Population structure and molecular genetic characterization of 5-flucytosine-susceptible and -resistant clinical Candida dubliniensis isolates from Kuwait

Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of C. dubliniensis isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two C. dubliniensis isolates previously analyzed and 58 additional isolates mostly collected during 2010–2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 C. dubliniensis isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.

都柏林假丝酵母菌（Candida dubliniensis）与白假丝酵母菌（Candida albicans）是两种亲缘关系密切的菌种。尽管都柏林假丝酵母菌的致病性较弱，但其对氟康唑（fluconazole）产生耐药性的倾向更高，且部分菌株对5-氟胞嘧啶（5-flucytosine, 5-FC）存在固有耐药性。此前研究证实，科威特地区所有对5-氟胞嘧啶耐药的分离株，均属于基于核糖体DNA（rDNA）内转录间隔区（internal transcribed spacer, ITS）分型的7种单倍型之一。本研究采用多位点序列分型（multilocus sequence typing, MLST）对都柏林假丝酵母菌分离株进行基因指纹分析，以明确5-氟胞嘧啶耐药与敏感菌株的群体结构，并将分析结果与全球分离株数据集的相关数据进行对比。本研究共纳入两类菌株：此前已完成相关分析的52株都柏林假丝酵母菌分离株，以及2010至2013年间采集的58株新增分离株（绝大多数为该时期样本），所有菌株均通过表型检测与分子生物学方法完成鉴定。基于内转录间隔区的单倍型通过单倍型特异性PCR和/或核糖体DNA的PCR-DNA测序进行鉴定。群体结构通过8位点多位点序列分型进行解析。采用E试验法（E-test）检测所有菌株对5-氟胞嘧啶、氟康唑、伏立康唑（voriconazole）以及两性霉素B（amphotericin B）的药敏敏感性。在110株都柏林假丝酵母菌分离株中共检测到5种ITS单倍型（ITSH），其中ITSH1最为常见（n=80株），其次为ITSH4（n=25株）；另有2株分别归属于ITSH5与ITSH8，1株归属于ITSH7。多位点序列分型共鉴定出16种二倍体序列型（diploid sequence types, DSTs），其中包括6种新型二倍体序列型。DST11（n=52）与DST14（n=25）为优势基因型，且与DST21一同仅分布于中东国家。其余二倍体序列型（除部分新型序列型外）分布范围更广，已在多个其他国家被检出。仅ITSH4菌株（n=25）归属于DST14，该类菌株对5-氟胞嘧啶耐药，且在CdFCA1基因中携带S29L氨基酸突变。ITSH5、ITSH7与ITSH8菌株分别归属于不同的二倍体序列型。由此可见，科威特临床分离的都柏林假丝酵母菌基因型异质性有限，多数分离株属于区域特异性二倍体序列型。所有对5-氟胞嘧啶耐药的都柏林假丝酵母菌分离株，均归属于ITSH4与基于多位点序列分型的DST14基因型。部分菌株可归类为额外ITS单倍型这一结论，同样得到多位点序列分型数据的支持。