氯喹对白念珠菌生物膜及其耐药性的作用研究

doi:10.13591/j.cnki.kqyx.2024.06.002

摘要/Abstract

摘要：

目的探究氯喹单独使用及联用传统抗真菌药物对白念珠菌生物膜及其耐药性的改变。方法采用白念珠菌标准株及耐药株进行实验,使用XTT减低法和棋盘稀释法分别检测氯喹单独使用及联用抗真菌药物后对两种白念珠菌生物膜的抑制作用,绘制时间-杀菌曲线,扫描电镜观察两种白念珠菌生物膜形态。结果 50 μmol/L及以上浓度氯喹对两种白念珠菌生物膜有直接抑制作用,且随着浓度增加,抑制作用增强。氯喹与两性霉素B联用存在协同抑菌作用。氯喹单独使用和联用两性霉素B的时间-杀菌曲线结果显示,白念珠菌在不同时相培养中生长趋势有所不同;形态学观察发现均可降低白念珠菌菌丝和生物膜形成能力。结论氯喹对白念珠菌生物膜存在直接抑制,可降低白念珠菌生物膜耐药性。氯喹与两性霉素B联用时具有协同作用。

关键词: 白念珠菌, 抗真菌药物, 氯喹

Abstract:

Objective To evaluate the effects of chloroquine alone and in combination with traditional antifungal agents on the Candida albicans biofilms and its drug resistance. Methods This study used standard strains of Candida albicans, and drug-resistant strains of Candida albicans. The inhibitory effects of chloroquine alone and in combination with antifungal drugs on biofilms of Candida albicans were detected by XTT reduction method and chessboard dilution method respectively. The morphological characteristics of biofilms were observed under scanning electron microscopy. Results Chloroquine at the concentration of 50 μmol/L or above showed a direct inhibitory effect and increased with concentration. Chloroquine combined with amphotericin B had a synergistic inhibitory effect. Results of the time-killing curve showed that the growth trends of biofilms treated with chloroquine alone and combined with amphotericin B varied in different time periods during the experimental culture. Morphological observation also revealed that chloroquine alone and in combination with amphotericin B could reduce the ability of Candida albicans to form hyphae and biofilms. Conclusion Chloroquine has an inhibitory effect on Candida albicans biofilms and can reduce its drug resistance. Furthermore, chloroquine shows a synergistic anti-fungal effect when combined with amphotericin B.

Key words: Candida albicans, antifungal agent, chloroquine

中图分类号:

R781.5

吴翘楚, 史般若, 缪昊宸, 魏昕. 氯喹对白念珠菌生物膜及其耐药性的作用研究[J]. 口腔医学, 2024, 44(6): 408-413.

WU Qiaochu, SHI Banruo, MIAO Haochen, WEI Xin. Effect of chloroquine on Candida albicans biofilms and its drug resistance[J]. Stomatology, 2024, 44(6): 408-413.

图/表 6

图1

表1

表2

表3

图2

图3

参考文献 29

[1]	Alikhani T, Daie Ghazvini R, Mirzaii M, et al. Drug resistance and biofilm formation in Candida species of vaginal origin[J]. Iran J Public Health, 2022, 51(4):913-918. doi: 10.18502/ijph.v51i4.9253 pmid: 35936523
[2]	Das S, Goswami AM, Saha T. An insight into the role of protein kinases as virulent factors, regulating pathogenic attributes in Candida albicans[J]. Microb Pathog, 2022, 164: 105418.
[3]	Feng WL, Yang J, Ma Y, et al. Aspirin and verapamil increase the sensitivity of Candida albicans to caspofungin under planktonic and biofilm conditions[J]. J Glob Antimicrob Resist, 2021, 24: 32-39.
[4]	Liu SQ, Jiang LL, Miao HC, et al. Autophagy regulation of ATG13 and ATG27 on biofilm formation and antifungal resistance in Candida albicans[J]. Biofouling, 2022, 38(9):926-939.
[5]	Shen JD, Ma M, Duan W, et al. Autophagy alters the susceptibility of Candida albicans biofilms to antifungal agents[J]. Microorganisms, 2023, 11(8):2015.
[6]	张琴琴, 马鸣, 花荣, 等. 法尼醇对白念珠菌生物膜葡聚糖的影响及白念珠菌耐药相关性[J]. 口腔医学, 2023, 43(6):488-493.
[7]	钱芳, 魏昕, 许雯倩, 等. XTT减低法检测法尼醇对白念珠菌生物被膜的抑制作用[J]. 口腔生物医学, 2014, 5(2):82-85.
[8]	Ramage G, Vande Walle K, Wickes BL, et al. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms[J]. Antimicrob Agents Chemother, 2001, 45(9):2475-2479. doi: 10.1128/AAC.45.9.2475-2479.2001 pmid: 11502517
[9]	Meletiadis J, Mouton JW, Meis JFGM, et al. In vitro drug interaction modeling of combinations of azoles with terbinafine against clinical Scedosporium prolificans isolates[J]. Antimicrob Agents Chemother, 2003, 47(1):106-117. doi: 10.1128/AAC.47.1.106-117.2003 pmid: 12499177
[10]	Lewis RE, Diekema DJ, Messer SA, et al. Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species[J]. J Antimicrob Chemother, 2002, 49(2):345-351.
[11]	Prichard MN, Prichard LE, Shipman C Jr. Strategic design and three-dimensional analysis of antiviral drug combinations[J]. Antimicrob Agents Chemother, 1993, 37(3):540-545. doi: 10.1128/AAC.37.3.540 pmid: 8384816
[12]	Jiang LL, Zheng LX, Sun K, et al. In vitro and in vivo evaluation of the antifungal activity of fluoxetine combined with antifungals against Candida albicans biofilms and oral candidiasis[J]. Biofouling, 2020, 36(5):537-548.
[13]	Koshikawa T, Abe M, Nagi M, et al. Biofilm-formation capability depends on environmental oxygen concentrations in Candida species[J]. J Infect Chemother, 2022, 28(5):643-650. doi: 10.1016/j.jiac.2022.01.010 pmid: 35115240
[14]	Priya A, Pandian SK. Biofilm and hyphal inhibitory synergistic effects of phytoactives piperine and cinnamaldehyde against Candida albicans[J]. Med Mycol, 2022, 60(8):myac039.
[15]	Vaňková E, Kašparová P, Dulíčková N, et al. Combined effect of lasioglossin LL-Ⅲ derivative with azoles against Candida albicans virulence factors: Biofilm Formation, phospholipases, proteases and hemolytic activity[J]. FEMS Yeast Res, 2020, 20(3):foaa020.
[16]	Ma TY, Yu QL, Ma CC, et al. Role of the inositol polyphosphate kinase Vip1 in autophagy and pathogenesis in Candida albicans[J]. Future Microbiol, 2020, 15: 1363-1377.
[17]	Bastidas RJ, Heitman J, Cardenas ME. The protein kinase Tor1 regulates adhesin gene expression in Candida albicans[J]. PLoS Pathog, 2009, 5(2):e1000294.
[18]	Silva RCMC, Tan L, Rodrigues DA, et al. Chloroquine inhibits pro-inflammatory effects of heme on macrophages and in vivo[J]. Free Radic Biol Med, 2021, 173: 104-116.
[19]	Song ZL, Liu YT, Xie CH, et al. Synthesis and pharmacological evaluation of choroquine derivatives bearing long aminated side chains as antivirus and anti-inflammatory agents[J]. Bioorg Chem, 2021, 116: 105346.
[20]	Cavassin FB, Baú-Carneiro JL, Vilas-Boas RR, et al. Sixty years of amphotericin B: An overview of the main antifungal agent used to treat invasive fungal infections[J]. Infect Dis Ther, 2021, 10(1):115-147.
[21]	Gao Y, Zhang ZH, Lun ZC, et al. Synergistic effects of fluconazole combined with doxycycline against dual-species cultures of Candida albicans and Staphylococcus epidermidis and the mechanisms of action[J]. Microb Drug Resist, 2022, 28(5):525-535.
[22]	Lewis RE, Kontoyiannis DP. Rationale for combination antifungal therapy[J]. Pharmacotherapy, 2001, 21(8 Pt 2):149S-164S. pmid: 11501988
[23]	Su S, Yan HY, Min L, et al. The antifungal activity of caspofungin in combination with antifungals or non-antifungals against Candida species in vitro and in clinical therapy[J]. Expert Rev Anti Infect Ther, 2022, 20(2):161-178.
[24]	Boonstra JM, Märtson AG, Sandaradura I, et al. Optimization of fluconazole dosing for the prevention and treatment of invasive candidiasis based on the pharmacokinetics of fluconazole in critically ill patients[J]. Antimicrob Agents Chemother, 2021, 65(3):e01554-e01520.
[25]	Sastré-Velásquez LE, Dallemulle A, Kühbacher A, et al. The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment[J]. PLoS Pathog, 2022, 18(12):e1011066.
[26]	Krishnan-Natesan S. Terbinafine: A pharmacological and clinical review[J]. Expert Opin Pharmacother, 2009, 10(16):2723-2733. doi: 10.1517/14656560903307462 pmid: 19874252
[27]	Hernández-Cervantes A, Znaidi S, van Wijlick L, et al. A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans[J]. Nat Commun, 2020, 11(1):6224. doi: 10.1038/s41467-020-20010-9 pmid: 33277479
[28]	Xiao J, Zeng Y, Rustchenko E, et al. Dual transcriptome of Streptococcus mutans and Candida albicans interplay in biofilms[J]. J Oral Microbiol, 2022, 15(1):2144047.
[29]	Wiederhold NP. Pharmacodynamics, mechanisms of action and resistance, and spectrum of activity of new antifungal agents[J]. J Fungi (Basel), 2022, 8(8):857.

SMIC₅₀	标准株	耐药株
氯喹/(μmol/L)	400	400
氟康唑/(μg/L)	512	1 024
两性霉素B/(μg/L)	1	2
卡泊芬净/(μg/L)	128	256
5-氟胞嘧啶/(μg/L)	256	1 024
特比萘芬/(μg/L)	128	256

白念菌株	联用药物	FICI	结果
标准株	氟康唑	0.515	IND
	两性霉素B	0.375	SYN
	卡泊芬净	0.250	SYN
	5-氟胞嘧啶	1.000	IND
	特比萘芬	1.008	IND
耐药株	氟康唑	1.000	IND
	两性霉素B	0.250	SYN
	卡泊芬净	1.000	IND
	5-氟胞嘧啶	0.750	IND
	特比萘芬	1.000	IND

白念菌株	联用药物	∑SYN	∑ANT	结果
标准株	氟康唑	0	-617	ANT(S)
	两性霉素B	325	-29	SYN(S)
	卡泊芬净	182	-76	SYN(M)
	5-氟胞嘧啶	15	-265	ANT(S)
	特比萘芬	2	-65	IND
耐药株	氟康唑	17	-972	ANT(S)
	两性霉素B	417	-11	SYN(S)
	卡泊芬净	107	-218	ANT(M)
	5-氟胞嘧啶	23	-437	ANT(S)
	特比萘芬	14	-78	IND