白假絲酵母菌臨床菌株對氟康唑耐藥性及其與CAP1基因相關性研究

談潘莉，汪浙炯，趙金方

白假絲酵母菌臨床菌株對氟康唑耐藥性及其與CAP1基因相關性研究

談潘莉，汪浙炯，趙金方

目的 了解白假絲酵母菌臨床菌株對氟康唑的耐藥率以及氟康唑耐藥與白假絲酵母菌CAP1基因相關性。方法 采用微量稀釋法檢測了245株白假絲酵母菌臨床菌株對氟康唑的敏感性。采用實時熒光定量RT-PCR(qRT-PCR)和流式細胞術分別檢測白假絲酵母菌CAP1基因mRNA(CAP1-mRNA)及其表達產物Cap1p。采用氟康唑濃度遞增(4～64 μg/mL)的YPD培養液連續培養法了解氟康唑誘導白假絲酵母菌耐藥性的作用，qRT-PCR和流式細胞術確定CAP1基因與氟康唑耐藥性形成的關系。結果 134株白假絲酵母菌對氟康唑敏感(MIC≤8 μg/mL)、36株劑量依賴敏感(16～32 μg/mL)、75株耐藥(≥64 μg/mL)，耐藥率為30.6%(75/245)。氟康唑耐藥白假絲酵母菌株CAP1-mRNA和Cap1p表達水平均明顯高于氟康唑敏感菌株(P<0.05)。氟康唑能誘導氟康唑敏感菌株產生耐藥性(MIC≥64 μg/mL)，其CAP1-mRNA和Cap1p表達水平也顯著升高(P<0.05)。結論 白假絲酵母菌臨床菌株對氟康唑有較高的耐藥率。氟康唑能誘導氟康唑敏感白假絲酵母菌株產生耐藥性。白假絲酵母菌對氟康唑耐藥性與CAP1基因表達上調密切相關。

白假絲酵母菌；氟康唑；耐藥性；CAP1基因；表達

由于人口老齡化及抗生素廣泛使用等原因，近年來真菌感染性疾病發病率不斷升高，尤其是侵襲性真菌(invasive fungi)感染性疾病，已成為嚴重威脅人類健康和生命安全的新問題[1]。國外文獻報道，侵襲性真菌感染性疾病最常見的病原體為假絲酵母菌屬(Candida)真菌，占所有病原真菌的70%～90%，我國也不例外[2-5]。假絲酵母菌屬真菌種類繁多、分布廣泛，目前發現至少有9個種可引起人和動物侵襲性真菌病，其中以白假絲酵母菌(Candidaalbicans)最為常見，當機體免疫功能低下時，白假絲酵母菌常作為條件致病菌引起局部或全身感染[6]。氟康唑(fluconazole)是臨床治療時最常用的抗假絲酵母菌感染性疾病藥物，但其耐藥現象也最為嚴重[7-8]。有文獻報道，CAP1基因及其產物轉錄因子Cap1p與白假絲酵母菌耐藥密切相關[9-10]。我們檢測了245株白假絲酵母菌臨床菌株對氟康唑的敏感性，檢測了氟康唑敏感或耐藥菌株CAP1-mRNA、Cap1p蛋白表達水平差異，同時探討了氟康唑誘導白假絲酵母菌耐藥及其與CAP1基因表達關系。

1 材料與方法

1.1 菌株來源及培養 采用假絲酵母菌顯色培養基(BioMerieux)從2013年1-12月本院各臨床科室送檢的咽拭、痰液、尿液、糞便標本中分離假絲酵母菌，各菌株經VITEK 2-compact全自動微生物分析儀(BioMerieux)進行鑒定，從中獲得白假絲酵母菌245株。氟康唑敏感的白假絲酵母菌ATCC22019株由本實驗室保存并提供。上述菌株接種于沙保瓊脂平板上，35 ℃培養2 d。

1.2 最低抑菌濃度測定 采用美國臨床實驗室標準化研究所(CLSI)介紹的微量稀釋法，檢測氟康唑對白假絲酵母菌的最低抑菌濃度(MIC)。挑取沙保平板上培養的白假絲酵母菌接種于YPD培養液中，35 ℃、400 r/min振蕩培養24 h。培養物經細胞計數板計數后，用RPMI-1640培養液(GiBco)配制成菌數為5×103CFU/mL的懸液。96孔細胞培養板中每孔加100 μL白假絲酵母懸液，然后加入100 μL RPMI-1640培養液稀釋的氟康唑注射液(法國輝瑞制藥有限公司，國藥準字J20100010)，使氟康唑終濃度分別為0.25、0.5、2、4、8、16、32、64和128 μg/mL，35 ℃培養24 h后觀察結果，以未見真菌生長的最低藥物濃度為該菌株的MIC。MIC≤8 μg/mL為敏感、16～32 μg/mL為劑量依賴敏感、≥64 μg/mL為耐藥[11]。實驗中采用白假絲酵母菌ATCC22019株為質控菌株。

1.3 CAP1-mRNA測定 挑取沙保平板上培養的白假絲酵母菌接種于YPD培養液中，35 ℃、400 r/min振蕩培養16 h，8 000 r/min離心5 min(4 ℃)。取白假絲酵母菌沉淀用Novelase消化液懸浮，室溫消化10 min后，參照Trizol試劑(Invitrogen)說明書介紹的步驟提取總RNA，紫外分光光度法測定其RNA濃度[12]。根據文獻報道的CAP1基因序列[13-14]，采用Primer Premier 5.0軟件設計檢測白假絲酵母菌CAP1-mRNA和18S-RNA的實時熒光定量RT-PCR(qRT-PCR)引物(表1)，各引物由上海Invitrogen公司合成。以1 μg總RNA為模板，采用逆轉錄試劑盒(TaKaRa)、SYBR Premix Ex.TaqTM熒光定量PCR試劑盒(TaKaRa)、上述引物及ABI-7500型實時熒光定量PCR儀檢測CAP1-mRNA，反應參數：95 ℃30 s；95 ℃5 s、60 ℃30 s，40個循環。以18S-RNA為內參照，采用ΔΔCt模型及REST2005軟件對qRT-PCR數據進行定量分析[15]。

Note: F--上游引物(Forward primers); R--下游引物(Reverse primers).

1.4 Cap1p表達水平測定 挑取沙保平板上培養的白假絲酵母菌接種于YPD培養液中，35 ℃、400 r/min振蕩培養16 h，8 000 r/min離心5 min(4 ℃)。取白假絲酵母菌沉淀用pH7.4、0.01 mol/L磷酸緩沖鹽水(PBS)懸浮，經細胞計數板計數后用PBS配制成菌數為1×106CFU/mL的懸液，然后按上法離心，如此重復2次。白假絲酵母菌沉淀懸于1 mL 1%去氧膽酸鈉-1%無水乙醇-PBS中，37 ℃、200 r/min振蕩孵育30 min，按上法離心后用1%去氧膽酸鈉-PBS懸浮、離心2次。取白假絲酵母菌沉淀加入1∶1 000稀釋的兔抗Cap1p-IgG(本實驗室提供)，4 ℃孵育30 min，按上法用PBS洗滌、離心3次后加入1∶1 000稀釋的FITC標記羊抗兔IgG(ImmunoResearch)，4 ℃孵育30 min，按上法用PBS洗滌、離心3次。白假絲酵母菌沉淀用0.5 mL PBS懸浮，然后用FACSCalibur流式細胞儀檢測20 000個菌細胞(激發波長488 nm)，用Cellquest軟件分析表達Cap1p蛋白(Cap1p+)菌細胞百分率及其平均熒光強度(mean fluorescence intensity，MFI)。

1.5 耐藥性誘導及CAP1-mRNA和Cap1p檢測 氟康唑敏感的134株白假絲酵母菌中，34株氟康唑MIC<2 μg/mL，其余100株氟康唑MIC為2～8 μg/mL。將上述100株白假絲酵母菌分別接種于YPD培養液中，35 ℃、400 r/min振蕩培養至濃度約為1×108CFU/mL，取1×106白假絲酵母菌接種于5 mL含4 μg/mL氟康唑的YPD培養液中，按上法振蕩培養并計數，再取1×106白假絲酵母菌接種于5 mL含8 μg/mL氟康唑的YPD培養液中，直至培養液中氟康唑濃度為64 μg/mL。將64 μg/mL氟康唑YPD白假絲酵母菌培養物8 000 r/min離心5 min(4 ℃)，取沉淀的白假絲酵母菌按上法進行MIC、CAP1-mRNA和Cap1p蛋白表達水平測定，實驗中設置氟康唑未誘導菌株對照。

1.6 統計學分析 分別采用SPSS19.0統計學軟件中的t檢驗，對實驗中獲得的CAP1-mRNA和Cap1p表達水平數據進行檢驗。

2 結 果

2.1 白假絲酵母菌臨床菌株的耐藥率 氟康唑對白假絲酵母菌質控菌株ATCC22019的MIC為0.25或0.5 μg/mL，表明本研究中微量稀釋法實驗質量及其結果可靠[11]。微量稀釋法檢測結果顯示，245株白假絲酵母菌對氟康唑的MIC范圍為0.25～128 μg/mL，其中134株對氟康唑敏感、36株劑量依賴敏感、75株耐藥，敏感率為54.7%(134/245)，耐藥率為30.6%(75/245)(表2)。

2.2 氟康唑耐藥臨床菌株CAP1基因高表達 qRT-PCR和流式細胞術檢測結果分別顯示，75株氟康唑耐藥白假絲酵母菌臨床菌株CAP1-mRNA和Cap1p表達水平均明顯高于134株氟康唑敏感白假絲酵母菌臨床菌株(t=2.784，2.154；P<0.05)(圖1)。

*：Expression of CAP1-mRNA and Cap1p levels in fluconazole-resistantC.albicansisolates compared with that in fluconazole- sensitiveC.albicansisolates (P<0.05).

圖1 白假絲酵母菌CAP1-mRNA和Cap1p表達水平比較

Fig.1 Comparison of CAP1-mRNA and Cap1p expression levels inC.albicansisolates

2.3 氟康唑誘導白假絲酵母菌耐藥性 100株氟康唑敏感白假絲酵母菌(MIC=2～8 μg/mL)依次在4、8、16、32和64 μg/mL氟康唑YPD培養液中均能良好生長，但生長速度減慢。微量稀釋法檢測結果顯示，氟康唑對64 μg/mL氟康唑YPD培養液中收集的菌株MIC≥64 μg/mL。

2.4 誘導耐藥菌株CAP1基因高表達 與氟康唑誘導前比較，100株氟康唑敏感白假絲酵母菌(MIC=2～8 μg/mL)最終經64 μg/mL氟康唑YPD培養液培養后，其CAP1-mRNA水平和Cap1p表達水平均顯著升高(t=3.675，2.772；P<0.05)(圖2)。

3 討 論

白假絲酵母菌是一種條件致病性真菌，可引起人和動物的局部或全身性感染[16-17]，所致疾病種類繁多，既可引起皮膚、黏膜、內臟、中樞神經系統感染性疾病，也可引起皮膚、呼吸道、消化道過敏性疾病[18]。氟康唑是臨床上治療白假絲酵母菌感染性疾病的首選藥物，但白假絲酵母菌對其耐藥性也最為嚴重，且對氟康唑耐藥菌株通常也對其他唑類抗真菌藥物交叉耐藥[7-8]。因此，了解白假絲酵母菌臨床菌株對氟康唑耐藥率及其耐藥機制具有重要的臨床意義。最近的國外和國內文獻報道，白假絲酵母菌臨床菌株對氟康唑的耐藥率分別為21.3%和15.0%[19-20]。本研究發現，白假絲酵母菌臨床菌株對氟康唑的耐藥率高達30.6%，提示藥物治療白假絲酵母菌感染性疾病時，應進行藥物敏感試驗并根據其結果選用合適抗真菌藥物，以提高臨床療效。

*：Expression of CAP1-mRNA and Cap1p levels inC.albicansisolates compared with that inC.albicansisolates before fluconazole induction (P<0.05).

圖2 氟康唑誘導后白假絲酵母菌CAP1-mRNA和Cap1p表達水平變化

Fig.2 Change of CAP1-mRNA and Cap1p expression levels ofC.albicansafter fluconazole induction

白假絲酵母菌對氟康唑耐藥機制主要有真菌多藥抗藥蛋白(multidrug resistant protein，MRP)表達水平上調、氟康唑靶酶14α-去甲基化酶(CYP51A1)突變所致親和力下降[21-22]。CAP1基因表達產物Cap1p是普遍存在于真核細胞中的AP-1家族成員，為含有bZip結構域的轉錄因子[14]。Alarco等首先發現釀酒酵母Cap1p能上調MFS超家族多藥抗藥基因FLR1的表達，從而使釀酒酵母產生對氟康唑產生抗藥性[23]；此后Alarco等又發現白假絲酵母菌Cap1p可上調多個MRP編碼基因表達，從而在白假絲酵母菌對氟康唑耐藥性形成過程中發揮重要作用[9]。有文獻報道，白假絲酵母菌長期暴露于低濃度氟康唑中，也可誘導其產生對氟康唑的耐藥性[24]。本研究發現，氟康唑耐藥的白假絲酵母菌臨床菌株CAP1-mRNA和Cap1p表達水平，均明顯高于氟康唑敏感的白假絲酵母菌臨床菌株(P<0.05)；氟康唑敏感的白假絲酵母菌能適應培養基中濃度逐步增加的氟康唑，轉變為對氟康唑耐藥的菌株，同時其CAP1-mRNA和Cap1p表達水平也顯著升高(P<0.05)。上述實驗結果提示，氟康唑能誘導白假絲酵母菌產生對其耐藥性，其機制與CAP1 基因密切相關；臨床上使用氟康唑治療白假絲酵母菌感染性疾病時，不僅應足量使用并注意合適療程，還需監測白假絲酵母菌對氟康唑敏感性變化，以免誘導白假絲酵母菌產生耐藥性。

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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)

The aim of this study is to investigate the resistant rate ofCandidaalbicans(C.albicans) isolates against fluconazole, and to determine the correlation between the fluconazole-resistance and CAP1 gene ofC.albicans. The microdilution test on 245C.albicansisolates 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-resistantC.albicansisolates were significantly higher than those in the fluconazole-sensitive isolates (P<0.05). When the fluconazole-sensitiveC.albicansisolates 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 thatC.albicansisolates have a higher fluconazole-resistant rate. Fluconazole can induce the resistance against itself in fluconazole-sensitiveC.albicansstrains. The fluconazole-resistance ofC.albicansis closely associated with the expression up-regulation of its CAP1 gene.

Candidaalbicans; fluconazole; resistance; CAP1 gene; expression

Zhao Jin-fang, Email:xinqiuzh@163.com

10.3969/cjz.j.issn.1002-2694.2015.04.007

趙金方，xinqiuzh@163.com

浙江中醫藥大學附屬第一醫院檢驗科，杭州 310006

R379.4

A

1002-2694(2015)04-0325-05

2014-10-03；

2014-11-08