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: [email protected] (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.
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