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Antimicrobial photodynamic therapy on drugresistant pseudomonas aeruginosainduced infection. an in vivo study
Photochemistry and Photobiology, 2012, 88: 590–595
Antimicrobial Photodynamic Therapy on Drug-resistant Pseudomonasaeruginosa-induced Infection. An In Vivo Study†
Maria C. E. Hashimoto1, Renato A. Prates1, Ilka T. Kato1, Silvia C. Nu
Martha S. Ribeiro*11Center for Lasers and Applications, Institute of Energetic and Nuclear Researches, IPEN–CNEN ⁄ SP,
2Institute of Health Researches, INPES-CETAO ⁄ SP, Sa˜o Paulo, SP, Brasil
3Department of Mathematical Sciences and Earth, Federal University of Sa˜o Paulo (UNIFESP),
Received 18 January 2012, accepted 2 March 2012, DOI: 10.1111/j.1751-1097.2012.01137.x
burns continues to be a great concern causing over 50% ofburn-related deaths in hospital environments (4).
Pseudomonas aeruginosa is considered one of the most important
A burn wound infection can evolve easily to high levels of
pathogens that represent life-threatening risk in nosocomial
bacteremia and consequently sepsis, leading these patients to
environments, mainly in patients with severe burns. Antimicrobial
death (5). Bloodstream infections are a major cause of
photodynamic therapy (aPDT) has been effective to kill bacteria.
morbidity and mortality in thermally injured patients. Studies
The purpose of this study was to develop a burn wound and
show that microorganisms isolated from burned patients in
bloodstream infection model and verify aPDT effects on it. In
specialized centers present an increasing resistance to anti-
vitro, we tested two wavelengths (blue and red LEDs) on a clinical
biotics, therefore this type of infection becomes very dangerous
isolate of P. aeruginosa strain with resistance to multiple
and difﬁcult to treat. In this setting, new alternatives of
antibiotics using HB:La+3 as photosensitizer. VerapamilÒ asso-
ciated to aPDT was also studied. In vivo, P. aeruginosa-infected
Antimicrobial photodynamic therapy (aPDT) involves the
burned mice were submitted to aPDT. Bacterial counting was
killing of organisms by light in the presence of a nontoxic
performed on local infection and bloodstream. Survival time of
photoactivable dye or photosensitizer (PS). Excitation of the
animals was also monitored. In this study, aPDT was effective to
PS by absorption of light of appropriate wavelength converts
reduce P. aeruginosa in vitro. In addition, VerapamilÒ assay
the PS to its photoactive triplet state, which in the presence of
showed that HB:La+3 is not recognized by ATP-binding cassete
oxygen will then generate reactive oxygen species, such as
(ABC) efﬂux pump mechanism. In the in vivo study, aPDT was
singlet oxygen and superoxide, resulting in cell death (6).
able to reduce bacterial load in burn wounds, delay bacteremia
Antimicrobial PDT has been suggested as an alternative
and keep the bacterial levels in blood 2–3 logs lower compared
approach for treating local infections as it has been shown that
with an untreated group. Mice survival was increased on 24 h.
a wide range of microorganisms, including resistant bacteria,
Thus, this result suggests that aPDT may also be a novel
viruses and yeasts, can be killed by aPDT (7,8).
prophylactic treatment in the care of burned patients.
Pseudomonas aeruginosa is a common nosocomial pathogen
that causes infections with a high mortality rate mainly due tothe high intrinsic resistance of microorganism to many
antimicrobials and the development of increased multidrug
Infections caused by opportunistic pathogens are the main
resistance in healthcare facilities (9). Antimicrobial PDT is a
cause of morbidity and mortality in immunocompromised
new approach for the treatment of drug-resistant bacterial
patients (1). Patients with severe burns present an immuno-
infections and the eradication in vitro and in vivo of P.
suppression condition and consequently higher susceptibility
aeruginosa using lethal photosensitization has been reported in
to infections due to the destruction of the cutaneous barrier
the literature (10–14). Indeed, Hamblin et al. developed a
that acts as protection against external agents and owing to the
mouse model to test the efﬁcacy of aPDT against infected
fact that coagulated and denatured proteins on the wound site
wounds induced by bioluminescent P. aeruginosa (12); how-
provide an optimal environment to microbial growth (2,3).
ever, so far, the effect of aPDT on MDR P. aeruginosa-
Accidents by burns deserve special attention as they are one of
infected burns in vivo has not been widely investigated.
the most common forms of trauma. Septicemia caused by
Within this context, this study was divided into two phases:
initially, we performed an in vitro assay and tested the viabilityof the use of aPDT in a clinical isolate of P. aeruginosa strainusing hypocrellin B: lanthanum (HB:La+3), which has shown
†This paper is part of the Symposium-in-Print on ‘‘Antimicrobial Photodynamic
to be an successful photosensitizer (15). After that, we
*Corresponding author email: firstname.lastname@example.org (Martha S. Ribeiro)
developed a burn wound infection model in mice and
Ó 2012 Wiley Periodicals, Inc.
Photochemistry and Photobiology Ó 2012 The American Society of Photobiology 0031-8655/12
investigated whether aPDT could reduce P. aeruginosa in the
Photochemistry and Photobiology, 2012, 88
infected site. Finally, we examined if aPDT applied in a single
culture, which was incubated for 30 min at 37°C. The cells were
or double session could prevent bloodstream infection and
harvested and washed in deionized water. Thereafter, they weresuspended in 10 l
M of HB:La+3 for 5 min and illuminated with red
LED at ﬂuences of 24, 48, 72 and 96 J cm)2. The experiments wereperformed in triplicate.
We used methylene blue (MB-150 lM) as a control since it has been
recognized as a substrate for efﬂux pumps (18). Preirradiation time was
Microbial strains. Pseudomonas aeruginosa strain used in this study was
5 min and a diode laser at k = 660 nm and intensity of 133 mW cm)2
isolated from a patient with septicemia following procedures deﬁned
was used to irradiate the cells. The cells were pretreated with Vp as
by CLSI (Clinical Laboratory Standards Institute). Antimicrobial
susceptibility test showed resistance to 12 types of antimicrobials,
Mouse model of full-thickness thermal burns. Adult female BALB ⁄ c
including reference drugs as cefotaxime, cefepime, meropenem and
mice (age 3–4 months; body mass of about 25 g) were used in the
ciproﬂoxacin (Table 1). Cells were cultured in tryptic soy broth (TSB)
study. All the animals were housed one per cage, maintained on a 12 h
at 37°C, and used for experiments in mid log growth phase to an
light and 12 h dark cycle, and had access to food and water ad libitum.
optical density at k = 600 nm of 0.6–0.8, which corresponds to 108
The procedures were approved by Institutional Ethic Committee on
colony forming unit (CFU mL)1; 16). This bacterial suspension was
Research Animal Care. The mice were anesthetized by intraperitoneal
centrifuged, washed with phosphate buffered saline (PBS), and then
injection of ketamine (90 mg kg)1) and xylasine (10 mg kg)1) cocktail
resuspended in PBS at a density of 3 · 108 CFU mL)1.
and received buthorfanol tartrate (2 mg kg)1) subcutaneously for pain
Photosensitizer and light source. Hypocrellin B with lanthanide ions
relief. The dorsal surface was shaved and burns were created by
(HB:La+3) was synthesized and characterized at Applied Biomedical
applying a preheated (95°C) steel device against dorsal surface of the
Optics Laboratory (UNIFESP ⁄ SP, Brazil; Fig. 1). Stock solutions of
mice for 10 s (nonlethal, full thickness and third degree burns). The
HB:La+3 at 1 mM were dissolved in PBS to a ﬁnal concentration of
device area was 6–6.5 cm2, corresponding to a burn of 14.5–15.7% of
10 lM. Light-emitting diode (LED; Eccoﬁbras ⁄ Sa˜o Carlos, Brazil)
the total body surface area according to Meeh’s formula (19).
emitting at k = 460 ± 20 nm and a LED (MMOptics ⁄ Sa˜o Carlos,
Establishment of infection. The infecting bacterium inoculum con-
taining 3 · 109 CFU mL)1 was prepared from stock cultures. Samples
were brought to room temperature, cultured in tryptic soy agar (TSA)
Photodynamic inactivation in vitro. HB:La+3 (10 lM) was used with
and growth at 37°C overnight. Infection was induced immediately
a blue and red LED in separated groups. Preirradiation time was 30 s
after burn creation by a subcutaneously inoculation of 100 lL of
and illuminations were carried out in 96-well plate for 1 min
(12 J cm)2), 2 min (24 J cm)2), 4 min (48 J cm)2), 6 min (72 J cm)2)
HB:La+3 lethality test. To verify if HB:La+3 could be lethal to
and 8 min (96 J cm)2). The experiments were performed in triplicate.
animals in tested conditions, 20 mice were submitted to burning and
HB:La+3 associated to efﬂux pump inhibitor on P. aeruginosa.
HB:La+3 (100 lL to 10 lM) was inoculated under the burn and
Pseudomonas aeruginosa cells were treated with verapamil (Vp), a
illuminated directly with blue LED and red LED, both with 24 J cm)2.
known calcium channel blocker and pump inhibitor (17), (Sigma–
All the animals were observed for 7 days.
Aldrich, MO) to verify if HB:La+3 could be a substrate of efﬂux
Antimicrobial PDT in burn wounds and bacterial counting in local
pumps. Final concentration of 10 lg L)1 Vp was added to bacterial
tissue. HB:La+3 (100 lL) was added 30 min after infection. After5 min to allow HB:La+3 to bind and penetrate into bacteria, thewounds were illuminated separately with blue LED or red LED
Table 1. Antimicrobials tested on clinical isolate of Pseudomonas
Mice were killed by cervical dislocation immediately after treatment
to measure the quantity of bacteria in local burn tissues and verify
local proliferation. Burn wound tissues were cut and homogenized in1 mL of PBS. Number of P. aeruginosa in 1 g of tissue (wet weight)
was determined by serial dilution plate count in triplicate on TSA
(triptic soy agar). Tissues surrounding the entire burn area were
excised using a sterile surgical scissor. The depth of the skin biopsy
extended all the way to the panniculus carnosus muscle on the back so
all epidermal and dermal components were removed. Immediately
following excision, the tissues were weighted and grinded with 1 mL of
sterile PBS, the aliquots were serially diluted in PBS to give dilutions of
10)1 to 10)5 times the original concentrations and streaked horizon-
tally on TSA plates as described by Jett et al. (20). Plates were
Antimicrobial PDT in burn wounds and bacterial load in blood-
stream. Antimicrobial PDT was performed as described elsehwere in a
single (30 min after infection) and double session (15 and 30 min after
infection) to verify if aPDT in burn wounds can avoid or delaybacteremia. In all experiments, the light source was placed vertically in
S, susceptible; R, resistent; I, intermediate.
contact with the animal skin, which was protected with sterile plastic
Figure 1. (A) Absorbance spectrum of hypocrellin B: lanthanum (HB:La+3); (B) Molecular structure of HB:La+3.
ﬁlm. To measure the quantity of bacteria in bloodstream, the mice
increase in bacterial killing of about 1 log compared with MB
were bled by the retro-orbital plexus 7, 10, 15, 18 and 22 h
postinfection in all groups. Numbers of CFU mL)1 in blood weredetermined by serial dilution plate count in triplicate on TSA.
A 200 lL of blood were collected and placed into 1.8 mL of TSB
Mouse model of third degree thermal burns and establishment of
with sodium sulfonate. After serial dilution from 10)1 to 10)4 times the
original concentration, 10 lL aliquots of each dilution were streakedonto an agar plate in triplicate and incubated to 37°C for 12 h to allow
In this in vivo study, we established the infection after full
Mouse survival follow-up and statistical analysis. The survival of the
thickness, third degree burns conﬁrmation by histology. We
animals of the previous experiments was monitored after aPDT. A log-
veriﬁed that mice submitted only to burns or bacterial
rank test veriﬁed the signiﬁcance of difference. For the other
inoculation survived the whole experimental period, whereas
experiments, bacterial colonies were counted and converted into
mice submitted to burns and contamination died within 18 h.
CFU for statistical analysis. Values are given as means, and error bars
We then tested HB:La+3 lethality following aPDT and
are standard deviations. Statistical comparison between means wascarried out using one-way analysis of variance (ANOVA). Mean
observed that burned not infected mice that received HB:La+3
comparisons were performed with the Tukey’s test. Signiﬁcance was
alone or associated to blue and red LED survived during the
Antimicrobial PDT in burns and bacterial load in situ
No bacterial decrease was observed in burned mice only
Results for lethal photosensitization of a clinically isolated
irradiated or inoculated with HB:La+3 as well as in burned
P. aerugionosa using HB:La+3 are shown in Fig. 2. After
mice without treatment (control group). However, aPDT using
2 min, corresponding to 24 J cm)2 LED ﬂuence, ca 5 logs of
HB:La+3 combined to blue or red LEDs showed about 2 logs
killing were achieved. This statistically signiﬁcant decrease in
of bacterial reduction (Fig. 4). In fact, the initial mean value of
bacterial load (P < 0.05) persisted until 8 min of irradiation(96 J cm)2). No statistically signiﬁcant differences were ob-served on decline of bacterial load between blue and red LED.
Control group (no irradiation and photosensitizer), LEDsgroups (only blue or red irradiation by 8 min) and HB:La+3group (only 10 lM photosensitizer for 8 min) did not show anybactericidal effect.
In Fig. 3, we show the effects of aPDT on bacterial reductionusing HB:La+3 and MB associated to Vp. After 24 J cm)2light dose had been delivered (2 min), there was a statisticallysigniﬁcant reduction of about 5 logs for HB:La+3 group, aswell as HB:La+3 combined to Vp (P < 0.05). This reduction
Figure 3. Effect in vitro of HB:La+3 and MB associated to verapamil
was kept until 96 J cm)2 (8 min) for both groups. No
on the reduction of P. aeruginosa exposed to red LED (LED intensity:
statistically signiﬁcant differences were observed between
200 mW cm)2) and red laser (laser intensity 133 mW cm)2), respec-
HB:La+3 group and HB:La+3 associated to Vp. On the other
tively, depending on the exposure time. Bars represent SD.
hand, using MB and Vp, we observe a statistically signiﬁcant
Figure 2. Effect in vitro of HB:La+3 on the reduction of P. aeruginosa
Figure 4. Comparison of viability of bacterial cells recovered in situ
exposed to blue and red LED depending on the exposure time (LED
from untreated infected burns with those treated by blue or red aPDT.
intensity: 200 mW cm)2). Bars represent SD.
Each data point is a mean ± SD of ﬁve animals.
Photochemistry and Photobiology, 2012, 88
Figure 6. Comparison of viability of bacterial cells recovered fromblood of untreated mice with those treated by blue or red aPDT at two
Figure 5. Comparison of viability of bacterial cells recovered from
sessions. Each data point is a mean ± SD of ﬁve animals.
blood of untreated mice with those treated by blue or red aPDT at onesession. Each data point is a mean ± SD of ﬁve animals.
infectious load collected from ﬁve animals was 7.8 logs.
Immediately after aPDT, the mean infectious burden wassigniﬁcantly reduced to 6.0 and 6.2 logs for blue and red LED,respectively. Despite statistically signiﬁcant differences be-tween control and treated groups (P < 0.05), no signiﬁcantdifferences were detected between blue and red LEDs. Theseanimals were submitted only to one session of aPDT.
Antimicrobial PDT in burns and bacterial load in bloodstream
Figure 5 demonstrates the number of viable cells of P. aeru-ginosa in bloodstream after aPDT on infected burns. As canbe observed, 7 h after bacterial inoculation, the control grouppresented an average of about 6 logs of bacterial load in thebloodstream, while aPDT blue and aPDT red groups did not
Figure 7. Survival plot for blue and red aPDT after one and two
present any bacterial growth at this time. Antimicrobial PDT
blue and aPDT red groups showed bacteremia only 10 h afterbacterial inoculation, and the bacteria levels were about 2 logslower compared with control group at this time. A 15 h after
bacterial inoculation, control group presented 7.1 logs ofbacterial burden, whereas aPDT blue and aPDT red groups
We show in Fig. 7 fraction surviving curves for untreated and
maintained about 4.8 logs. After 18 h, although bacterial
aPDT-treated mice. It can be observed that all animals of the
levels in aPDT groups increased to 5.5 logs, they were kept
control group died within 18 h of bacterial inoculation. In
2 logs lower than the control group. This behavior was
contrast, aPDT groups survived until 48 h following infection.
observed up to 22 h after the bacterial inoculation. There
An interesting remark is that aPDT-blue treated mice survived
was not any statistically signiﬁcant difference when control,
longer than aPDT-red treated mice. In fact, only 10% of the
HB:La+3 alone and LED alone groups were compared among
animals from aPDT red group survived within 36 h after
them as well as between aPDT blue and aPDT red groups.
infection, but in aPDT blue group 70% of the animals survived
Nonetheless, signiﬁcant differences were detected when aPDT
within this same period. However, despite signiﬁcant differ-
groups were compared with control, HB:La+3 and LEDs
ences between control and aPDT groups (P < 0.05), no
statistically signiﬁcant differences were observed between
We then investigated if the observation above described one
aPDT blue and aPDT red groups (P > 0.05).
session of aPDT delayed bacteremia in mice and retained ca2 logs bacterial load level lower than control group, could be
more effective when two sessions of aPDT were applied.
Disappointingly, two sessions of aPDT were not successful to
In this study, we used a local and bloodstream murine model
avoid bacteremia and reverse bacterial load in bloodstream
of a clinical isolate of P. aeruginosa to induce infection in
(Fig. 6). However, bacterial recovering following two sessions
burned mice. Our results indicate that the model was suitable
of aPDT was signiﬁcantly lower than control group (about
for studying aPDT and showed success in delaying bacteremia
3 logs, P < 0.05; compare Figs. 5 and 6).
In previous pilot studies, we investigated in vitro and in vivo
Consequently, aPDT delayed bacteremia and kept bacterial
effects of methylene blue (MB) and toluidine blue (TBO),
load in the bloodstream lower than untreated group until the
respectively (data not shown) and both photosensitizers
end of the experiment. Double session of aPDT diminished
showed a limiting bacterial killing activity. We then investi-
even more bacteria in the bloodstream, but survival of aPDT-
gated the effects of HB:La+3 on P. aeruginosa susceptibility
treated mice in single or double session was similar.
in vitro. Hypocrellins have been shown to act as photosensi-
In common with our results, mice decease was not avoided
tizer agents because they exhibit rapid preparation, easy
when excisional wounds infected with P. aeruginosa were
puriﬁcation, low aggregation tendency, high quantum yield
treated with BF6-mediated aPDT (29). On the other hand, in a
for singlet oxygen generation and fast clearance in vivo (21). In
less-aggressive P. aeruginosa wound infection, 90% of mice
addition, HB:La+3 enhances singlet oxygen quantum yield, it
survived following aPDT treatment using a poly-L-lysine-ce6
does not present photobleaching and it was able to reduce
conjugate (12). In another reported study, similar bacterial
100% Candida albicans following 30 s of exposure to red or
reduction in situ (more than 2 logs) was observed when burn
wounds infected with P. aeruginosa were treated with
Our data show that 10 lM of HB:La+3 combined to blue or
red LED achieved about 5 logs of killing after 2 min of
Pseudomonas aeruginosa is a highly virulent and invasive
irradiation without complete eradication, different than Tof-
bacterium that rapidly reaches the bloodstream causing sepsis,
foli et al. (22). It is well known that Gram-negative bacteria
but previous studies have demonstrated that aPDT can
are more resistant to aPDT. In particular, P. aeruginosa is one
inactivate P. aeruginosa virulence factors (10,30,31). We
of the Gram-negative bacteria more difﬁcult to kill by aPDT
suggest that inactivation of virulence factors responsible for
(23,24). In fact, a few reports show a high index of P. aeru-
bacterial invasion and tissue damage is contributing to a better
ginosa killing using phenothiazines in vitro (13,14,23), and in
response of mice face to infection and, for this reason, it was
those cases, higher photosensitizer concentrations (13,23) and
possible to delay bacteremia, maintaining lower bacterial levels
longer exposure times (about 30 min; 13,14) are necessary. P.
in bloodstream and increasing mice survival. Nonetheless,
aeruginosa used in our study was recovered and isolated from a
despite bacterial load in bloodstream has been diminished even
hemodynamic catheter of a sepsis patient, and it was resistant
more (about 1 log) with two sessions of aPDT on burn wound,
to 12 groups of antibiotics. This wild strain also produces more
the interval for the second treatment was not enough to
mucus compared to ATCC 27853 strain (data not shown).
prevent recurrence and reduce bacterial load in the blood to a
This characteristic can prevent HB:La+3 diffusion through the
cell and can protect P. aeruginosa against aPDT (25). In
Regarding the light source, blue and red LEDs showed the
addition, literature supports that efﬂux mechanisms are the
same behavior in bacterial viable cells recovered from mice
major components of microbial resistance and inhibitors of
blood. Interestingly, despite no statistically signiﬁcant differ-
efﬂux pumps could enhance bactericidal effect by aPDT
ences between aPDT red and aPDT blue groups (P = 0.0502),
our results showed that aPDT-blue treated mice started to die
The resistance-nodulation-cell division (RND) pump family
5 h later than aPDT-red treated mice. Furthermore, survival in
plays a key role in resistance of P. aeruginosa. However, ABC
aPDT red group was 60% less than in aPDT blue group 36 h
pumps can also be presented in this Gram-negative bacterium
after infection. Although we do not have data to support it,
in a small number (27). For this reason, we decided to verify
this increase in the survival of aPDT-blue treated mice may
this pump activity using methylene blue, a substrate for ABC
result from two effects: bacterial death or host defense. As
pumps and verapamil (28). As expected, a small but statisti-
absorption of HB:La+3 is more intense in blue region (see
cally signiﬁcant increase in bacterial killing was observed
Fig. 1), a plausible hypothesis for our results is that more PS
(about 1 log). We evaluated whether HB:La+3 could also be a
molecules are activated following blue LED irradiation with
substrate for ABC pumps. Contrary to MB, our results clearly
consequent sublethal damage to bacteria. Indeed, it has been
demonstrated that Vp associated to HB:La+3 did not enhance
reported that the oxidative damage promoted by sublethal
P. aeruginosa killing in vitro. This ﬁnding suggests that
photodynamic inactivation inhibits virulence determinants and
HB:La+3 is not recognized by ABC efﬂux pumps.
reduces in vivo pathogenicity of Candida albicans (32). Further
The next set of experiments aimed to develop an animal
studies are necessary to conﬁrm our proposition.
model of infection to investigate the effects of aPDT using
From the data presented in this study, it can be concluded
HB:La+3 combined to blue or red LEDs on P. aeruginosa-
that killing in vitro of P. aeruginosa using HB:La+3 associated
infected burns. It is worth mentioning that, in a pilot study, we
to blue LED or red LED was similar, and ABC efﬂux pump
inoculated a clinical isolate of P. aeruginosa onto the burns,
inhibitor did not increase killing when associated to HB:La+3.
following methodology reported by Huang et al. (13). How-
The burn model used in this study induced a third degree burn
ever, in this case, it was not possible to establish septicemia.
and it was possible to develop local and disseminated infection
Hence, we decided to inject bacterial cells subcutaneously (19).
by subcutaneous inoculation of a clinical isolate of P.
Once the mouse model of infection was established, we tested
aeruginosa with resistance to multiple antibiotics. HB:La+3
HB:La+3 lethality in vivo. Our ﬁndings did not show any lethal
under the parameters tested was not noxious to mice.
effect on mice by the use of HB:La+3 since only infected micedied within 18 h.
We also observed that mice aPDT-treated showed a
statistically signiﬁcantly lower bacterial burden on burn
Put together, these ﬁndings suggest that aPDT could be an
wounds and in bloodstream. In situ, bacterial burden was
alternative approach for the treatment of P. aeruginosa-
reduced by 2 logs following aPDT, using blue or red LED.
infected burns, as it was able to reduce P. aeruginosa in situ,
Photochemistry and Photobiology, 2012, 88
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Press Release Renault and ALD Automotive announce their commitment to zero emissions mobility ALD Automotive and Renault are today announcing their partnership project for an electric vehicle. ALD Automotive, a major international player in financing and vehicle fleet management, has confirmed its interest in Renault zero emissi
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