Antibacterial Efficacy of Oregano-Carbon Nanotubes Coated Silk Sutures: An In Vitro Experimental Study

Menna S Gaber; Nehal Loutfy Abouraya; Shaymaa I Habib

doi:10.1016/j.identj.2025.100893

. 2025 Jul 23;75(5):100893. doi: 10.1016/j.identj.2025.100893

Antibacterial Efficacy of Oregano-Carbon Nanotubes Coated Silk Sutures: An In Vitro Experimental Study

Menna S Gaber ^1,^⁎, Nehal Loutfy Abouraya ¹, Shaymaa I Habib ¹

PMCID: PMC12305716 PMID: 40706475

Abstract

Introduction

Microbial infections following surgery or other medical procedures are a major health issue. To avoid or reduce the risk of infections, research is now concentrated on creating novel drug-free materials with antibacterial qualities. Sutures coated with antimicrobial agents are one way to prevent surgical site infections.

Aim

To develop and characterize an oregano-carbon nanotubes/polycaprolactone (PCL)-chitosan composite coating and evaluate the antimicrobial activity of sutures coated with this formulation.

Methods

The dipping solution was prepared, and characterization was performed using Fourier Transform infrared spectroscopy, gas chromatography–mass spectrometry, transmission electron microscope and scanning electron microscope. Antibacterial activity against E. coli and S. aureus was evaluated after 1, 3 and 5 days using the colony-forming unit (CFU).

Results

Oregano-carbon nanotubes/PCL-chitosan composite coated sutures showed higher antibacterial activity compared to uncoated ones at all tested time intervals.

Conclusion

Oregano-carbon nanotubes/PCL-chitosan composite could be a promising candidate as a natural-derived suture coating to guard against surgical site infection.

Clinical relevance

This study highlights the potential use of oregano-carbon nanotubes/PCL-chitosan composite as a suture coating.

Key words: Oregano, Carbon nanotubes, Chitosan, Polycaprolactone, Sutures, Antibacterial property

Introduction

Surgical site infections (SSIs) are one of the most critical parameters after surgical intervention, particularly when foreign items like sutures and implants are present at the site of injury. These foreign items cause adherence and colonization of harmful bacteria on the surface of surgical sutures leading to infection.¹ Silk sutures are widely employed for oral and dental surgeries. Silk suture has high strength, low bacterial adhesion and good handling properties. Unfortunately, they lack antibacterial properties to guard against SSIs.²

Several antiseptics and antibiotics have been suggested for coating surgical sutures, but they have cytotoxic effects and are unsafe.³^,⁴ Numerous investigations have demonstrated that the use of triclosan-coated sutures reduces the amount of various bacterial species in vitro as well as animal or clinical trial wound infections. Nevertheless, several reports advise care in their usage due to adverse effects on wound healing, the emergence of resistance and effects on thyroid hormones.⁴

It has been acknowledged that natural compounds, such as plant extracts, may provide antimicrobial coatings for sutures owing to their safety and efficacy.⁵^,⁶ Recently, oregano essential oil (OEO) has drawn interest due to wide range of biological activities, especially those that are antibacterial, antioxidant, anti-inflammatory, antispasmodic, antifungal and antiviral. These are mostly caused by the presence of thymol and carvacrol, which disrupt the cell membrane of the pathogen and result in structural and functional damage.7, 8, 9 Torres et al.¹⁰ added OEO to chitosan scaffolds, and the results showed antibacterial activity against E. coli. Also, Tahric et al.¹¹ investigated the antimicrobial capabilities of OEO against gram-positive and gram-negative bacteria and results revealed inhibition of biofilm formation and an antibacterial effect against the studied bacteria.

Carbon nanotubes (CNTs), which are allotropes of carbon with a diameter less than or equal to 100 nm, are well known for their antibacterial activity because they cause significant structural damage to the cell wall and membrane of microorganisms or produce toxic substances.¹² To attain sustained release of the antibacterial agent for a longer duration and avoid burst release if it is applied alone, a carrier is required. Therefore, the aim of this in vitro study was to develop a natural-based suture coating incorporating OEO and CNTs, loaded within an appropriate carrier system to enhance long-term antibacterial activity for the prevention of SSI.

Materials and methods

Preparation of different solutions

Oregano essential oil preparation

The dry leaves of the Origanum vulgare plant were used in this study as a precursor for the OEO preparation via a hydrodistillation method using a modified Clevenger system. Oregano leaves (50 g) were ground, after which 500 millilitres of distilled water were added and the mixture was heated to boiling. During oil evaporation, steam moved upward through the tube at the top of the container. Then the vapor full of essential oil was condensed and dehydrated with anhydrous sodium sulphate powder. Finally, the pure oil was stored at 4 °C in a dark container until further use.¹³

Preparation of polycaprolactone and chitosan solutions

Two grams of PCL polymer pellets (Mw 80 KDa, Sigma-Aldrich chemistry) were weighted using a digital balance (Scaltec Instruments) and added to 10 mL of glacial acetic acid to obtain a concentration of 20% w/v. The mixture was kept on the magnetic stirrer overnight to ensure complete dissolution of the polymer.¹⁴ At the same time, 0.3 g of low-molecular-weight chitosan powder (90% degree of deacetylation, Loba Chemie) was dissolved in 95% acetic acid solution to prepare chitosan solution with concentration 3% w/v.¹⁵

Preparation of the dipping solution

The dipping solution was prepared by mixing equal volumes (1 mL each) of PCL, chitosan, and OEO solutions, as determined by a pilot study to ensure proper coating flow. Thereafter, 0.3 g of multi-walled carbon nanotubes (MWCNTs; 10–40 nm, Nanogate) was added, this being the maximum concentration achievable without causing precipitation or phase separation, as confirmed by the pilot study. The resulting mixture was stirred using a magnetic stirrer for 1 hour and then sonicated for 30 minutes at room temperature.

Coating procedure

Black multifilament braided sterile silk surgical sutures size 3-0 (Trusilk) were used in this study. The sutures were cut, soaked in the prepared dipping solution and stirred using a magnetic stirrer at a speed of 400 rpm. Subsequently, the coated sutures were air-dried for 30 minutes. This process was repeated three times to ensure a uniform and continuous coating over the suture surface.¹⁶

Characterization of the prepared solutions and sutures coating

Gas chromatography–mass spectrometry

A mass spectrometer and gas chromatograph were used to evaluate the produced oil extract. A mechanism for ionizing electrons with an ionization energy of 70 eV was employed. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. By comparing the components' relative retention times and mass spectra with those of the NIST, WILLY library data of the GC/MS system, the components of OEO were identified.¹⁷

Fourier Transform infrared spectroscopy (FTIR)

The functional groups and chemical composition of each component; OEO, chitosan, PCL, CNTs and the dipping solution were determined by Fourier Transform Infrared Spectrometer (Nicolet 6700, Thermo Scientific) using the KBr method. FTIR spectra were collected over the range of 4,000–400 cm⁻¹ in the transmittance mode.

Transmission electron microscope

The morphology, structure and size of CNTs were assessed by transmission electron microscope (HR-TEM, JEOL JEM-2100) at an operating accelerating voltage of 200 kV.

Scanning electron microscope

The surface morphology of silk sutures was investigated before and after coating using a scanning electron microscope (Quanta FEG250, Thermo Fisher Scientific) equipped with an Energy Dispersive X-ray analysis Unit (EDAX) (Quanta 250 FEG/ EDS) at a magnification of ×300. Before analysis, suture samples (1 cm length) were mounted onto aluminium stubs, secured with carbon adhesive tape, and gold sputtered using a sputter coater (EMITECH, K550X sputter coater). Moreover, the diameter of the sutures was measured before and after coating using image analysis to determine the thickness of the formed coating layer at a magnification of ×500.

Assessment of the antibacterial activity of the coated sutures

S. aureus strain ATCC 6538 and E. coli strain ATCC 8739 were selected in this study as representative of gram-positive bacteria and gram-negative bacteria, respectively. They were obtained from the Microbiological Resources Centre (Cairo Mircen) for the agar dilution method. Bacteria were inoculated in Mueller Hinton Broth (MHB) and incubated for 24 hours at 37 °C to a bacterial density of 0.5 McFarland. The sample size of the antibacterial activity of sutures was calculated according to a study done by Syaflida et al.¹⁸ The difference in the amount of bacterial colony adherent was 300 ± 173.6, so by using power 80% and 5% significance level and an effect size equal to 1.73, 6 sutures were needed in each group. The samples were assigned into 2 groups: group 1 with uncoated sutures (control) and group 2 with coated sutures.

A total of 24 samples were prepared (12 samples/group, 6 samples/bacterial species). Sutures were cut to 6 cm and transferred individually to a tube containing 1 mL of previously prepared bacterial suspension of density 1.5 × 10⁸ CFU/mL. The sutures were then incubated for different intervals: 24 hours, 3 days and 5 days at 37 °C. After each predetermined time period, the extracted bacterial suspension was diluted serially to 7 dilutions. Next, aliquots of 0.1 mL from each tube were seeded in duplicates onto MHB Agar plates to be grown for counting and incubated for 48 h at 37 °C. Then, plates with colonies between 30 and 300 CFU/mL were selected, the colonies were counted and mean values of CFU/mL were obtained. A group without any samples was set as the blank control to calculate the antibacterial efficiency according to the following equation¹⁹:

Antibacterial activity (%) = \frac{(N c - N s)}{N c} \times 100

where Ns is the number of bacterial colonies in the retrieved bacterial suspension following the suture samples' 24-hour, 3 days and 5 days incubation periods, and Nc is the blank control.¹⁹

Statistical analysis

Data were analyzed using Medcalc software, version 22 for Windows (MedCalc Software). Continuous data were explored for normality using the Kolmogrov Smirnov test and the Shapiro Wilk test. Results showed normal distribution and were described as mean and standard deviation. Intergroup comparison of continuous data was performed using an independent t-test with statistical significance set at P ≤ .05, while intragroup comparison was performed using paired t-test with statistical significance set at P ≤ .05 or repeated measures ANOVA with statistical significance set at P ≤ .0166 when appropriate.

Results

Characterization of the prepared solutions and sutures coating

Gas chromatography–mass spectrometry

The volatile components of the OEO were identified via GC-MS analysis (Figure 1A). Thirty compounds were reported in the form of retention time and percentage of peak area. Results showed that thymol (12.87 min, 24.9%), carvacrol (21.38 min, 17.9%) and ɑ-pinene (9.65 min, 2.07%) were the most significant volatile chemicals found.

Fig 1 — A, The total ion chromatogram of oregano essential oil. B, FTIR spectra of PCL, chitosan, CNTs, OEO, and dipping solution.

Fourier Transform infrared spectroscopy (FTIR)

Results of FTIR analysis are shown in Figure 1B. The FTIR spectrum of PCL shows the characteristic bands of carbonyl stretching C=O around 1,728 cm⁻¹. The band at 2,985 cm⁻¹ corresponds to asymmetric CH₂ stretching while 2,893 cm⁻¹ is assigned to symmetric CH₂ stretching. The band at 1,234 cm⁻¹ corresponds to asymmetric C-O-C stretching and 1,118 cm⁻¹ is assigned to symmetric C-O-C stretching. The observed OH peak at 3,703 cm^-1 is attributed to the dissolution of PCL in acetic acid to form a film before the analysis. Regarding chitosan, the spectrum showed a broad peak at 3,435 cm^–1, which is assigned to the N-H and hydrogen-bonded O-H stretch vibrational frequencies. Additionally, in the C–H stretching region of the FTIR spectrum, the more intense peak observed at 2,918 cm⁻¹ corresponds to the asymmetric stretching vibration of CH₂ groups, while the less intense peak at 2,851 cm⁻¹ is attributed to the symmetric stretching mode of CH₂. Moreover, the sample displayed the characteristic CH₂ scissoring band at 1,465 cm⁻¹. An amide bond was also identified in the spectrum, with the C=O stretching vibration appearing at 1627 cm⁻¹.

For CNTs, the FTIR spectrum exhibited a peak at 3,433 cm⁻¹, attributed to O–H stretching of hydroxyl groups. The peak at 2,918 cm⁻¹ corresponds to C–H stretching vibrations from the CNT surface, while the peak at 1,629 cm⁻¹ is assigned to C=C stretching. Additionally, the peak at 1,429 cm⁻¹ indicates C=O stretching in the carboxyl group, and the peak at 1,107 cm⁻¹ corresponds to C–O stretching vibrations.

With respect to OEO chemical composition, the spectrum revealed a peak at 2,923 cm^-1 that indicated the presence of the aromatic C–H stretching vibration. Moreover, O–H bonds appeared at 3,423 cm^-1, along with C–O stretching and C–N bending vibrations observed at 1,158 and 1,019 cm^-1, respectively. The peak is at 1,567 cm^-1 assigned to N–H bending while the C–C stretching of the aromatic ring of carvacrol, the major OEO component, appeared at 1,418 cm^-1. The peak at 1,277 cm^-1 is strongly related to the characteristic absorption of the C–O–C stretching vibration, and a characteristic band appeared at 888 cm^-1 for C-H bending. The transmission peaks of the dipping solution showed the characteristic peaks of all constituent elements. Moreover, the spectrum showed slight peaks’ shifts compared to the constituent elements, in addition to the appearance of new peaks that were not present in the constituent elements at 2,727 cm^-1, 1,891 cm^-1, 1,514 cm^-1 and 1,065 cm^-1.

Transmission electron microscope

Micrograph of CNTs demonstrated that the CNTs are multi-walled nanotubes with a hollow elongated tubular structure, with lengths of up to 5 μm and a diameter range of 10 to 40 nm.

Scanning electron microscope

Scanning electron micrographs, as shown in Figure 2, confirmed the formation of a continuous and relatively homogenous coating on the surface of sutures that were dipped in the oregano-carbon nanotubes/PCL-chitosan composite solution. In contrast to the smooth surface of uncoated sutures, the coated sutures exhibited a rougher texture. Image analysis further revealed that the coating thickness ranged from 230.7 μm to 338 μm.

Fig 2 — Scanning electron micrographs of the surface of (A) coated and (B) uncoated sutures showing the presence of homogenous coating on the surface (the red arrows) at magnification ×300. Scanning electron micrographs of the cross section of (C) coated and (D) uncoated sutures showing the thickness of the coating (blue braces) at magnification ×500.

Antibacterial activity

Results of the antibacterial activity of the oregano-carbon nanotubes/PCL-chitosan composite coated sutures are presented in Table 1, Table 2 and Table 1, Table 2. Results revealed a statistically significant higher antibacterial activity against both E. coli and S. aureus for the coated sutures compared to uncoated sutures at all tested time intervals, P < .0001. Results also showed that the antibacterial activity increased significantly with time against E. coli and S. aureus with the highest antibacterial effect revealed at day 3 and 5 with no significant difference between them. The colonies of E. coli in CFU/mL in the control group at day 1, 3 and 5, respectively, were 29 × 10⁶, 27 × 10⁶ and 26 × 10⁶ and intervention group at day 1, 3 and 5, respectively, were 6.6 × 10⁶, 5.9 × 10⁶ and 5.5 × 10⁶. The colonies of S. aureus in the control group at day 1, 3 and 5 were, respectively, 27 × 10⁶, 25 × 10⁶ and 22 × 10⁶ and intervention group at day 1, 3 and 5 were, respectively, 5.9 × 10⁶, 4.8 × 10⁶ and 4.1 × 10⁶.

Table 1.

Mean and standard deviation values of log CFU of E. coli and S. aureus showing intergroup and intragroup comparisons within each group between time points.

Intervention/Time	Coated		Uncoated		P
	Mean	SD	Mean	SD
Day 1	6.84aB	0.025	7.44^aA	0.024	<.0001*
Day 3	6.77^bB	0.007	7.42^aA	0.025	<.0001*
Day 5	6.74^bB	0.007	7.39^aA	0.027	<.0001*
P	<.0001*		.063

Intervention/Time
	Mean	SD	Mean	SD

Day 1	6.77^aB	0.025	7.43^aA	0.032	<.0001*
Day 3	6.65^bB	0.024	7.33^aA	0.018	<.0001*
Day 5	6.59^cB	0.022	7.34^aA	0.029	<.0001*
P	<.0001*		.088

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Means that do not share the same small superscript vertically are statistically significant. Means that do not share the same capital superscript horizontally are statistically significant. *corresponds to statistically significant (P < .05).

Table 2.

Mean and standard deviation values of the antibacterial activity percentage of the coated sutures on E. coli and S. aureus showing intergroup and intragroup comparisons between the three time intervals.

Intervention/Time	E. coli		S. aureus		P
	Mean	SD	Mean	SD
Day 1	75.57b^A	1.31	77.47b^A	1.62	.0507
Day 3	77.78^abB	0.37	81.73a^A	1.01	.0031*
Day 5	78.46a^B	0.38	82.27a^A	0.91	.0026*
P	<.0001*		<.0001*

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Discussion

Numerous efforts have been made to combat and prevent SSIs. Strategies to reduce the frequency of SSIs mostly concentrate on device-based solutions, like antibacterial sutures, and system-based solutions, like enhanced antiseptic programs.²⁰^,²¹ The need for more natural and biocompatible alternatives aroused researchers' interest toward herbal substances. Origanum vulgare, also known as oregano, is among these herbs and may be useful as an antimicrobial agent. To provide approaches for treating wound infections, these substances can be added to natural or synthetic polymers that are compatible with host cells. Natural essential oils’ bacteriostatic qualities are drawing increasingly more interest because they hinder the development of drug resistance in bacteria.²²^,²³ The antibacterial properties of OEO against both gram-positive S. aureus and gram-negative E. coli bacteria have been proven either alone or synergistically with regular antibiotics.²⁴^,²⁵

In this study, CNTs were selected and added to the OEO as part of the prepared dipping solution for silk sutures. CNTs have many advantageous properties that make them suitable for biomedical applications, such as biocompatibility, ultra-light weight, chemical inertness, high tensile strength and a wide range of antibacterial and antifungal properties.²⁶

To allow sustainable and controlled release of CNTs and OEO, a carrier was prepared from PCL and chitosan. PCL is characterised by its biocompatibility, biodegradability and simplicity of modification and presents good mechanical properties that make it ideal for tissue engineering and coating surgical sutures. However, its inherent hydrophobicity poses a limitation. To address this, its combination with chitosan, a hydrophilic polymer, has been proposed to enhance its properties and regulate its degradation rate27, 28, 29 as reported by Liu et al.³⁰ and Sadeghi et al.³¹

In the current study, essential oil was extracted from Origanum vulgare leaves using the water distillation method. This technique offers several advantages, including the ability to process finely powdered plant materials, the absence of organic solvents, cost effectiveness of the distillation apparatus, ease of setup and suitability for field applications. Successful extraction was confirmed by GC/MS analysis, which identified the polyphenolic compounds characteristic of oregano essential oil, namely carvacrol and thymol.

Several methods can be used to create drug-eluting sutures including electrospinning, grafting and dip coating. The dip-coating method applied in this work is a simple, non-toxic and an effective method to coat different substrates. This was proven by Ravishankar et al.¹⁶ and Selvaraju et al.³² who found that the dipping method provided a homogeneous continuous layer of the coat on the sutures’ surface as observed in this study by SEM (Figure 2). In our study the coating was successfully prepared showing an antibacterial effect while maintaining the integrity of the surgical sutures.

In this study, E. coli and S. aureus were selected to evaluate the antibacterial activity of the coated sutures because they are common pathogens responsible for infections in skin and soft tissues, surgical sites, bones and joints. The National Nosocomial Infections Surveillance System reports that E. coli and S. aureus are two of the most often isolated bacteria at surgical sites.¹⁶

Regarding the antibacterial activity assessment, the agar dilution method was used because it was proven to be reliable in determining MICs, easy to read, straightforward, affordable and with well-defined parameters.³³ Many studies have investigated the antibacterial effect for only a short duration of no more than 24 hours.¹⁶^,34, 35, 36 Very few studies have explored the antibacterial effect over a longer duration, which is more clinically significant in providing protection against SSIs. The 5-day period was chosen because this period typically requires antibiotic coverage post-operatively.³⁷

Results revealed a statistically significant higher antibacterial activity for the coated sutures against both E. coli and S. aureus compared to the uncoated sutures at all time intervals. The antibacterial effect was significant from day 1, which is crucial for preventing early wound infections, thereby confirming the successful release of OEO and CNTs from the carrier. Results also showed that the antibacterial activity increased significantly with time against the studied bacterial species.

This enhanced antibacterial activity in the coated group may be attributed to both CNTs and the chemical composition of OEO. As identified in the GC-MS analysis, carvacrol and thymol are the predominant polyphenols in OEO. These compounds disrupt and depolarise the bacterial cytoplasmic membrane, leading to cell leakage and eventual bacterial cell death.³⁸

The results of the present study were in agreement with Orlando et al.³⁹ and Tejada-Munoz et al.,⁴⁰ who found that OEO has an antibacterial activity against gram-negative E. coli and gram-positive S. aureus. Moreover, Torres et al.¹⁰ reported that the Origanum vulgare essential oil incorporated into chitosan-based scaffolds was an effective inhibitor for E. coli strains. Meanwhile, Moskvitina et al.¹² recorded high antibacterial activity for carboxylated CNTs against both E. coli and S. aureus after 24 hours, as confirmed by SEM, where nanotubes could adsorb on aggregates enveloping the bacterial cells, damaging the cell wall. The antibacterial effect of CNTs against S. aureus has also been reported by Asaftei et al.,⁴¹ with nanotube diameters ranging from 50 to 150 nm, used either alone or in combination with antibiotics to achieve a synergistic effect. This study has two limitations: it is an in vitro study and needs further animal studies for clinical validation and to assess the anti-inflammatory effect of the coating in vivo; and the effect of the coating on bacterial biofilm formation was not tested.

Conclusions

Oregano-carbon nanotubes /PCL-chitosan composite could be a promising candidate as a natural-derived suture coating to guard against surgical site infection.

Authors’ contribution

Conceptualisation: Habib, Abouraya. Data acquisition: Gaber. Data analysis and interpretation: Gaber. Design: Habib, Abouraya. Investigation: Gaber. Supervision: Habib, Abouraya. Validation: Habib, Abouraya. Visualisation: Habib, Abouraya. Writing—original draft: Gaber. Writing—review and editing: Gaber, Habib, Abouraya

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.identj.2025.100893.

Appendix. Supplementary materials

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.jpg^{(1MB, jpg)}

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PERMALINK

Antibacterial Efficacy of Oregano-Carbon Nanotubes Coated Silk Sutures: An In Vitro Experimental Study

Menna S Gaber

Nehal Loutfy Abouraya

Shaymaa I Habib

Abstract

Introduction

Aim

Methods

Results

Conclusion

Clinical relevance

Introduction

Materials and methods

Preparation of different solutions

Oregano essential oil preparation

Preparation of polycaprolactone and chitosan solutions

Preparation of the dipping solution

Coating procedure

Characterization of the prepared solutions and sutures coating

Gas chromatography–mass spectrometry

Fourier Transform infrared spectroscopy (FTIR)

Transmission electron microscope

Scanning electron microscope

Assessment of the antibacterial activity of the coated sutures

Statistical analysis

Results

Characterization of the prepared solutions and sutures coating

Gas chromatography–mass spectrometry

Fig. 1.

Fourier Transform infrared spectroscopy (FTIR)

Transmission electron microscope

Scanning electron microscope

Fig. 2.

Antibacterial activity

Table 1.

Table 2.

Discussion

Conclusions

Authors’ contribution

Conflict of interest

Acknowledgments

Funding

Footnotes

Appendix. Supplementary materials

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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