Effects of Piper betle Leaf Extract-Coated Suture Material on Clinical Strains of Staphylococcus aureus and Staphylococcus pseudintermedius Isolated from Skin-Infected Dogs

Simple Summary

Non-absorbable sutures are predisposed to bacterial adhesion, increasing the risk of surgical site infections. Alternative prevention, such as using an extract from Piper betle leaves as a biocidal coating agent on suture materials, is an option. This study assessed the effectiveness of P. betle leaf extract-coated sutures against staphylococci. The extract was obtained from ethanolic extraction and analyzed for phytochemicals. Four treatments, including uncoated, antibiotic/extract-free-coated, P. betle leaf extract-coated, and gentamicin-coated sutures, were tested. Analysis of P. betle leaf extract revealed hydroxychavicol as the main phytochemical. The P. betle leaf extract-coated suture showed a stable hydroxychavicol amount on the coated suture during the experiment period. The P. betle leaf extract-coated sutures showed antibacterial, antibiofilm, and anti-adhesion activities to tested staphylococci. Furthermore, a low dose of the P. betle leaf extract-coating agent on the suture had less of a cytotoxicity effect.

Abstract

Non-absorbable sutures provide a site for bacterial attachment and increase the risk of surgical site infections. An alternative prevention of infections requires plant-extract coatings on sutures. The objectives of this study were to develop P. betle leaf extract-coated non-absorbable sutures and to investigate their activities on staphylococci. P. betle leaves were extracted and analyzed for the phytochemicals. P. betle extract was coated on sutures, including polyester and polypropylene. The stability of hydroxychavicol on coated sutures was evaluated. Four treatments were designed, including (1) uncoated, (2) antibiotic/extract-free-coated, (3) extract-coated, and (4) gentamicin-coated sutures. Each treatment was tested for antibacterial, antibiofilm, and anti-adhesion activities on Staphylococcus aureus and Staphylococcus pseudintermedius. In addition, the cytotoxicity of extract-coated sutures was tested. Analysis of the extract identified hydroxychavicol (40.07%) as the primary phytochemical. Stability tests indicated higher hydroxychavicol on Day 1 of extract-coated polyester compared to polypropylene, and the levels decreased on the subsequent days (p < 0.05). Antibacterial activity of extract-coated polyester showed antibacterial effects during the experiment period (5.16 ± 2.35 mm), while polypropylene showed no effectiveness. Additionally, biofilm inhibition was found to be 36.63 ± 27.08% and 37.34 ± 26.98% in tested staphylococci for extract-coated polyester and polypropylene, respectively. Anti-adhesion showed that the extract-coated sutures had a higher ability to decrease tested bacteria attachment (56.25–60.42% living cell reduction). The cytotoxicity study revealed that extract-coated sutures of ≤1.5 mg/1.5 cm had a 99% survival rate. The findings indicate that the coated sutures showed antibacterial, antibiofilm, and anti-adhesion effects against staphylococci causing canine skin infections and might lead to alternative surgical use in veterinary medicine.

1. Introduction

The occurrence of surgical site infection significantly contributes to increasing morbidity and costs within veterinary medicine [1]. The risk of surgical site infections is an important concern in veterinary procedures; consequently, managing these infections is crucial for reducing the incidence and adverse effects [2]. The surgical site infection burden in dogs was reported in previous studies in a range of 1–10% [2,3]. Currently, surgical site infection has been receiving increased attention because of a rise in antibiotic resistance [4]. The prevalence of surgical site infections in dogs and cats has been documented, ranging between 3 and 10% [2], with the most common bacteria identified including staphylococci (Staphylococcus pseudintermedius and Staphylococcus aureus) [2,5], Escherichia coli [6], Streptococcus spp. [6,7], and Pseudomonas spp. [5,7]. In addition, S. aureus and S. pseudintermedius, particularly the methicillin-resistant strains, have the ability to adhere to surfaces, resulting in the development of biofilms on suture materials [8,9], which contributes to their resistance to antibiotics. Surgical instruments, such as suture materials, may provide a substrate for bacterial adhesion and biofilm development, potentially leading to treatment failure and heightened antibiotic resistance [10].

The emergence of antibiotic resistance in animals leads to beneficial consequences through promoting the utilization of alternative therapies and preventive strategies [11]. Alternative antibacterial agents, including extracts from medicinal plants, are being investigated for the potential to reduce the increasing rate of antibiotic resistance. Studies on various medicinal plants have shown their antibacterial efficacy against pathogenic bacteria, including Piper betle [12], Peganum harmala [13], Knema retusa [14], and Curcuma longa [15]. Piper species are aromatic plants in which secondary metabolites demonstrate beneficial effects for humans and are frequently used in traditional medicine to treat various dermatological diseases [16]. Several species of Piper are found in Thailand, such as P. argyritis, P. betle, P. betloides, P. boehmeriifolium, P. caninum, P. colubrinum, and P. dominantinervium [17]. The extract and essential oil of P. betle leaves contain several bioactive compounds, including polyphenols and terpenes [18], which have antibacterial, anti-inflammatory, antioxidant, and anticancer properties [19]. The major bioactive compounds included in P. betle are eugenol and hydroxychavicol [20], which are identified as potential phytochemicals with antibacterial activity [21,22]. Therefore, the application of medical materials using plant-derived compounds may act as both antimicrobial and other bioactive activities, including anti-adhesion and antibiofilm activities.

Non-absorbable suture materials used in veterinary medicine provide an opportunity for bacterial accumulation and the development of biofilm [9]. The application of non-absorbable sutures significantly increases the risk of bacterial infection at the surgical site, particularly around the skin incision [8]. The most effective method for preventing infection is to maintain a clean surgical site; however, this might not always be feasible; therefore, actions should be implemented to minimize bacterial contamination at the suture insertion. Nowadays, the utilization of antibacterial agent-coated suture materials has become widespread in veterinary medicine. The recent common antibacterial agent-coated suture material in veterinary surgery is triclosan-coated suture material [23]; however, the resistance of triclosan to bacteria has been reported, especially in P. aeruginosa [24]. From the development of antibiotic resistance in Staphylococcus causing infection after surgical procedures in dogs, the development of an alternative biocide, which is derived from a natural substance, such as P. betle extract, for coating suture material is required. Therefore, the objectives of this study were to develop P. betle leaf extract-coated non-absorbable suture materials and to investigate the biological activities of the coated sutures on staphylococci isolated from canine skin infection strains.

2. Materials and Methods

2.1. Ethical Considerations

The procedures for dog handling and sample collection were approved by the Walailak University Institutional Animal Care and Use Committee (WU-IACUC) under the approval ID of WU-ACUC-67029. In addition, the protocols for laboratories were approved by the Walailak University Institutional Biosafety Committee (WU-IBC) with an approval ID of WU-IBC-67-027. The isolated bacteria (S. aureus WU1-1, S. aureus WU13-1, S. pseudintermedius WU48-1, S. pseudintermedius WU55-1, and S. pseudintermedius D08-1) used in this study were collected from the skin lesions of dogs using the swabbing technique. It was noticed that clinical isolates of S. aureus WU1-1, S. aureus WU13-1, S. pseudintermedius WU48-1, and S. pseudintermedius WU55-1 were not collected in this study, but these isolates were obtained from our other study [25]. In addition, S. pseudintermedius D08-1 was a strain that was isolated from this study.

2.2. Isolation and Identification of Clinical Strains of S. aureus and S. pseudintermedius from Skin Swab Samples

Skin lesions of dogs with suspected bacterial infections were aseptically swabbed using a sterile cotton stick, and then the cotton swab was put into a sterile glass tube. The samples were immediately sent to the laboratory, and the cotton stick was spread on mannitol salt agar (MSA; HiMedia^®, HiMedia Laboratories, Mumbai, India) and incubated at 37 °C for 18–24 h. The suspected colony of staphylococci was selected and sub-cultured on MSA until a pure culture was obtained. A single colony of pure culture on MSA was picked up and transferred to a tryptic soy agar (TSA; HiMedia^®, HiMedia Laboratories, Mumbai, India) and incubated at the same previous condition. Bacteria grown on TSA were sent for bacteria identification using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique of the Bruker Biotyper Microflex LT/SH Maldi-MS system (MALDI Biotyper^®, Bruker Daltonik GmbH, Bremen, Germany) at the Office of Scientific Instruments and Testing, Prince of Songkla University, Thailand. The isolates that were revealed as S. aureus and S. pseudintermedius with a score value ≥ 2.00 were selected for further testing steps. The isolates that were revealed to score ≥ 2.00 were highly identified at the bacterial species level [26]. In addition, S. aureus ATCC 25923 was used as a reference strain for inoculation and tested for each experiment. Moreover, additional clinical strains were investigated, including S. aureus WU1-1, S. aureus WU13-1, S. pseudintermedius WU48-1, S. pseudintermedius WU55-1, and S. pseudintermedius D08-1. The tested isolates were kept in 25% glycerol for further testing at Walailak University, Thailand.

2.3. Ethanolic P. betle Leaf Extraction

Fresh mature green betel (P. betle Linn.) leaves were collected in the southern area of Thailand. Leaves of P. betle were identified by a botanist using a referent guideline as the previous study that used the leaves from the same culture farm [27]. The leaves were washed with distilled water and then dried in a hot air oven at a temperature ranging from 40 °C to 45 °C for 3 days. The dried leaves were ground in a dry blender and then kept in an airtight glass bottle. A total of 50 g of powder was soaked in 200 mL of absolute ethanol for 7 days. The specimen was filtrated, and the solution was evaporated using a rotary evaporator (Buchi Rotavapor^® R-300, Buchi Labortechnik, Flawil, Switzerland) at 40 °C under 140 mbar until the sample was viscous. Crude extract specimens were additionally air-dried at 40 °C in a hot air oven to eliminate the remaining solvent, and the extract weight was measured daily until it reached a stable mass. Crude extract was stored at −20 °C until it was used for further steps.

2.4. Measurement of Phytochemical Composition in P. betle Leaf Extract

Crude extract was dissolved in absolute ethanol for a final concentration of 200 mg/mL and subjected to analysis of the whole phytochemical compounds in the sample using Gas Chromatography–Mass Spectrometry (GC-MS; Agilent 7890B GC-5977B MSD System, Agilent Technologies, Inc., Santa Clara, CA, USA) at the Office of Scientific Instruments and Testing, Prince of Songkla University, Thailand. In addition, the P. betle extract solution was measured for hydroxychavicol as a main bioactive compound using High-Performance Liquid Chromatography (HPLC; Agilent 1260 Infinity III LC Systems, Agilent Technologies, Inc., Santa Clara, CA, USA). For HPLC, a known concentration of a hydroxychavicol standard solution was prepared and used as a reference. A total concentration of 1 mg/mL of referent hydroxychavicol was prepared in dimethyl sulfoxide (DMSO). The referent stock of hydroxychavicol was diluted to a set of 0.1, 0.3, 0.5, and 0.7 mg/mL. Each concentration of dilution was injected for graph plotting, and the area under the curve was calculated. In addition, a simple linear graph and an equation were generated to use for the hydroxychavicol amount in crude extract calculation. Then, 10 µL of dissolved solution was used for HPLC analyses. The flow rate of the mobile phase was set at 1 mL/min at room temperature, and chromatograms were performed at 280 nm for 20 min. The amount of each bioactive compound (mg) in solvent (mL) of each duration time was converted to the amount of each bioactive compound (mg) in the weight of the coated suture (mg).

2.5. Determination of Minimal Inhibitory Concentration (MIC) of Crude Extract in the Selected Staphylococci

The determination of the minimum inhibitory concentration (MIC) of the crude extract was conducted using the broth microdilution method, following the VET01S: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tested for Bacteria Isolated from Animals, 3rd Edition, and the Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 9th Edition, from the Clinical and Laboratory Standards Institute (CLSI), with some modifications [28,29]. Briefly, the extract was diluted in a 2-fold dilution ranging from 0.0625 to 4.00 mg/mL in a 96-well plate using Mueller–Hinton broth (MHB; HiMedia^®, HiMedia Laboratories, Mumbai, India). Then, a total of 100 µL of 1 × 10⁶ CFU/mL of tested bacterial suspension was added into each well and incubated at 37 °C for 18 h. S. aureus ATCC25923 was used as a reference strain for MIC determination, while gentamicin (Glentham^® Life Sciences, Corsham, UK) and 1% DMSO were used as positive and negative controls, respectively. A total of 0.03% of resazurin (Glentham^® Life Sciences, Corsham, UK) was added to each well and then incubated at 37 °C for 1 h. The well that completely inhibited the growth by the extract was defined as the MIC value.

2.6. Experimental Treatment Design

In this study, the experiments were designed with 4 intervention groups, including (1) no coating agent on the suture (without a base agent, crude extract, and gentamicin); (2) an antibiotic/extract-free coating agent on the suture (the main ingredient was a coating agent with 5 mL of sterile distilled water); (3) a crude extract-containing coating agent on the suture (the main ingredient was 50 mg of P. betle leaf extract in a total of 60 mL of coating solution); and (4) an antibiotic drug-containing coating agent on the suture (the main ingredient was 5 mg of gentamicin in a total of 60 mL of coating solution). The suture was cut into 3.0 cm and used for anti-staphylococcal testing, while lengths of 1.5 cm were designated for bioactive compound stability, antibiofilm testing, anti-adhesion testing, and cytotoxicity determination. For each experiment except cytotoxicity determination, the result of each treatment was measured on the preparation day (Day 1) and the subsequent day after preparation (Day 3, Day 5, Day 7, Day 14, Day 20, and Day 30).

2.7. Preparation of Non-Absorbable Suture Materials and Coating Agents

Polypropylene (Prolene^TM Ethicon^®, Raritan, NJ, USA) and polyester (Ethibond Excel^TM Ethicon^®, Raritan, NJ, USA) with a size of USP (3-0) were selected as monofilament and multifilament sutures, respectively. Each suture was pretreated in absolute ethanol to remove oil from the suture surface for 2 h and then dried at 50 °C for 30 min in a hot air oven. The pretreated sutures were soaked in 75% ethanol at room temperature for 15 min for disinfection of contaminated bacteria in a closed container. After discarding ethanol, the disinfected suture was dried in the container at 50 °C for 1 h to completely remove any remaining ethanol and was then kept at room temperature until it was used in the coating step. The gentamicin-added coating agent was used as a positive control, while the antibiotic- and crude extract-free coating agent was used as a negative control for all experiments. Tween 80, glycerol, and sterile distilled water were homogeneously mixed as the emulsifier part of the coating agent. In addition, polycaprolactone (Merck^®, Sigma-Aldrich, St. Louis, MO, USA) completely dissolved in ethyl acetate was used as the polymer part of the coating agent-based solution. The polymer part was stirred continuously while the emulsifier part was added. After the mixed solution became homogeneous, the main ingredient for each treatment was added to the mixture. According to the preliminary results of MICs of selected staphylococci, the crude extract was used at 10 mg/mL, and gentamicin was used at 1.5 mg/mL. The disinfected suture was dipped in the homogenous coating solution for 5 s and then placed on a sterile petri dish. The sutures that were used on the day of preparation (Day 1) were left for 1 h before use, while the sutures that were used on the subsequent day were kept at 4 °C until used.

2.8. Stability of Main Bioactive Compounds in P. betle-Coated Suture Materials

The 1.5 cm crude extract-coated suture was dissolved in 1 mL of absolute ethanol for 24 h at room temperature. Then, the dissolved solution was vigorously vortexed, and the mixture was filtered using a nylon syringe filter with a 0.22 µm pore size. The filtered solution was used for hydroxychavicol measurement using a UV-Vis spectrophotometer at 280 nm. A set of standard hydroxychavicol, ranging from 0.1 to 1.0 mg/mL, was prepared and measured for OD280 for linear graph plotting and linear equation generation. In addition, the concentration of hydroxychavicol composited in coated sutures was measured using HPLC under the same condition described previously.

2.9. The Effect of P. betle Leaf-Coated Sutures on Anti-Staphylococcus

The anti-staphylococcal effect was determined using the agar-plate method by inhibition zone measurement. The selected staphylococci were cultured in Mueller Hinton broth (MHB; HiMedia^®, HiMedia Laboratories, Mumbai, India) separately and then incubated at 37 °C for 16–18 h. The turbidity of the incubated MHB was adjusted to 0.5 McFarland, and then the inoculum was plated on a Mueller Hinton agar (MHA; HiMedia^®, HiMedia Laboratories, Mumbai, India). The 3 cm uncoated and coated sutures of each treatment were placed on the inoculated MHA plate, and the plate was incubated at 37 °C for 16–18 h. The inhibition length of each treatment was measured at 5 positions from the terminal side, including at 0.5 cm, 1.0 cm, 1.5 cm, 2.0 cm, and 2.5 cm of the suture. The obtained zone length of each suture was averaged for the mean inhibition zone value of each treatment.

2.10. The Effect of P. betle Leaf-Coated Sutures on Antibiofilm Formation

The effect of antibiofilm formation was adapted by using a crystal violet assay, which was tested in a 24-well plate. The protocol for antibiofilm formation for suture materials was modified from a previous study [30]. Tested staphylococci were cultured in tryptic soy broth (TSB; HiMedia^®, HiMedia Laboratories, Mumbai, India) supplemented with 1% dextrose (Loba Chemie™, Loba Chemie Ltd., Mumbai, India) and then incubated at 37 °C for 18–24 h. In each treatment, 2 sutures were aseptically placed at the bottom of the well, and bacterial inoculum that was adjusted to 1 × 10⁶ CFU/mL was added into each well. The plate was incubated at 37 °C for 24 h. The sutures were then transferred to a new 24-well plate and washed twice using 1X phosphate buffer saline (PBS; pH = 7.4). Moreover, PBS was discarded, and the plate was then dried at room temperature for 1 h before adding 500 µL of 2.5% glutaraldehyde for 30 min. After discarding the fixative agent and drying, 500 µL of 0.05% crystal violet (QRëCTM, Auckland, New Zealand) was added to each well and incubated at 37 °C for 30 min. The dye was removed, and each well was washed using sterile distilled water twice, and the plate was dried at 40 °C in a hot air oven. Each well was dissolved for the stained dye on the suture using 1 mL of 95% ethanol for 1 h. In addition, 800 µL of dissolved solution was transferred into a new 24-well plate and measured for OD570 using a spectrophotometer (Multiskan SkyHigh Microplate Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA) and the SkanIt Software version 6.1RE (Thermo Scientific SkanIt Software, Thermo Fisher Scientific Inc., Waltham, MA, USA). A blank control was prepared using tested media without inoculum incubated with sutures, and the tested media was used as a negative control.

2.11. The Effect of P. betle Leaf-Coated Sutures on Antibacterial Adhesion

Tested staphylococci were cultured in TSB supplemented with 1% glucose and incubated at 37 °C for 18–24 h. Then, the inoculum solution was adjusted to 1.5 × 10⁶ cells/mL. Furthermore, two 1.5 cm sutures from each treatment were placed into each well of a 24-well plate. A total of 500 µL of adjusted inoculum was added into each tested well and then incubated at 37 °C for 18–24 h. The suture was placed in a new microcentrifuge tube with 1 mL of sterile 1X PBS (pH = 7.4) and vortexed vigorously for 30 s. A total of 100 µL of suspension was used for the pouring plate technique with melted MSA agar. The solidified MSA was incubated at 37 °C for 18–24 h, and the colony was counted and calculated as CFU/cm. Moreover, another set of tested sutures was rinsed twice with 1X PBS (pH = 7.4) and then fixed with 2.5% glutaraldehyde for 1 h. The fixed suture was dehydrated in a series of 20–100% ethanol and completely dried using a critical point dryer (CPD; K850 Critical Point Drier, Quorum Technologies, Kent, UK). The dried specimen was then coated with gold particles using a gold sputter coater (Cressington Sputter Coater 108 Auto, Cressington Scientific Instruments Inc., Watford, UK) and examined under a scanning electron microscope (SEM; MERLIN^® Compact, SEM-Zeiss, Munich, Germany).

2.12. Cytotoxic Effect of P. betle Leaf Extract-Coated Suture Materials

P. betle leaf extract-coated sutures were tested for cytotoxicity to human keratinocytes (HaCaT; ATCC^®, American Type Culture Collection, Manassas, VA, USA). Briefly, HaCaT cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). P. betle leaf extract-coated and antibiotic/P. betle leaf extract-free coating agent-coated sutures were tested in each well containing HaCaT cells. The different extract concentrations, including 0 mg, 0.75 mg, 1.5 mg, 2.25 mg, 4.5 mg, and 9.0 mg per 1.5-cm suture length, were chosen to test. The selection of extract concentrations was based on the experimentally determined amounts of extract released from the coated sutures and the antibacterial activity tested in the previous steps. Then, HaCaT cells with sutures were incubated at 37 °C for 24 h under a humidified condition with 5% of carbon dioxide. Additionally, the blank used as an untreated control was the well that cultured HaCaT cells without sutures, while 10% DMSO served as the cytotoxicity control. The suture was removed from the well, and 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was added to each well to harvest the cells. Harvested cells were enumerated using a hemacytometer and subsequently seeded into 24-well plates at a final density of 7.5 × 10⁴ cells per well in 1 mL of complete media. The plate was incubated at 37 °C for 24 h under a humidified condition and 5% carbon dioxide prior to further experimental steps. The cultured cells were observed for their density and morphology under an inverted phase-contrast microscope (Olympus CKX53, Olympus^®, Tokyo, Japan) at 20× magnification. Furthermore, a final concentration of 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (MTT; Merck^®, Sigma-Aldrich, St. Louis, MO, USA) was added into 1 mL of culture media in each well and incubated at 37 °C for 4 h under a humidified condition and 5% carbon dioxide. Then, the supernatant of each well was discarded, and 500 µL of DMSO was added to dissolve the formazan crystal, and the plate was gently shaken for 10 min under a dark condition. A total of 200 µL of solution from each well was transferred into a new 96-well plate, and the plate was measured for absorbance at 570 nm using a microplate reader (Spark^®, Tecan, Männedorf, Switzerland). The viability percentage of HaCaT cells was calculated as follows: (absorbance at 570 nm of treatment − absorbance at 570 nm of cytotoxicity control)/(absorbance at 570 nm of treatment − absorbance at 570 nm of blank) × 100.

2.13. Statistical Analysis

The data from each experiment was recorded and manipulated in Microsoft Excel 365. Data are presented as descriptive statistics, including means and standard deviations. In addition, the distribution of the outcome variable was tested with a normality test using the Shapiro–Wilk test to judge for normal distribution. Independent t-test and one-way analysis of variance (ANOVA) were used to test the normally distributed data, while the Mann–Whitney U test and Kruskal–Wallis test were performed in the case of other data distributions. Statistical running was analyzed using R-programming language version 4.5.2. (https://www.r-project.org, accessed on 1 November 2026). All statistical analyses were performed under a 95% confidence interval, and a p-value < 0.05 was considered significant.

3. Results

3.1. Phytochemicals in Ethanolic Extract of P. betle Leaves

The ethanolic extract of P. betle leaves was analyzed for their phytochemical composition using GC-MS. The main phytochemical was hydroxychavicol, which was found in approximately 40% of the samples. In addition, the other main compounds were eugenol and eugenol derivatives, including 5-allyl-2-hydroxyphenyl acetate, acetyl eugenol, and chavibetol (m-eugenol). The phytochemical composition in the extract of P. betle leaves is presented in

Table 1. Phytochemicals of the ethanolic extract of P. betle leaves using GC-MS.

No	Compound Name	Type	Chemical Formula	Relative Amount
1	Hydroxychavicol	Phenolics	C9H10O2	40.07%
2	5-allyl-2-hydroxyphenyl acetate	Phenolics	C11H12O3	16.23%
3	Dihydro-eugenol acetate	Phenolics	C12H14O3	13.20%
4	Methyl eugenol	Phenolics	C10H12O2	9.23%
5	Acetoxychavicol acetate	Phenolics	C13H14O4	3.21%
6	Acetic acid	Carboxylic acid	C2H4O2	2.25%
7	Palmitic acid	Fatty acid	C16H32O2	1.94%
8	Neophytadiene	Terpene	C20H38	1.75%
9	Linolenic acid	Fatty acid	C18H30O2	2.08%
10	Phytol	Terpene	C20H40O	1.14%
12	Quinacridone	Pigment	C20H12N2O2	1.18%
13	γ-muurolene	Terpene	C15H24	0.78%
14	3,7,11,15-tetramethylhexadec-2-en-1-yl acetate	Terpene	C22H42O2	0.76%
16	Octadec-9-enoic acid	Fatty acid	C18H34O2	0.68%
17	Chavicol	Phenolics	C9H10O	0.57%
18	Caryophyllene	Terpene	C15H24	0.48%
19	Phytol acetate	Terpene	C22H42O2	0.83%
20	Others (<0.5% each)	-	-	3.37%

. Moreover, the extract was measured for the amount of hydroxychavicol using HPLC. The linear curve of the hydroxychavicol standard solution was used to generate a linear equation for hydroxychavicol quantity calculation and is presented in Figure 1. The equation demonstrated a strong correlation between the concentration of hydroxychavicol (mg/mL) and the area under the curve (mAU), with a coefficient of 99.94%. In addition, the retention time of the hydroxychavicol standard solution was from 4.673 to 5.00 min. This retention time corresponds to the highest curve identified in the extract sample. Retention time and area under the curve graphs of the hydroxychavicol standard solution and the extract using HPLC at a wavelength of 280 nm are demonstrated in Figure 2. The calculated concentration of hydroxychavicol in P. betle leaf extract was approximately 0.24 mg/mL.

3.2. Hydroxychavicol Stability in Extract from P. betle Leaf Coated on Suture Materials

The coated suture was soaked in absolute ethanol to dissolve the coating solution containing P. betle leaf extract. The dissolved solutions were measured for absorbance at 280 nm for hydroxychavicol, and a linear curve was generated using the obtained absorbance at 280 nm and the standard hydroxychavicol amount. A linear equation with R² = 97.66% was used for hydroxychavicol amount calculation in dissolved solutions (Figure 3). The results show that the amount of hydroxychavicol coated on polyester was greater than on polypropylene. In addition, the amount of hydroxychavicol on both polyester and polypropylene was higher on the day of preparing the coating agent (Day 1) compared to the subsequent days (p < 0.05). Nevertheless, the amount of hydroxychavicol started to decrease on Day 3. From Day 3 to Day 30, there were no differences in hydroxychavicol levels. The trend of hydroxychavicol amounts in both polyester and polypropylene is presented in Figure 4. Moreover, the amount of hydroxychavicol for 7 days was 0.23 ± 0.04 mg per 1 cm and 0.15 ± 0.04 mg per 1 cm for polyester and polypropylene, respectively.

3.3. Anti-Staphylococcal Effect of Extract from P. betle Leaf Coated on Suture Materials

For both S. aureus and S. pseudintermedius, the coating agent base showed no effect on the anti-staphylococcal effect, the same as the suture without the coating agent at and after the preparation days. In addition, the P. betle leaf extract-coated polypropylene had no anti-staphylococcal effect. Overall, the results of the inhibition zone for each treatment in both S. aureus and S. pseudintermedius show a similar pattern. The trend of inhibition zones for each treatment tested in S. aureus and S. pseudintermedius is presented in Figure 5 and Figure 6, respectively. The inhibition zone of P. betle leaf extract-coated polyester (5.16 ± 2.35 mm) was found in most tested isolates compared to polypropylene. In addition, the inhibition lengths of P. betle leaf extract-coated polyester were 5.41 ± 3.09 mm and 4.91 ± 1.19 mm in S. aureus and S. pseudintermedius, respectively. The extract-coated polyester showed anti-staphylococcal effects on the preparing day and the subsequent day after preparation, and the antibacterial effects on the preparing day were nearly similar to the day after preparation (p ≥ 0.05). Comparing between extract-coated and gentamicin-coated treatments, the antibacterial effects on tested staphylococci were higher in gentamicin-coated treatments; however, there was no statistically significant difference on both the day of preparation and the subsequent day after preparation. Similarly, the antibacterial effect of gentamicin-coated polypropylene was expressed both on the preparation day and the subsequent day after preparation.

3.4. Antibiofilm Effect of Extract from P. betle Leaf Coated on Suture Materials

The effect of P. betle extract coated on polyester sutures presented a similar trend in both S. aureus and S. pseudintermedius. The rates of biofilm inhibition in tested staphylococci of P. betle leaf extract-coated sutures were 36.63 ± 27.08% for polyester (45.96 ± 27.82% for S. aureus and 25.36 ± 22.43% for S. pseudintermedius) and 37.34 ± 26.98% in polypropylene (48.80 ± 26.34% for S. aureus and 26.12 ± 22.92% for S. pseudintermedius), respectively. On the preparation day, the gentamicin-coated suture showed a higher biofilm inhibition than the extract-coated type. However, this finding was not significantly different in the biofilm inhibition percentage between the two groups. Conversely, the results of biofilm inhibition in polypropylene are different for S. aureus compared to S. pseudintermedius. The biofilm inhibition of the gentamicin-coated suture was greater than that of the extract-coated group, with no statistical difference in S. aureus. For S. pseudintermedius, the effect of the extract-coated treatment was higher biofilm inhibition than the gentamicin-coated group; however, this comparison showed no statistical significance. Figure 7 and Figure 8 present comparisons of biofilm inhibition between P. betle leaf extract-coated and gentamicin-coated treatments tested on S. aureus and S. pseudintermedius, respectively.

3.5. Anti-Adhesion Effect of Extract from P. betle Leaf Coated on Suture Materials

The effect of anti-adhesion on S. aureus in both polyester and polypropylene was beneficial in P. betle leaf extract-coated sutures during the experimenting period. For polyester, the extract-coated treatment had a higher anti-adhesion effect on S. aureus than the gentamicin-coated treatment, which presented fewer viable S. aureus cells attached to the coated sutures. However, the gentamicin-coated group was found to have a lower effect of anti-adhesion than the extract/antibiotic-free-coated treatment in S. aureus. In addition, the anti-adhesion of extract-coated polypropylene in S. aureus was higher than the gentamicin-coated treatment. For S. pseudintermedius, the anti-adhesion effect of the gentamicin-coated treatment was conversely higher than P. betle leaf extract in both polyester and polypropylene during the experiment period. Overall, the number of staphylococcal cells on P. betle leaf extract-coated sutures was 2.95 × 10⁴ ± 44.67 CFU/cm for polyester (2.88 × 10⁴ ± 45.71 CFU/cm in S. aureus and 2.82 × 10⁴ ± 43.65 CFU/cm in S. pseudintermedius) and 1.32 × 10⁴ ± 10.97 CFU/cm for polypropylene (1.32 × 10⁴ ± 11.75 CFU/cm in S. aureus and 1.29 × 10⁴ ± 11.48 CFU/cm in S. pseudintermedius), respectively. Comparisons of the number of viable staphylococcal cells on each suture type resulting from the anti-adhesion effect on S. aureus and S. pseudintermedius are presented in Figure 9 and Figure 10, respectively. Furthermore, the attached bacterial cells of both S. aureus and S. pseudintermedius were observed under a scanning electron microscope. The scanning electron microscopic results found that the anti-adhesion of P. betle extract-coated polyester predominantly decreased in attached staphylococcal cells on the polyester surface in both S. aureus and S. pseudintermedius. Nevertheless, the anti-adhesion of P. betle extract-coated polypropylene was extremely less in cell attachment in both coated and uncoated sutures. The adhesion of staphylococcal cells on each uncoated, extract/antibiotic-free-coated, and P. betle extract-coated surface is presented in Figure 11. In addition, the proportions of live staphylococcal cells were investigated. Compared to the uncoated group, the reducing percentage of staphylococcal living cells affected by P. betle extract-coated polyester and polypropylene were 57.06% (56.25% reduction in S. aureus and 60.42% reduction in S. pseudintermedius) and 85.02% (89.44% reduction in S. aureus and 90.02% reduction in S. pseudintermedius), respectively.

3.6. Cytotoxicity of Extract from P. betle Leaf Coated on Suture Materials on the HaCaT Cell Line

P. betle leaf extract-coated and extract/antibiotic-free coating agent-coated sutures were tested with the HaCaT cell line. The survival percentages of HaCaT cells in the control and 10% DMSO groups were 100 ± 2.43% and 5.33 ± 2.29%, respectively. The survival rate of HaCaT cells was P. betle extract dose dependent. Figure 12 presents the cytotoxicity assessment of extract/antibiotic-free-coated sutures and P. betle leaf extract-coated sutures tested with HaCaT keratinocytes, compared with the control and 10% DMSO. The tested amounts of coated extract at 0 mg per 1.5 cm with a survival rate of 96.89 ± 2.94% (p = 0.14), 0.75 mg per 1.5 cm with a survival rate of 99.63 ± 1.53% (p = 0.99), and 1.5 mg per 1.5 cm with a survival rate of 99.77 ± 2.90% (p = 0.99) were not different from the control group. In addition, the tested amount of extract at 9 mg per 1.5 cm with a survival rate of 5.12 ± 1.62% showed a comparable survival rate for HaCaT cells, nearly similar to the 10% DMSO group (p = 1.00). Nevertheless, the amounts of extract coated at 2.25 mg per 1.5 cm (survival rate of 29.35 ± 3.08%) and 4.50 mg per 1.5 cm (survival rate of 14.73 ± 2.30%) were statistically significant from others (p < 0.05). Moreover, the quantification of HaCaT cell viability after 24 h treatment using the MTT assay is presented in Figure 13.

4. Discussion

The major phytochemical found in the ethanolic extract of P. betle leaves was hydroxychavicol, which is similar to studies in other regions of Thailand [31,32], India [33], and Indonesia [34]. P. betle extract revealed antibacterial activity in several pathogens isolated from animals, such as mastitis-inducing S. aureus [35], canine pyoderma-inducing S. pseudintermedius [36], avian pathogenic E. coli [12], and mastitis-inducing E. coli [37]. Several biological activities of hydroxychavicol have been confirmed, such as antimicrobial, anticarcinogenic, antioxidant, and anti-inflammatory activities [38]. Hydroxychavicol is a natural pharmacological molecule grouped in the allylbenzene class [39]. Moreover, hydroxychavicol was mostly reported as having both bacteriostatic and bactericidal effects in the formulas of betel leaf extract and betel leaf essential oil, respectively [40]. Even if the mechanism of hydroxychavicol in eukaryotic cells depended on redox balance interference [41], the mechanism in bacterial cells has been unclear [39]. In addition, the suppression of SulA protein expression and disturbance of FtsZ ring formation have been documented as possible mechanisms of hydroxychavicol actions leading to DNA damage and disruptions in cell division, respectively [39,42]. The family of proteins involved in cell division was reported as a target molecule of hydroxychavicol, particularly the FtsA protein of E. coli, with highly stable interaction [43]; however, a study of the hydroxychavicol effects on cell division proteins of staphylococci has not been reported yet. Furthermore, hydroxychavicol induced cell death of E. coli, which was at a higher rate in the gshA mutant than the wild type due to DNA damage from the repair deficiency mechanism [39]; nevertheless, the effect on the genotypic aspect of staphylococci has not been investigated. In Gram-positive bacteria, hydroxychavicol showed an effect on Streptococcus mutans, which induced disintegration of the plasma cell membrane and disruption of cell membrane permeability [44,45]. A previous study predicted the mechanism of hydroxychavicol action obtained from leaf extract on S. aureus as cell death caused by cell membrane damage and interferences of protein and DNA function [46]. Nevertheless, the mentioned mechanism might also be a possible hydroxychavicol action on staphylococci, and further investigation should be conducted for hypothesis confirmation.

The amount of hydroxychavicol was followed during the experiment period, and a small quantity of the bioactive compound was insignificantly decreased compared to the day of preparation with other subsequent days. This finding was similar to a previous study on hydroxychavicol storage conditions in which the compound showed a lower degradation rate due to its specific structure. The structure of hydroxychavicol, particularly the different number and position of functional groups, might affect the stability of bioactive compound degradation [47]. Both monofilament (polypropylene) and multifilament (polyester)-coated P. betle extracts showed the same hydroxychavicol quantity trend. However, the amount of P. betle extract on the multifilament suture was greater than in the monofilament type. This is due to the different characteristics of both the suture and the coating agent-based solution. For multifilament, the suture contained several grooves among each single filament, which promotes the site for extract contact on the material. In addition, this groove also assisted the polycaprolactone in adhering to the suture and storing a greater amount of the bioactive ingredient. Moreover, the extract and polycaprolactone additionally blocked the groove area for bacterial adhesion. Of the different characteristics of polyester and polypropylene, the amount of extract coated in the different concentrations affected the different concentrations of the bioactive compound (hydroxychavicol). Thus, the different concentrations of bioactive compounds in polyester and polypropylene altered the different outcomes of hydroxychavicol activity that resulted in the differences in antibacterial, antibiofilm, and anti-adhesion activities in S. aureus and S. pseudintermedius. In humans, polycaprolactone has been permitted by the Food and Drug Administration (FDA) [48]; however, approval in animals has not been declared. Various advantages of polycaprolactone have been identified, including tissue biological compatibility, mechanical characteristics, and processing ability [49,50,51]. Furthermore, the inherent characteristics of caprolactone involve copolymerization and surface treatments, which are applied in combination with bioactive chemical filling [52,53,54]. A previous study of polycaprolactone-coated sutures found that sutures coated with pure polycaprolactone revealed better breaking strength and elongation; however, pure coating of polycaprolactone resulted in less releasing of bioactive filling agents, such as ciprofloxacin [55]. Of this, a caprolactone-based coating solution could be applied for bioactive compound release control; nevertheless, the behavior of control in hydroxychavicol should be additionally studied.

P. betle leaf extract-coated sutures presented antibacterial activity on both S. aureus and S. pseudintermedius. In addition, the coating agent-based solution and suture materials found no antibacterial effects on tested bacteria. Hydroxychavicol and eugenol primarily act as the main antibacterial phytochemicals against staphylococci [36]. Of this, the effects were particularly from P. betle leaf extract. Ethanolic extract of P. betle leaves was reported to have antibacterial activity against S. aureus, particularly the methicillin-sensitive S. aureus MSSA strain [56,57]. In addition, the effect of P. betle extract showed antibacterial activity on MRSA [58,59]. Crude extract of P. betle leaves showed an antibacterial activity against staphylococci isolated from dogs with pyoderma, including S. pseudintermedius and S. pseudintermedius [36]. Moreover, a previous study found that P. betle extracts had higher antibacterial activity against methicillin-sensitive S. pseudintermedius (MSSP) than methicillin-resistant S. pseudintermedius (MRSP) isolated from Thai dogs [31]. Notably, an extract of P. betle leaves was reported to have a bactericidal effect [60,61]. The antibiofilm properties of P. betle extract were documented to be effective against S. aureus, even at subinhibitory doses [56]. Moreover, a study of S. pseudintermedius isolated from Thai dogs indicated that P. betle extract exhibited higher antibiofilm formation activity in MSSP compared to MRSP [31]. In addition, the extracts that revealed inhibition activity of biofilm formation of more than 50% were defined as having high antibiofilm activity, and those with ≤50% as having low antibiofilm activity [62]. In this study, the antibiofilm formation effect of the P. betle extract-coated suture showed quite high antibiofilm activity against S. aureus; however, there was low antibiofilm activity against S. pseudintermedius. Regarding a goal of developing P. betle-coated sutures, the testing intended to reduce the production of biofilm from the tested staphylococci, and the experiment showed the inhibition of biofilm formation. The eradication of mature biofilm needed a greater concentration of plant extract than that utilized for the inhibition of biofilm [60], and our objective was dealt with in the abovementioned. The key mechanism for inhibiting biofilm formation has been identified as quorum-sensing interference and the rupturing of several signaling pathways through the interactions of lipopolysaccharides and exopolysaccharides [63]. Moreover, downregulation of the icaA gene might be the mode of action of P. betle extract affecting the biofilm production in S. pseudintermedius [60]. Regarding biofilm formation, attachment of bacterial cells was the first step of biofilm development [64,65]. Furthermore, inhibition of staphyloxanthin production by P. betle was observed in biofilm cells of S. aureus, which typically produce higher levels of this pigment compared to planktonic cells [66]. Of this, the effect of P. betle extract suggested a reduction in the biofilm-forming ability of S. aureus [57]. The percentage of biofilm formation inhibition by P. betle extract-coated sutures exhibited a consistent trend across both polyester and polypropylene for the same species. However, there were different inhibition rates between different species. The variation might be due to the reported mechanisms found in staphylococci from the downregulation of the icaA gene. However, the level of downregulation might be different in each species, but a comparison study between these species has not been reported. Of this, a comparison study of icaA gene expression between these species should be additionally investigated to explain the varying levels of biofilm formation inhibition affecting P. betle extract and hydroxychavicol. Our results reveal that the anti-adhesion of P. betle-coated sutures showed an effect against adhesion on tested staphylococci, which had a greater effect on S. pseudintermedius than S. aureus. The anti-adhesion effect of P. betle on E. coli demonstrated that the extract of P. betle exhibits anti-adhesion properties against bacteria [12,67]. Furthermore, P. betle extract has been reported as having anti-adhesion properties against various microorganisms, including pathogenic protozoa [68] and pathogenic yeast [69]. This supports our findings that P. betle extract has an effective anti-adhesion activity for microbial attachment to abiotic surfaces, such as suture materials.

For cytotoxicity of P. betle-coated sutures, our study found that P. betle extract showed dose-dependent cytotoxicity on HaCaT cells. Most cytotoxicity studies of P. betle extract were performed on cancer cell lines, such as HeLa cell (uterus adenocarcinoma), KB cell (papilloma), and MCF-7 cell (mammary gland adenocarcinoma) lines [70,71,72]. Nevertheless, there are a few cytotoxicity studies of P. betle extract conducted on skin cells, such as gingival keratinocytes (GK) [73] and the HaCaT cells [74]. A study in gingival keratinocytes found that cell morphology changes showed different degrees of cell death depending on extract concentration [73]. Moreover, a low cytotoxicity P. betle extract concentration on gingival keratinocytes was found at less than 1600 µg/mL [73]. A previous study of P. betle extract on HaCaT cells correspondingly found that the cytotoxicity effect depended on the concentration of the tested extract, and a concentration less than 10 µg/mL showed cell viability to be more than 88% [74]. Furthermore, the 50% inhibitory concentration (IC₅₀) of P. betle extract on HaCaT cells was approximately 50 µg/mL [74]. The limitation of this study was the in vitro experiments on skin cell lines derived from dogs or other lab animal species. The study of P. betle extracts and its main bioactive compounds, such as hydroxychavicol and eugenol, should be further investigated, particularly in normal skin cell lines from animals, including mouse epithelial cells (JB6Cl30-7b, RT101, or T36274), mouse epidermal cells (JB6Cl41-5a), dog epidermal keratinocytes (CPEK), and dog primary dermal epithelial cells. Future studies should be performed for safety evaluation of P. betle-coated sutures applied on the skin of animal models, such as mice or rabbits. Moreover, this study focused on Gram-positive bacteria, particularly Staphylococcus species, as the primary pathogens causing surgical site infections. Although Gram-negative bacteria possess clinical significance with a lower rate of infection than Gram-positive bacteria, the study of Gram-negative bacteria causing surgical site infection should be conducted as an additional investigation in future studies to cover for all bacteria causing surgical site infection in dogs.

5. Conclusions

This study demonstrated the development of P. betle leaf extract-coated polyester and polypropylene as novel antibacterials supports suture materials for inhibiting staphylococci causing skin infection in dogs. The hydroxychavicol that acts as the main antibacterial property on the coated suture was stable on the tested suture during the experiment period. In addition, the P. betle leaf extract-coated suture also showed antibiofilm formation and an anti-adhesion effect of tested staphylococci. The cytotoxic effect of P. betle leaf extract-coated sutures was dependent on the dose of the coated extract in keratinocyte cell lines in vitro. The results suggest that P. betle leaf extract-coated sutures might have veterinary benefits by inhibiting the growth, biofilm, and adhesion of staphylococci causing skin infections in dogs. Furthermore, P. betle leaf extract-coated sutures might be applied in veterinary surgery in the future. A limitation of this study was the use of in vitro experiments on skin cell lines from dogs. Future research should focus on the safety evaluation of P. betle-coated sutures applied to animal models like mice or rabbits. This study concentrated on Gram-positive bacteria, especially Staphylococcus species, while Gram-negative bacteria are clinically relevant even if they have a lower rate than Gram-positive bacteria. Future research should also examine Gram-negative bacteria to ensure a comprehensive understanding of all bacterial causes of surgical site infections in dogs.

6. Patents

This research received petty patents, number 2503004862, regarding the “Formulation and preparation process of a non-absorbable suture coating agent containing Piper betle leaf extract” from Thailand.