多位点序列分析法在白念珠菌性阴道炎唑类耐药菌株分子流行病学研究中的运用

doi:10.3969/j.issn.1673-8640.2018.03.011

摘要/Abstract

摘要： 目的

运用多位点序列分析（MLST）法对白念珠菌性阴道炎患者阴道分离的对唑类抗菌药物耐药的白念珠菌临床菌株进行分子流行病学调查,对比耐药株和敏感株之间的基因型差异,分析其分子流行病学特征并研究耐药株的遗传多样性和种群关系。

方法

对上海市嘉定区中心医院2015年7月—2016年6月的白念珠菌性阴道炎患者的白带样本进行真菌培养、鉴定及体外药物敏感性试验,共收集60株对氟康唑、伏立康唑和伊曲康唑中1种或多种药物耐药的菌株;另收集同期对3种药物均敏感的菌株38株。采用MLST法对耐药菌株和敏感菌株进行分析,采用BioNumerics 7.6软件进行亲缘性和聚类分析。

结果

耐药菌株组存在14种序列类型（ST）,其中9种已报道,主要型别为ST310、ST1364、ST2551和ST135;敏感菌株组存在19种ST,其中12种已报道,主要型别为ST2048和ST1474。耐药菌株组的ST数/菌株数比值低于敏感菌株组,且2个组的ST数与菌株数均呈线性正相关关系（耐药菌株组：r=0.995,P=0.005;敏感菌株组：r=0.989,P=0.011）。亲缘性分析显示耐药菌株各基因型间的亲缘性要近于敏感菌株。聚类分析显示耐药菌株的种群形成了多级分支,ST310、ST1364和ST2551同处于第1分支,ST135为第2分支,其主要ST型别之间形成了较为明显的聚类分布。

结论

MLST法结果显示上海市嘉定区中心医院临床分离的白念珠菌为多克隆性,唑类药物耐药菌株的基因型分布较敏感菌株更为集中。耐药菌株各基因型间的亲缘性要近于敏感菌株,且其种群分类的基因型呈明显的聚类分布。

关键词: 白念珠菌, 唑类抗菌药物, 耐药菌株, 多位点序列分析

Abstract:

Objective To investigate the molecular epidemiological characteristic of azole-resistant Candida albicans from patients with Candida albicans vaginitis by multilocus sequence typing（MLST）,and to analyze the genotype difference between resistant and sensitive isolates and the relationship between genetic diversity and population. Methods The leucorrhoea specimens were collected from patients with Candida albicans vaginitis in Shanghai Jiading District Central Hospital from July 2015 to June 2016 for culturing,identification and susceptibility test in vitro. A total of 60 isolates being resistant or multi-resistant to fluconazole,itraconazole and/or voriconazole and 38 sensitive isolates were collected. All of them were analyzed by MLST. By BioNumerics 7.6 software,affinity and cluster analysis was performed. Results There were 14 sequence types（ST） in drug resistant group,9 of which had been reported,and the main types were ST310,ST1364,ST2551 and ST135. There were 19 ST in drug sensitive group,12 of which had been reported,and the main types were ST2048 and DST1474. The ratio of ST of drug resistant group to the number of isolates was lower than that of drug sensitive group,and the numbers of ST in the 2 groups showed a linear positive correlation with the number of isolates（drug resistant group：r=0.995,P=0.005;drug sensitive group：r=0.989,P=0.011）. Affinity analysis showed that the affinity of each genotype of drug resistant isolates was close to that of sensitive isolates. Cluster analysis showed that the population of drug resistant isolates formed multistage branches. ST310,ST1364 and ST2551 were in the 1st branch,and ST135 was in the 2nd branch. There was obvious clustering distribution in main ST. Conclusions Using MLST,the isolates of Candida albicans found in Shanghai Jiading District Central Hospital are multiple cloning,and the genotype distribution of drug resistant isolates is more concentrated than that of sensitive isolates. The affinity of each genotype of drug resistant isolates is close to that of sensitive isolates,and the genotypes of their populations are clustered.

Key words: Candida albicans, Azole, Drug resistant isolate, Multilocus sequence typing

中图分类号:

R379.4

徐炜新, 孙杰. 多位点序列分析法在白念珠菌性阴道炎唑类耐药菌株分子流行病学研究中的运用[J]. 检验医学, 2018, 33(3): 233-238.

XU Weixin, SUN Jie. Multilocus sequence typing in the molecular epidemiological study of azole-resistant Candida albicans from patients with Candida albicans vaginitis[J]. Laboratory Medicine, 2018, 33(3): 233-238.

图/表 7

参考文献 11

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抗菌药物	敏感	剂量依赖敏感	耐药
氟康唑	≤8	16~32	≥64
伊曲康唑	≤0.125	0.25~0.50	≥1
伏立康唑	≤1	2	≥4

抗菌药物	敏感	剂量依赖敏感	耐药
氟康唑	≤8	16~32	≥64
伊曲康唑	≤0.125	0.25~0.50	≥1
伏立康唑	≤1	2	≥4

管家基因	引物序列（5'~3'）	产物长度（bp）
AAT1a-F	ACTCAAGCTAGATTTTTGGC	373
AAT1a-R	CAGCAACATGATTAGCCC
ACC1-F	GCAAGAGAAATTTTAATTCAATG	407
ACC1-R	TTCATCAACATCATCCAAGTG
ADP1-F	GAGCCAAGTATGAATGATTTG	443
ADP1-R	TTGATCAACAAACCCGATAAT
MPⅠb-F	ACCAGAAATGGCCATTGC	375
MPⅠb-R	GCAGCCATGCATTCAATTAT
SYA1-F	AGAAGAATTGTTGCTGTTACTG	391
SYA1-R	GTTACCTTTACCACCAGCTTT
VPS13-F	TCGTTGAGAGATATTCGACTT	403
VPS13-R	ACGGATGGATCTCCAGTCC
ZWF1-F	GTTTCATTTGATCCTGAAGC	491
ZWF1-R	GCCATTGATAAGTACCTGGAT

管家基因	引物序列（5'~3'）	产物长度（bp）
AAT1a-F	ACTCAAGCTAGATTTTTGGC	373
AAT1a-R	CAGCAACATGATTAGCCC
ACC1-F	GCAAGAGAAATTTTAATTCAATG	407
ACC1-R	TTCATCAACATCATCCAAGTG
ADP1-F	GAGCCAAGTATGAATGATTTG	443
ADP1-R	TTGATCAACAAACCCGATAAT
MPⅠb-F	ACCAGAAATGGCCATTGC	375
MPⅠb-R	GCAGCCATGCATTCAATTAT
SYA1-F	AGAAGAATTGTTGCTGTTACTG	391
SYA1-R	GTTACCTTTACCACCAGCTTT
VPS13-F	TCGTTGAGAGATATTCGACTT	403
VPS13-R	ACGGATGGATCTCCAGTCC
ZWF1-F	GTTTCATTTGATCCTGAAGC	491
ZWF1-R	GCCATTGATAAGTACCTGGAT

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