基于蜡螟感染模型研究亚抑菌浓度氟康唑对光滑念珠菌毒力的影响

中国真菌学杂志　 2023, Vol. 18 　Issue (4): 291-295.

基于蜡螟感染模型研究亚抑菌浓度氟康唑对光滑念珠菌毒力的影响

程鹏^1,2, 阿祥仁², 周健武², 牟晓明², 田丽娟², 马小亚¹

1. 青海大学医学院, 西宁 810001;
2. 青海省人民医院医学检验科, 西宁 810007

收稿日期:2022-12-06 出版日期:2023-08-28 发布日期:2023-09-02
通讯作者: 阿祥仁,E-mail:xiangrena_001@163.com E-mail:xiangrena_001@163.com
作者简介:程鹏,男(汉族),硕士研究生在读.E-mail:1172975751@qq.com
基金资助:
青海省2022年创新平台建设专项项目(2022-ZJ-T01)

Study on the effect of fluconazole with subinhibitory concentration on the virulence of Candida glabrata based on the Galleria mellonella infection model

CHENG Peng^1,2, A Xiangren², ZHOU Jianwu², MU Xiaoming², TIAN Lijuan², MA Xiaoya¹

1. Medical College of Qinghai University, Xining 810001, China;
2. Department of Medical Laboratory, Qinghai Provincial People's Hospital, Xining 810007, China

Received:2022-12-06 Online:2023-08-28 Published:2023-09-02

摘要/Abstract

摘要： 目的基于蜡螟感染模型探究亚抑菌浓度(sub-MIC)氟康唑(fluconazole,FLU)对光滑念珠菌毒力的影响。方法收集青海省人民医院2021年4月至2022年10月临床分离非重复的光滑念珠菌作为实验菌株,采用ATB法测定FLU对光滑念珠菌的最低抑菌浓度(MIC)。采用蜡螟感染模型检测光滑念珠菌的毒力,并采用病理学和病原学方法评估建模是否成功。穿刺及不同sub-MIC FLU对健康蜡螟的影响:对照组不做任何处理,实验组分为三组,即PBS组(注射10 μL PBS)、FLU亚组(分别注射10 μL FLU药液,使感染蜡螟体内FLU浓度为0.5 mg/kg、1 mg/kg、2 mg/kg、4 mg/kg、8 mg/kg、16 mg/kg)和穿刺组(微量注射器穿刺但不注射)。不同sub-MIC的FLU对光滑念珠菌感染蜡螟的影响:采用2.50×10⁶酵母/只剂量感染蜡螟,2 h后,对照组再注射10 μL PBS,实验组再注射不同浓度 FLU工作液,观测对光滑念珠菌感染的蜡螟的治疗效果。结果 FLU对9株临床分离的光滑念珠MIC为8~32 mg/L,光滑念珠菌ATCC2001的MIC为32 mg/L。实验组蜡螟肠组织病理切片中检测到酵母样孢子,肠组织培养出光滑念珠菌,对照组中肠组织病理切片和真菌培养均未发现真菌。穿刺可造成蜡螟损伤,但所致死亡率低于10%。1/2MIC FLU可提高光滑念珠菌感染蜡螟生存率,具有统计学意义(P＜0.05),而1/4MIC、1/8MIC、1/16MIC FLU对光滑念珠菌感染蜡螟生存率较PBS无明显的干预作用,差异无统计学意义(P>0.05)。结论 1/2MIC FLU对光滑念珠菌感染的蜡螟具有一定的治疗作用,但浓度低于1/2MIC对感染蜡螟无明显的保护作用。

关键词: 光滑念珠菌, 蜡螟, 亚抑菌浓度, 氟康唑

Abstract: Objective Based on the Galleria mellonella infection model, the effect of sub-MIC fluconazole on the virulence of Candida glabrata was investigated. Methods Non repetitive Candida glabrata strains clinically isolated from Qinghai Provincial People’s Hospital from April 2021 to October 2022 were collected, and the minimum inhibitory concentration (MIC) of fluconazole against Candida glabrata was determined by ATB method. The virulence of Candida glabrata was tested by the Galleria mellonella infection model, and the success of the model was evaluated by pathological and pathogenic methods. Effects of puncture and different sub-MIC FLUs on healthy Galleria mellonella: the control group did not do any treatment, and the experimental group was divided into three groups, namely PBS group (10 μL PBS), FLU subgroup(inject 10 μL fluconazole solution respectively, making the FLU concentration in the body of infected Galleria mellonella 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, and 16 mg/kg) and puncture group (micro syringe puncture but no injection). Use 2.50×10⁶ yeasts /larva infected with Galleria mellonella, after 2 h, the control group was injected with 10 μL PBS, the experimental group was injected with different concentrations of FLU working solution, observing the therapeutic effect on the Galleria mellonella infected by Candida glabrata. Results The MIC of fluconazole to 9 clinically isolated Candida glabrata was 8 mg/L~32 mg/L, and that of Candida glabrata ATCC2001 was 32 mg/L. Yeast-like spores were detected in the pathological section of the intestinal tissue of the Galleria mellonell in the experimental group, and Candida glabra was cultured in the intestinal tissue.No fungus was found in the pathological section and fungal culture of the intestinal tissue in the control group. Puncture could cause damage to the Galleria mellonella, but the mortality was less than 10%. 1/2 MIC fluconazole could improve the survival rate of Candida glabrata infected Galleria mellonella with statistical significance(P<0.05),while 1/4 MIC, 1/8 MIC,1/16 MIC fluconazole had no significant intervention effect on the survival rates of Candida glabrata infected Galleria mellonella compared with PBS and the difference was not statistically significant (P>0.05). Conclusion 1/2 MIC fluconazole had inhibitory effect on the virulence of Candida glabrata, but the concentration below 1/2 MIC had no obvious protective effect.

Key words: Candida glabrata, Galleria mellonella, subinhibitory concentration, fluconazol

中图分类号:

R379.4

程鹏, 阿祥仁, 周健武, 牟晓明, 田丽娟, 马小亚. 基于蜡螟感染模型研究亚抑菌浓度氟康唑对光滑念珠菌毒力的影响[J]. 中国真菌学杂志, 2023, 18(4): 291-295.

CHENG Peng, A Xiangren, ZHOU Jianwu, MU Xiaoming, TIAN Lijuan, MA Xiaoya. Study on the effect of fluconazole with subinhibitory concentration on the virulence of Candida glabrata based on the Galleria mellonella infection model[J]. Chinese Journal of Mycology, 2023, 18(4): 291-295.

参考文献

[1] GABALDÓN T, GÓMEZ-MOLERO E, BADER O. Molecular typing of Candida glabrata[J]. Mycopathologia, 2020,185(5):755-764.
[2] 刘静, 郑秋实, 黄广华. 光滑念珠菌的生物学、流行病学以及耐药机制研究进展[J]. 菌物学报,2020,39(11):2014-2024.
[3] PAPPAS P G, KAUFFMAN C A, ANDES D R, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of America[J]. Clin Infect Dis, 2016,62(4):e1-50.
[4] EL-HOUSSAINI H H, ELNABAWY O M, NASSER H A, et al. Influence of subinhibitory antifungal concentrations on extracellular hydrolases and biofilm production by Candida albicans recovered from Egyptian patients[J]. BMC Infect Dis, 2019,19(1):54.
[5] CHEN J, ZHOU H, HUANG J, et al. Virulence alterations in Staphylococcus aureus upon treatment with the sub-inhibitory concentrations of antibiotics[J]. J Adv Res, 2021,31:165-175.DOI: 10.1016/j.jare.2021.01.008.
[6] 赵望, 孙毅, 曾同祥. 大蜡螟作为病原真菌动物实验模型的相关研究[J]. 中国真菌学杂志,2020,15(4):248-252.
[7] AMES L, DUXBURY S, PAWLOWSKA B, et al. Galleria mellonella as a host model to study Candida glabrata virulence and antifungal efficacy[J]. Virulence, 2017,8(8):1909-1917.
[8] MARCOS-ZAMBRANO L J, BORDALLO-CARDONA M Á, BORGHI E, et al. Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella[J]. Med Mycol, 2020,58(1):83-92.
[9] IGNASIAK K, MAXWELL A. Galleria mellonella (greater wax moth) larvae as a model for antibiotic susceptibility testing and acute toxicity trials[J]. BMC Res Notes, 2017,10(1):428.
[10] BERKOW E L, LOCKHART S R, OSTROSKY-ZEICHNER L. Antifungal susceptibility testing: Current approaches[J]. Clin Microbiol Rev, 2020,33(3):e00069-19.
[11] SMITH D F Q, CASADEVALL A. Fungal immunity and pathogenesis in mammals versus the invertebrate model organism Galleria mellonella[J]. Pathog Dis, 2021,79(3):ftab013.
[12] 谢珊珊, 陈烁, 叶梦思, 等. 亚抑菌浓度卡泊芬净对白念珠菌生物膜形成的影响及机制研究[J]. 中国抗生素杂志, 2022,47(11):1-7.
[13] BERNARDI S, ANDERSON A, MACCHIARELLI G, et al. Subinhibitory antibiotic concentrations enhance biofilm formation of clinical Enterococcus faecalis isolates[J]. Antibiotics (Basel), 2021,10(7):874.
[14] PESEE S, ANGKANANUWAT C, TANCHAROENSUKJIT S, et al. In vitro activity of caspofungin combined with fluconazole on mixed Candida albicans and Candida glabrata biofilm[J]. Med Mycol, 2016,54(4):384-393.
[15] MéNARD G, ROUILLON A, CATTOIR V, et al. Galleria mellonella as a suitable model of bacterial infection: past, present and future[J]. Front Cell Infect Microbiol, 2021,11:782733.DOI: 10.3389/fcimb.2021.782733.