口腔白念珠菌耐药性与基因分型及耐药基因表达的分析

doi:10.3969/j.issn.1673-8640.2017.011.009

摘要/Abstract

摘要：

目的了解口腔白念珠菌耐药情况及其基因型分布,初步探讨其耐药表型、基因型、生物膜与耐药基因（CDR1、ERG11）表达之间的联系。方法选用ATB Fungus 3药物敏感性试剂盒对所收集的125株口腔白念珠菌进行药物敏感性试验;采用结晶紫法检测白念珠菌的生物膜形成能力;通过重复序列聚合酶链反应（PCR）对白念珠菌进行基因分型;采用实时荧光定量PCR检测耐药基因CDR1和ERG11的表达。结果在125株白念珠菌中筛选出7株耐药株。白念珠菌对伊曲康唑的耐药率为4.8%（6/125）,对氟康唑的耐药率为0.8%（1/125）,对氟胞嘧啶的耐药率为0.8%（1/125）。重复序列PCR将菌株分为4个型别,其中1型19株、2型2株、3型2株、4型2株,各型之间在耐药率、生物膜形成能力、ERG11和CDR1的表达上差异无统计学意义（P值分别为0.645、0.769、0.686、0.782）。耐药组的生物膜形成能力和ERG11表达能力强于敏感组（P值分别为0.001、0.002）,而CDR1的表达差异无统计学意义（P=0.836）。结论绝大部分口腔白念珠菌对抗真菌药物敏感,少数对抗真菌药物耐药,以对伊曲康唑耐药为主。各菌株生物膜形成能力存在一定的差异,耐药株的生物膜形成能力强于敏感株。口腔白念珠菌重复序列PCR分型与耐药之间没有必然的联系。耐药株耐药基因ERG11的表达总体强于敏感株。

关键词: 白念珠菌, 耐药性, 生物膜, 基因分型

Abstract:

Objective To investigate the drug resistance and genotypes of oral Candida albicans,and to investigate the correlation between drug resistance,genotypes,biofilm-forming capability and the expressions of drug-resistant genes（CDR1 and ERG11）. Methods Antifungal susceptibility testing was performed by ATB Fungus 3 strips among 125 isolates of Candida albicans. The biofilm-forming capability was determined by crystal violet method. Genotyping of Candida albicans was performed by repeated sequence polymerase chain reaction（PCR）. The expressions of drug-resistant genes（CDR1 and ERG11） were determined by real-time fluorescence quantitation PCR. Results A total of 7 drug-resistant isolates were identified from the 125 isolates of Candida albicans. The drug resistance rates against itraconazole,fluconazole and 5-fluorouracil were 4.8%（6/125）,0.8%（1/125） and 0.8%（1/125）,respectively. By repeated sequence PCR,19 isolates were type 1,2 isolates were type 2,2 isolates were type 3,and 2 isolates were type 4. There was no statistical significance in drug resistance rate,biofilm-forming capability and CDR1 and ERG11 expressions among the 4 different types（P=0.645,0.769,0.686 and 0.782）. The biofilm-forming capability and ERG11 expression were higher in drug-resistant isolates than those in sensitive isolates（P=0.001 and 0.002）. However,there was no statistical significance in CDR1 expression between drug-resistant isolates and sensitive isolates（P=0.836）. Conclusions Most of the oral Candida albicans are sensitive to antifungal agents,and a minority of them are resistant to antifungal agents,mainly itraconazole. There is difference for biofilm-forming capability among them. The biofilm-forming capability of drug-resistant isolates is stronger than that of sensitive ones. There was no correlation between repeated sequence PCR genotyping of oral Candida albicans and drug resistance. ERG11 expression is higher in drug-resistant isolates than that in sensitive isolates.

Key words: Candida albicans, Drug resistance, Biofilm, Genotyping

中图分类号:

R446.5

孙康德, 陈铖, 陈旭, 张家胜, 虞中敏, 陈福祥. 口腔白念珠菌耐药性与基因分型及耐药基因表达的分析[J]. 检验医学, 2017, 32(11): 979-984.

SUN Kangde, CHEN Cheng, CHEN Xu, ZHANG Jiasheng, YU Zhongmin, CHEN Fuxiang. Drug resistance and genotypes of oral Candida albicans and expressions of drug-resistant genes of oral Candida albicans[J]. Laboratory Medicine, 2017, 32(11): 979-984.

图/表 5

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基因	引物序列（5'~3'）
ERG11	正向 TTACCTCATTATTGGAGACGTGATG
	反向 CACGTTCTCTTCTCAGTTTAATTTCTTTC
CDR1	正向 TTTAGCCAGAACTTTCACTCATGATT
	反向 TATTTATTTCTTCATGTTCATATGGATTGA
18s rRNA	正向 TCTTTCTTGATTTTGTGGGTGG
	反向 TCGATAGTCCCTCTAAGAAGTG

基因	引物序列（5'~3'）
ERG11	正向 TTACCTCATTATTGGAGACGTGATG
	反向 CACGTTCTCTTCTCAGTTTAATTTCTTTC
CDR1	正向 TTTAGCCAGAACTTTCACTCATGATT
	反向 TATTTATTTCTTCATGTTCATATGGATTGA
18s rRNA	正向 TCTTTCTTGATTTTGTGGGTGG
	反向 TCGATAGTCCCTCTAAGAAGTG

菌株编号	敏感株A值	菌株编号	耐药株A值
7	0.430	6	0.730
8	0.155	27	1.051
9	0.291	38	0.777
14	0.321	42	0.802
18	0.337	70	1.154
20	0.199	115	1.315
24	0.315	142	1.134
29	0.672
39	0.533
43	0.593
48	0.330
50	0.641
51	0.400
53	0.665
54	0.663
58	0.373
59	0.315
63	0.222

菌株编号	敏感株A值	菌株编号	耐药株A值
7	0.430	6	0.730
8	0.155	27	1.051
9	0.291	38	0.777
14	0.321	42	0.802
18	0.337	70	1.154
20	0.199	115	1.315
24	0.315	142	1.134
29	0.672
39	0.533
43	0.593
48	0.330
50	0.641
51	0.400
53	0.665
54	0.663
58	0.373
59	0.315
63	0.222

菌株编号	敏感株		菌株编号	耐药株
菌株编号	ERG11	CDR1	菌株编号	ERG11	CDR1
7	0.965 112	3.630 833	6	64.860 650	28.370 460
8	0.486 783	2.536 197	27	0.934 706	1.491 116
9	0.456 785	3.824 363	38	0.711 336	2.471 095
14	0.644 768	6.769 090	42	48.214 050	4.932 684
18	0.225 430	1.985 239	70	75.620 110	16.345 870
20	0.215 991	3.230 778	115	2.238 671	3.148 120
24	0.212 846	1.615 657	142	0.982 531	1.631 217
29	0.144 198	3.071 844
39	0.145 219	1.368 895
43	0.218 079	1.958 766
48	0.162 715	1.656 068
50	0.569 264	4.228 862
51	0.447 614	4.737 794
53	0.557 316	6.200 157
54	0.625 842	2.900 463
58	0.227 322	2.469 059
59	0.922 972	8.543 139
63	0.534 140	27.195 560