Background

      Burn wound infection is one of the common and serious healthcare problems [1]. Many factors such as type, amount of the microbial burden colonizing in burn wounds, as well as, the ability of microbial bioflm formation are the risk factors in morbidity and mortality in burn patients [2]. Burn wounds are colonized by Grampositive bacteria derived from skin commensals, followed later by Gram-negative bacteria and yeasts [3]. Acinetobacter baumannii is an important opportunistic bacterium capable of developing a wide range of infections in burn wounds [4]. It emerged as the common and invasive bacteria under its robust antibacterial resistance and virulence factor. Te inappropriate over use of antibiotics in empirical therapy led to multidrug-resistant A. baumannii outbreaks all over the world [5, 6]. Terefore, a key challenge is to fnd new non-antibiotic therapies to eliminate A. baumannii from colonizing in burn wounds. A. baumannii senses light through the photoreceptor protein “blue light sensing A”, which is encoded by the blsA gene. blsA expression prevents the formation of bioflm as one of the most important virulence factors and ultimately leads to increased susceptibility of bacteria to antimicrobial agents. Te involvement of CsuE (csuE) and AbaI (abaI) proteins in the bioflm formation of A. baumannii is well-established. CsuE as one of the main A. baumannii adhesions involved in initial attachment as the frst step for colonization and subsequent bioflm formation. AbaI, is an essential factor for production of the acyl-homoserine lactone signal, which plays critical role in bioflm development. Te loss of functional csuE and abaI activities in A. baumannii are corresponding decrease in growth and bioflm formation rate in softtissue infection [7].

      Recently, it was reported that curcumin-nisin (CurNis) formulation is a non-toxic compound with antimicrobial and anti-infammatory activities [8]. Nisin as an antimicrobial peptide can be used synergistically in combination with conventional therapeutic agents and activate the adaptive immune response and have an immunomodulatory role. Also, the potential for using nisin to treat local site-specifc infections such as skin infections has been reported [9]. Curcumin, a natural polyphenol compound derived from the turmeric rhizomes (Curcuma longa L.), through its polyphenol nature and the presence of methoxy and hydroxyl groups, is attributed to many properties, in particular antioxidant, anti-infammatory,antimicrobial, antimutagenic, and anti-angiogenic ones[10, 11]. Te great potential efects of curcumin against chronic diseases including irritable bowel syndrome (IBS) and renal protection from high doses of naproxen in the rat model are due to its antioxidant and anti-infammatory activities [12]. Tere is one meta-analysis based on randomized clinical trials, performed by Jakubczyk et al.[11, 12], describing an intervention of pure curcumin,which increased total antioxidant capacity (SMD=2.696,Z=2.003, CI=95%, p=0.045). Despite the therapeutic uses of these compounds, they have a short half-life, poor pharmacokinetics, poor bioavailability, low solubility, and instability, which limits their therapeutic efect in  vivo [13]. Terefore, using a biodegradable polymer such as poly (L-lactic acid) for the development of nanocarrier can promote their efective therapeutic usage [14].

      Studies also showed that Cur has signifcant antimicrobial efects at very low molarity following activation by light and/or ultrasound in the process of antimicrobial photodynamic therapy (aPDT) and antimicrobial sonodynamic therapy (aSDT) [13, 15–19]. aPDT is a potential alternative approach, which is the photoinactivation of microorganisms and thereby kill cells by reactive oxygen species (ROS) generated by a harmless visible light-activated nontoxic photosensitizer in the presence of oxygen. According to the literature, aPDT has antimicrobial efects on bacteria isolated from infected human burn wounds and improves wound healing in mice and humans [20–23]. Sun et  al. reported aPDT may be an appropriate alternative to standard therapies for infected wounds [24]. aSDT, as a novel ultrasound-driven treatment has been found to be very efective in killing microorganisms due to its strong penetrating power through a sonochemical process [17]. Several recent reports have mentioned satisfactory results with aSDT in inhibiting microorganisms due to its non-invasive nature [17,18, 25]. Te main advantage of aSDT over aPDT is the increased penetration of ultrasound irradiation to the target site compared to light [17]. aPDT and aSDT are the local therapies and can reduce side efects of systemic administration of antimicrobial agents. Another advance in aPDT and aSDT is the management of multidrugresistant bacterial infections and the produced ROS by them cause broad-spectrum oxidative damage to the target cells Interestingly, aPDT and aSDT are promising strategies to which to date no resistant strain has been reported [18, 25].

     It has been shown that silver sulfadiazine (SSD) is an efective topical antibiotic for treatment and protect of second-degree (partial-thickness) and third-degree (fullthickness) burns, respectively, which should be apply for several consecutive days [26]. Along with the benefcial antimicrobial properties of SSD, its major disadvantage is release of silver (Ag+) ions in high doses and the toxic efects on keratinocytes and fbroblasts [27]. Due to the increasing need for the efective treatment of burn wound infections, more attention in this study has been focused on the simultaneous use of aPDT and aSDT, which is called antimicrobial photo-sonodynamic therapy (aPSDT). Herein, we explored the anti-bioflm efcacy of aPSDT using CurNis-based poly (L-lactic acid) nanoparticles (CurNisNp) as a photo-sonosensitizer to eliminate the bioflms of A. baumannii on surfaces of wounds in the animal model. It was hypothesized that CurNisNp-mediated aPSDT will remove the A. baumannii bioflms and improve wound healing more than aPDT and/or aSDT and provide an experimental basis for future clinical application in the treatment of burn wound infections.

Results

Confrmation of synthesized CurNisNp

      Surface morphology of synthesized CurNis in nano scale was confrmed using FESEM analysis. As shown in Fig.  1a, the surface of CurNisNp appeared to be homogeneous with a spherical shape indicating acceptable compatibility between Cur-Nis and poly (L-lactic acid). Te average diameter, PDI, and zeta potential of CurNisNp were approximately 78.6±17.9nm, 0.171±0.04, and−30.7±4.84mV, respectively (Fig.  1b, c). Ultraviolet-Visible (UV–Vis) spectra of pure Cur, Nis, and CurNisNp are presented in Fig. 1d. Accordingly, the absorption spectra of synthesized CurNisNp showed an absorbance peak at 439nm.

In vitro drug release

      Standard plot obtained from UV-absorbance analysis of free Cur-Nis was used to estimate from the release kinetics of CurNis-entrapped nanoparticle. In  vitro release of CurNis from CurNisNp showed a rapid initial burst at the first hour followed by a sustained release for 14 days. Around 20–40% of the total entrapped CurNis was released within 10 h and around 80% was released at 4 days. This in vitro release profile showed that continuous release of CurNis was continued until the 14th day (Fig. 2).

Cytotoxicity assessment of CurNisNp on normal human skin fbroblast cell line (MHFB‑1) cells

       Cytotoxicity analysis using MTT assay kit demonstrated that CurNisNp at the highest concentration (500μg/mL) was not signifcantly toxic to MHFB-1 (Fig.  3a). In the fow cytometry assay shown in Fig. 3b, the cell apoptosis rate was very negligible. Acridine orange (AO) and ethidium bromide (EB) fuorescent staining is a method to analyze the viable cells, early apoptosis, and late apoptosis, as well as the morphologic changes in cells. As the results shown, untreated and treated MHFB-1 cells showed viable cells with green color and intact nuclei(Fig.  3c). Taken together, these data demonstrate that CurNisNp had no cytotoxic efect on cells.

SSR doses of CurNisNp, irradiation time of LED,and ultrasound intensity against A. baumannii

       Te microbroth dilution assay were performed to detect the SSR dose of CurNisNp (CurNisNpSSR) against A. baumannii. Te data indicate that the dose-dependent reduction in cell viability was induced by increasing the concentrations of CurNisNp. When 31.2 to 500μg/mL CurNisNp were used, the cell viability of A. baumannii signifcantly decreased compared to the control group (P<0.05),whereas there was no remarkable reduction when CurNisNp was decreased from 15.6 to 0.9μg/mL (P>0.05). So, the maximum CurNisNpSSR against A. baumannii was found to be 15.6μg/mL with the median (interquartile range [IQR]) log10 CFU/mL of 7.1 (6.8–8.9) (Fig. 4a).

      Also, the cell viability of A. baumannii was decreased with the enhancement of irradiation time of LED and irradiation intensity of ultrasound waves. Te log10 CFU/mL reductions in cell viability of A. baumannii were demonstrated at ultrasound intensity of 53.4 and 61.6mW/cm2 , respectively (P<0.05). Additionally, there was a signifcant decrease in A. baumannii cell viability following exposure to LED with the energy density of 300–420J/cm2 for 5min (P<0.05). Taking into account the cell survival rate, the maximum SSR doses of irradiation time of LED (LEDSSR) and irradiation intensity of ultrasound waves (USSSR) were 4min (energy density of 252–336J/cm2 ) and 45.2mW/cm2 , respectively (P>0.05) with the median (IQR) log 10 CFU/mL of 7.4 (6.9–8.6) and 8.3 (7.1–9.4), respectively (Fig. 4b, c).

Antimicrobial efects of CurNisNp, LED irradiation, ultrasound waves, and their combination against A.baumannii in planktonic growth

      Te results in Fig. 5 showed that all treatment groups could decrease the cell viability of A. baumannii in planktonic growth compared with the control group (P<0.05). As shown in Fig.  5, the median (IQR) log10 CFU/mL of CurNisNp SSR plus LEDSSR, CurNisNpSSR plus US SSR, LEDSSR plus LEDSSR , and CurNisNpSSR plus LEDSSR plus USSSR were 6.2 (4.8–7.1), 5.4 (4.5–7.1), 6.8 (5.2–7.6), and 3.7 (2.9–5.1), respectively.

Anti‑bioflm efects of treatment groups against A.baumannii

       Anti-bioflm activities of diferent treatment groups against A. baumannii were determined using colorimetric assay. Te anti-bioflm activity of diferent treatment groups was evaluated by colorimetric assay.Te mean±standard deviation (SD) and median (IQR) of OD value of A. baumannii bioflm following the different treatments are presented in Table  1. All the aPDTSSR, aSDTSSR, and aPSDTSSR groups based on CurNisNp had anti-bioflm efects against A. baumannii and could considerably reduce the bacterial counts in bioflm structures in comparison with the control group (P<0.05). CurNisNp-aPSDTSSR showed a signifcantly higher anti-bioflm activity than the other treatment groups (P<0.05). Although the bioflms of A. baumannii are displayed to be more susceptible to CurNisNpaSDTSSR than CurNisNp-aPDTSSR, this diference is not signifcant (P>0.05). According to the results in Table 1, although there is no signifcant diference in the reduction of bioflm between the SSD group with CurNisNpaPDTSSR (P=0.129) and CurNisNp-aSDTSSR (P=0.968),but CurNisNp-aPSDTSSR compared to SSD was able to signifcantly reduce the A. baumannii bioflm (P=0.001).

Intracellular ROS generation

        To investigate whether ROS is involved in aPDT and aSDTinduced antimicrobial efect, the levels of intracellular ROS generation in treated A. baumannii cells were assessed. A. baumannii cells when incubated in presence in CurNisNp alone and SSD, showed no remarkable ROS generation (2.3-fold and 0.2-fold, respectively; P>0.05), while, a signifcant ROS production was observed when cells were treated by CurNisNp and irradiated with ultrasound waves (13.7-fold; P<0.05) and LED (15.2-fold; P<0.05). Also, no considerable ROS generation was observed when cells were exposed to aPDT and aSDT (P>0.05).

In vivo assessment of wound healing

     Wound healing process and the percentage of wound contracture rate was shown in Fig. 6. By day 5, all groups showed thick scabs. After day 10, wound healing was accelerated with application ofaPSDTSSR compared to SSD and control groups. As shown in Fig.  6, aPSDTSSR signifcantly enhanced wound closure and re-epithelialization during burn wound healing process (P<0.05). On day 14, the wound surfaces were almost closed and wound sizes were smallest in aPSDTSSR groups.

In vivo antibacterial efects of CurNisNp‑mediated aPSDT on infections of burn wounds

      We used mouse, a well-known burn wound model, to explore the antibacterial efects of CurNisNp-mediated aPSDT. In this study, the antibacterial efects of aPSDTSSR and SSD groups were studied on burn wound infections in a mouse model. Te results show that the time-dependent reduction in cell viability was induced with the passing of the days. According to the results in Fig. 7, at the 5th, 10th, and 15th days, successful antibacterial efects against treated A. baumannii by aPSDTSSR and SSD were achieved (P<0.05). aPSDTSSR group could reduce the amount of A. baumannii by up to 4.72 ±0.15 log10 CFU/mL compared with the no treatment group on the 15th day (P<0.05), while SSD was able to reduce the amount of bacteria by up to 6.93± 1.30 log10 CFU/mL in comparison with the control group (P<0.05).

Efects of CurNisNp‑mediated aPSDT on virulence gene expression patterns in A. baumannii

      To explore the mechanism underlying the anti-virulence induced by CurNisNp-mediated aPSDT, the gene expression patterns in A. baumannii was determined using quantitative real-time PCR. After treatment of A. baumannii using aPSDTSSR and SSD groups,the changes in the expression of virulence genes were evaluated at various time intervals. As shown in Fig. 8, changes in gene expression are time-dependent. In aPSDTSSR-treated A. baumannii, mRNA expression was considerably upregulated to 3.1-, 5.4-, 9.6-, and 15.0-folds in blsA on the 1st, 5th, 10th, and 15th days, respectively (P < 0.05). In contrast, the expression of genes involved in the formation of microbial bioflm (abaI and csuE) was signifcantly downregulated (P < 0.05). Also, the change in gene expression in the SSD group was similar to aPSDTSSR and no signifcant diference was observed between them (P > 0.05). According to the results, a signifcant reduction in abaI and csuE expression and a considerable increase in blsA expression was observed on all test days (P < 0.05).

Histopathological analysis

       To investigate whether CurNisNp are involved in aPSDTSSR-induced wound healing, the evaluation of infammation, fbroblasts, blood vessels, and re-epithe lialization in cross-sectioned tissue obtained from each treated burn wound were assessed by the hematoxylineosin (HE) staining under a general optical microscope. Histologically, the dermal infammatory cell infltration, abscess formation, absence of collagen distinction, and loss of epidermis were observed in the wounds on 1st day (Fig. 9). Te proliferation of marginal epithelium of ulcer was initiated on the 5th day post aPSDTSSR and SSD. On 10th day, continuing re-epithelialization was observed in both aPSDTSSR and SSD groups compared to the control group. In addition, dermal closure and granulation-tissue formation were initiated. Complete tissue re-epithelialization, fbroblastic proliferation, presence of modeled dense collagen mesh, and moderate fbrosis were the important fndings on the 15th day. Day 15 observations suggest that the wound healing efect of aPSDTSSR is related to the activation of granulation tissue formation, as well as, collagen regeneration occurred more efciently in aPSDTSSR than in the SSD group.

Discussion

      aSDT is analogous to aPDT except that drug activation is obtained via ultrasound instead of light [17]. Similar to aPDT, aSDT process generates ROS through the ultrasound-mediated stimulated sonosensitizer in the presence of O2 and the ultrasound-activated cavitation efects can destruct the microbial cells [15, 17]. Following the development of the biological activity of aPDT and aSDT in recent years, many sensitizers were found to have both photodynamic and sonodynamic antimicrobial efects. Terefore, aPSDT has been taken into consideration to obtain better treatment outcomes by reducing the dose of both light energy/ultrasound irradiation and sensitizers, in resulting reducing the cytotoxicity efects [28].

       An appropriate photo-sonosensitizer should have low toxicity to the host cells and high toxicity to the target cells when associated with ultrasound irradiation and/or illumination. Te results of the current study showed that there was negligible cytotoxicity against MHFB-1 cells, suggesting that the synthesized CurNisNp had the least toxicity against eukaryotic cells and good biocompatibility with the host cells.

       Recently, the photo-sono-biological and photo-sonokilling potential of Cur has been explored due to its capability of producing ROS, a free radical, and can play a role in intracellular compounds, activating transcription factors, and altering cytokines [15–19]. In this study, poly (L-lactic acid) as a nanocarrier was used to overcome the poor bioavailability of Cur-Nis. poly (L-lactic acid) is one of the most successfully used biodegradable polymers for drug delivery system and produce the non-toxic biodegradable metabolite monomers following its hydrolysis within the body [29]. Te obtained CurNisNp was welldispersed in an aqueous solution with a narrow particle size distribution. Additionally, when Cur-Nis is encapsulated in the poly (L-lactic acid), it is segregated from the aqueous phase and can release continuously until 2 weeks [8, 30].

      Bioflm-related infections are signifcantly resistant to clearance by the host immune system and various antimicrobial agents [31]. It is evident that bioflm-forming ability can be considered one of the main virulence factors of A. baumannii. Bioflm-producing A. baumannii strains manifest an altered growth rate and transcribe genes that provide them with inherent resistance to living in strict environments, routine antibiotics, and the host immune system [4, 32].

       Te bioflm killing/degradation in  vitro was assessed with the colorimetric method based on crystal violate assay. A signifcant reduction was observed at CurNisNp-mediated aPDTSSR (71.4%), aSDTSSR (76.8%), and aPSDTSSR (93.6%) when were compared with the control group.

       Te objective of this study was to determine whether ultrasound irradiation in aSDT can be used to enhance CurNisNp-mediated aPDT to eliminate the microbial bioflms of A. baumannii in  vitro and in  vivo. According to the literature, no studies have been published regarding the concurrent application of aPSDT in eradicating A. baumannii bioflms. Although a limited number of studies have evaluated the efect of aPSDT on other microorganisms, their results are consistent with the results of this study [33–36].

       Te results of Pourhajibagher et  al. [33] showed that aPSDT could provide a means of circumventing the limitations of decontamination of the dental implant surfaces. In their ex  vivo study, chitosan nanoparticles indocyanine green-mediated aPSDT decreased the polymicrobial periopathogenic bioflms on surfaces of the titanium dental implants to 90.5%. Te inactivation of Candida albicans bioflms using combined aPDT/aSDT in oropharyngeal candidiasis was highlighted by Alves et al. [18]. Xu et al. [37] reported that synergistic aPDT and aSDT improved the inhibition rate of antibioticresistant bacteria in infectious diseases compared with either the aPDT or aSDT alone.

       During the aPSDT, phot-sonosensitizer attaches to the surface of microbial cells, penetration into the cells, and will be activated by relatively low-intensity ultrasound and visible light irradiation. When phot-sonosensitizer treated microbial cells are received the ultrasound waves in aqueous micro-environments, result in microbubbles and cavitation which implosive collapse of gas-flled microbubbles. After that, released energy can be transferred to the oxygen and generate ROS. On the other hand, the excited photo-sonosensitizer reacts with biomolecules through electron transfer to form radicals, which react with oxygen to generate ROS (Type I); and/ or react directly with oxygen through energy transfer, generating singlet oxygen (Type II). Te present data provide indirect evidence that one of the mechanisms of aPSDT is probably based on induction of ROS production and this will result in target-cell death [4, 36, 38–40].

       Several studies confrmed that the intracellular ROS production was greatly increased in aPDT and/or aSDT compared with the ultrasound, light, and photosen sitizer/or sonosensitizer alone [41–45]. We evaluated intracellular ROS generation by DCFH-DA fuorescence and showed revealed CurNisNp can be activated using both ultrasound and light to produce more ROS for aPSDT against A. baumannii bioflms. Moreover, ROS generation was greatly increased in aPSDTSSR compared with aPDTSSR and aSDTSSR.

       Te relationship between bioflm formation and related genes was evaluated previously [46]. In this study, we also compared the levels of virulence-associated genes expression such as blsA, abaI, and csuE in treated A. baumannii by aPDTSSR, aSDTSSR, and aPSDTSSR. Te evidence obtained suggests that blsA plays a positive role in the virulence of A. baumannii due to its global efect on A. baumannii physiology and results fnally in increased sensitivity of bacteria to the antimicrobial agents [47]. Te production of pili which is required for the initial steps of bioflm formation and development of A. baumannii, is intermediated by the csuE [48]. Also, AbaI, encoded by abaI, as a quorum-sensing molecule that catalyzes the synthesis of AHL signals, is a required factor for the production of bioflm on abiotic surfaces [49]. In this study, there was a signifcant downregulation in abaI and csuE expression, as well as a considerable upregulation in blsA expression in A. baumannii during aPSDTSSR in comparison with the other groups. On the other hand, the fndings show that changes in gene expression were time-dependent. On the 15th day, the highest and lowest changes in gene expression were reported. Our results demonstrated that CurNisNp-aPSDTSSR could kill bacteria and eliminate A. baumannii bioflms, as well as, promote wound healing in mice. Additionally, not only there was no signifcant diference between CurNisNp-aPSDTSSR and SSD groups in anti-bioflm and anti-virulence activities, but also wound healing and re-epithelialization occurred more efciently in aPSDTSSR than in the SSD group. Our fndings warrant detailed examination of the interactions between the CurNisNp-aPSDT as an alternative therapy for the successful treatment of burn wound infections.

Conclusion

       In conclusion, this in  vitro and in  vivo study demonstrated that CurNisNp-aPSDT without any cytotoxicity on normal human skin fbroblast cell line could reduce the cell viability of A. baumanni via ROS generation. CurNisNp-aPSDT deceased the bioflm growth in A. baumannii by altering the expression of genes involved in bacterial pathogenesis and promote wound healing by the acceleration of skin re-epithelialization more than SSD as the standard treatment group. In addition, the results indicated that CurNisNp-aPSDT might be a promising complementary strategy to treat burn wound infections.

Methods

Synthesis of CurNisNp as the photo‑sonosensitizer

     CurNisNp was prepared by the double emulsion-difusion-evaporation method [8]. Briefy, an equal amount of curcumin and nisin (5mg) was dissolved in 200μL 1% polyvinyl alcohol (PVA). 50mg poly (L-lactic acid) was added to the compound and the mixture was then sonicated at 30W for 1min to form a primary emulsion. Te emulsion was added dropwise to 16mL 2% PVA and 1% sucrose. Following the sonication of nanosuspension at 30W, 40% duty cycle for 3min, the secondary emulsion was continuously stirred until all the solvents were evaporated. Te nanosuspension was centrifuged(at 16,000×g for 15min) and then washed three times.5% mannitol was added to the nanosuspension and the formulation lyophilized to obtain the dry powder of CurNisNp.

Characterization of CurNisNp

      Te surface morphology of CurNisNp was studied by feld emission scanning electron microscopy (FESEM; ZEISS, German). Te size distribution profles of nanometer-sized particles, zeta potential, and polydispersity index (PDI) of the CurNisNp were carried out using a MALVERN Zetasizer Ver. 6.01 (Malvern Instruments,UK) at approximately 25°C. Also, the absorption spectrum of CurNisNp was carried out by a UV-visible spectrophotometer, scanning the absorbance spectra in the range of 350–600nm wavelength.

In vitro Cur‑Nis release study

      In vitro release study of Cur-Nis from CurNisNp was performed as described by Omobhude et al. [30]. Briefy, 10mg of CurNisNp was suspended in 10ml PBS and a homogenous solution was kept on a rotatory shaker at 37°C and 200×g. Te samples were collected after centrifugation at 16,000×g for 15min and the UV-absorbance analysis of supernatant was carried out at various time intervals.

Screening of CurNisNp cytotoxicity by MTT assay, fuorescent staining, and fow cytometry analysis

      Te cellular cytotoxicity of the prepared CurNisNp was tested on normal human skin fbroblast cell line (MHFB- 1; IBRC C11179) as described previously [50]. Te cell was purchased from Iranian Biological Resource Center(Tehran, Iran) and grown in the Dulbecco’s Modifed Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum, 2mML-glutamine, 100μg/mL amphotericin B and 1% penicillin/ streptomycin antibiotic solution (all purchased from Sigma-Aldrich, Steinheim, Germany) in a humidifed environment with 5% CO2 at 37°C. MHFB-1 cells were seeded in 96-well microtiter plates at a density of 2.0× 104 cells per well. After 24h, cells were washed with PBS and the highest concentration of CurNisNp (500μg/mL) was added to triplicate wells and kept for 24h at 37°C, with 5% CO2/95% air in a humidifed incubator. On one of the microtiter plates, a 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Sigma-Aldrich, Steinheim, Germany) was used to evaluate the viability of the cells with a microplate reader at 570nm according to the manufacturer’s instructions. On another microtiter plate, the cells were resuspended in 100μL binding bufer and incubated with 5μL of Annexin V-FITC and propidium iodide (PI) as a coun terstain for 15min at room temperature, according to the manufacturer’s recommendations. Finally, percent of alive cells was evaluated by fow cytometry (Becton Dickinson) and data were analyzed using Flowjo software V7.For detection of alive cells by fuorescent staining, paraformaldehyde (4% in PBS)-fxed cells were stained with dual acridine orange (AO; 100μg/mL)/ ethidium bromide (EB; 100μg/ mL) fuorescent staining solution for 10min as described previously [51]. Te alive cells were assessed under the fuorescent microscope (OLYMPUS BX53, Japan).

Light source

     Te light source used a light-emitting diode (LED,DY400–4, Denjoy, China) at the wavelength of 435±10nm with an output intensity of 1000–1400mW/cm2. Te medium temperature is stable at room temperature (25±2°C).

Ultrasonic irradiation system

     An ultrasound system was used as described previously [13]. Te ultrasound parameters employed for treatment purposes were set as follows: a frequency of 1MHz and pulsed repetition frequency of 100Hz.

Bacterial strain and growth conditions

      A. baumannii ATCC 29212 strain obtained from Iranian Biological Resource Center has grown aerobically in brain heart infusion (BHI) broth (Laboratorios Conda S.A., Spain) at 37 °C. To examine the antimicrobial efcacy of CurNisNp- based aPSDT, A. baumannii suspension of approximately 1.5× 108 colony-forming unit (CFU)/mL was prepared using both spectrophotometry (optical density [OD] 600nm: 0.08–0.13) and colony  counting.

Determination of antimicrobial efects of CurNisNp, LED irradiation, ultrasound waves, and their combination against A. baumannii in planktonic growth

      Te antimicrobial efects of CurNisNp, LED irradiation, ultrasound waves, and their combination, which would show as +/+/+,+/+/−, +/−/+, −/+/+, −/+/−,−/−/+, +/−/−, −/−/− were determined as follows:

5. Determination of antimicrobial efects of CurNisNp against A. baumannii

      aSDT was done as described previously [36]. Briefy,100μL of CurNisNpSSR was added to 100μL/well of the A. baumannii (1.5× 106CFU/mL) and the microtiter plate was incubated in the dark room (5min; 25°C). After that, the sonication was performed with USSSR. Eventually, the log10 CFUs/mL were counted based on the methods described above.

6. Determination of the antimicrobial efects of ultrasound waves plus LED irradiation against A. baumannii

      To determine the antimicrobial response to ultrasound waves plus LED, 200μL of the A. baumannii(1.5× 105CFU/mL) was added to each well and the microtiter plate was incubated in the dark room for 5Ten, the wells were exposed with LEDSSR and then USSSR. Following growth on the BHI agar plates, the log10 CFUs/mL were counted based on the methods mentioned above.

7. Determination of the antimicrobial efects of CurNisNp plus LED irradiation plus ultrasound waves (aPSDT) against A. baumannii

     100μL of CurNisNpSSR was added to the 100μL/well of the A. baumannii (1.5× 106CFU/mL) in a 96-well micro titer plate. After incubation of microtiter plate for 5 min in the dark room, the bacterial suspension was exposed to LEDSSR and then immediately USSSR. Te treated A. baumannii cells were cultured onto BHI agar and the log10 CFUs/mL were then counted as described above.

Experimental design

     Te test groups consisted of A. baumannii subjected to:

A. CurNisNpSSR (CurNisNp at SSR dose)

B. LEDSSR (Irradiation time of light source at SSR dose)

C. USSSR (Ultrasound intensity at SSR dose)

D. aPDTSSR (CurNisNp at SSR dose + Irradiation time

of light source at SSR dose)

E. aSDTSSR (CurNisNp at SSR dose + Ultrasound intensity at SSR dose)

F. aPSDTSSR (CurNisNp at SSR dose + Irradiation time of light source at SSR dose + Ultrasound intensity at SSR)

G. Control (A. baumannii suspension without treatment)

H. Silver sulfadiazine (SSD) (1% w/w as the standard treatment)

Crystal violet bioflm assay

      Static bioflm formation was assayed in the wells of a 96-well microtiter. Briefy, A. baumannii cells in BHI broth (total volume 300 μL) at the concentration of 1.5× 108CFU/mL were added to wells of a 96-well micro titer plate and incubated at 37 °C for 72h. After treatment of A. baumannii bioflms according to the experimental design described, the bioflms in a 96-well microtiter plate were stained with 0.1% crystal violet for 20min, dissolved in 95% ethanol, and absorbances were measured spectrophotometrically at 570nm by a microplate reader (BioTek, Germany). To assess the treatment efciency of an experimental study on bioflm killing/degradation, the percentage of bioflm killing/degradation was determined as follows:

Measurement of intracellular ROS

      Te intracellular ROS was estimated using fuorescent2 ′,7′-dichlorofuorescein diacetate (DCFHDA) method [55]. Briefy, after centrifugation of the treated A. baumannii cell suspensions at 300×g for 30min, the supernatant was collected and treated with10μM DCFH-DA for 1h. Te fuorescence intensity of DCF was then quantifed with excitation and emission wavelengths of 485 and 530nm, respectively.

Mouse model of A. baumannii‑infected burn wound

      All protocols in the animal experiments were approved by the Animal Care Ethics Committee of Tehran University of Medical Sciences (Application no. IR.TUMS. MEDICINE.REC.1399.944). It should be considered that according to the in adherence to international and guidelines for ethical conduct in the care and use of animals [56], in order to observe the ethics of working with experimental animals, after achieving a favorable and acceptable test group in in  vitro, the in  vivo study was done only in that group.

     8 to 12-week-old female  BALB/c  mice,weighing between 20g and 25g(pasteur  Institute,Tehran,Iran) were housed in individual cages under sanitary conditions with a 12-h light/dark cycle and access to the standardized pellet diet and water ad  libitum. Each mouse was anesthetized by an intraperitoneal injection of 2% xylazine at a dose of 5mg/kg and 10% ketamine at a dose of 100mg/kg. Te dorsal skin of the mice was shaved (3 cm×4cm) with an electric razor and cleaned with povidone-iodine (10%) and ethanol (70%). As described our previous study [48], a full-thickness, third-degree burn model was prepared. A 50μL suspension of A. baumannii (1.5× 108CFU/mL) was dripped into each wound. Te wound was fxed with sterile gauze, and the mice were housed in individual cages. As previously reported [47], the duration from inoculation to successful modeling is 24h. Eight mice were then treated by aPSDT at SSR doses of CurNisNp, irradiation time of LED, and ultrasound intensity after induction of A. baumannii, eight mice were treated by SSD, and the other burned mice served as controls (n =4) and received physiological saline instead of any therapeutic treatment. Te aPSDTSSR and SSD treatment were done completely in the darkroom and repeated for up to 15days.

Evaluation of wound healing potential

     Te progressive changes of burned area were photographed every 5days and analyzed using size analysis Software-Image J. Te percentage of the wound contracture rate was determined according to the following formula:

Assessment of the virulence‑associated genes expression by quantitative real‑time PCR

       Te residual suspensions of treated wound samples were used for RNA extraction. Te total RNA extraction was performed using the GeneAll Hybrid-R RNA purifcation kit (Seoul, Korea) according to the manufacturer’s recommendations. Te elimination of genomic DNA and cDNA synthesis were done by RNase-free DNase I treatment and RevertAid First Strand cDNA Synthesis Kit, respectively (both purchased from Termo Scientifc GmbH, Germany). Te nucleotide sequences of primers for quantitative real-time PCR as described in Table 2. A housekeeping gene (16S rRNA) as the most stable gene was used for the normalization of the reactions. Reaction plates were processed under the following conditions: an initial denaturation of 5min at 95 °C, followed by 35 cycles of 95 °C for 15 s, annealing for 10 s at 60 °C, and 72 °C for 10 s. Fold diferences in RNA expression were calculated by the 2−∆∆Ct method using the Relative Expression Software Tool (REST) 2009 software (version 2.0.13; Qiagen, Valencia, CA, USA), and the changes greater than or equal to two-fold were considered signifcant [57].

Histopathological examinations of burn wound infections

      The tissues cross-sections (5–10 mm) from each wound obtained on the 1st, 5th, 10th, and 15th days after aPSDTSSR and SSD were processed for histopathological study. Tissue samples were fixed in 10% buffered formalin in PBS for 72 h. Paraffin-embedded tissue sections of 3–5 μm thick were prepared and stained with hematoxylin-eosin (HE). A general optical microscope (Olympus, Tokyo, Japan) was used to assess the inflammation, fibroblasts, blood vessels, and re-epithelialization.

Statistical analysis

     All experiments were repeated fve times. Te data of quantitative variables were presented as the median, interquartile range (IQR), and mean±standard deviation (SD) based on the studies of Pérez-Granda et al. [58] and Alonso et al. [59]. Data were evaluated using one-way analysis of variance (ANOVA) and values P  <0.05 were considered statistically signifcant.

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