2.1.

Bacterial Strain and Culture

We used a strain of P. aeruginosa, ATCC 27853 (Microbiologics, Inc., Minnesota), which was reported to be resistant to several antibiotics, including penicillin G, kanamycin, and vancomycin.¹¹ A stock bacterial suspension was maintained by growing the bacteria in the brain heart infusion broth (05508, Nissui Pharmaceutical Co., Tokyo, Japan). A suspension of P. aeruginosa was cultured in a static incubator for 18 h and a 37°C shaking incubator at $140\text{strokes}/\min$ for 6 h. The bacteria were then centrifuged at 3000 rpm for 20 min, resuspended in phosphate buffer saline (PBS) at a density of $3\times {10}^9\mathrm{CFU}/\mathrm{ml}$, and stored in a deep freezer at $-80°\mathrm{C}$ before use.

2.2.

Photosensitizer and Additives

A hydrophilic cationic PS, MB (319112; Sigma Aldrich, St. Louis, Missouri), was used at a concentration of $965\mu \mathrm{M}$ in saline. Hydrophilic cationic PSs can bind with negatively charged lipopolysaccharide molecules in the outer membrane of Gram-negative bacteria.¹² To the MB solution, 10-mM EDTA disodium dihydrate [2NA(EDTA.2Na)] (345-01865; Dojindo Laboratories Co., Kumamoto, Japan), 10% ethanol (057-00451; Fujifilm Wako Pure Chemical Co., Osaka, Japan), and 25% DMSO (043-07216; Fujifilm Wako Pure Chemical Co., Osaka, Japan) were added. The concentrations of ethanol, at 10%, and EDTA, at 10 mM, were based on the results of our previous in vitro study on the aPDT against P. aeruginosa in biofilms.¹³ Meanwhile, the concentration of DMSO was determined based on the observation that 25% DMSO displayed no dark toxicity on the bacteria in the biofilm in vitro (data not shown). Here, we compared the efficacies of aPDT with only MB; aPDT with MB, EDTA, and ethanol (MB/EDTA/ethanol); and aPDT with MB, EDTA, ethanol, and DMSO (MB/EDTA/ethanol/DMSO). In addition, we assessed the efficacies for the rats treated with only saline (control) and those treated with the MB/EDTA/ethanol/DMSO solution in the dark to assess the dark toxicity of the PS mixture, and the results were compared with those for the above-described three aPDT groups.

2.3.

Light Source

A $3\times 6$ array of LEDs with a center wavelength of 665 nm and a spectral width of 17.2 nm in FWHM (Effect Corporation, Tokyo, Japan) was used as the light source for aPDT [Fig. 1(a)]. The area of burned skin ($2\times 6{\mathrm{cm}}^2$), on which the PS mixture solution was applied, was illuminated at a distance of 2.5 cm from the surface of the glass plate covering the LEDs. The average light intensity, which was measured using a powermeter (Vega P/N, 7Z01560; Ophir Optronics Solutions Ltd., Jerusalem, Israel), was $45\mathrm{mW}/{\mathrm{cm}}^2$ at the wound surface. The intensity distribution on the wound surface was highly uniform with a variation smaller than 5%.

Fig. 1

Setup and timeline of the in vivo aPDT experiment. (a) A rat under LED illumination. (b) The regions for PS mixture applications ($2\times 6{\mathrm{cm}}^2$) and infections ($1\times 5{\mathrm{cm}}^2$) on the burn wound ($4\times 10{\mathrm{cm}}^2$). (c) The timeline of the experiment.

2.4.

Establishment of Burn Wound Infection

All animal procedures were approved by the Ethics Committee of Animal Care and Experimentation in the National Defense Medical College, Japan (permission number 17020). Male Sprague–Dawley rats (7 to 8 weeks old and 210 to 280 g) (Japan SLC, Inc., Shizuoka, Japan) were used. First, the rats were anesthetized by the intraperitoneal injection of $50\mathrm{mg}/\mathrm{kg}$ pentobarbital. Then, their dorsal hair was clipped with a hair clipper and shaved with an electric shaver and washed. Before a burn was induced, $0.01\mathrm{mg}/\mathrm{kg}$ buprenorphine was intramuscularly injected into the rat thigh muscle for pain relief. A deep burn of $\sim 20\%$ of the total body surface area ($4\times 10{\mathrm{cm}}^2$) was induced by exposing the dorsal skin of the rat to 98°C heated water for 10 s through a Walker–Mason template.¹⁴ Immediately after the burns were induced, the rats were resuscitated with an intraperitoneal injection of saline solution at $25\mathrm{mg}/\mathrm{kg}$. At 15 min postburn, the infection was established by applying a $100\text{-}\mu \mathrm{l}$ suspension of P. aeruginosa at ${10}^8\mathrm{CFU}/\mathrm{ml}$ onto the central area ($1\times 5{\mathrm{cm}}^2$) of the burn [Fig. 1(b)].

2.5.

Grouping of Animals, aPDT, and Humane Endpoint

The rats with burn wound infection were divided into five treatment groups with four ($n=4$ for each group): (1) only saline (control), (2) a PS mixture (MB/ethanol/EDTA/DMSO) in the dark (PS-only), (3) aPDT with only MB (aPDT I); (4) aPDT with MB/ethanol/EDTA (aPDT II), and (5) aPDT with MB/ethanol/EDTA/DMSO (aPDT III).

Figure 1(c) shows the timeline chart of the experimental procedures for the aPDT I to III groups. At 30 min after infection, a $125\text{-}\mu \mathrm{l}$ PS mixture (only MB, MB/ethanol/EDTA, or MB/ethanol/EDTA/DMSO) was applied onto an area of $2\times 6{\mathrm{cm}}^2$ at the center of the burn, including the infected area [Fig. 1(b)]. Ten minutes later, the burn area was illuminated with the LED light for 20 min. The PS mixture application and illumination were consecutively repeated three times as one daily treatment. For the control rats, saline was applied, whereas for the PS-only rats, the PS mixture (MB/ethanol/EDTA/DMSO) was applied; these groups had the same timeline as the three aPDT groups but received no illumination. The treatment was conducted every 24 h for 3 days (days 0, 1, and 2). After the daily treatment, the wound surface was covered with a moist wound dressing sheet (Zuiko Medical Corp., Osaka, Japan) and wrapped in bandages (Daiei Co., Ltd, Osaka, Japan) until the next day’s treatment for all groups of rats. Meanwhile, all of the rats were kept one per cage and allowed easy access to water and food. Finally, the humane endpoint was set to the loss of more than 25% of the body weight on day 0.

2.6.

Sample Collections

Before and after each day’s treatment, swabbing of two randomly chosen regions of interest (ROIs) on the infected wound surface [Fig. 1(b)] was done with two sterile saline-wetted cotton swabs to evaluate the CFUs on the wound surface. Immediately after the treatment on day 2, two biopsy samples that included all skin layers and subcutaneous tissue, were randomly collected from the infected area ($1\times 5{\mathrm{cm}}^2$) on each rat using an 8 mm in diameter biopsy punch. The biopsy samples were used for histological analysis to assess bacterial depth distributions.

2.7.

Colony-Forming Assays

The CFUs on the wound surface were assessed by obtaining two cotton swabs, one taken from one ROI and the other taken from a different ROI, and inserting them separately into 1 ml of sterile saline and vortexed to detach the bacteria. The halves of those suspensions, at $500\mu \mathrm{l}$ each, were mixed together to make a 1-ml sample. Each surface swabbing sample was serially diluted with saline and spread onto nutrient agar media (05514; Nissui Pharmaceutical Co., Tokyo, Japan) in a dish. Since we previously observed that for the same P. aeruginosa strain, the nutrient agar media showed a higher detectability than the selective media [nalidixic acid-cetrimide (NAC) agar], we used nutrient agar media in this study. When bacteria other than P. aeruginosa were involved in the samples, we could distinguish them on the basis of the morphologies, sizes, and colors of their colonies. In such cases, we subcultured those colonies on an NAC agar plate for identification. We excluded those bacteria that were confirmed not to be P. aeruginosa from evaluation. However, we seldom observed other types of bacteria, probably indicating a clean experimental environment. After incubating the dishes at 37°C for 18 h, the colonies on the media surface were counted to obtain the CFU/ml values.

2.8.

Histological Analysis of Bacterial Depth Distributions

The depth distributions of the bacteria in the burned tissues were determined with Gram staining, a fast and effective and standard staining technique for examining clinical biopsy samples. Gram staining technique can identify both large aggregates of clustered bacteria and individual detached bacteria in biofilms.¹⁵ One problem of Gram staining is the relatively low color contrast between bacteria and their background tissue. We expected that fluorescence-based detection techniques, such as immunofluorescence imaging¹⁶ and live imaging using GFP-tagged P. aeruginosa,¹⁷ enabled higher-contrast detection. However, our preliminary data showed that autofluorescence considerably disturbed the detection of the GFP fluorescence signals. We also tested the use of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and CFDA (5(6)-carboxyfluorescein diacetate) to detect bacteria in the tissues, but similar problems (disturbance by autofluorescence) arose. Therefore, we chose the Gram-staining technique. It should be noted that both alive and dead bacteria could be detected by this method.

The biopsied samples obtained from the wound of each rat were fixed in 4% paraformaldehyde in a PBS (163-20145; Fujifilm Wako Pure Chemical Co., Osaka, Japan) overnight and preserved in 70% ethanol until paraffin embedding. Then, the specimens were processed in Tissue-Tek VIP^® 6 (VIP 6-J0; Sakura Finetek Japan Co., Ltd., Tokyo, Japan) within a week, embedded in paraffin using a Tissue-Tek TEC Plus Dispensing Console (TEC-P-DC-J0; Sakura Finetek Japan Co., Ltd., Tokyo, Japan), sliced into $7\text{-}\mu \mathrm{m}$ sections using a sliding microtome (TTM-200; Sakura Finetek Japan Co., Ltd., Tokyo, Japan), and placed on glass slides (MAS-02; Matsunami Glass Ind. Ltd., Tokyo, Japan).

The sections were Gram-stained with Gram–Hucker’s Stain solution I (4116-2), II (4117-2), and III (4118-2) (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) after de-paraffinization with xylene and a decreasing ethanol gradient. Next, an increasing ethanol gradient and xylene were used to dehydrate the sections. The specimens were then covered with a cover glass and observed under a Carl Zeiss Inverted Microscope (Axiovert 200; Carl Zeiss, Göttingen, Germany) at $1000\times$ magnification to assess the depth distributions of the bacteria. For each Gram-stained section, four vertical regions with a width of $600\mu \mathrm{m}$ and a minimum horizontal separation of $700\mu \mathrm{m}$ were analyzed. In each vertical region, a narrower vertical ROI with a width of $120\mu \mathrm{m}$ was randomly chosen, and the number of bacteria was counted for every $100\mu \mathrm{m}$ depth section from the region on the skin surface down to the muscle layer. For cluster forming bacteria, we estimated the approximate numbers. When the total numbers of bacteria for each depth section were exceeded, e.g., 200 and 300, we scored the numbers as “$>200$,” “$>300$,” respectively. As described above, two biopsied samples were obtained from each rat, and bacterial counting was conducted for eight ROIs in each rat. Thus, the total number of ROIs was 32 for each group of rats ($n=4$), and the averaged numbers for 32 ROIs were used to compare the groups.

2.9.

Statistical Analysis

Statistical analysis of the data was performed using GraphPad Prism 6 software. Nonparametric two-way ANOVA was used to compare the average numbers of bacteria for each depth section between the groups. Statistical significance was determined for the results of each group in comparison with those of the control group or those of the aPDT I group. A difference with a $P\text{-}\text{value}<0.05$ was considered statistically significant.

3.1.

Bacterial Numbers on the Wound Surface

No rats reached the defined humane endpoint in this study. Figure 2 shows the time courses of the average numbers of CFUs on the wound surfaces, which were evaluated for swabbing samples, in all groups of rats. Before the treatment on day 0, the average CFUs were similar for all groups at around $4.6{\log }_{10}\mathrm{CFU}/\mathrm{ml}$. After the treatments, no bacteria were detected in any treatment groups, including the PS-only group, indicating the efficacy of aPDT with any mixtures as well as the dark toxicity of the PS mixture. However, the bacteria on the wound surfaces were rapidly regrown by the timepoint before the treatment on day 1 for all treatment groups, although their numbers were smaller than that of the control group by 0.9 to $1.7{\log }_{10}$. The bactericidal effects of the treatments on day 1 were different depending on the groups; CFU reductions were $1.6{\log }_{10}$ for aPDT I (MB only), $3.4{\log }_{10}$ for aPDT II (MB/ethanol/EDTA), and $4.2{\log }_{10}$ for aPDT III (MB/ethanol/EDTA/DMSO). The efficacy of aPDT with only MB was lower than that of the treatment with only the PS mixture, i.e., the dark toxicity. After the treatments on day 1, efficient bacterial regrowth occurred again in all treatment groups. At the timepoint before the treatment on day 2, the bacterial numbers for the PS-only, aPDT I, and aPDT II groups were similar to that of the control group, showing monotonical increases in the number of bacteria at the timepoints before the treatment of each day during days 0 to 2. On the other hand, the bacterial number for the aPDT III group was slightly smaller than that at the timepoint before the treatment on day 1. For aPDT group III, the treatment on day 2 greatly reduced the number of bacteria ($>5.3\text{-}{\log }_{10}$ reduction), resulting in no bacterial detection on the wound surface.

Fig. 2

Time courses of bacterial numbers on the wound surface in different groups of rats ($n=4$ in each group). On each day, both of the numbers before (squares) and after (circles) treatment are shown except for the control; the corresponding two plots are connected with a vertical dashed line.

For other treatment groups, on the other hand, bactericidal effects of the treatments on day 2 were limited to $\sim 1.2\text{-}\log$ reduction, indicating time-dependent decreases in the efficacy. These results showed the importance of adding DMSO to the PS mixture in controlling the numbers of bacteria on the wound surfaces.

3.2.

Bacterial Depth Distributions

Figure 3 shows the representative images of the Gram-stained sections of the wounds after the treatment on day 2 for all groups, with three columns of images taken at different magnifications for each group. The magnification of the images in the first column image was $200\times$. A depth scale with box segments with a depth of $100\mu \mathrm{m}$ and a depth of $120\mu \mathrm{m}$ is displayed, and the depth origin ($0\mu \mathrm{m}$) is set at the base of the epidermis (black dotted line). The negative depths (0 to $-200\mu \mathrm{m}$) represent epidermis, including the stratum corneum and a region above this. The depth regions of 0 to $\sim 600\mu \mathrm{m}$ and $\sim 600$ to $\sim 1400\mu \mathrm{m}$ correspond to dermis and hypodermis, respectively. Higher magnification images of three representative segments: the epidermis ($-100$ to $0\mu \mathrm{m}$), the uppermost region of the dermis (0 to $100\mu \mathrm{m}$), and the deepest region of the dermis (500 to $600\mu \mathrm{m}$) are shown in the second column. Further magnified images of black dashed line boxes in the second column images are shown in the third column, in which bacteria are indicated with blue arrows. Regions defined with blue dashed lines indicate clustered bacteria with/without biofilms.

Fig. 3

Representative Gram-stained images of P. aeruginosa-infected burned skin sections of rats after five different treatments on day 2: (a) saline only (control), (b) MB/ethanol/EDTA/DMSO in the dark (PS-only), (c) aPDT with only MB (aPDT I), (d) aPDT with MB/ethanol/EDTA (aPDT II), and (e) aPDT with MB/ethanol/EDTA/DMSO (aPDT III). Three columns of images were taken at different magnifications for each group. The magnification of the images in the first column is $200\times$. A depth scale with box segments with a depth of $100\mu \mathrm{m}$ and a width of $120\mu \mathrm{m}$ is displayed, and the depth origin ($0\mu \mathrm{m}$) is set at the base of the epidermis (black dotted line). The negative depths (0 to $-200\mu \mathrm{m}$) represent the epidermis, including the stratum corneum and a region above it. The regions of 0 to $\sim 600\mu \mathrm{m}$ and $\sim 600$ to $\sim 1400\mu \mathrm{m}$ correspond to the dermis and hypodermis, respectively. Higher magnification images of three representative segments, the epidermis ($-100$ to $0\mu \mathrm{m}$), the uppermost region of the dermis (0 to $100\mu \mathrm{m}$), and the deepest region of the dermis (500 to $600\mu \mathrm{m}$) are shown in the second column. The magnified images of the black dashed line boxes in the second column are shown in the third column with the indicated bacteria (blue arrows). The regions defined with blue dashed lines indicate clustered bacteria with or without biofilms.

In the wounds of the control rats [Fig. 3(a)], massive high-density clustered bacteria with biofilms were seen in and on the epidermis (first column). There were also many bacteria in the uppermost dermal region (middle, third column), and the deepest dermal region (bottom, third column). In the wounds of the PS-only group rats [Fig. 3(b)], high-density clustered bacteria with biofilms were observed in or on the epidermis; however, their areas were smaller than those in the control group. There were many clustered bacteria in the uppermost dermal region (middle, third column). Unexpectedly, only a limited number of bacteria was observed in the deepest dermal region (bottom, third column). Meanwhile, the wounds of the rats with aPDT I (MB only) and aPDT II (MB/ethanol/EDTA) exhibited similar distributions of bacteria [Figs. 3(c) and 3(d)]. Neither high-density clustered bacteria nor biofilms were observed in or on the epidermis. However, there were high-density clustered bacteria, probably with biofilms, in the uppermost dermal regions [middle, third column; Figs. 3(c) and 3(d)]. Some bacteria were seen in the deepest dermal regions, but their numbers appeared to be smaller than those in the control group [bottom, third column; Figs. 3(c) and 3(d)]. In the wounds of the rats with aPDT III (MB/ethanol/EDTA/DMSO), neither bacteria nor biofilms were found clearly in any regions [Fig. 3(e)].

Figure 4 shows the average depth distributions of bacteria ($n=4$ for each group) after the treatment on day 2, with the bacterial distributions in the region deeper than $800\mu \mathrm{m}$ shown at a higher magnification. In the control group [Fig. 4(a)], there were large numbers of bacteria in and on the epidermis, with $\sim 480$ in the segment of 0 to $-100\mu \mathrm{m}$ (the epidermis) and $\sim 280$ in the segment of $-100$ to $-200\mu \mathrm{m}$ (the region above the stratum corneum). Meanwhile, the treatment with only the PS mixture (in the dark) considerably reduced the numbers of bacteria in the epidermis and the region above the stratum corneum to $\sim 180$ and $\sim 90$, respectively; however, there were no significant differences in the numbers between the control and PS mixture groups. In contrast, the numbers of bacteria in the epidermis and the region above the stratum corneum were significantly decreased by aPDT of any type. The bacterial count was decreased to $\sim 90$ and 0, respectively, in the aPDT I group [Fig. 4(c), $P<0.0001$]; to $\sim 10$ and 0, respectively, in the aPDT II group [Fig. 4(d), $P<0.0001$]; and to $\sim 10$ and 0, respectively, in the aPDT III group [Fig. 4(e), $P<0.0001$], respectively, compared with those in the control group. However, in the wounds of the aPDT I group [Fig. 4(c)], there were many bacteria, $\sim 490$, in the segment of 0 to $100\mu \mathrm{m}$ (the uppermost region of the dermis). On the other hand, the number of bacteria in this segment was significantly smaller in the wounds of the aPDT II group, at $\sim 330$ [Fig. 4(d); $P<0.001$] and the aPDT III group, at $\sim 10$ [Fig. 4(e); $P<0.0001$], than those in the aPDT I group.

Fig. 4

Depth distributions of P. aeruginosa in the burned skins of rats that were treated under five different treatment conditions: (a) saline only (control), (b) MB/ethanol/EDTA/DMSO in the dark (PS-only), (c) aPDT with only MB (aPDT I), (d) aPDT with MB/ethanol/EDTA (aPDT II), and (e) aPDT with MB/ethanol/EDTA/DMSO (aPDT III). The depth at $0\mu \mathrm{m}$ indicates the base of the epidermis. The numbers of bacteria were counted for every $100\text{-}\mu \mathrm{m}$ depth section with the width of $120\mu \mathrm{m}$ from the skin surface down to the muscle layer based on the Gram-stained tissue sections. The values indicate the average numbers of the bacteria for 32 ROIs in total (four rats, two sections per rat, four ROIs per section). ****$P<0.0001$, compared with the bacterial number in the corresponding region of the control group. ^####$P<0.0001$ and ^###$P<0.001$, compared with the bacterial number in the corresponding region in the aPDT I group.

For the region deeper than $100\mu \mathrm{m}$, the aPDT I and II groups showed similar bacterial distributions, and the distribution of the aPDT III group was smaller than that of the aPDT I and II groups. Unexpectedly, the PS-only group showed a smaller bacterial distribution than the aPDT I group or aPDT II group. Among all of the groups, the aPDT III group had the smallest bacterial numbers for the deepest region, i.e., the region deeper than $1200\mu \mathrm{m}$ in the hypodermis.