低强度脉冲超声联合制霉菌素可协同抑制兔阴道白色念珠菌生物被膜感染

doi:10.12122/j.issn.1673-4254.2025.02.10

摘要/Abstract

摘要：

目的探索低强度脉冲超声（LIPUS）联合制霉菌素（NYS）对阴道白色念珠菌生物被膜感染的治疗效果。方法建立体外白色念珠菌生物被膜模型，同时将体外培养成熟的生物被膜用自动微移液管接种至20只雌性新西兰大白兔阴道内，以建立兔阴道白色念珠菌生物被膜感染模型，实验分为Control组、NYS组、US组和联合治疗组。体外实验采用XTT法检测生物被膜最低抑菌浓度（MIC），结晶紫染色定量生物被膜，激光共聚焦显微镜（CLSM）观察生物被膜活性，扫描电镜（SEM）观察形态学变化，DCFH-DA检测活性氧（ROS）。体内实验通过观察兔外阴症状，HE染色和透射电镜（TEM）观察治疗前后阴道组织病理结构变化。结果体外实验显示，联合治疗组MIC₅₀和MIC₈₀相比NYS组均降低2倍（P<0.05），此外，联合治疗组生物被膜清除率分别比NYS组和US组增加约26%和68%（P<0.001），同时联合治疗组生物被膜活性更低、结构破坏更严重，ROS产量更多。体内实验表明，与NYS组和US组比较，联合治疗组兔外阴症状与炎症浸润显著改善（P<0.05），阴道残留菌丝/菌株减少，上皮超微结构明显改善。结论 LIPUS联合NYS对体内外白色念珠菌生物被膜有显著的协同抗真菌作用。

关键词: 低强度脉冲超声, 白色念珠菌, 生物被膜, 制霉菌素, 阴道炎

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

谢梦瑶, 杨敏, 李欣, 杜永洪. 低强度脉冲超声联合制霉菌素可协同抑制兔阴道白色念珠菌生物被膜感染[J]. 南方医科大学学报, 2025, 45(2): 296-303.

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.

图/表 6

图1 体外白色念珠菌生物被膜形成过程

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).

图2 结晶紫染色定量检测白色念珠菌生物被膜

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.

图3 CLSM观察白色念珠菌生物被膜活性

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.

图4 SEM观察白色念珠菌生物被膜结构

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.

图5 白色念珠菌生物被膜ROS检测

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).

图6 兔阴道白色念珠菌生物被膜感染治疗效果

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.

参考文献 30

1	Macias-Paz IU, Pérez-Hernández S, Tavera-Tapia A, et al. Candida albicans the main opportunistic pathogenic fungus in humans[J]. Rev Argent Microbiol, 2023, 55(2): 189-98.
2	瞿嵘, 蔡绍曦, 佟万成, 等. 呼吸机相关性肺部念珠菌感染危险因素及耐药分析[J]. 南方医科大学学报, 2009, 29(1): 57-9.
3	de Oliveira DC, Silva LB, da Silva BV, et al. A new acridone with antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans[J]. J Appl Microbiol, 2019, 127(5): 1362-72.
4	Pierce CG, Vila T, Romo JA, et al. The Candida albicans biofilm matrix: composition, structure and function[J]. J Fungi, 2017, 3(1): 14.
5	Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance[J]. J Bacteriol, 2001, 183(18): 5385-94.
6	Muzny CA, Schwebke JR. Biofilms: an underappreciated mechanism of treatment failure and recurrence in vaginal infections[J]. Clin Infect Dis, 2015, 61(4): 601-6.
7	Achkar JM, Fries BC. Candida infections of the genitourinary tract[J]. Clin Microbiol Rev, 2010, 23(2): 253-73.
8	Chew SY, Than LTL. Vulvovaginal candidosis: contemporary challenges and the future of prophylactic and therapeutic approaches[J]. Mycoses, 2016, 59(5): 262-73.
9	Harriott MM, Lilly EA, Rodriguez TE, et al. Candida albicans forms biofilms on the vaginal mucosa[J]. Microbiology, 2010, 156(Pt 12): 3635-44.
10	Lacroix G, Gouyer V, Gottrand F, et al. The cervicovaginal mucus barrier[J]. Int J Mol Sci, 2020, 21(21): 8266.
11	郭霜, 满江位, 姜春倩, 等. 低强度脉冲超声的生物物理学效应及相关机制的研究进展[J]. 中国医学物理学杂志, 2019, 36(5): 605-9.
12	Greish K. Enhanced permeability and retention of macromolecular drugs in solid tumors: a royal gate for targeted anticancer nanomedicines[J]. J Drug Target, 2007, 15(7/8): 457-64.
13	Roy J, Pandey V, Gupta I, et al. Antibacterial sonodynamic therapy: current status and future perspectives[J]. ACS Biomater Sci Eng, 2021, 7(12): 5326-38.
14	Mendling W, Brasch J, Cornely OA, et al. Guideline: vulvovaginal candidosis (AWMF 015/072), S2k (excluding chronic mucocutaneous candidosis)[J]. Mycoses, 2015, 58(): 1-15.
15	Coutinho A, Prieto M. Cooperative partition model of nystatin interaction with phospholipid vesicles[J]. Biophys J, 2003, 84(5): 3061-78.
16	Yang M, Cao YC, Zhang ZF, et al. Low intensity ultrasound-mediated drug-loaded nanoparticles intravaginal drug delivery: an effective synergistic therapy scheme for treatment of vulvovaginal candidiasis[J]. J Nanobiotechnology, 2023, 21(1): 53.
17	Song RT, Yan F, Cheng M, et al. Ultrasound-assisted preparation of exopolysaccharide/nystatin nanoemulsion for treatment of vulvovaginal candidiasis[J]. Int J Nanomedicine, 2020, 15: 2027-44.
18	Donders G, Sziller IO, Paavonen J, et al. Management of recurrent vulvovaginal candidosis: narrative review of the literature and European expert panel opinion[J]. Front Cell Infect Microbiol, 2022, 12: 934353.
19	Sobel JD. Vulvovaginal candidosis[J]. Lancet, 2007, 369(9577): 1961-71.
20	Yu H, Chen SP, Cao P. Synergistic bactericidal effects and mechanisms of low intensity ultrasound and antibiotics against bacteria: a review[J]. Ultrason Sonochem, 2012, 19(3): 377-82.
21	Powell LC, Adams JYM, Quoraishi S, et al. Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms[J]. Front Cell Infect Microbiol, 2023, 13: 1122340.
22	Janeth Rimachi Hidalgo K, Carmello JC, Carolina Jordão C, et al. Antimicrobial photodynamic therapy in combination with nystatin in the treatment of experimental oral candidiasis induced by Candida albicans resistant to fluconazole[J]. Pharmaceuticals, 2019, 12(3): 140.
23	Wang T, Ma W, Jiang ZN, et al. The penetration effect of HMME-mediated low-frequency and low-intensity ultrasound against the Staphylococcus aureus bacterial biofilm[J]. Eur J Med Res, 2020, 25(1): 51.
24	Xu PY, Kumar Kankala R, Wang SB, et al. Sonodynamic therapy-based nanoplatforms for combating bacterial infections[J]. Ultrason Sonochem, 2023, 100: 106617.
25	Pericolini E, Gabrielli E, Ballet N, et al. Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis[J]. Virulence, 2017, 8(1): 74-90.
26	Sun CQ, Peng J, Yang LB, et al. A cecropin-4 derived peptide C18 inhibits Candida albicans by disturbing mitochondrial function[J]. Front Microbiol, 2022, 13: 872322.
27	Bujdáková H. Management of Candida biofilms: state of knowledge and new options for prevention and eradication[J]. Future Microbiol, 2016, 11(2): 235-51.
28	Sui X, Yan L, Jiang YY. The vaccines and antibodies associated with Als3p for treatment of Candida albicans infections[J]. Vaccine, 2017, 35(43): 5786-93.
29	Ouyang J, Tang ZM, Farokhzad N, et al. Ultrasound mediated therapy: Recent progress and challenges in nanoscience[J]. Nano Today, 2020, 35: 100949.
30	Greifová G, Májeková H, Greif G, et al. Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative Lactobacillus reuteri [J]. Folia Microbiol, 2017, 62(6): 515-24.

[1]	张志飞, 廖洪建, 杨敏, 胡璨, 杜永洪. 左氧氟沙星联合纤维素酶在体外能有效根除卡介苗生物被膜感染[J]. 南方医科大学学报, 2023, 43(2): 257-264.
[2]	唐文韬, 邓娟, 贺思程, 李君粉, 周艺情, 王嫣. 低强度脉冲超声抑制脓毒症大鼠脾脏淋巴细胞的凋亡[J]. 南方医科大学学报, 2023, 43(10): 1789-1795.
[3]	付雷雯, 乐婷婷, 王玲, 郭辉杰, 刘志华, 杨钧, 陈清, 胡静. 耳念珠菌不同条件下生长特性及体内毒性[J]. 南方医科大学学报, 2020, 40(07): 1049-1055.
[4]	罗东，汪威，陈俊林，刘宝茹，陈锦云，王嫣，陈文直. 低强度脉冲超声对阿霉素联合环磷酰胺化疗后大鼠造血功能的影响[J]. 南方医科大学学报, 2019, 39(07): 836-.
[5]	曲良超，严金秀，蒋章颉，宋志平，罗佛全，彭清华. 低强度脉冲超声预处理通过激活胆碱能抗炎通路抑制HMGB1表达减轻肺缺血再灌注损伤[J]. 南方医科大学学报, 2018, 38(09): 1061-.