目的：研究光滑念珠菌和近平滑念珠菌对氟康唑耐药的易感性及其诱导耐药株的耐药机制。方法：选取对氟康唑敏感的光滑念珠菌、近平滑念珠菌临床株各1株以及对氟康唑敏感的光滑念珠菌标准株ATCC2001、近平滑念珠菌标准株ATCC22019,利用浓度递增法,在体外用氟康唑诱导使其成为耐药株,采用实时定量PCR检测外排泵相关基因、其转录因子和靶酶编码基因表达,并对光滑念珠菌PDR1转录因子、近平滑念珠菌MRR1转录因子和ERG11基因进行PCR扩增和测序。结果：光滑念珠菌较近平滑念珠菌更易被氟康唑诱导为耐药株,CDR1的过度表达为其主要的耐药机制,对PDR1转录因子测序后发现了新的突变位点Y932C、V847F。近平滑念珠菌诱导耐药株的MDR1表达显著增加,其MRR1转录因子中亦存在新的突变位点P295S、I799S。结论：光滑念珠菌和近平滑念珠菌对氟康唑的耐药易感性不同,耐药机制也存在差异。新发现的转录因子突变位点有待验证其功能。
Objective: To investigate the susceptibility and mechanism of resistance to fluconazole in Candida glabrata and Candida parapsilosis isolates. Methods: Four strains(one clinical C. glabrata, one clinical C. parapsilosis, ATCC2001 and ATCC22019) were induced to be resistance strains in vitro by increasing concentrations of fluconazole. The expression levels of transporter genes and their transcription factor, and azole antifungal target gene were identified by real time-PCR. Meanwhile, the sequence of PDR1 gene of C. glabrata and MRR1, ERG11 gene of C. parapsilosis were determined by PCR based DNA sequencing. Results: C. glabrata was more prone to be resistant to fluconazole than C. parapsilosis. CDR1 was significantly overexpressed in resistant strains of C. glabrata, while MDR1 was significantly overexpressed in resistant strains of C. parapsilosis. PDR1 mutations Y932C, V847F were identified in resistant strains of C.glabrata, and MRR1 mutations P295S, I799S were found in resistant strains of C. parapsilosis. Conclusions: C. glabrata and C. parapsilosis showed different susceptibility to fluconazole resistance, and difference also occurred in resistance mechanism. And the function of new identified mutations in transcription factor needs further verification.
[1] Camplesi M Junior, Silva HM, Arantes AM, et al.Invasive fungal infection in patients with hematologic disorders in a Brazilian tertiary care hospital[J]. Rev Soc Bras Med Trop,2017,50(1):80-85.
[2] Perlroth J, Choi B, Spellberg B.Nosocomial fungal infections: epidemiology, diagnosis, and treatment[J]. Med Mycol,2007,45(4):321-346.
[3] Wang H, Xiao M, Chen SC, et al.In vitro susceptibilities of yeast species to fluconazole and voriconazole as determined by the 2010 National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) study[J]. J Clin Microbiol,2012,50(12):3952-3959.
[4] Sanglard D.Emerging Threats in Antifungal-Resistant Fungal Pathogens[J]. Front Med (Lausanne),2016,3:11.
[5] Versalovic J, Koeuth T, Lupski JR.Distribution of repeti-tive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes[J]. Nucleic Acids Res,1991,19(24):6823-6831.
[6] Rex JH, Walsh TJ, Sobel JD, et al.Practice guidelines for the treatment of candidiasis. Infectious Diseases Socie-ty of America[J]. Clin Infect Dis,2000,30(4):662-678.
[7] Cantón E, Pemán J, Quindós G, et al.Prospective multicenter study of the epidemiology, molecular identification, and antifungal susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis isolated from patients with candidemia[J]. Antimicrob Agents Chemother,2011,55(12):5590-5596.
[8] Cleveland AA, Farley MM, Harrison LH, et al.Changes in incidence and antifungal drug resistance in candide-mia: results from population-based laboratory surveillance in Atlanta and Baltimore, 2008-2011[J]. Clin Infect Dis,2012,55(10):1352-1361.
[9] Pfaller MA, Jones RN, Castanheira M.Regional data analysis of Candida non-albicans strains collected in United States medical sites over a 6-year period, 2006-2011[J]. Mycoses,2014,57(10):602-611.
[10] Bennett JE, Izumikawa K, Marr KA.Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis[J]. Antimicrob Agents Chemother,2004,48(5):1773-1777.
[11] Sanguinetti M, Posteraro B, Fiori B, et al.Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance[J]. Antimicrob Agents Chemother,2005,49(2):668-679.
[12] Torelli R, Posteraro B, Ferrari S, et al.The ATP-binding cassette transporter-encoding gene CgSNQ2 is contribu-ting to the CgPDR1-dependent azole resistance of Candida glabrata[J]. Mol Microbiol,2008,68(1):186-201.
[13] Ferrari S, Ischer F, Calabrese D, et al.Gain of function mutations in CgPDR1 of Candida glabrata not only media-te antifungal resistance but also enhance virulence[J]. PLoS Pathog,2009,5(1):e1000268.
[14] Shin JH, Chae MJ, Song JW, et al.Changes in karyotype and azole susceptibility of sequential bloodstream isolates from patients with Candida glabrata candidemia[J]. J Clin Microbiol,2007,45(8):2385-2391.
[15] Souza AC, Fuchs BB, Pinhati HM, et al.Candida parapsilosis resistance to fluconazole: molecular mechanisms and in vivo impact in infected Galleria mellonella Larvae[J]. Antimicrob Agents Chemother,2015,59(10):6581-6587.
[16] Zhang L, Xiao M, Watts MR, et al.Development of fluconazole resistance in a series of Candida parapsilosis isolates from a persistent candidemia patient with prolonged antifungal therapy[J]. BMC Infect Dis,2015,15:340.
[17] Silva AP, Miranda IM, Guida A, et al.Transcriptional profiling of azole-resistant Candida parapsilosis strains[J]. Antimicrob Agents Chemother,2011,55(7):3546-3556.
[18] Branco J, Silva AP, Silva RM, et al.Fluconazole and voriconazole resistance in Candida parapsilosis Is conferred by gain-of-function mutations in MRR1 transcription factor gene[J]. Antimicrob Agents Chemother,2015, 59(10):6629-6633.
