Abstract
Wound infections represent a significant cause of delayed wound healing. An active wound dressing was developed by incorporating extracts of Rhodomyrtus tomentosa leaf and Quercus infectoria gall in a carboxymethyl cellulose hydrogel (RQ hydrogel). In a modified artificial wound bed mimicking chronic wound infections, RQ hydrogel could inhibit up to 7 log CFU/mL of pathogens in both single- and dual-species biofilms. Antibacterial efficacy was further assessed in three different ex vivo porcine skin wound infection models. Model I: Ex vivo skin infection, the skin was exposed to dual cutures of Staphylococcus aureus/Acinetobacter baumannii. Topical application of RQ hydrogel resulted in 5 log CFU/mL reduction within 6 h. Model II: Ex vivo burn wound infection, more than 3 log CFU/mL of dual-species cultures were inhibited after treatment with RQ hydrogel. Model III: Ex vivo biofilms, preformed biofilms were established by culturing the pathogens on pig skin for 24 h in culture media containing wound exudates, followed by the application of RQ hydrogel on the preformed biofilms. Reduced bacterial biofilms were observed as indicated by decreased fluorescence intensity. Moreover, scanning electron microscopy confirmed the ex vivo inhibitory effects of RQ hydrogel.
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Introduction
Biofilms are complex communities of microorganisms encased in an extracellular matrix. They are a major cause of delayed wound healing that may lead to chronic and persistent infections and result in significant health care burdens1. Microbes can easily infect open wounds as the barrier to bacterial penetration provided by the skin is lost. Once attached to the wound surfaces, microorganisms can rapidly colonize and form biofilms. The presence of biofilm matrix then plays a significant role in protecting microorganisms from antibiotic treatment and host immune defense mechanisms. Wound infections can be caused by mono-species bacteria, especially normal skin flora Gram-positive organisms such as Staphylococcus epidermidis and Staphylococcus aureus. In addition, Pseudomonas aeruginosa, Gram-negative bacteria, is frequently isolated from wound samples and is considered the most common pathogen causing wound infections2. However, polymicrobial infections of wounds have been increasingly recognized and were reported to be more resistant to healing than single-species infections in a murine model3. Co-infection with S. aureus and A. baumannii has been reported as they were mostly recovered from clinical samples of 208 burn patients. The co-infection results in the overrepresentation of ABC transporters and alters several genes required for S. aureus survival and successful establishment in in vivo infection4. Another study involving 200 diabetic patients with foot ulcers reported polymicrobial infections in 115 samples. S. aureus isolates from polymicrobial wounds showed a higher prevalence of pvl and luk-DE genes, which are key contributors to bacterial pathogenicity, compared with S. aureus isolated from monomicrobial wounds5. Co-culture of clinical isolates from polymicrobial infections involving S. aureus and Acinetobacter baumannii has been reported to exhibit a commensal relationship. However, an increase in hemolytic activity was observed when A. baumannii isolates were cultured in proximity to the S. aureus LS1 strain6.
Because of the emergence of multidrug resistant pathogens, medicinal plants and natural extracts are gaining increasing attention as promising alternative approaches to conventional antibiotics for treating infected wounds. Natural extracts not only provide antimicrobial effects but also present other biological properties that facilitate wound healing, such as antioxidant and anti-inflammatory activities. Polysaccharide and ethanolic extracts of Anoectochilus formosanus exhibited antioxidant activity, wound-healing properties and antibacterial activity with minimal inhibitory concentrations (MICs) ranging from 1.25 to 2.5 mg/mL7. Wounds treated with Commiphora gileadensis extract showed high percentages of inflammatory cell infiltration, re-epithelization, and tissue granulation8. One of the main challenges in using plant extracts in clinical settings is the high concentration needed to achieve therapeutic effects, as well as their generally narrow antimicrobial spectrum. Combining multiple natural extracts may help enhance efficacy and reduce the dosage required for effective treatment.
Rhodomyrtus tomentosa is a medicinal herb that exhibits antibacterial, antioxidant, and anti-inflammatory properties. In traditional medicine, it has been used to treat diarrhea and some oral diseases. Rhodomyrtone, a pure compound isolated from R. tomentosa leaf extract plays an important role in the antibacterial activity of R. tomentosa, showing activity comparable to vancomycin9. Rhodomyrtone has demonstrated potent antibacterial activity against Gram-positive bacteria in multiple studies. It effectively inhibited the growth of S. aureus ATCC 25923 and S. epidermidis ATCC 35984, with MIC of 0.39 µg/mL. In comparison, vancomycin exhibited MICs of 0.62 µg/mL and 0.78 µg/mL against the same strains, respectively10. Furthermore, an evaluation of rhodomyrtone against 110 clinical isolates of methicillin-resistant S. aureus revealed an MIC₉₀ value of 1 mg/L, which was comparable to the efficacy observed with vancomycin11. Moreover, R. tomentosa extract has been used in various applications including agriculture, cosmetics, food, and medicine. However, the extract has shown inhibitory effects only on Gram-positive bacteria. For the treatment of infected wounds, the extract must be combined with another compound since wound infections usually present mixed species of Gram-positive and Gram-negative bacteria. Quercus infectoria gall extract was selected for the present study as it possesses anti-inflammatory (IC₅₀ = 34 μg/mL)12 and antioxidant activities (IC₅₀ = 30.78 μg/mL)13 that may assist wound healing.
Therefore, a carboxymethyl cellulose-based hydrogel was prepared that entrapped extracts of R. tomentosa and Q. infectoria (RQ hydrogel) for delivery on infected wounds. RQ hydrogel is proposed as an alternative, low-cost, tissue-resembling dressing for the treatment of infected wounds. The antibacterial, antibiofilm and antioxidant activities of RQ hydrogel were investigated. Ex vivo skin infection models were established to confirm the microbial inhibitory effects of the hydrogels.
Materials and methods
Materials
Carboxymethyl cellulose (CMC) was obtained from Chanjao Longevity Co., Ltd., Thailand. Polyethylene glycol 400 (PEG 400) and propylene glycol and were from Jkk chemical Co., Ltd., Thailand. Microbial reference strains were from American Type Culture Collection (Manassas, VA, USA). Bacterial and fungal culture media were purchased from Becton, Dickinson, and Company (Sparks, MD, USA). Dimethyl sulfoxide (DMSO) was obtained from Fisher Scientific. Petri dishes and multiwell plates were from SPL Life Sci- ence (Gyeonggi-do, Republic of Korea). All other chemicals were purchased from Merck KGaA (Darmstadt, Germany) unless otherwise specified. Quercus infectoria galls were purchased from TPC Herb (Bangkok, Thailand), an herb company approved by the Thai Food and Drug Administration. Gall extract was produced by Thai–China Flavours and Fragrances Industry Co., Ltd.,Thailand (Batchno.C641012OANN). Rhodomyrtus tomentosa leaves were collected from Hat Yai, Songkhla, Thailand (6°55′31.0"N 100°19′09.0"E) in March 2021. Permission to collect R. tomentosa was obtained from Natural Product Research Center of Excellence, Prince of Songkla University, Thailand. The plant was identified by Mr. Jarensak Sea Wai, and a classified reference voucher specimen (NPRC0057) was deposited at the Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
Preparation of Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel
The carboxymethyl cellulose hydrogel base was prepared by mixing 2% v/v of carboxymethyl cellulose with 15% v/v of propylene glycol, 25% v/v of polyethylene glycol 400 and 58% v/v of sterile water. The hydrogel base was then loaded with R. tomentosa ethanolic leaf extract at a concentration of 0.07 g/mL and Q. infectoria ethanolic gall extract at a concentration of 0.5 g/mL. The resulting final concentrations of R. tomentosa and Q. infectoria in the hydrogel were 0.007 g/mL and 0.05 g/mL, respectively. The hydrogel base was prepared as a control blank by adding the ethanol solvent used for plant extraction.
Physical characterization of Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel
Fourier transform infrared spectroscopy (FTIR)
FTIR was conducted to analyse the spectra of Q. infectoria gall extract, R. tomentosa leaf extract, hydrogel base, and the developed RQ hydrogel. The measurements were carried out using a Nicolet iS5 spectrometer (Thermo Scientific, USA). A total of 64 scans were recorded for each sample, including the background, over a frequency range of 4000–500 cm⁻1.
Swelling ratio analysis
The swelling behaviour of the hydrogel base and the developed hydrogel was assessed based on a previously reported method with slight modifications13. Dried hydrogel samples were weighed and placed in pre-weighed tea bags, which were subsequently immersed in distilled water. At each time intervals, the tea bags were removed, gently wipe with filter paper to remove surface moisture, and subsequently weighed. The swelling ratio was calculated using the following equation: Swelling ratio (%) = (Wt—W0)/W0 × 100.
Viscosity properties
The rheological behaviour of hydrogel base and RQ hydrogel was performed using Discovery hybrid rheometer TA Instruments HR-2 with a Peltier system. Curves of the hydrogels’ viscosity were obtained by applying a shear rate in the range from 1 to 1000 s−1.
Release kinetics
In vitro release of natural extracts from the developed hydrogel was performed at 37 °C. Phosphate-buffered saline (pH 7.4) as a release medium. Gallic acid, a natural phenolic compound found in plants and rich in Q. infectoria gall extract was used as a marker for detecting the release kinetics of the hydrogel. The diffusion of gallic acid was examined using Franz diffusion cell system (contact area: 1.77 cm2, Hanson model 57–6 M). The diffusion membrane was Spectra/Por®3 RC Dialysis with 3.5 kDa MWCO and size 2.5 cm (Spectrum Laboratories, USA and Canada). The amount of released gallic was determined using high-performance liquid chromatography (Aligent Technologies, Santa Clara, CA, USA). The release profiles were studied using several models including zero-order, first-order, Higuchi, and Korsmeyer-Peppas models to determine the values of a correlation coefficient.
Scanning electron microscope
SEM was carried out to observe physical morphology of the hydrogel base and after loading with Q. infectoria gall and R. tomentosa leaf extracts. The hydrogels were freeze dried to obtain dry powder and observed under SEM (Hitachi TM3030Plus Tabletop SEM, USA).
Determination of total phenolic and flavonoid contents of the hydrogel formulations
Total phenolic contents were determined using a modified Folin–Ciocalteu method. Twenty-microliter of appropriately diluted R. tomentosa and Q. infectoria extracts or gallic acid standard solution, were mixed with 100 μL of Folin–Ciocalteu reagent and 80 μL of sodium carbonate in a 96-well plate. The plate was then mixed and placed in darkness for 30 min. Total phenolic contents were determined by measuring absorbance values at 765 nm. To determine flavonoids, 20 μL of diluted hydrogel were mixed with 180 μL of 2% w/v aluminium chloride in a microtiter plate. The mixture was incubated in darkness for 10 min and absorbance was measured at 415 nm. Total flavonoid content was calculated from a quercetin calibration curve.
Antioxidant activity
The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method was used to assess the antioxidant activity of RQ hydrogel. The assay was performed in a 96-well plate by mixing 20 μL of RQ hydrogel in 180 μL of 0.1 mM DPPH radical solution. The plate was incubated in darkness at room temperature for 30 min and absorbance was measured at 517 nm. Methanol was used as a blank and Trolox was used as a standard control.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The minimum inhibitory concentration (MIC) of RQ hydrogel was determined using the broth microdilution method of the Clinical and Laboratory Standard Institute14 against Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 35984, Acinetobacter baumannii ATCC 19606, and Pseudomonas aeruginosa ATCC 27853. RQ hydrogel and the hydrogel base were two-fold diluted in a 96-well microtiter plate containing Mueller–Hinton broth (MHB, Difco, France) and 50 μL of bacterial suspensions (106 CFU/mL) were added to wells to obtain a final concentration of 5 × 105 CFU/mL. The plate was incubated for 24 h at 37 °C. The MIC was the lowest concentration that showed a complete inhibition of visible bacterial growth. Bacterial suspensions from microtiter plate wells at or above the MIC were dropped on Mueller–Hinton agar and incubated at 37 °C overnight. The lowest concentration that produced a complete inhibition of bacterial growth was defined as the minimum bactericidal concentration (MBC).
Antibiofilm formation
The effects of RQ hydrogel on biofilm formation were investigated against E. faecalis ATCC 29212, S. aureus ATCC 25923, S. epidermidis ATCC 35984, A. baumannii ATCC 19606, and P. aeruginosa ATCC 27853. The RQ hydrogel treatment was added at subinhibitory concentrations (0.0625–0.5xMIC) into wells of a 96-well plate containing tryptic soy broth (TSB) supplemented with 0.25% glucose. The concentrations were selected based on the MIC values previously determined for each pathogen (Table 2), which ranged from 3.90–250 µg/mL. Bacterial suspensions of approximately 106 CFU/mL were added to the wells and the plate was incubated at 37 °C for 24 h. After incubation, the wells were washed twice with PBS, air-dried and stained with 0.1% crystal violet for 30 min. The wells were then washed and air-dried again. The stained biofilms were dissolved with dimethyl sulfoxide (DMSO) and inhibitory activity was determined by quantifying the optical density of biofilms at 570 nm.
Antibacterial activity against 24-h preformed biofilms
Each bacterial suspension of E. faecalis ATCC 29212, S. aureus ATCC 25923, S. epidermidis ATCC 35984, A. baumannii ATCC 19606, and P. aeruginosa ATCC 27853 was added to wells of a 96-well microtiter plate containing TSB supplemented with 0.25% glucose. After incubation at 37 °C for 24 h, planktonic cells were removed, and the wells were rinsed twice with PBS. The 24-h preformed biofilms were challenged with RQ hydrogel at concentrations of 2–8 × MIC. The plates were then incubated at 37 °C for 24 h. Supernatants were removed and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution at 0.5 mg/mL was added to each well. The plate was incubated in darkness for further 2 h at 37 °C. The MTT solution was removed, formazan crystals were dissolved in DMSO, and the absorbance of the solution was measured at 570 nm. Bacterial cell viability was expressed as a relative percentage: (mean A570 of treated well/mean A570 of untreated well) × 100.
Antibacterial activity in a modified artificial wound bed model
To prepare biofilm cultures mimicking infected wounds, single-species bacterial suspensions of S. aureus ATCC 25923 and A. baumannii ATCC 19606, and a dual-species S. aureus/A. baumannii culture were mixed in a brain heart infusion broth containing 1% gelatine, 25% bovine serum, 5% packed red blood cells, and 0.5% agar. A Three cm-long and 0.5 cm-wide tube was placed in a conical tube and the prepared biofilm cultures were poured into the tube. The tube was incubated at 37 °C for 24 h. Meanwhile, artificial wound beds were produced according to a previous study15 with some modifications. Tryptic soy broth supplemented with 1% (w/v) gelatin and 1.2% (w/v) agar was poured into Petri dishes. After the agar solidified, a second layer was added, and a magnetic stirring bar was placed on the second layer of agar. After solidification, the stirring bar was aseptically removed, leaving an oval indentation that represented a wound bed. To investigate antibacterial activity in this model, a 24 h culture was removed from the tube and placed on the wound bed. The RQ hydrogel was then placed on the culture and the plate was incubated at 37 °C overnight. An area of gel about 5 × 5 mm2 around the wound bed and the biofilm in the wound bed were removed for bacterial enumeration.
Ex-vivo wound infection model by repeated exposure to bacteria
Porcine skin was obtained from a local slaughterhouse in Hat Yai, Songkhla, Thailand. The protocol was approved by the Institutional Animal Care and Use Committee, Prince of Songkla University (Protocol No. 2024-SCI35-130). Pieces of porcine skin (1 × 1 cm) were placed on Petri dishes containing sterile wet cotton balls to prevent dehydration. A dual-species S. aureus/A. baumannii bacterial suspension of approximately 108 CFU/mL was swabbed onto the skin with sterile cotton swab. The skin was left at 37 °C for 2 h to allow infection. Then, the RQ hydrogel was applied on the skin at 0, 6, and 12 h after infection. Bacteria were counted at 6, 12, and 24 h. Enumeration was performed by wiping the skin surface with a cotton swab, which was then placed in a sterile tube containing 0.85% NaCl. The solutions were serially diluted and plated on TSA. The plates were incubated for further 24 h at 37 °C before colony counting.
Ex-vivo model of burn wound infections
A porcine skin ex-vivo infected wound model was prepared according to a previous study16. Pig skin was purchased from a slaughterhouse in Hat Yai, Songkhla Province, Thailand. Hair was removed from the skin. The skin was disinfected with 70% ethanol. Fat layers were then removed with a surgical scalpel. The skin was disinfected again in 70% ethanol. The prepared skin was kept at -20 °C until used. To investigate antibacterial activity in the ex vivo model, the porcine skin was defrosted at room temperature, cut into 1 × 1 cm pieces, and placed in a 12-well plate. Burn wounds were created on the skin using an electric soldering iron. Single-species suspensions of 108 CFU/mL of S. aureus ATCC 25923 and A. baumannii ATCC 19606, and a dual-species S. aureus/A. baumannii suspension were introduced into the wells and the plate were incubated at 37 °C for 2 h. Hydrogels were then placed on the wounds. After incubation at 37 °C for 24 h, bacteria were recovered from the wound model, plated on agar and incubated for a further 24 h for bacterial enumeration.
Wound exudate collection
All purulent exudate samples used in this study were leftover materials collected from routine diagnostic procedures at the Department of Pathology, Songklanagarind Hospital. The samples, originally obtained for clinical purposes, were anonymized and could not be traced back to individual patients. The use of the samples did not interfere with any routine clinical workflows. Collection and pooling were performed by authorized personnel designated by the head of the department. The protocol was approved by the Human Research Ethics Committee, Faculty of Medicine, Prince of Songkla University (Ethical Approval No. REC 66–156-19–2) and met the criteria for exemption. Informed consent was waived by the Human Research Ethics Committee, Faculty of Medicine, Prince of Songkla University. All experiments were conducted in accordance with relevant guidelines and regulations.
Visualization of ex vivo biofilms
Biofilms of a dual-species culture of S. aureus/A. baumannii were produced on pig ear skin following a previously described method17. Sterile filter paper disks were cut and put into a 24-well plate. The pig ear skin was cut into 1 × 1 cm pieces, sterilized by dipping in 70% ethanol, and placed into the wells. The purulent exudate was freeze-dried and sterilized using UV radiation. The exudate was diluted in sterile water and added into the wells. A dual-species S. aureus/A. baumannii bacterial suspension of 108 CFU/mL was then added into the wells and incubated at 37 °C for 24 h. RQ hydrogel was applied on the infected skin and the plate was incubated for further 24 h. After incubation, the treated skin was removed, stained with SYTO-9 and PI dye, and observed under fluorescence microscope. Another set of samples were prepared for observation by SEM. Before visualization and imaging by SEM, skin samples were fixed with 2.5% glutaraldehyde for 6 h, dehydrated in an alcohol series from 30 to 100%, dried using critical point drying, and coated with gold.
In vitro biocompatibility testing
The cytotoxicity of the hydrogels was evaluated using an indirect assay on L929 fibroblast cells (Chinese Academy of Preventive Medical Sciences, Beijing, China), following a modified method based on ISO 10993–5:2009. After culturing L929 cells to reach 80–90% confluence, the developed hydrogel was added into inserts of the transwell plates. A hydrogel base without plant extracts was used as a control. After 24 h incubation, the inserts and culture media were removed and MTT solution at 0.5 mg/ml was added to each well. The plates were then incubated for further 4 h before measuring cell viability using spectrophotometry.
Statistical analysis
All experiments were performed in triplicate, and results are presented as mean ± standard deviation (SD). Hydrogel base was included as negative controls in all experiments, unless otherwise specified. Statistical analysis was conducted using Student’s t-test, with p < 0.05 considered statistically significant.
Results
Physical characterization of Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel
FTIR analysis was performed on the Q. infectoria gall, R. tomentosa leaf extract, hydrogel base, and RQ hydrogel to confirm the successful integration of the extracts into the hydrogel (Fig. 1A). The FTIR spectrum of the QI extract (a) showed characteristic peaks for hydrogen-bonded hydroxyl groups (3238 cm⁻1), aromatic C–H bonds (2870, 1704 cm⁻1), and carbonyl groups (1610 cm⁻1)18. The spectrum of the R. tomentosa extract (b) displayed peaks at 3300, 2922, 1708, and 1608 cm⁻1. The hydrogel base (c) exhibited peaks corresponding to intermolecular and intramolecular hydrogen-bonded hydroxyl groups (3438 cm⁻1), C–H alkyl groups (2871 cm⁻1), carboxyl groups (1646 cm⁻1), and C–O–C bonds (1083 cm⁻1)19. In the FTIR spectrum of the combined RQ hydrogel (d), no significant shifts in peak positions were observed, indicating that the extracts were successfully incorporated into the hydrogel without disrupting the functional groups of the hydrogel base.
Physical characterization of RQ hydrogel. (A) Fourier transform infrared (FTIR) spectra (B) swelling behaviour, (C) viscosity properties, and (D) cumulative release profiles of gallic acid from RQ hydrogel. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel.
The swelling behavior of the RQ hydrogel was evaluated by measuring its weight before and after immersion in distilled water over a 60-min period. As demonstrated in Fig. 1B, both the hydrogel base and the RQ hydrogel exhibited a rapid increase in swelling within the first 2 min which the swelling ratios of the hydrogel base and RQ hydrogel were calculated to be 92.73% and 103.35%, respectively. Thereafter, the swelling ratios of both hydrogels continued to increase gradually throughout the observation period. The slightly higher swelling capacity of the RQ hydrogel may be attributed to the presence of hydrophilic functional groups from the bioactive compounds found in Q. infectoria galls20, which is consistent with the FTIR analysis. The ability to absorb fluids and wound exudates is a critical property of wound dressings, as it facilitates exudate removal and controlled drug release, thereby promoting the wound healing process.
The viscosity of the developed hydrogel was evaluated to determine whether the incorporation of natural extracts affected its gelling properties. This analysis is essential to understand how the viscosity of the hydrogel system responds to varying shear rates. Both the hydrogel base and the RQ hydrogel exhibited a decrease in viscosity with increasing shear rate, demonstrating typical non-Newtonian, shear-thinning behavior (Fig. 1C). The reduction in viscosity is likely attributed to the alignment and disentanglement of polymer chains and crosslinkers21. Notably, there was no significant difference in the viscosity profiles of the hydrogel base and the RQ hydrogel, suggesting that the addition of natural extracts did not alter the rheological properties of the hydrogel.
To investigate the release mechanism of bioactive compounds from the developed hydrogel, the diffusion of gallic acid from the RQ hydrogel was evaluated using a Franz cell diffusion assay. The in vitro release data were fitted to various kinetic models, including zero-order, first-order, Higuchi, and Korsmeyer–Peppas models. The correlation coefficient (R2) was calculated for each model, with a higher R2 value indicating a better fit to the release kinetics. As summarized in Table 1, the release profiles followed the order of fit: Higuchi > Korsmeyer–Peppas > First-order > Zero-order. The release of gallic acid from the hydrogel was best described by the Higuchi model, with an R2 value of 0.9736 and a release rate constant (kₕ) of -0.0013 (Fig. 1D). The Higuchi model is commonly used to characterize the release of water-soluble or poorly soluble drugs from insoluble or swellable porous matrices. The present study confirmed that gallic acid diffused from the hydrogel matrix in accordance with the Higuchi mechanism which has also been reported by our previous20. This type of controlled release is highly desirable for wound dressings, as it allows for prolonged delivery of bioactive compound, reducing the need for frequent dressing changes.
The morphological structure of the developed hydrogel was examined using SEM. As shown in Fig. 2, SEM images revealed the freeze-dried morphology of both hydrogel formulations. The hydrogel base exhibited a dense structure with a smooth, regular, and homogeneous surface, while the RQ hydrogel displayed a rougher and more compact morphology. The irregular surface structure of the RQ hydrogel may contribute to its higher water absorption capacity compared with the hydrogel base. Previous studies have reported that rougher surfaces can enhance drug entrapment and promote more efficient release profiles.
Scanning electron images of RQ hydrogel and hydrogel base. The representative photographs were taken from three independent observations. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel.
Antioxidant activity
The free radical scavenging capacity of RQ hydrogel was investigated using DPPH assays. The antioxidant effects of the treatment were observed even at the lowest concentration tested. At 50 mg/mL, RQ hydrogel displayed 93% radical scavenging activity. At 0.39–25 mg/mL, radical scavenging activity was approximately 91%. Compared with the hydrogel base, the percentage inhibition of DPPH radicals of RQ hydrogel was significantly different. The hydrogel base at 50 mg/mL showed DPPH radical scavenging activity of 23% while the antioxidant activity of the remaining concentrations of hydrogel base was about 12% (p < 0.05) (Fig. 3).
Percent inhibition of DPPH radical scavenging activity at different concentrations of hydrogels. The results are presented as means ± SD from three independent experiments performed in triplicate, p < 0.05. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel.
Antibacterial activity
The antibacterial activity of RQ hydrogel was investigated using the broth microdilution method. As shown in Table 2, RQ hydrogel could inhibit all the tested pathogens with MIC and MBC values of 3.90–31.25 mg/mL and 15.62–250 mg/mL, respectively. The hydrogel base had no antimicrobial effect as no MIC value could be determined.
Antibiofilm activity
The antibiofilm activity of RQ hydrogel was evaluated by measuring the biofilm growth of pathogens after treatment with sub-MICs using the crystal violet assay. RQ hydrogel exhibited concentration-dependent inhibition of biofilm formation of Gram-positive and Gram-negative bacteria (Fig. 4). A significant reduction in biofilm formation of all the tested bacteria was observed after treatment with 0.25 and 0.5 × MICs (p < 0.05). At 0.125 × MIC, biofilm formation by E. faecalis, S. aureus, A. baumannii, and P. aeruginosa was significantly inhibited. At 0.0625 × MIC, RQ hydrogel could only inhibit the biofilms produced by S. aureus and A. baumannii. In comparison with the hydrogel loaded with Q. infectoria alone (Fig. S1), the RQ hydrogel at 0.125 × MIC significantly inhibited biofilm formation by E. faecalis, S. aureus, and S. epidermidis (p < 0.05). A. baumannii biofilms were also significantly reduced following treatment with RQ hydrogel at 0.25 and 0.5 × MIC, whereas no significant difference was observed in P. aeruginosa biofilm formation.
Effects of Quercus infectoria/Rhodomyrtus tomentosa extract-loaded hydrogel on biofilm formation of important pathogenic bacteria following incubation for 24 h. The MICs of the hydrogel against E. faecalis, S. aureus, S. epidermidis, A. baumannii, and P. aeruginosa were 15.62, 7.81, 3.90, 15.62, and 31.25 μg/mL, respectively. The results are presented as means ± SD from three independent experiments performed in triplicate, p < 0.05.
Antibacterial activity in established biofilms
The eradication of 24-h preformed biofilms was evaluated using an MTT metabolic cell activity assay (Fig. 5). All concentrations of RQ hydrogel (2–8 × MIC) significantly reduced bacterial cell viability in established biofilms of all the tested bacteria (p < 0.05). The killing effects were dose dependent. RQ hydrogel demonstrated greater activity against S. aureus and S. epidermidis by inhibiting the metabolic activity of bacteria in preformed biofilms by 63.7–77.6%, whereas the viability of E. faecalis, A. baumannii, and P. aeruginosa was reduced by 16.7–60.6%. In addition, the developed hydrogel significantly inhibited the growth of bacteria in S. epidermidis and A. baumannii preformed biofilms at all the tested concentrations, compared with Q. infectoria-loaded hydrogel. For S. aureus and P. aeruginosa, RQ hydrogel at 2 and 8 × MIC, and 4 and 8 × MIC, respectively, resulted in significant bacterial reduction (Fig. S2).
Bacterial cell viability in 24-h preformed biofilms of important pathogenic bacteria after treatment with Quercus infectoria/Rhodomyrtus tomentosa extract-loaded hydrogel. The MICs of the hydrogel against E. faecalis, S. aureus, S. epidermidis, A. baumannii, and P. aeruginosa were 15.62, 7.81, 3.90, 15.62, and 31.25 μg/mL, respectively. The results are presented as means ± SD from three independent experiments performed in triplicate, p < 0.05.
Effects of RQ hydrogel on single and dual-species biofilms
A modified artificial wound bed was prepared and exposed to single and dual-species biofilms to mimic chronic wound infections. As shown in Fig. 6a, after treatment with RQ hydrogel, the single-species biofilms of E. faecalis, S. aureus, S. epidermidis and A. baumannii, and the dual-species S. aureus/A. baumannii biofilm could not be detected. Meanwhile the bacterial counts of cultures treated with the hydrogel base ranged from 8.2 to 12.1 CFU/mL. RQ hydrogel was less effective against P. aeruginosa, which was present at 6 CFU/mL after treatment. However, the bacterial count of treated P. aeruginosa was still significantly lower than the bacterial counts in the control (p < 0.05). Meanwhile, a significant reduction of microorganisms in the wound bed after treatment with RQ hydrogel were also recorded (p < 0.05). The strongest effect was detected against S. epidermidis, which was reduced by about 7 CFU/mL. The biofilm cultures of S. aureus, A. baumannii, E. faecalis, P. aeruginosa, and the dual-species culture were reduced by 6.0, 4.1, 1.6, 0.9, and 5.3 CFU/mL (Fig. 6b). Representative images of pathogenic bacterial colonies after treatment with hydrogel base and RQ hydrogel are shown in Fig. S3. Compared with the hydrogel loaded with Q. infectoria alone, RQ hydrogel significantly inhibited the growth of single bacterial cultures of E. faecalis, S. aureus, S. epidermidis, and A. baumannii, as well as dual cultures of S. aureus/A. baumannii (p < 0.05).
Bacterial viability in single and dual-species biofilms recovered from hydrogel dressings (A) and artificial wound beds (B) in an in vitro simulated chronic infected wound after treatment for 24 h. The results are presented as means ± SD from three independent experiments performed in triplicate, p < 0.05. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel. Qi hydrogel; Quercus infectoria extract-loaded hydrogel.
Ex vivo infection model
The antibacterial effects of the developed hydrogel were confirmed on a second ex vivo porcine skin infection model based on a dual-species S. aureus/A.baumannii culture. In this model, bacteria were recovered from the infected skin at 6, 12 and 24 h while the skin was re-infected with 106 CFU/mL of the dual-species culture at 6 and 12 h. RQ hydrogel displayed potent bactericidal activity, significantly reducing bacterial growth by more than 5 log CFU/mL at all time intervals (p < 0.05). At 6 and 12 h, the bacterial counts recovered from RQ hydrogel-treated skin were lower than the limit of detection (102 CFU/mL) while the bacterial counts from the control were more than 107–108 CFU/mL. At 24 h, the bacterial count on skin treated with RQ hydrogel was about 104 CFU/mL while the count on the control was more than 109 CFU/mL (Fig. 7). Moreover, treatment with the RQ hydrogel for 12 and 24 h resulted in a significant reduction of the tested bacteria, compared with with Q. infectoria-loaded hydrogel (p < 0.05).
Recovered bacteria from a porcine skin infection model. The porcine skin was infected with a dual bacterial culture of Staphylococcus aureus and Acinetobacter baumannii and treated with RQ hydrogel at 0, 6 and 12 h. Bacteria were recovered from the infected skin after incubation for 6, 12, and 24 h. The presented results are means ± SD from three independent experiments performed in triplicate, p < 0.05. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel. Qi hydrogel; Quercus infectoria extract-loaded hydrogel.
Ex vivo wound infection model
The ex vivo antibacterial activity of RQ hydrogel was investigated using an infected porcine burn wound model16. The infected skin was in contact with a single or dual-species culture for 2 h and then treated with RQ hydrogel for 24 h. The single-species cultures of S. aureus and A. baumannii were selected as representative Gram-positive and Gram-negative strains, respectively. Bacterial viability was significantly decreased in RQ hydrogel-treated porcine skin compared with the control (Fig. 8) (p < 0.05). The reduction in recovered S. aureus, A. baumannii, and mixed S. aureus/A. baumannii cultures were 3.5, 3.9, and 4.0 CFU/mL, respectively. When compared with the hydrogel loaded with Q. infectoria alone, the RQ hydrogel significantly inhibited the growth of S. aureus by approximately 2.4 log CFU/mL (p < 0.05). In contrast, no significant difference was observed for the growth of A. baumannii between Q. infectoria-loaded hydrogel and the hydrogel base.
Recovered bacteria from an infected ex vivo burn wound model after treatment with hydrogels for 24 h. The presented results are means ± SD from three independent experiments performed in triplicate, p < 0.05. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel. Qi hydrogel; Quercus infectoria extract-loaded hydrogel.
Fluorescence microscopy and scanning electron microscopy
Pieces of pig ear skin were incubated for 24 h in microwells containing sterile purulent exudate and then treated with RQ hydrogel. In order to visualize the inhibition of biofilm growth, samples were observed under fluorescence and scanning electron microscopes. The control group demonstrated a higher SYTO-9 green fluorescence intensity, indicating the presence of high numbers of live bacteria. In contrast, the group treated with RQ hydrogel showed reduced biofilm density and increased red fluorescence of propidium iodide dye (Fig. 9A). The results were consistent with the SEM images which clearly illustrated the bactericidal effects of RQ hydrogel, compared with the control. The eradication of biofilm from the porcine skin was evident as only very few bacteria cells and a reduced biofilm matrix were observed (Fig. 9B).
Ex vivo 24-h preformed biofilms of a dual-species Staphylococcus aureus/Acinetobacter baumannii culture were treated with Quercus infectoria/Rhodomyrtus tomentosa extract-loaded hydrogel. The effects of the treatment were observed under the confocal laser scanning microscope after staining with LIVE/DEAD bacterial viability fluorescent dye (A) and under the scanning electron microscope (B). The representative photographs were taken from three independent observations. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel.
Cytotoxicity testing
To ensure the safety of the developed hydrogel, its biocompatibility was assessed using L929 fibroblast cells. The RQ hydrogel exhibited no cytotoxic effects, as cell viability remained approximately 100% at the highest tested concentration (250 mg/mL), compared with untreated control cells. Similar results were observed in cells treated with the hydrogel base (Fig. 10). The findings indicated that the developed hydrogel possesses good biocompatibility and is suitable to use as a topical wound dressing for the treatment of infected wounds.
Cytotoxicity of RQ hydrogel on L929 fibroblast cells after treatment for 24 h. Cell viability was determined MTT assay. The results are presented as means ± SD from three independent experiments performed in triplicate, p < 0.05. RQ hydrogel; Rhodomyrtus tomentosa/Quercus infectoria extract-loaded hydrogel.
Discussion
Wound infections represent a major clinical challenge, as they can substantially impair the healing process and contribute to the development of chronic wounds. These conditions are frequently associated with increased patient discomfort, diminished quality of life, and elevated healthcare costs. The effective management of chronic wound infections is complex and typically requires a multidisciplinary approach. Using antibiotics can cause drug resistant microorganisms that may lead to more difficult to treat infections22,23. Several active wound dressings by incorporating with antimicrobial compound and growth factor promoting wound healing process have been reported. A bilayer scaffold incorporating platelet-rich fibrin as a source of growth factors to regulate the wound healing process, and simvastatin to promote neovascularization and tissue regeneration, significantly accelerated wound recovery in an in vivo model24. Another double-layer micro/nanofiber dressing developed by the same group demonstrated a rapid wound healing rate. It was composed of soy protein, which promotes tissue regeneration and cell differentiation, and pomegranate peel extract, which provides antioxidant, anti-inflammatory, and antibacterial properties25. A novel bilayer wound dressing incorporating quaternized chitosan and curcumin as active components achieved 96% wound closure within 14 days, indicating a significant acceleration in the healing process26. The current study proposed an active hydrogel incorporating Q. infectoria galls and R. tomentosa leaf extracts as an alternative strategy to treat wound infections. The developed hydrogel not only presented pronounced and broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria but also antioxidant activity that could promote wound healing.
Antioxidant effects play an important role in wound healing by removing excess reactive oxygen species (ROS) from chronic infected wounds to reduce oxidative stress. A hydrogel matrix composed of a double network of polyvinyl alcohol (PVA) and agarose containing hyperbranched polylysine and tannic acid, a strong antioxidant molecule promotes wound healing, and suppression of scar formation27. A multifunctional hydrogel produced by incorporation of hyaluronic acid methacrylate matrix with tannic acid (TA) and okra extract as active compounds possess potent antioxidant ability in vitro and provide positive effects in combat bacterial infection, reducing inflammation and promoting wound healing in vivo28. The RQ hydrogel developed in the current study showed antioxidant activity by inhibiting DPPH free radicals. The antioxidant effects were from the presence of high contents of flavonoids and phenolics in RQ hydrogel. Phenolic compounds reduce ROS by acting as hydrogen donors and singlet oxygen quenchers to scavenge free radicals. A previous study of R. tomentosa leaf extract reported an IC50 value of about 7.8 μg/mL in the DPPH assay29. The highest antioxidant activity reported was found in a methanol extract of R. tomentosa leaf, which inhibited 86% of DPPH radicals at an extract concentration of 1000 μg/mL30. In addition, the IC50 of Q. infectoria gall extract was reported to be 0.5 μg/mL in an antioxidant activity assay31. For comparison, at the lowest RQ hydrogel concentration tested (0.39 mg/L) in the present study, the percent inhibition of DPPH radicals was 92%. The potent antioxidant activity was a result of the combined effects of the R. tomentosa and Q. infectoria extracts.
Rhodomyrtone is an active compound in Rhodomyrtus tomentosa extract which has demonstrated excellent antibacterial activity against pathogens such as Bacillus cereus, Enterococcus faecalis, S. aureus, S. epidermidis, Streptococcus mutans, S. pneumoniae, and S. pyogenes10. Interestingly, Unique antibacterial mechanisms of rhodomyrtone have been proposed as the pure compound might accumulate in bacterial cell walls and cell membranes and inhibit the synthesis of nucleic acids, proteins, bacterial cell walls, and lipids32. It could uncouple cell membrane potentially releasing ATP, lipids and cytoplasmic proteins33 or disrupt nucleoid segregation checkpoints in the early stage of bacterial cell division34. Novel reported mechanisms of action of rhodomyrtone include temporary binding to phospholipid head groups of cell membrane and formation of protein-trapping membrane vesicles35. Moreover, rhodomyrtone can modulate innate immune responses in assisting the clearance of S. aureus36. The unique and novel mode of action makes it difficult for microorganisms to develop resistance. The mentioned properties make R. tomentosa leaf extract a very suitable active agent for wound treatment. However, rhodomyrtone is active only with Gram-positive bacteria while some major pathogens in wound infections are Gram-negative organisms. Therefore, Q. infectoria gall extract, a broad-spectrum antimicrobial compound, was included in the hydrogel wound dressing. The gall extract exhibits strong antibacterial activity against Gram-negative bacteria including A. baumannii, P. aeruginosa, and Klebsiella pneumoniae with MICs ranging from 0.05 to 0.1 mg/mL37. Q. infectoria gall extract also exerts anti-inflammatory activity38 and has been used to treat wounds for decades in traditional medicine. The ability of Q. infectoria gall extract to promote wound healing has already been proved in animal models and clinical trials39,40. In addition, the gall extract has been used in combination with other active agents such as copper nanoparticles41 and Pistacia atlantica extract42 to improve their biological properties for wound dressing applications.
Once bacteria attach to a wound, they can rapidly colonize, proliferate and form biofilms that prolong wound infections. A biofilm-challenged wound heals in about 6 weeks, which is 2 weeks longer than a normal wound43. During biofilm formation, microorganisms communicate with each other through quorum sensing, which regulates the metabolic cell activity of planktonic bacteria and induces biofilm formation. The RQ hydrogel demonstrated antibiofilm activity that could prevent biofilm formation by several serious pathogens. Since the concentration of RQ hydrogel used in the biofilm formation assay did not kill the microorganisms, the inhibition of biofilm formation may have been due to phytochemicals in the extracts that affected bacterial quorum sensing. This possibility is lent weight by reports that R. tomentosa strongly inhibited the quorum sensing of Chromobacterium violaceum44 and Q. infectoria gall extract inhibited quorum sensing-controlled virulence factors45.
It is well established that the healing of chronic wounds is hindered by the presence of biofilms, which harbor multiple microorganisms and lead to infections that are difficult to treat. Biofilm formation is a complex, multi-step process that includes microbial adhesion, proliferation, microcolony formation, biofilm maturation, and dispersion. Once biofilms are established, they are very difficult to eradicate and bacteria in biofilms are usually resistant to treatment with antibiotics. Extracellular polymeric substances play a significant role in protecting microorganisms from environmental stressors such as antibiotics and human defense mechanisms. Eliminating biofilms or eradicating microorganisms within wound biofilms typically requires high concentrations or combinations of antimicrobial agents, or additional interventions such as physical debridement or photothermal irradiation. A hydrogel incorporating polyimidazolium, a highly potent cationic antibacterial polymer and N-acetylcysteine, a powerful antioxidant, has been shown to accelerate the closure of biofilm-infected wounds in both a three-dimensional ex vivo human skin equivalent model and a murine diabetic wound model46. A wound dressing composed of Ag⁺ and biofilm-targeting agents, including ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride, significantly reduced viable bacteria in in vitro biofilms, and decreased both bacterial viability and extracellular polymeric substances in a murine wound biofilm model47. In the current study We investigated bacterial cell viability in 24-h preformed biofilms after treatment with RQ hydrogel. In an MTT metabolic cell activity assay, the combined effects of R. tomentosa and Q. infectoria extracts were demonstrated by the strong bactericidal activity against microorganisms in the biofilms. Subsequently, biofilms of single- and dual-species cultures were selected to test the inhibitory effects of RQ hydrogel against microorganisms in a simulated chronically infected artificial wound bed. S. aureus was used as a representative strain of Gram-positive bacteria since it is a common pathogen found in chronic wounds while A. baumannii was a representative strain of Gram-negative bacteria as it is a pathogen that is resistant to most available antibiotics.
Burn wounds are serious complications due to their extensive surface area, depth, and increased risk of infection. These injuries pose significant medical challenges and are associated with a high mortality rate48. Furthermore, burn patients in intensive care units frequently present co-infections of S. aureus and A. baumannii and it was found that essential genes required for the fitness of S. aureus during single infection converted to non-essential genes during co-infection with A. baumannii49. Co-infections of S. aureus and other microorganisms have shown increased severity and poor clinical outcomes50. However, interaction mechanisms of both bacteria in cocultures have not been well documented. To investigate the antibacterial effects of RQ hydrogel in a more relevant environment, an ex vivo burn wound model was tested using a co-infection of S. aureus and A. baumannii. The model was modified from previous reports that demonstrated bacterial migration into a deeper region of porcine tissues16, indicating the mimicry of an in vivo infection. The burn wound was created on porcine skin using an electric soldering iron and then infected with the cocultures for 2 h before treatment with hydrogels. RQ hydrogel could still significantly reduce bacteria of both single- and dual-species cultures in the burn wound.
A porcine skin infection model was investigated by re-infection with microorganisms at 6 and 12 h to observe whether the RQ hydrogel could remain active against wound infections. As expected, even after 24 h incubation, RQ hydrogel could kill a dual-species culture of S. aureus and A. baumannii. The excellent antibacterial activity in the ex vivo models was due not only to the combined effects of R. tomentosa and Q. infectoria, but also to the entrapment of both compounds for delivery to the infection site. To confirm the effects of the RQ hydrogel, fluorescence and scanning electron micrographs were captured using another ex vivo model. This model was modified and according to a previous report. Biofilms of a dual-species S. aureus/A. baumannii culture were pre-formed on superficial burn wounds of pig ear skin in the presence of wound exudate. Due to its ability to eliminate both single- and mixed-species cultures in the three ex vivo models, RQ hydrogel was a suitable dressing for local application and treatment of wound infections.
Biocompatibility is a critical consideration for the clinical application of novel biomaterials. The cytotoxicity of the developed hydrogel was evaluated using L929 fibroblast cells, following ISO 10993 guidelines for the biological evaluation of medical devices. Fibroblasts are widely used in in vitro biocompatibility testing due to their essential role in wound healing, including the degradation of fibrin clots and the formation of structural frameworks that support other cells involved in the healing process51. The pronounced effects observed in ex vivo models confirmed the efficacy and safety of the developed hydrogel, supporting its potential as an alternative strategy for treating chronic wound infections.
Conclusions
The current study proposed a hydrogel formulation containing a combination of R. tomentosa leaf and Q. infectoria gall extracts to enhance antibacterial efficacy against both Gram-positive and Gram-negative bacteria commonly involved in wound infections. The active hydrogel possessed pronounced antioxidant activity due to its high phenolic and flavonoid contents. Its antibacterial efficacy was confirmed through in vitro experiments and a modified artificial wound bed model. Additionally, ex vivo models simulating wound infections demonstrated its significant inhibitory effects on both single- and dual-species cultures. The biological activity of the hydrogel was evaluated in both in vitro and ex vivo models mimicking chronic wound infections, with the goal of reducing the use of animal testing. The results clearly demonstrated that the proposed active hydrogel could be a promising alternative wound dressing for the treatment of infected wounds. However, to support future clinical applications in humans, in vivo studies in animal models are currently underway to comprehensively assess the efficacy and safety of the RQ hydrogel.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We are grateful to Thomas Duncan Coyne (International and Public Relations, Faculty of Science, Prince of Songkla University) for critically reading and editing the manuscript.
Funding
This work was supported by: National Research Council of Thailand (Grant No. N42A670131).
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S.L. designed the experiments, performed the experiments, analyzed the data, prepared figures and tables, and wrote the manuscript. S.P. designed the experiments, performed the experiments, analyzed the data, and prepared figures and tables. S.K. performed the experiments. S.B. performed the experiments. S.W. performed the experiments. A.C. performed the experiments. M.D. performed the experiments, analyzed the data, and prepared figures. S.P.V. edited and critically reviewed the manuscript and approved the final draft. All authors read and approved the final manuscript.
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Lethongkam, S., Paosen, S., Kurakaeo, S. et al. Hydrogel dressing containing natural extracts inhibits dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii in ex vivo wound infection models. Sci Rep 15, 34599 (2025). https://doi.org/10.1038/s41598-025-18150-3
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DOI: https://doi.org/10.1038/s41598-025-18150-3
