peptide and PDT. Polycationic conjugates of chlorin e6 and
poly-L-lysine was made and successfully penetrate otherwise the impermeable outer membrane of Gram (-) [33].
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A. Méndez-Vilas (Ed.)
Cationic fullerenes were also used to deliver photosensitisers and after a short incubation followed by white light
illumination, they have a broad-spectrum antimicrobial activity and can rapidly kill more than 99.99% of bacterial and
fungal cells [34]. This approach of using cationic fullerenes was also successfully to rescue mice from lethal wound
infection with Proteus mirabilis and Pseudomonas aeruginosa [35], which has great clinical potential. Using this
delivery approach to deliver cationic photosensitizers appears to be the most popular and best approach towards PDI of
bacteria research recently.
5. Mechanism of PDI damage to bacteria
It has long been regarded that porphyrin binding to the cytoplasmic membrane is a prerequisite for the photsensitization
of Gram (+) [36] and Gram (-) bacteria [23], regardless whether they are aerobic or anaerobic [37]. However, there are
reports of PDI of Gram (-) bacteria in which that the photosensitizers did not even come into contact with the bacterium
or penetrate the bacterium to be effective. These reports suggest that if singlet oxygen can be generated in sufficient
quantities near the outer membranes of the cells, it can diffuse into the cells to cause damage to the vital structures [16].
Though it was found that Gram (+) bacteria were more sensitive than Gram (-) bacteria to the killing of singlet oxygen
[38], the role of singlet oxygen was confirmed by measuring the decrease in cytotoxicity as the distance between the
singlet oxygen source and the bacteria was increased [16]. It was also concluded that the structure of the cell wall thus
plays an important role in susceptibility to singlet oxygen [38].When using photosensitizer Rose Bengal for PDI of
different bacteria, it was again found that several Gram (+) species were inactivated about 200 times more quickly (99%
inactivation) than a Gram (-) Salmonella typhimurium strain [39]. As it was estimated that the diffusion distance of
singlet oxygen is approximately 0.02 m [14], the failure of some photosensitizers bind to Gram (-) species to produce
any killing, would mean that the reactive species produced on photoactivation were unable to diffuse towards the
critical target sites of the bacteria. Taking these observations into account, through generation of singlet oxygen [16, 38]
there are two basic mechanisms that have been proposed for the lethal damage caused to bacteria by PDI as reviewed by
Hamblin and Hasan [40]: (i) damage to the cytoplasmic membrane, allowing leakage of the cellular contents or
inactivation of membrane transport systems and enzymes and (ii) DNA damage.
6. Preclinical studies focused on animal models
Table 1 lists preclinical studies (animal models) of PDI to eradicate various bacteria, various photosensitizers tested by
photosensitizer alone, combination or conjugated forms and the outcomes are promising. Nowadays, methylene blue
seems to be the popular photosensitizer investigated which could be an extension of the previous success from in vitro
findings [41, 42]. However, other newer photosensitizers and delivered by carriers such as fullerenes are also being
developed. Table 1 is not meant to be extensive, but those relevant to the development of using PDT to kill bacteria and
significant findings are listed here. Readers are encouraged to always refer to the PubMed for latest findings.
Table 1 Preclinical studies of PDI to eradicate bacteria
Bacteria and
testing models
Pseudomonas
aeruginosa
infected the dorsal
skin in mice
Photosensitizer
MRSA
infected
the burn wounds
of guinea pigs and
biopsy
after
treatment to see
culture growth
Deuteroporphyrin IX
dihydrochloride
(deuteroporphyrin)
and
hemin
used
separately
or
in
mixture
670
Specific
and
nonspecific tin (IV)
chlorin e6-monoclonal
antibody conjugates
Light source and
treatment regimen
630 nm light with a
power density of 100
mW cm-2 for 1600
seconds
In
vitro
photosensitization was
performed on bacterial
cultures
in
liquid
medium
by
illumination with two
100 W incandescent
lamps that provided a
light intensity of 37
Em-2 s-1, equalling
1100 lux.
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Outcomes
Reference
Greater than 75% decrease in
the number of viable bacteria
at sites treated with a specific
conjugate, whereas normal
bacterial growth was observed
in animals that were untreated
or treated with a nonspecific
conjugate.
Deuteroporphyrin alone was
strongly bactericidal only after
photosensitization,
hemin
alone
was
moderately
bactericidal
but
light
independent, a combination of
both deuteroporphyrin and
hemin was extremely potent
even in the dark and did not
require
illumination
to
eradicate
the
bacteria.
[43]
[44]
Science against microbial pathogens: communicating current research and technological advances
_______________________________________________________________________________
A. Méndez-Vilas (Ed.)
A real-time
monitoring of
infection in
excisional wound
of the back of
mice, infected with
non-pathogenic
strain of E. coli
that expressed the
lux operon from
Photorhabdus
luminescens; these
cells emitted a
bioluminescent
signal that allowed
the infection to be
rapidly quantified,
using a low-light
imaging system
An
excisional
wound on the
mouse back was
contaminated with
one
of
two
bioluminescent
Gram-negative
species, Proteus
mirabilis
and
Pseudomonas
aeruginosa
Poly-L-lysine chlorine
6 conjugate
Oral candidiasis in
an
immunosuppressed
murine
model,
mimicking what is
found in human
patients;
SCID
mice
were
inoculated orally
with
Candida
albicans by swab 3
times a week for a
4-week period
Epidemic
methicillinresistant
Staphylococcus
aureus (EMRSA16) infected in
excisional
and
Methylene blue
C60
fullerene
functionalized
with
three
dimethylpyrrolidinium
groups (BF6)
Methylene blue
Mice were illuminated
with 665 nm light
delivered by a 1 W
diode laser coupled
with a 200 mm fiber
that gave a circular spot
of 3 cm diameter on the
mice and equally
illuminated wounds
with an irradiance of
100 mW cm-2. Mice
were given a total
fluence of 160 J cm-2 in
four 40 J cm-2 aliquots,
with imaging taking
place after each aliquot
of light. The total
illumination time was
27 min.
of
180
J
cm-2
broadband white light
(400-700 nm)
Mice received a topical
oral
cavity
administration of 0.05
mL methylene blue at
250-500 g mL-1, 10
min later, they were
irradiated with 664 nm
light from a diode laser
light with a cylindrical
diffuser; swabs were
taken to determine
colony forming unit of
bacteria.
360 J cm-2 of laser light
(670 nm) in the
presence of 100 g mL1
of methylene blue was
given.
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Reduction of more than 99%
of the viable bacteria was
noted after the mixture, an
effect that lasted for up to 24
h.
Light-dose dependent loss of
luminescence in the wound
treated with conjugate and
light, which was not seen in
untreated wounds. Treated
wounds healed as well as
control wounds, showing that
the PDT did not damage the
host tissue.
[45]
Fullerene-mediated PDT of
mice
infected
with
P.
mirabilis led to 82% survival
compared with 8% survival
without treatment (p < 0.001).
PDT of mice infected with P.
aeruginosa did not lead to
survival, but when PDT was
combined with a suboptimal
dose
of
the
antibiotic
tobramycin (6 mg kg-1 for 1
day) there was a synergistic
therapeutic effect with a
survival of 60% compared
with a survival of 20% with
tobramycin alone (p < 0.01).
Methylene
blue
dosedependent PDI of Candida
albicans was found: 250-400
g mL-1 reduced fungal
growth but did not eliminate
Candida albicans; 450-500 g
totally
eradicated
mL-1
Candida albicans from the
oral cavity.
[35]
Compared to the control,
significant 25-fold and 14-fold
reduction in the number of
viable EMRSA was seen in
excision
wounds
and
superficial
wounds
respectively.
[47]
[46]
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A. Méndez-Vilas (Ed.)
superficial
wounded mice
Oral candidiasis in
rats infected with
Candida albicans
Methylene blue
The light source used
was a galliumaluminum-arsenide
(GaAlAs) laser with a
wavelength of 660 nm,
output power of 100
mW, energy density of
245 J cm-2, and time of
69 seconds. The
irradiation laser was
applied to the tongue
dorsum by contact.
After 5 days, different
treatments were
administered: laser and
photosynthesizer
methylene blue 0.1 mg
mL-1 (L+P+); laser
only (L+P-);
photosensitizer only
(L-P+); and
physiologic solution
only (L-P-). Samples of
the oral cavity were
collected for a count of
colony-forming units
per mL.
The number of C. albicans
recovered from the oral cavity
of the rats was similar
between the groups (P =
0.106). The L+P+ group
showed fewer microscopic
lesions of candidiasis than the
L-P- group (P = 0.001),
suggesting that PDT showed
some effect on experimental
candidiasis in rats. The L+P+
group presented lower
proteinase activity compared
with the other groups, with
significant difference between
the groups L+P+ and L-P+ (P
= 0.018).
[48]
7. Clinical applications
Hamblin’s group has published an extensive review on the clinical applications of PDT for infectious diseases such as
acne, human papillomatosis virus, dental infection and gastric Helicobacter pylori infection [49]. A clinical report
published later shows the promising antifungal PDT effects of using methylene blue in 10 patients infected with
chromoblastomycosis [50]. The patients selected did not receive antifungal treatment in the past 6 months before the
treatment and did not present any additional disease or predisposing condition for other infections. A 20% methylene
blue preparation in Eucerin cream was applied to the skin lesions for 4 h under a gauze occlusive dressing, which was
then removed prior to the irradiation with red light emitting diode (LED, GaAlAs, wavelength absorption 660 nm,
energy dose of 28 J cm-2) for 15 min. All of the ten patients treated presented reduction in the compromised area after
six PDT applications, considering clinical and microscopical aspects. Though the complete healing was not achieved in
any patient, and the mycological tests were still positive, except for one patient; these findings show the potential of coadjuvant therapy with conventional antifungal chemotherapy which can improve patient quality of life by reducing the
infection area and degree as well as the length of time for antifugal therapy.
8. Conclusion and future perspectives
Though there are obvious advantages of using PDT to combat microbial pathogens such as killing drug resistant
bacteria, lack of induction of PDT resistance, the approach is not without challenges [49]. Some of the challenges are
cessation of PDI on bacteria when the light is turned off, and the selectivity for microbial cells over host. Several means
are being developed in this field, such as when using PDT to kill bacteria, in vivo bioluminescence imaging has been
developed to allow real-time monitoring of PDI of bacteria [45, 51], new animal models are being developed to test
against the standard antibiotics and new photosensitizers [52]. In order to make PDI treatment to be of clinical use, light
and photosensitizer must be able to be delivered successfully to the target tissue, therefore attempts of using fiber optics
to deliver light to activate nebulized methylene blue to lung have been made [53]. Antimicrobial PDT will become more
important in the future as antibiotic resistance is expected to continue to increase. Therefore, we expect the solution in
part to come from PDT using photosensitizers with suitable light source and appropriate delivery means.
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Science against microbial pathogens: communicating current research and technological advances
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A. Méndez-Vilas (Ed.)
Acknowlegements The support by Formatex Research Center, Spain for invitation and publication of this chapter is gratefully
acknowledged.
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