白假丝酵母菌临床菌株对氟康唑耐药性及其与CAP1基因相关性研究
谈潘莉, 汪浙炯, 赵金方
浙江中医药大学附属第一医院检验科,杭州 310006
赵金方,xinqiuzh@163.com
摘要

目的 了解白假丝酵母菌临床菌株对氟康唑的耐药率以及氟康唑耐药与白假丝酵母菌CAP1基因相关性。方法 采用微量稀释法检测了245株白假丝酵母菌临床菌株对氟康唑的敏感性。采用实时荧光定量RT-PCR(qRT-PCR)和流式细胞术分别检测白假丝酵母菌CAP1基因mRNA(CAP1-mRNA)及其表达产物Cap1p。采用氟康唑浓度递增(4~64 μg/mL)的YPD培养液连续培养法了解氟康唑诱导白假丝酵母菌耐药性的作用,qRT-PCR和流式细胞术确定CAP1基因与氟康唑耐药性形成的关系。结果 134株白假丝酵母菌对氟康唑敏感(MIC≤8 μg/mL)、36株剂量依赖敏感(16~32 μg/mL)、75株耐药(≥64 μg/mL),耐药率为30.6%(75/245)。氟康唑耐药白假丝酵母菌株CAP1-mRNA和Cap1p表达水平均明显高于氟康唑敏感菌株( P<0.05)。氟康唑能诱导氟康唑敏感菌株产生耐药性(MIC≥64 μg/mL),其CAP1-mRNA和Cap1p表达水平也显著升高( P<0.05)。结论 白假丝酵母菌临床菌株对氟康唑有较高的耐药率。氟康唑能诱导氟康唑敏感白假丝酵母菌株产生耐药性。白假丝酵母菌对氟康唑耐药性与CAP1基因表达上调密切相关。

关键词: 白假丝酵母菌; 氟康唑; 耐药性; CAP1基因; 表达
中图分类号:R379.4 文献标志码:A 文章编号:1002-2694(2015)04-0325-05
Resistance of Candida albicans isolates against fluconazole and the resistance correlation with CAP1 gene
TAN Pan-li, WANG Zhe-jiong, ZHAO Jin-fang
Department of Laboratory Medicine, the First Affiliated Hospital of Zhejiang Chinese Medicine University, Hangzhou 310006, China
Corresponding author: Zhao Jin-fang, Email:xinqiuzh@163.com
Abstract

The aim of this study is to investigate the resistant rate of Candida albicans ( C. albicans) isolates against fluconazole, and to determine the correlation between the fluconazole-resistance and CAP1 gene of C. albicans. The microdilution test on 245 C. albicans isolates showed that 134 strains were fluconazole-sensitive (MIC≤8 μg/mL), 36 strains were fluconazole-dose-dependent sensitive (16-32 μg/mL) and 75 strains were fluconazole-resistant (≥64 μg/mL) that produced a 30.6% (75/245) fluconazole-resistant rate. The real-time fluorescent quantitative RT-PCR (qRT-PCR) and flow cytometric examination confirmed that the mRNA (CAP1-mRNA) and Cap1p levels expressed by CAP1 gene in the fluconazole-resistant C. albicans isolates were significantly higher than those in the fluconazole-sensitive isolates ( P<0.05). When the fluconazole-sensitive C. albicans isolates were continuously cultured in YPD liquid medium containing fluconazole with progressively-increased dosages (4-64 μg/mL), the isolates produced the resistance against fluconazole (MIC≥64 μg/mL) as well as the CAP1-mRNA and Cap1p expression levels were significantly elevated ( P<0.05). All the data indicate that C. albicans isolates have a higher fluconazole-resistant rate. Fluconazole can induce the resistance against itself in fluconazole-sensitive C. albicans strains. The fluconazole-resistance of C. albicans is closely associated with the expression up-regulation of its CAP1 gene.

Keyword: Candida albicans; fluconazole; resistance; CAP1 gene; expression

由于人口老龄化及抗生素广泛使用等原因, 近年来真菌感染性疾病发病率不断升高, 尤其是侵袭性真菌(invasive fungi)感染性疾病, 已成为严重威胁人类健康和生命安全的新问题[1]。国外文献报道, 侵袭性真菌感染性疾病最常见的病原体为假丝酵母菌属(Candida)真菌, 占所有病原真菌的70%~90%, 我国也不例外[2, 3, 4, 5]。假丝酵母菌属真菌种类繁多、分布广泛, 目前发现至少有9个种可引起人和动物侵袭性真菌病, 其中以白假丝酵母菌(Candida albicans)最为常见, 当机体免疫功能低下时, 白假丝酵母菌常作为条件致病菌引起局部或全身感染[6]。氟康唑(fluconazole)是临床治疗时最常用的抗假丝酵母菌感染性疾病药物, 但其耐药现象也最为严重[7, 8]。有文献报道, CAP1基因及其产物转录因子Cap1p与白假丝酵母菌耐药密切相关[9, 10]。我们检测了245株白假丝酵母菌临床菌株对氟康唑的敏感性, 检测了氟康唑敏感或耐药菌株CAP1-mRNA、Cap1p蛋白表达水平差异, 同时探讨了氟康唑诱导白假丝酵母菌耐药及其与CAP1基因表达关系。

1 材料与方法
1.1 菌株来源及培养

采用假丝酵母菌显色培养基(BioMerieux)从2013年1-12月本院各临床科室送检的咽拭、痰液、尿液、粪便标本中分离假丝酵母菌, 各菌株经VITEK 2-compact全自动微生物分析仪(BioMerieux)进行鉴定, 从中获得白假丝酵母菌245株。氟康唑敏感的白假丝酵母菌ATCC22019株由本实验室保存并提供。上述菌株接种于沙保琼脂平板上, 35 ℃培养2 d。

1.2 最低抑菌浓度测定

采用美国临床实验室标准化研究所(CLSI)介绍的微量稀释法, 检测氟康唑对白假丝酵母菌的最低抑菌浓度(MIC)。挑取沙保平板上培养的白假丝酵母菌接种于YPD培养液中, 35 ℃、400 r/min振荡培养24 h。培养物经细胞计数板计数后, 用RPMI-1640培养液(GiBco)配制成菌数为5× 103 CFU/mL的悬液。96孔细胞培养板中每孔加100 μ L白假丝酵母悬液, 然后加入100 μ L RPMI-1640培养液稀释的氟康唑注射液(法国辉瑞制药有限公司, 国药准字J20100010), 使氟康唑终浓度分别为0.25、0.5、2、4、8、16、32、64和128 μ g/mL, 35 ℃培养24 h后观察结果, 以未见真菌生长的最低药物浓度为该菌株的MIC。MIC≤ 8 μ g/mL为敏感、16~32 μ g/mL为剂量依赖敏感、≥ 64 μ g/mL为耐药[11]。实验中采用白假丝酵母菌ATCC22019株为质控菌株。

1.3 CAP1-mRNA测定

挑取沙保平板上培养的白假丝酵母菌接种于YPD培养液中, 35 ℃、400 r/min振荡培养16 h, 8 000 r/min离心5 min(4 ℃)。取白假丝酵母菌沉淀用Novelase消化液悬浮, 室温消化10 min后, 参照Trizol试剂(Invitrogen)说明书介绍的步骤提取总RNA, 紫外分光光度法测定其RNA浓度[12]。根据文献报道的CAP1基因序列[13, 14], 采用Primer Premier 5.0软件设计检测白假丝酵母菌CAP1-mRNA和18S-RNA的实时荧光定量RT-PCR(qRT-PCR)引物(表1), 各引物由上海Invitrogen公司合成。以1 μ g总RNA为模板, 采用逆转录试剂盒(TaKaRa)、SYBR Premix Ex.TaqTM荧光定量PCR试剂盒(TaKaRa)、上述引物及ABI-7500型实时荧光定量PCR仪检测CAP1-mRNA, 反应参数:95 ℃30 s; 95 ℃5 s、60 ℃30 s, 40个循环。以18S-RNA为内参照, 采用Δ Δ Ct模型及REST2005软件对qRT-PCR数据进行定量分析[15]

表1 qRT-PCR引物序列及产物长度 Tab.1 Sequences of primers in qRT-PCR and length of products
1.4 Cap1p表达水平测定

挑取沙保平板上培养的白假丝酵母菌接种于YPD培养液中, 35 ℃、400 r/min振荡培养16 h, 8 000 r/min离心5 min(4 ℃)。取白假丝酵母菌沉淀用pH7.4、0.01 mol/L磷酸缓冲盐水(PBS)悬浮, 经细胞计数板计数后用PBS配制成菌数为1× 106 CFU/mL的悬液, 然后按上法离心, 如此重复2次。白假丝酵母菌沉淀悬于1 mL 1%去氧胆酸钠-1%无水乙醇-PBS中, 37 ℃、200 r/min振荡孵育30 min, 按上法离心后用1%去氧胆酸钠-PBS悬浮、离心2次。取白假丝酵母菌沉淀加入1∶ 1 000稀释的兔抗Cap1p-IgG(本实验室提供), 4 ℃孵育30 min, 按上法用PBS洗涤、离心3次后加入1∶ 1 000稀释的FITC标记羊抗兔IgG(ImmunoResearch), 4 ℃孵育30 min, 按上法用PBS洗涤、离心3次。白假丝酵母菌沉淀用0.5 mL PBS悬浮, 然后用FACSCalibur流式细胞仪检测20 000个菌细胞(激发波长488 nm), 用Cellquest软件分析表达Cap1p蛋白(Cap1p+)菌细胞百分率及其平均荧光强度(mean fluorescence intensity, MFI)。

1.5 耐药性诱导及CAP1-mRNA和Cap1p检测

氟康唑敏感的134株白假丝酵母菌中, 34株氟康唑MIC< 2 μ g/mL, 其余100株氟康唑MIC为2~8 μ g/mL。将上述100株白假丝酵母菌分别接种于YPD培养液中, 35 ℃、400 r/min振荡培养至浓度约为1× 108 CFU/mL, 取1× 106白假丝酵母菌接种于5 mL含4 μ g/mL氟康唑的YPD培养液中, 按上法振荡培养并计数, 再取1× 106白假丝酵母菌接种于5 mL含8 μ g/mL氟康唑的YPD培养液中, 直至培养液中氟康唑浓度为64 μ g/mL。将64 μ g/mL氟康唑YPD白假丝酵母菌培养物8 000 r/min离心5 min(4 ℃), 取沉淀的白假丝酵母菌按上法进行MIC、CAP1-mRNA和Cap1p蛋白表达水平测定, 实验中设置氟康唑未诱导菌株对照。

1.6 统计学分析

分别采用SPSS19.0统计学软件中的t检验, 对实验中获得的CAP1-mRNA和Cap1p表达水平数据进行检验。

2 结 果
2.1 白假丝酵母菌临床菌株的耐药率

氟康唑对白假丝酵母菌质控菌株ATCC22019的MIC为0.25或0.5 μ g/mL, 表明本研究中微量稀释法实验质量及其结果可靠[11]。微量稀释法检测结果显示, 245株白假丝酵母菌对氟康唑的MIC范围为0.25~128 μ g/mL, 其中134株对氟康唑敏感、36株剂量依赖敏感、75株耐药, 敏感率为54.7%(134/245), 耐药率为30.6%(75/245)(表2)。

表2 245株白假丝酵母菌临床菌株对氟康唑的敏感性 Tab.2 Susceptibility of 245 C. albicans isolates against fluconazole
2.2 氟康唑耐药临床菌株CAP1基因高表达

qRT-PCR和流式细胞术检测结果分别显示, 75株氟康唑耐药白假丝酵母菌临床菌株CAP1-mRNA和Cap1p表达水平均明显高于134株氟康唑敏感白假丝酵母菌临床菌株(t =2.784, 2.154; P< 0.05)(图1)。

图1 白假丝酵母菌CAP1-mRNA和Cap1p表达水平比较Fig.1 Comparison of CAP1-mRNA and Cap1p expression levels in C. albicans isolates

2.3 氟康唑诱导白假丝酵母菌耐药性

100株氟康唑敏感白假丝酵母菌(MIC=2~8 μ g/mL)依次在4、8、16、32和64 μ g/mL氟康唑YPD培养液中均能良好生长, 但生长速度减慢。微量稀释法检测结果显示, 氟康唑对64 μ g/mL氟康唑YPD培养液中收集的菌株MIC≥ 64 μ g/mL。

2.4 诱导耐药菌株CAP1基因高表达

与氟康唑诱导前比较, 100株氟康唑敏感白假丝酵母菌(MIC=2~8 μ g/mL)最终经64 μ g/mL氟康唑YPD培养液培养后, 其CAP1-mRNA水平和Cap1p表达水平均显著升高(t =3.675, 2.772; P< 0.05)(图2)。

图2 氟康唑诱导后白假丝酵母菌CAP1-mRNA和Cap1p表达水平变化Fig.2 Change of CAP1-mRNA and Cap1p expression levels of C. albicans after fluconazole induction

3 讨 论

白假丝酵母菌是一种条件致病性真菌, 可引起人和动物的局部或全身性感染[16, 17], 所致疾病种类繁多, 既可引起皮肤、黏膜、内脏、中枢神经系统感染性疾病, 也可引起皮肤、呼吸道、消化道过敏性疾病[18]。氟康唑是临床上治疗白假丝酵母菌感染性疾病的首选药物, 但白假丝酵母菌对其耐药性也最为严重, 且对氟康唑耐药菌株通常也对其他唑类抗真菌药物交叉耐药[7, 8]。因此, 了解白假丝酵母菌临床菌株对氟康唑耐药率及其耐药机制具有重要的临床意义。最近的国外和国内文献报道, 白假丝酵母菌临床菌株对氟康唑的耐药率分别为21.3%和15.0%[19, 20]。本研究发现, 白假丝酵母菌临床菌株对氟康唑的耐药率高达30.6%, 提示药物治疗白假丝酵母菌感染性疾病时, 应进行药物敏感试验并根据其结果选用合适抗真菌药物, 以提高临床疗效。

白假丝酵母菌对氟康唑耐药机制主要有真菌多药抗药蛋白(multidrug resistant protein, MRP)表达水平上调、氟康唑靶酶14α -去甲基化酶(CYP51A1)突变所致亲和力下降[21, 22]。CAP1基因表达产物Cap1p是普遍存在于真核细胞中的AP-1家族成员, 为含有bZip结构域的转录因子[14]。Alarco等首先发现酿酒酵母Cap1p能上调MFS超家族多药抗药基因FLR1的表达, 从而使酿酒酵母产生对氟康唑产生抗药性[23]; 此后Alarco等又发现白假丝酵母菌Cap1p可上调多个MRP编码基因表达, 从而在白假丝酵母菌对氟康唑耐药性形成过程中发挥重要作用[9]。有文献报道, 白假丝酵母菌长期暴露于低浓度氟康唑中, 也可诱导其产生对氟康唑的耐药性[24]。本研究发现, 氟康唑耐药的白假丝酵母菌临床菌株CAP1-mRNA和Cap1p表达水平, 均明显高于氟康唑敏感的白假丝酵母菌临床菌株(P< 0.05); 氟康唑敏感的白假丝酵母菌能适应培养基中浓度逐步增加的氟康唑, 转变为对氟康唑耐药的菌株, 同时其CAP1-mRNA和Cap1p表达水平也显著升高(P< 0.05)。上述实验结果提示, 氟康唑能诱导白假丝酵母菌产生对其耐药性, 其机制与CAP1 基因密切相关; 临床上使用氟康唑治疗白假丝酵母菌感染性疾病时, 不仅应足量使用并注意合适疗程, 还需监测白假丝酵母菌对氟康唑敏感性变化, 以免诱导白假丝酵母菌产生耐药性。

The authors have declared that no competing interests exist.

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