Low-intensity pulsed ultrasound combined with nystatin treatment synergistically inhibits vaginal Candida albicans biofilm infection in rabbits

doi:10.12122/j.issn.1673-4254.2025.02.10

Abstract

Abstract:

Objective To explore the efficacy of low-intensity pulsed ultrasound (LIPUS) combined with nystatin for treatment of vaginal Candida albicans biofilm infection. Methods In vitro cultured Candida albicans biofilm were treated with LIPUS, nystatin, or both, and the minimum inhibitory concentration (MIC) of nystatin was determined. Crystal violet staining, confocal laser microscopy (CLSM) and scanning electron microscopy were used to quantify the biofilm and observe the activity and morphological changes of the biofilms; DCFH-DA was used to detect the changes in reactive oxygen species (ROS). Twenty female New Zealand White rabbits with vaginal inoculation of Candida albicans biofilm were randomized into 4 groups for treatment with normal saline, LIPUS, nystatin, or both LIPUS and nystatin. The changes in vulvar symptoms of the rabbits were observed, and the histopathological and ultrastructural changes of the vagina before and after treatment were observed using HE staining and transmission electron microscopy. Results In the combined treatment group, the MIC₅₀ and MIC₈₀ of nystatin in Candida albicans biofilms were both reduced by 50% compared with those in nystatin group, and the biofilm clearance rate increased by 26% and 68% compared with nystatin and LIPUS groups, respectively. Compared with nystatin and LIPUS treatment alone, the combined treatment produced stronger effects for inhibiting biofilm activity, causing structural disruption and promoting ROS production. In the rabbit models, the combined treatment more effectively improved vulvar symptoms and inflammatory infiltration, reduced residual vaginal hyphae/strains, and improved ultrastructure of the vaginal epithelium than LIPUS and nystatin treatment alone. Conclusion LIPUS combined with nystatin produces a significant synergistic antifungal effect against Candida albicans biofilm both in vitro and in vivo.

Key words: low-intensity pulsed ultrasound, Candida albicans, biofilm, nystatin, vaginitis

Mengyao XIE, Min YANG, Xin LI, Yonghong DU. Low-intensity pulsed ultrasound combined with nystatin treatment synergistically inhibits vaginal Candida albicans biofilm infection in rabbits[J]. Journal of Southern Medical University, 2025, 45(2): 296-303.

Figures/Tables 6

Fig.1 Crystalline violet staining and microscopic observation of changes in the structure of Candida albicans biofilm from 2 h to 72 h in culture (scale bar=520 μm).

Fig.2 Quantification of Candida albicans biofilm by crystalline violet staining. A: Crystalline violet staining to observe the changes in the structural characteristics of biofilm after different treatments (Scale bar=360 μm). B: Detection of A570 nm of crystal violet stained biofilm by enzyme marker. C: Biofilm reduction rate after different treatments. ***P<0.001.

Fig.3 Confocal laser scanning microscopy for observing 3D and orthogonal views of Candida albicans biofilm activity after different treatments (scale bar=100 μm). Green and red fluorescence signals represent live and dead fungi, respectively.

Fig.4 Scanning electron microscopy for observing Candida albicans biofilm ultrastructure. ①Partial magnification of the dense biofilm structure destroyed and Candida albicans exposed inside the membrane after nystatin (NYS) treatment; ② Partial magnification of severely destroyed structure of Candida albicans inside the biofilm after NYS combined with ultrasound treatment. Red arrows represent exposed Candida albicans. Scale bar: 5 μm; ①, ② Scale bar=2 μm.

Fig.5 Production of ROS by Candida albicans biofilm in each group. A: Confocal laser scanning microscopy for observing ROS fluorescence (green) intensity produced by the biofilm after different treatment (scale bar=100 μm). B: Flow cytometry detection of ROS fluorescence intensity produced by the biofilms in each group (the horizontal coordinate represents the fluorescence intensity and the vertical coordinate represents the number of cells).

Fig.6 Assessment of treatment efficacy against Candida albicans vaginal biofilm infections in the rabbits. A: Vulvar symptoms of the rabbits after 7 days of continuous treatment. B: HE staining of the vaginal tissues after 7 days of treatment (black arrows indicate Candida albicans aggregation and red arrows indicate submucosal inflammatory infiltration. scale bar=100 μm). C: Transmission electron microscopy of observing vaginal epithelial ultrastructure (scale bar=2 μm). Yellow arrows indicate Candida albicans infection, green arrows indicate infiltrated neutrophils, and blue arrows indicate swollen mitochondria.

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