Efficacy of In‑Situ Chitosan‑Silver Nanoparticle Solutions Against Methicillin‑Resistant Staphylococcus aureus (MRSA)

Abstract

Background: Developing new therapeutics against methicillin‑resistant Staphylococcus aureus (MRSA) is a pressing medical challenge. Antiseptics, unlike antibiotics, can target resistant strains while preserving normal microbiota.

Materials and Methods: We evaluated the antibacterial potency of chitosan‑silver nanoparticle (Ag NP) solutions prepared in situ, varying the chitosan to Ag ratio. Ag NPs were synthesized via a green chemistry route using ginger rhizome extract and ascorbic acid; surface modification with cetrimonium bromide (CTAB) was also explored to enhance dispersibility. The resulting nanoparticles and chitosan‑Ag NP mixtures were characterized by X‑ray diffraction (XRD), transmission electron microscopy (TEM), Fourier‑transform infrared spectroscopy (FTIR), and UV–Vis spectroscopy.

Results and Conclusions: XRD, FTIR, UV–Vis, and TEM confirmed the high purity and expected composition of chitosan and Ag NPs. Chitosan‑Ag NP solutions demonstrated superior antimicrobial efficacy compared to their individual components. In contrast, in‑situ preparation of chitosan‑Ag NP/CTAB solutions led to precipitation and could not be evaluated. These findings suggest that chitosan‑Ag NP mixtures are a promising strategy to combat drug‑resistant bacteria.

Background

Infections remain a leading cause of global morbidity and mortality, driven in part by widespread antibiotic use that selects for resistant organisms such as MRSA. MRSA infections encompass a spectrum from superficial skin lesions to life‑threatening conditions like pneumonia, endocarditis, and septicemia, imposing a substantial economic burden (≈$3–4 billion annually). New antibacterial strategies are urgently needed. Antiseptics offer sustained activity against resistant strains without disrupting host microbiota. Combining distinct antibacterial mechanisms—e.g., metals and natural polymers—via hybrid nanomaterials presents a novel approach to defeat resistant bacteria.

Silver has long been recognized for its antimicrobial properties, and silver nanoparticles (Ag NPs) exhibit potent antibacterial and antifungal effects. They can act synergistically with conventional antibiotics but may also suffer from aggregation, uncontrolled ion release, and cytotoxicity. Incorporating Ag NPs into biopolymers such as chitosan can enhance stability and antimicrobial activity. Chitosan, derived from chitin, is biocompatible, bacteriostatic, and suitable for medical applications. Surface modification of Ag NPs with CTAB can improve dispersion stability, though its impact on antibacterial efficacy is not fully understood.

The aim of this study was to identify the optimal chitosan‑Ag NP ratio that yields maximum activity against clinical MRSA isolates.

Methods

Materials

Silver nitrate, L‑ascorbic acid, and CTAB (C16H33N(CH3)3Br) were purchased from Sigma‑Aldrich. Ginger rhizome was sourced locally. Chitosan (200 kDa, 82 % deacetylation) was obtained from Bioprogress. Ultrapure water (resistivity > 17 MΩ cm⁻¹) and standard microbiological media were used throughout.

In‑Situ Preparation of Chitosan/Ag NP Solutions

Ag NPs were first synthesized by reducing silver nitrate with ginger extract and ascorbic acid under reflux at 60 °C for 1.5 h. The resulting particles were washed by centrifugation until neutral pH. For CTAB‑modified Ag NPs (Ag/CTAB NPs), 3 ml of a 76.4 mg ml⁻¹ Ag NP dispersion was mixed with 20 ml of 6.7 mg ml⁻¹ CTAB solution and sonicated for 3 h. CTAB loading was quantified by UV–Vis at 190 nm.

Chitosan solutions (1 % w/v) were prepared by dissolving 1 g of chitosan in 100 ml of 2 % acetic acid for 24 h at room temperature. Chitosan/Ag NP solutions were then formed by adding varying volumes of chitosan to Ag NP dispersions, generating a range of chitosan and Ag NP concentrations (Table 1).

Physicochemical Characterization

XRD was performed on an Empyrean diffractometer (Cu Kα, 1.54 Å). TEM imaging used a JEM‑ARM‑200F at 200 kV. FTIR spectra were recorded on a Tensor 27 spectrometer with KBr pellets (1 mg sample / 200 mg KBr). UV–Vis absorption was measured on a Lambda 950 spectrometer (200–800 nm). All measurements were replicated at least three times.

Microbiological Tests

Seventy patient swabs (nasal and throat) were cultured on blood agar. Fifty Staphylococcus aureus strains were isolated and confirmed by standard biochemical tests. Antibiotic susceptibility was assessed by the Kirby–Bauer disk diffusion method on Mueller–Hinton agar for azithromycin, levofloxacin, clarithromycin, ciprofloxacin, and methicillin. MRSA strains were defined per CLSI guidelines.

Minimum inhibitory concentrations (MICs) of chitosan, Ag NPs, and chitosan‑Ag NP solutions were determined by broth macrodilution. Seven two‑fold serial dilutions of each nanoparticle preparation were prepared in nutrient broth, and 1, 2, or 3 ml of 1 % chitosan was added to each tube. After inoculation with 100 µl of a standardized bacterial suspension (≈1.5 × 10⁸ CFU ml⁻¹) and 24 h incubation at 37 °C, the lowest concentration preventing visible growth was recorded as the MIC.

Results

Characterization of Ag NPs and Chitosan

CTAB adsorption onto Ag NPs yielded an adsorptivity of 70.0 mg g⁻¹, corresponding to a CTAB content of ~6.5 %. XRD patterns displayed characteristic silver peaks at 38.15°, 44.33°, 64.48°, 77.47°, and 81.54° 2Θ, confirming crystalline Ag. A broad peak (12.00–21.06°) indicated residual organic material. Chitosan exhibited semicrystalline peaks near 9° and 20° 2Θ. FTIR spectra revealed expected functional groups for chitosan (broad O‑H/N‑H stretch 3450–3200 cm⁻¹, amide I at 1652 cm⁻¹) and Ag NPs (CO/CH bending, C=C/C=O stretching). UV–Vis spectra of Ag NPs showed a surface plasmon resonance peak at ~387 nm, shifting to ~417 nm after CTAB modification, indicating improved dispersion stability. TEM images revealed predominantly spherical Ag NPs sized 10–12 nm.

Antibacterial Activities

Pure Ag NPs and Ag/CTAB NPs displayed MICs of 9.6 µg ml⁻¹ against all 10 MRSA strains. Chitosan alone exhibited an MIC of 6 µg ml⁻¹, with 60 % of strains inhibited at 3.3–5 µg ml⁻¹. The chitosan‑Ag NP mixture achieved MICs as low as 1.2 µg ml⁻¹ for Ag NPs and 3.3 µg ml⁻¹ for chitosan, representing 2‑ to 4‑fold reductions compared to individual components. Attempts to prepare a chitosan‑Ag/CTAB mixture resulted in precipitation and could not be evaluated.

Discussion

Our findings confirm that in‑situ mixing of chitosan with Ag NPs synergistically enhances antibacterial activity against MRSA. The reduced MICs suggest improved interaction with bacterial cells, potentially through combined mechanisms: silver ion release and chitosan’s polycationic surface disrupting membranes. CTAB, while stabilizing the nanoparticles, diminished antibacterial efficacy and induced precipitation, indicating that surface chemistry critically influences therapeutic potential. Future studies should assess cytotoxicity of the chitosan‑Ag NP formulation to determine safe clinical dosing.

Conclusions

In‑situ prepared chitosan‑Ag NP solutions exhibit markedly superior activity against MRSA, reducing required concentrations by up to fourfold relative to their individual constituents. This strategy offers a promising, potentially personalized approach to counter antibiotic‑resistant infections. Further toxicity evaluation will clarify clinical applicability.