海南热带假丝酵母菌多样性、三唑类耐药株起源及传播规律研究

Understanding the epidemiology of pathogenic fungi is crucial for designing strategies for preventing and controlling fungal diseases and disease outbreaks. Hainan Province is located in the tropical region of China and is a top travel destination, especially during the winter. Thus, studying tropical pathogens is not only important for local residents but also for people from other parts of China and outside of China. Candida tropicalis (Ct) is one of the major tropical pathogens capable of causing invasive infections and high mortality rates. Our previous studies have found that Ct is commonly found in the oral cavities of local residents in Hainan and has unique novel genetic diversities and high frequencies of antifungal drug resistance. However, little is known about the environmental reservoir of Ct in Hainan, the source(s) of genetic diversity of Ct in human hosts, the origin of drug-resistant strains, and how such strains are spread. Building on results from previous studies, here we propose to collect Ct from a diversity of environmental and geographic niches in Hainan, analyze their antifungal drug susceptibility, and obtain their multilocus sequence genotypes by sequencing the 6 housekeeping genes. The data will be used to analyze their Ct genetic diversity,population structure such as gene flow and mode of reproduction, intra- and inter-population molecular variation, and the relationships between ecological populations in both genotypes and antifungal drug susceptibilities. Our study will provide robust information on the origins and spread of genotypes and drug resistance of Ct in human populations. The data will help develop effective prevention and control diseases caused by this pathogen.

掌握病原真菌的流行分布规律可有效的防控该病原体的爆发。热带假丝酵母(Candida tropicalis，Ct)是潜在的引起侵入性感染和致高死亡率的主要热带病原体之一，我们前期研究发现Ct广泛存在于海南本地人群口腔中并具有独特的新型遗传多样性及较高的抗真菌药物耐药性。然而，本地人体宿主Ct遗传多样性的来源、耐药株来源、传播规律及与其环境中Ct分布的相关性如何，尚知之甚少。因此本课题拟在前期研究的基础上，采集岛内不同环境和地理位置Ct标本，检测其抗真菌药物敏感性，用分子遗传技术扩增Ct6个管家基因片段以分析其多位点序列基因型；通过比较环境与人体不同来源Ct遗传多样性，种群遗传结构包括基因流频度，生殖模式和传播方式等，群体内和群体间分子变异情况及生态种群内基因型特点与耐药菌株的关系，探讨人体宿主Ct基因型与耐药性起源、传播与环境来源Ct的遗传相关性，为该病原体有效的防控奠定基础。

掌握热带假丝酵母菌（Candida tropicalis, Ct）的流行分布规律以及耐药株的起源与传播规律对有效防控Ct的爆发至关重要。为此，本项目结合前期临床与健康人群的Ct数据，采集相应市县环境标本和部分人体标本，对分离获得Ct进行多位点序列分型（MLST）研究，结合全球已发表的数据，利用群体遗传分析相关技术对Ct遗传多样性，耐药株起源及传播规律进行相应的研究。研究共采集了海南8个市/县自然环境标本968份，分离培养获得106个已知种和38个新种共984株酵母菌。Ct分离率21%(207/984)居环境酵母菌的首位，且26.1%Ct对氟康唑耐药；211例口腔癌患者口腔标本中Ct分离率为13.3%，对两性霉素B，氟康唑，伊曲康唑，酮康唑及氟胞嘧啶的耐药率分别为7.1%，14.3%，10.7%，7.1%和10.7%。成功测定了203株环境来源Ct 6个基因片段（ICL1，MDR1，SAPT2，SAPT4，XYR1及ZWF1a）共2677bp，共获得74（2.51%）个SNP位点，其中23个为新发现位点。203株Ct分属于112种二倍体序列类型（DST），其中81种是新发现的。群体遗传分析发现，基因流存在于某些地区、国家和大洲的Ct种群之间，造成Ct具有丰富的地区和国家种群遗传多样性。基于海南人体与环境来源Ct MLST数据的AMOVA分析表明，93%的遗传变异来源于种群内，而种群间仅贡献7%。环境与人体来源Ct耐药株共享DST表明环境耐药株可能对人类健康产生重大的负面影响。耐药株在进化树之间的广泛分布以及基因型相同敏感性不同菌株的存在表明海南Ct之间的氟康唑耐药性最可能通过独立突变多次发生。全基因组测序初步筛选出可能与耐药相关的突变基因，并发现海南黄皮挥发油成分及含量与其抗Ct等假丝酵母菌活性具有相关性。本项目的开展为后续开发更好的策略来防治因Ct引起的各类疾病奠定了基础。