In vitro assessment of berberine-loaded carboxymethyl chitosan hydrogel: A promising antimicrobial candidate for S. aureus-induced bovine mastitis treatment

Abstract

Bovine mastitis poses significant challenges to the global dairy industry, leading to substantial economic losses and public health concerns. Staphylococcus aureus, a prevalent causative agent of bovine mastitis, depends on effective adhesion and biofilm formation to establish infections. Berberine (BER), a naturally occurring phytochemical, demonstrates broad-spectrum antibacterial activity but suffers from poor bioavailability. This study developed a composite berberine-carboxymethyl chitosan/sodium alginate hydrogel to address these limitations. The hydrogel was characterized using scanning electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. In vitro assessments revealed that the BER hydrogel eradicated S. aureus biofilms (42% eradication at 156.26 μg/mL), inhibited bacterial adhesion, and reduced inflammatory cytokines (IL-6 and TNF-α) in S. aureus-infected MAC-T cells, with compliant biosafety biocompatibility (hemolysis rate <5%) and sustained drug release (100% over 6 h), though pH-dependent release kinetics necessitate microenvironment-specific formulation refinement. In conclusion, the BER hydrogel represents a potential therapeutic candidate for S. aureus-induced bovine mastitis.

Fig 1. Schematic illustration of BER hydrogel preparation and its anti-inflammatory effects on S. aureus-infected MAC-T cells.

Fig 2. Preparation and SEM images of the BER hydrogel.

(A). The formation process of BER hydrogel. (B). injectability of the hydrogel. (C). SEM images of the cross sections of BER hydrogel.

Fig 3. Physicochemical characterization of the BER hydrogel.

(A). FTIR spectra, (B). XRD patterns of the BER extract, CMCS/SA hydrogel and BER hydrogel. The pore sizes (C) and the Zeta potential (D) of BER hydrogel. The swelling ratio (E) of BER hydrogel were determined in PBS (pH = 7.4) at 37°C; and cumulative release of BER from the BER hydrogel (F) in PBS (pH = 3.0), PBS (pH = 7.4), and PBS (pH = 8.9) within 6 h at 37°C.

Fig 4. Effects of BER hydrogel on cell morphology and its biofilm of S. aureus.

(A). The MIC of BER hydrogel, the columns 1-9 were 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2,500 μg/mL of BER hydrogel, respectively, while the column N (from left to right) (negative control) S. aureus was used. (B). SEM analysis of cell morphology of S. aureus treated with 0.5 MIC BER hydrogel for 24 h. (C). SEM analysis of cell morphology of normal S. aureus. (D). SEM analysis of cell morphology of S. aureus treated with CMCS/SA hydrogel. (E)The biofilm eradication rate of BER hydrogel in the concentration range of 0.5-2MIC (n = 6/group). Data were analyzed by one-way ANOVA. p-value indicates the difference among the groups; p < 0.05 indicates a significant difference. Groups with different letters had significant differences (p < 0.05).

Fig 5. The biocompatibility and anti-inflammatory injury of BER hydrogel in S. aureus -induced mastitis MAC-T cell model.

(A). The hemolysis test of BER hydrogel. (B). The cell viability of MAC-T cells stimulated by 9.77-78.13 μg/mL BER hydrogel for 24 h. (C, D). The effects of BER hydrogel on the secretion of IL-6 and TNF-α in S. aureus- stimulated cells. (E, F). The gene expression of IL-6 and TNF-α in S. aureus stimulated cells. (G). The effects of BER hydrogel on the adhesion rate of S. aureus. Independent t-tests were conducted for data analysis of cell viability, other data were analyzed by one-way ANOVA. p -value indicates the difference among the groups; p < 0.01 indicates an extremely significant difference. Groups with different letters had significant differences (p < 0.01).