Article(id=1256263561068798000, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, articleNumber=null, orderNo=null, doi=10.13346/j.mycosystema.250096, pmid=null, cstr=32115.14.j.mycosystema.250096, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1743523200000, receivedDateStr=2025-04-02, revisedDate=null, revisedDateStr=null, acceptedDate=1752422400000, acceptedDateStr=2025-07-14, onlineDate=1777446173207, onlineDateStr=2026-04-29, pubDate=1771689600000, pubDateStr=2026-02-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1777446173207, onlineIssueDateStr=2026-04-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1777446173207, creator=13701087609, updateTime=1777446173207, updator=13701087609, issue=Issue{id=1256263559323967535, tenantId=1146029695717560320, journalId=1255847803461844995, year='2026', volume='45', issue='2', pageStart='250058', pageEnd='250280', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1777446172791, creator=13701087609, updateTime=1777447435276, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1256268854674710546, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1256268854678904851, tenantId=1146029695717560320, journalId=1255847803461844995, issueId=1256263559323967535, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=250096, endPage=, ext={EN=ArticleExt(id=1256263562977206332, articleId=1256263561068798000, tenantId=1146029695717560320, journalId=1255847803461844995, language=EN, title=Identification of the Cyp51 mutation site of the azole resistance gene in Aspergillus flavus, columnId=1256263562373226548, journalTitle=Mycosystema, columnName=Research paper, runingTitle=null, highlight=null, articleAbstract=

The molecular characterization and phenotype identification of resistance mutations of azole resistance genes cyp51A, cyp51B and cyp51C of 6 clinical Aspergillus flavus isolates from Xinjiang are carried out. The isolates were identified based on internal transcribed spacer (ITS) and BenA gene sequencing. The Sensititre Yeastone Fungal Drug Sensitivity Kit was used to detect the in vitro antifungal susceptibility of 7 drugs, and the MIC/MEC results were interpreted according to the standards recommended by CLSI M57S. The cyp51A, cyp51B and cyp51C genes of A. flavus strains were amplified and sequenced by PCR, and the sequencing results were compared with the reference strain of A. flavus to identify the resistance mutation phenotype. All 6 clinical isolates were identified as A. flavus. The results of antifungal susceptibility test showed that the isolated A. flavus strains were completely sensitive to echinocandins, itraconazole and posaconazole; of which 1 strain was resistant to amphotericin (8 μg/mL) and 2 strains were resistant to voriconazole (4 μg/mL). Sequencing of the cyp51A, cyp51B, and cyp51C genes of 2 azole-sensitive and 4 azole-resistant strains revealed that both cyp51A and cyp51C in sensitive and resistant strains showed synonymous and non-synonymous point mutations, and no mutation was found in cyp51B. However, the P276T mutation site in cyp51C gene was only found in one resistant isolate. Echinocandins, itraconazole and posaconazole showed better antifungal activity against A. flavus, while voriconazole and amphotericin showed lower antifungal activity against A. flavus. Probably cyp51C gene mutation of A. flavus is associated with azole resistance.

, correspAuthors=Xiaodong WANG, authorNote=null, correspAuthorsNote=
*E-mail:
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#Co-first author

, authorsList=Akeda YUSUP, Zubaida AZIZ, Kadirya KASIM, Xiaodong WANG), CN=ArticleExt(id=1256263563979644990, articleId=1256263561068798000, tenantId=1146029695717560320, journalId=1255847803461844995, language=CN, title=黄曲霉中唑类耐药基因Cyp51突变位点鉴定, columnId=1256263563312771301, journalTitle=菌物学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

对新疆临床分离的6株黄曲霉进行分子鉴定及唑类耐药基因cyp51Acyp51Bcyp51C耐药突变表型鉴定。采用PCR扩增ITS和BenA基因,扩增产物进行测序,blast比对后进行菌种的分子鉴定;采用Sensititre Yeastone真菌药敏试剂盒检测7种药物的体外抗真菌药物敏感性,MIC/MEC结果解释标准依据CLSI M57S推荐;设计特异性引物,采用PCR扩增黄曲霉菌株的cyp51Acyp51Bcyp51C基因并测序,测序结果与黄曲霉参考菌株比对鉴定耐药突变表型。真菌药敏试验MIC/MEC值显示,6株菌株均对棘白菌素类高度敏感(MEC≤0.015 μg/mL)、对伊曲康唑和泊沙康唑敏感率为100%;1株对两性霉素B耐药(MIC=8 μg/mL),2株对伏立康唑耐药(MIC=4 μg/mL)。基因测序结果显示,cyp51Acyp51C在敏感株和耐药株中均出现了同义和非同义点突变,cyp51B中未发现突变。然而,cyp51C基因中P276T突变位点只存在于耐药菌株中。棘白菌素类、伊曲康唑和泊沙康唑对黄曲霉的体外抑菌活性较好,伏立康唑和两性霉素对黄曲霉的抑菌活性较差;cyp51C P276T突变可能是黄曲霉对伏立康唑耐药的新潜在机制。

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Chinese Journal of Mycology, 13(1): 57-60 (in Chinese), articleTitle=Epidemiology of invasive aspergillosis in China, refAbstract=null), Reference(id=1256263591418782021, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, doi=null, pmid=null, pmcid=null, year=2022, volume=32, issue=20, pageStart=3047, pageEnd=3051, url=null, language=null, rfNumber=[19], rfOrder=18, authorNames=郭鹏豪, 黄旭东, 吴劲松, 蔡文莹, 陈雪琴, 温见翔, 廖康, journalName=中华医院感染学杂志, refType=null, unstructuredReference=郭鹏豪, 黄旭东, 吴劲松, 蔡文莹, 陈雪琴, 温见翔, 廖康, 2022. 广东省68家医院下呼吸道标本分离曲霉菌分布及其药物敏感性. 中华医院感染学杂志, 32(20): 3047-3051, articleTitle=广东省68家医院下呼吸道标本分离曲霉菌分布及其药物敏感性, refAbstract=null), Reference(id=1256263592194728265, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, doi=null, pmid=null, pmcid=null, year=2018, volume=13, issue=1, pageStart=57, pageEnd=60, url=null, language=null, rfNumber=[20], rfOrder=19, authorNames=徐媛, 陈敏, 廖万清, journalName=中国真菌学杂志, refType=null, unstructuredReference=徐媛, 陈敏, 廖万清, 2018. 中国侵袭性曲霉菌病流行病学现状. 中国真菌学杂志, 13(1): 57-60, articleTitle=中国侵袭性曲霉菌病流行病学现状, refAbstract=null)], funds=[Fund(id=1256263578777149661, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, awardId=TSYC202301B029, language=EN, fundingSource=Xinjiang Uygur Autonomous Region “Tianshan Elite Medical and Health High-level Talent Training Program Young Backbone Talents”(TSYC202301B029), fundOrder=null, country=null), Fund(id=1256263580232573155, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, awardId=TSYC202301B029, language=CN, fundingSource=新疆维吾尔自治区“天山英才医药卫生高层次人才培养计划中青年骨干人才”(TSYC202301B029), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1256263564873031745, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, xref=null, ext=[AuthorCompanyExt(id=1256263564889808962, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, companyId=1256263564873031745, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Department of Dermatology, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang, China), AuthorCompanyExt(id=1256263564910780484, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, companyId=1256263564873031745, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=新疆医科大学第一附属医院皮肤科,新疆 乌鲁木齐 830011)])], figs=[ArticleFig(id=1256263576080212149, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=EN, label=Table 1, caption=

PCR and RT-PCR primers

, figureFileSmall=null, figureFileBig=null, tableContent=
基因
Gene
引物名称
Primer
序列
Sequence (5ʹ→3ʹ)
Cyp51A Cyp51A-F1 CAAGAACAGCCTGCACAGAG
Cyp51A-R1 GGGTGGATCAGTCTTATTA
Cyp51A-F2 GCAATCATCGTCCTAAATC
Cyp51A-R2 CTGTCCATTCTTGTAGGTA
Cyp51A-F3 GCATGAGGGAGATCTATATG
Cyp51A-R3 CCTATAATTGCTGGTTTCG
Cyp51A-F4 TGAAGCTATTCAATGTAGAC
Cyp51A-R4 ACTGCTGATGGTGTGCTAAG
Cyp51B Cyp51B-F5 ATGGGCATCCTAGCTGTCATTC
Cyp51B-R5 GGCGGTGTATATGGTAATCTC
Cyp51B-F6 CCCTTGGTATTTCATTGGTTCCC
Cyp51B-R6 TTTCATGTTACCATGGGCCC
Cyp51B-F7 TTTCATGTTACCATGGGCCC
Cyp51B-R7 TTCAGAGCTAACAGCGATGGC
Cyp51B-F8 CGGAAGAACATTTCCCTGATCC
Cyp51B-R8 CGTCTGGCAATATCATGCAC
Cyp51C Cyp51C-F9 CAATGGTGCTGACAAACCTG
Cyp51C-R9 CAAAGGAGCGACACATAAG
Cyp51C-F10 GGTAATGTCTGGTCATAGG
Cyp51C-R10 ATGAGCTTGGAATTGGG
Cyp51C-F11 CGAATTCATCCTCAATGG
Cyp51C-R11 GTCTCTCGGATCACATT
Cyp51C-F12 GGAACTCTACCAAGAGCA
Cyp51C-R12 GCTCATCATAATGCATGAGG
), ArticleFig(id=1256263576491253949, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=CN, label=表1, caption=

基因扩增及测序引物

, figureFileSmall=null, figureFileBig=null, tableContent=
基因
Gene
引物名称
Primer
序列
Sequence (5ʹ→3ʹ)
Cyp51A Cyp51A-F1 CAAGAACAGCCTGCACAGAG
Cyp51A-R1 GGGTGGATCAGTCTTATTA
Cyp51A-F2 GCAATCATCGTCCTAAATC
Cyp51A-R2 CTGTCCATTCTTGTAGGTA
Cyp51A-F3 GCATGAGGGAGATCTATATG
Cyp51A-R3 CCTATAATTGCTGGTTTCG
Cyp51A-F4 TGAAGCTATTCAATGTAGAC
Cyp51A-R4 ACTGCTGATGGTGTGCTAAG
Cyp51B Cyp51B-F5 ATGGGCATCCTAGCTGTCATTC
Cyp51B-R5 GGCGGTGTATATGGTAATCTC
Cyp51B-F6 CCCTTGGTATTTCATTGGTTCCC
Cyp51B-R6 TTTCATGTTACCATGGGCCC
Cyp51B-F7 TTTCATGTTACCATGGGCCC
Cyp51B-R7 TTCAGAGCTAACAGCGATGGC
Cyp51B-F8 CGGAAGAACATTTCCCTGATCC
Cyp51B-R8 CGTCTGGCAATATCATGCAC
Cyp51C Cyp51C-F9 CAATGGTGCTGACAAACCTG
Cyp51C-R9 CAAAGGAGCGACACATAAG
Cyp51C-F10 GGTAATGTCTGGTCATAGG
Cyp51C-R10 ATGAGCTTGGAATTGGG
Cyp51C-F11 CGAATTCATCCTCAATGG
Cyp51C-R11 GTCTCTCGGATCACATT
Cyp51C-F12 GGAACTCTACCAAGAGCA
Cyp51C-R12 GCTCATCATAATGCATGAGG
), ArticleFig(id=1256263576642248896, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=EN, label=Table 2, caption=

The MIC/MEC values (µg/mL) of antifungal agents against clinical isolates of Aspergillus flavus

, figureFileSmall=null, figureFileBig=null, tableContent=
样本
Sample
伊曲康唑
Itraconazole
伏立康唑
Voriconazole
泊沙康唑
Posaconazole
阿尼芬净
Anidulafungin
米卡芬净
Micafungin
卡泊芬净
Caspofungin
两性霉素B
Amphotericin B
01 0.12 0.5 0.25 ≤0.015 0.015 0.015 4
02 0.25 1 0.25 ≤0.015 ≤0.008 ≤0.008 4
03 0.25 0.5 0.25 ≤0.015 ≤0.008 ≤0.008 4
04 0.25 0.5 0.25 ≤0.015 ≤0.008 ≤0.008 8
05 0.25 4 0.12 ≤0.015 ≤0.008 ≤0.008 2
06 0.25 4 0.25 ≤0.015 ≤0.008 0.015 4
), ArticleFig(id=1256263577078456518, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=CN, label=表2, caption=

黄曲霉对抗真菌药物的MIC/MEC值(µg/mL)

, figureFileSmall=null, figureFileBig=null, tableContent=
样本
Sample
伊曲康唑
Itraconazole
伏立康唑
Voriconazole
泊沙康唑
Posaconazole
阿尼芬净
Anidulafungin
米卡芬净
Micafungin
卡泊芬净
Caspofungin
两性霉素B
Amphotericin B
01 0.12 0.5 0.25 ≤0.015 0.015 0.015 4
02 0.25 1 0.25 ≤0.015 ≤0.008 ≤0.008 4
03 0.25 0.5 0.25 ≤0.015 ≤0.008 ≤0.008 4
04 0.25 0.5 0.25 ≤0.015 ≤0.008 ≤0.008 8
05 0.25 4 0.12 ≤0.015 ≤0.008 ≤0.008 2
06 0.25 4 0.25 ≤0.015 ≤0.008 0.015 4
), ArticleFig(id=1256263577296560330, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=EN, label=Table 3, caption=

The amino acid mutation sites of cyp51A, cyp51B and cyp51C genes in clinical isolates of Aspergillus flavus

, figureFileSmall=null, figureFileBig=null, tableContent=
样本号
Sample No.
唑类敏感/耐药
Azole sensitive/resistant
氨基酸突变位点
Amino acid mutation sites
Cyp51A
01 S - - F188 Y247 - - -
02 S P61 K136 F188 Y247 P315 P394 N462
03 S - - F188 Y247 - P394 -
04 S P61 - F188 - - P394 -
05 R P61 K136 F188 - - P394 -
06 R F188 - - Y247 - P394 -
Cyp51B
01 S undetected
02 S undetected
03 S undetected
04 S undetected
05 R undetected
06 R undetected
Cyp51C
01 S - - - - - L326 L348 S386 E421 N423D
02 S M54T G58 S240A D254G - L326 L348 S386 E421 N423D
03 S - - - - - - L348 S386 E421 N423D
04 S M54T G58 S240A - - L326 L348 - - -
05 R M54T G58 S240A D254G P276T - - - E421 N423D
06 R M54T G58 S240A D254G - L326 L348 S386 E421 N423D
), ArticleFig(id=1256263577908928721, tenantId=1146029695717560320, journalId=1255847803461844995, articleId=1256263561068798000, language=CN, label=表3, caption=

黄曲霉cyp51A、cyp51B和cyp51C基因氨基酸突变位点

, figureFileSmall=null, figureFileBig=null, tableContent=
样本号
Sample No.
唑类敏感/耐药
Azole sensitive/resistant
氨基酸突变位点
Amino acid mutation sites
Cyp51A
01 S - - F188 Y247 - - -
02 S P61 K136 F188 Y247 P315 P394 N462
03 S - - F188 Y247 - P394 -
04 S P61 - F188 - - P394 -
05 R P61 K136 F188 - - P394 -
06 R F188 - - Y247 - P394 -
Cyp51B
01 S undetected
02 S undetected
03 S undetected
04 S undetected
05 R undetected
06 R undetected
Cyp51C
01 S - - - - - L326 L348 S386 E421 N423D
02 S M54T G58 S240A D254G - L326 L348 S386 E421 N423D
03 S - - - - - - L348 S386 E421 N423D
04 S M54T G58 S240A - - L326 L348 - - -
05 R M54T G58 S240A D254G P276T - - - E421 N423D
06 R M54T G58 S240A D254G - L326 L348 S386 E421 N423D
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黄曲霉中唑类耐药基因Cyp51突变位点鉴定
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艾柯代·玉苏甫 # , 祖拜旦木·艾则孜 # , 哈地利亚·哈斯木 , 王晓东 *
菌物学报 | 研究论文 2026,45(2): 250096
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菌物学报 | 研究论文 2026, 45(2): 250096
黄曲霉中唑类耐药基因Cyp51突变位点鉴定
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艾柯代·玉苏甫#, 祖拜旦木·艾则孜#, 哈地利亚·哈斯木, 王晓东*
作者信息
  • 新疆医科大学第一附属医院皮肤科,新疆 乌鲁木齐 830011
Identification of the Cyp51 mutation site of the azole resistance gene in Aspergillus flavus
Akeda YUSUP, Zubaida AZIZ, Kadirya KASIM, Xiaodong WANG*
Affiliations
  • Department of Dermatology, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang, China
出版时间: 2026-02-22 doi: 10.13346/j.mycosystema.250096
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对新疆临床分离的6株黄曲霉进行分子鉴定及唑类耐药基因cyp51Acyp51Bcyp51C耐药突变表型鉴定。采用PCR扩增ITS和BenA基因,扩增产物进行测序,blast比对后进行菌种的分子鉴定;采用Sensititre Yeastone真菌药敏试剂盒检测7种药物的体外抗真菌药物敏感性,MIC/MEC结果解释标准依据CLSI M57S推荐;设计特异性引物,采用PCR扩增黄曲霉菌株的cyp51Acyp51Bcyp51C基因并测序,测序结果与黄曲霉参考菌株比对鉴定耐药突变表型。真菌药敏试验MIC/MEC值显示,6株菌株均对棘白菌素类高度敏感(MEC≤0.015 μg/mL)、对伊曲康唑和泊沙康唑敏感率为100%;1株对两性霉素B耐药(MIC=8 μg/mL),2株对伏立康唑耐药(MIC=4 μg/mL)。基因测序结果显示,cyp51Acyp51C在敏感株和耐药株中均出现了同义和非同义点突变,cyp51B中未发现突变。然而,cyp51C基因中P276T突变位点只存在于耐药菌株中。棘白菌素类、伊曲康唑和泊沙康唑对黄曲霉的体外抑菌活性较好,伏立康唑和两性霉素对黄曲霉的抑菌活性较差;cyp51C P276T突变可能是黄曲霉对伏立康唑耐药的新潜在机制。

黄曲霉  /  抗真菌药物敏感性  /  唑类耐药  /  cyp51基因

The molecular characterization and phenotype identification of resistance mutations of azole resistance genes cyp51A, cyp51B and cyp51C of 6 clinical Aspergillus flavus isolates from Xinjiang are carried out. The isolates were identified based on internal transcribed spacer (ITS) and BenA gene sequencing. The Sensititre Yeastone Fungal Drug Sensitivity Kit was used to detect the in vitro antifungal susceptibility of 7 drugs, and the MIC/MEC results were interpreted according to the standards recommended by CLSI M57S. The cyp51A, cyp51B and cyp51C genes of A. flavus strains were amplified and sequenced by PCR, and the sequencing results were compared with the reference strain of A. flavus to identify the resistance mutation phenotype. All 6 clinical isolates were identified as A. flavus. The results of antifungal susceptibility test showed that the isolated A. flavus strains were completely sensitive to echinocandins, itraconazole and posaconazole; of which 1 strain was resistant to amphotericin (8 μg/mL) and 2 strains were resistant to voriconazole (4 μg/mL). Sequencing of the cyp51A, cyp51B, and cyp51C genes of 2 azole-sensitive and 4 azole-resistant strains revealed that both cyp51A and cyp51C in sensitive and resistant strains showed synonymous and non-synonymous point mutations, and no mutation was found in cyp51B. However, the P276T mutation site in cyp51C gene was only found in one resistant isolate. Echinocandins, itraconazole and posaconazole showed better antifungal activity against A. flavus, while voriconazole and amphotericin showed lower antifungal activity against A. flavus. Probably cyp51C gene mutation of A. flavus is associated with azole resistance.

Aspergillus flavus  /  antifungal susceptibility  /  azole-resistance  /  cyp51 gene
艾柯代·玉苏甫, 祖拜旦木·艾则孜, 哈地利亚·哈斯木, 王晓东. 黄曲霉中唑类耐药基因Cyp51突变位点鉴定. 菌物学报, 2026 , 45 (2) : 250096 - . DOI: 10.13346/j.mycosystema.250096
Akeda YUSUP, Zubaida AZIZ, Kadirya KASIM, Xiaodong WANG. Identification of the Cyp51 mutation site of the azole resistance gene in Aspergillus flavus[J]. Mycosystema, 2026 , 45 (2) : 250096 - . DOI: 10.13346/j.mycosystema.250096
曲霉菌是环境中广泛存在的机会性致病真菌,可引起肺、眼、鼻、外耳道、皮肤等的感染。侵袭性曲霉病(invasive aspergillosis, IA)是曲霉菌感染最严重的一种类型,主要累及免疫功能受损患者,死亡率高达60% (Chowdhary et al. 2013;Chris & David 2015)。尽管烟曲霉仍是IA最常见的病原体,但近年来非烟曲霉导致的感染比例显著上升,尤以黄曲霉Aspergillus flavus Link居多,可达27.5% (徐媛等 2018)。值得注意的是,黄曲霉感染更易累及免疫正常的宿主,且常引发侵袭性鼻窦炎和角膜炎(Alessandro 2009;Al-Wathiqi et al. 2013)。唑类药物的作用机理是抑制麦角甾醇14α-去甲基化酶(CYP51),阻止麦角甾醇生物合成通路,导致麦角甾醇合成减少,发挥抗真菌作用(Patterson et al. 2016)。然而,随着农业和临床唑类药物的广泛使用,黄曲霉耐药株的检出率逐年上升(Vermeulen et al. 2013)。目前,烟曲霉的唑类耐药机制已较为明确,主要涉及cyp51A基因突变(如TR34/L98H)及外排泵过表达等。相比之下,对黄曲霉耐药研究相对有限。近年研究表明,黄曲霉对唑类耐药可能与其特有的cyp51基因(cyp51Acyp51Bcyp51C)突变相关,但不同基因亚型的作用存在显著差异(Sharma et al. 2018;Lucio et al. 2020)。新疆作为我国重要的农业和畜牧业基地,抗真菌药物使用模式与环境压力具有地域特殊性。本研究旨在探讨临床分离的黄曲霉的药敏特征及分析cyp51Acyp51Bcyp51C基因与唑类耐药的关系,揭示黄曲霉唑类耐药的可能机制,为区域性抗真菌药物的合理使用提供科学依据。
本研究的6株黄曲霉临床分离株筛选自2011年至2023年保存于新疆医科大学第一附属医院皮肤科真菌实验室真菌标本库的111株曲霉菌属分离株。全部菌株从临床标本中培养分离并经过形态学初步鉴定为黄曲霉。
将-80 ℃低温冰箱液体冷冻保存的菌株恢复室温复苏后,用无菌接种环挑取少量菌液接种于PDA培养基,置于28 ℃恒温培养箱培养3-5 d。
挑取一定量的菌落置于组织研磨器中进行研磨,并按照Ezup柱式真菌基因组DNA抽提试剂盒(生工生物工程(上海)股份有限公司)说明书进行菌种DNA提取。
PCR扩增核糖体DNA内转录间区(internal transcribed space, ITS)和BenA (微管蛋白)基因。PCR引物:ITS1 (5ʹ-TCCGTAGGTGAACCTGCG G-3ʹ)、ITS4 (5ʹ-TCCTCCGCTTATTGATATGC-3ʹ)和BT2a (5ʹ-GGTAACCAAATCGGTGCTGCTTT C-3ʹ)、BT2b (5ʹ-ACCCTCAGTGTAGTGACCCT TGGC-3ʹ)。PCR反应条件:95 ℃预变性5 min;94 ℃变性30 s、57 ℃退火30 s、72 ℃延伸90 s,共30个循环;72 ℃延伸10 min。扩增产物进行1.5%琼脂糖凝胶电泳,产物纯化后测序。将测序得到的基因序列在GenBank进行BLAST比对后鉴定菌种。
使用商品化的体外抗真菌药物敏感性检测试剂盒 Sensititre YeastOne真菌药敏板(Thermo Fisher)进行测量,实验步骤按照试剂盒说明书进行。我们测试了全部菌株对3类抗真菌药总计7种药物的敏感性,(a)多烯类:两性霉素B;(b)唑类:伊曲康唑、伏立康唑和泊沙康唑;(c)棘白菌素类:卡泊芬净、阿尼芬净和米卡芬净。两性霉素B的最终检测浓度范围为0.12- 8 μg/mL;卡泊芬净、米卡芬净、伏立康唑、泊沙康唑为0.008-8 μg/mL;伊曲康唑为0.015- 16 μg/mL;阿尼芬净:0.015-8 μg/mL。接种后的药敏板置于35 ℃培养箱孵育24-48 h。试剂盒以克柔念珠菌ATCC6258作为质量控制菌株。依据标准参考临床实验室标准协会(CLSI) M57S来评估药物的敏感性(Procop et al. 2022)。
提取6株黄曲霉菌株基因组DNA,按照真菌基因组DNA抽提试剂盒说明书进行。PCR扩增cyp51Acyp51Bcyp51C基因并测序,使用的引物序列见表1。PCR反应总体系为25 μL:上下游引物各1 μL,dNTP (mix) 1 μL,Taq酶0.2 μL,模板DNA 1 μL,PCR缓冲液2.5 μL,ddH2O 18.3 μL。PCR反应条件:95 ℃预变性5 min;94 ℃变性30 s,63 ℃退火30 s,72 ℃延伸30 s,共30个循环;72 ℃延伸10 min。利用SnapGene7.0.2软件将扩增的产物基因序列与GenBank数据库提供的参考菌株黄曲霉NRRL3357进行比对,以确定基因突变位点。
所有菌株经ITS和BenA测序,与GenBank数据库中黄曲霉序列比对,一致性≥98%,表明所有菌株均为黄曲霉。
6株临床分离黄曲霉对7种抗真菌药物的敏感性见表2。棘白菌素类药物:6株临床黄曲霉菌种均对阿尼芬净、卡泊芬净和米卡芬净具有较低水平的最低有效浓度(minimum effective concentration, MEC),总体MECs≤0.015 μg/mL。多烯类药物:根据CLSI M57S的黄曲霉两性霉素B的ECV值为4 μg/mL,即≥8 μg/mL提示非野生型(耐药);有1株黄曲霉对AMB耐药。唑类药物:黄曲霉对伊曲康唑和泊沙康唑的敏感率为100%;此外,发现2株对伏立康唑耐药(MIC> 2 μg/mL)。
通过PCR扩增获得与预期目的片段大小相同的产物,对该产物进行序列测定,与黄曲霉敏感菌的cyp51Acyp51Bcyp51C基因序列(GenBank登录号为NRRL3357)比对后发现,在敏感株和耐药菌株中的cyp51A基因共有7个同义突变位点(P61、K136、F188、Y247、P315、P394和N462);Cyp51B没有任何氨基酸替换;Cyp51C中检测到10个同义和非同义突变位点,9个突变位点(M54T、G58、S240A、D254G、L326、L348、S386、E421和N423D)也存在于敏感菌株中。1株伏立康唑耐药菌株中携带P276T点突变,这个突变仅存在于该菌株中(表3)。
近年来侵袭性曲霉病的发病率逐年上升,但治疗侵袭性真菌感染的药物种类有限,耐药菌株常导致治疗失败,给临床带来挑战。虽然黄曲霉占曲霉感染中的第2位,但关于黄曲霉的研究及临床信息相对较少,所以对黄曲霉进行药物敏感性试验及耐药机制的研究,对寻找新的治疗靶点意义重大。本研究中,棘白菌素类药物对黄曲霉体外抗菌活性最高,米卡芬净、阿尼芬净和卡泊芬净MEC均为0.015 μg/mL,能有效抑制全部菌株生长,这个结果与研究报道一致(郭鹏豪等 2022),进一步支持棘白菌素作为侵袭性曲霉病补救治疗的临床价值。唑类药物中抗菌效果最高的是伊曲康唑和泊沙康唑,优于伏立康唑,这与Wang et al. (2022)报道的黄曲霉唑类耐药情况基本一致。本实验6株黄曲霉中有1株对两性霉素B耐药,占所有黄曲霉菌株的16.67%。对中国安徽地区的曲霉临床分离株的药物敏感性研究显示,该地区黄曲霉对两性霉素B的耐药率为20.8% (Wang et al. 2022)。最近欧洲的一项研究,从26 909株曲霉属分离株中,在36.8%的土曲霉、14.9%的黄曲霉、5.2%的黑曲霉和2.01%的烟曲霉分离株中检测到对两性霉素B的耐药性。不同研究中的两性霉素B耐药菌株所占比例有所差异,可能与菌株的地区分布、标本来源和检测方法有关。Wang et al. (2018)的研究证实,Sensititre YeastOne真菌药敏检测试剂盒对于两性霉素B的检测结果比CLSI M38-A2方法的结果高3.3倍,提示需结合临床疗效对耐药阈值进行区域性验证。
甾醇14-去甲基化酶(CYP51)是唑类抗真菌药物作用的靶酶,其基因突变与多种真菌唑类药物耐药相关。本研究对4株敏感及2株耐药菌株的cyp51基因进行了测序,cyp51A基因中7个(P61、K136、F188、P315、Y247、P394和N462)突变位点在敏感株与耐药株中均存在,不是既往文献报道的K197N、D282E、M288L、Y132N和T469S等多个与黄曲霉对唑类耐药有关的突变位点(Krishnan-Natesan et al. 2008),提示其为黄曲霉种群多态性特征,可能不直接参与耐药。Cyp51B基因测序显示未发现任何突变位点,这与之前的研究结果一致(Sharma et al. 2018;Lucio et al. 2020),再次证明cyp51B基因序列较为保守,不易发生突变,黄曲霉耐药可能与cyp51B基因突变无关。迄今为止,只有黄曲霉cyp51C的一个点突变(Y319H)提示与唑类耐药密切相关(Paul et al. 2015)。本研究中cyp51C基因存在10个突变位点,在敏感和耐药菌株中均有9个(M54T、G58、S240A、D254G、L326、L348、S386、E421和N423D)突变位点,这些突变位点与耐药无关(Sharma et al. 2018;Choi et al. 2019),P276T突变仅在1株耐药菌株中检出,未见于敏感菌株,需扩大样本量验证其与伏立康唑耐药的因果关系。另一株伏立康唑耐药株未携带P276T突变或其他已知cyp51突变,提示可能存在其他耐药机制,如外排泵基因cdr1B过表达,生物膜形成、氧化应激应答增强等表型适应性途径降低药物敏感性。Liu et al. (2012)首次在伏立康唑耐药的黄曲霉临床分离株中发现cyp51C的T788G突变(S240A),并通过异源表达实验证明该突变可使伏立康唑MIC值升高 4倍,提示其可能参与耐药表型。本研究在敏感株和耐药株中存在cyp51C中的S240A。Paul et al. (2018)在印度耐药株中检测到S240A突变,但该突变在敏感株和耐药株中普遍存在。这表明S240A点突变可能与黄曲霉唑类耐药无直接因果关系,但其可能与其他突变或调控机制(如外排泵过表达)协同导致唑类耐药。本研究的样本量较小,且未构建点突变菌株进行验证性实验。后续研究可通过CRISPR-Cas9基因编辑技术构建P276T点突变菌株,评估其对伏立康唑MIC值的影响。
综上所述,本地区黄曲霉的耐药机制可能与cyp51C的点突变有关,然而,黄曲霉对唑类耐药现象并非受单一机制的调控,且临床很多患者存在滥用抗真菌药物的现象,导致黄曲霉对唑类药物的耐药性不断提高,而新型抗真菌药开发缓慢,改善黄曲霉对唑类的耐药问题迫在眉睫,因此,我们需要进一步研究黄曲霉致病机制和耐药机制,寻找新的药物治疗靶点,探索新的治疗体系,提高患者生存率。
艾柯代·玉苏甫:论文构思及撰写;祖拜旦木·艾则孜:论文修改、原始数据收集;哈地利亚·哈斯木:论文构思及指导;王晓东:论文验证与审核。
该研究不存在任何潜在利益冲突的商业或财务关系。
  • 新疆维吾尔自治区“天山英才医药卫生高层次人才培养计划中青年骨干人才”(TSYC202301B029)
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2026年第45卷第2期
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doi: 10.13346/j.mycosystema.250096
  • 接收时间:2025-04-02
  • 首发时间:2026-04-29
  • 出版时间:2026-02-22
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  • 收稿日期:2025-04-02
  • 录用日期:2025-07-14
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Xinjiang Uygur Autonomous Region “Tianshan Elite Medical and Health High-level Talent Training Program Young Backbone Talents”(TSYC202301B029)
新疆维吾尔自治区“天山英才医药卫生高层次人才培养计划中青年骨干人才”(TSYC202301B029)
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    新疆医科大学第一附属医院皮肤科,新疆 乌鲁木齐 830011

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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