烟曲霉菌临床分离株生物膜形成能力与基因分型研究

Biofilm forming ability and genotypes of clinically isolated Aspergillus fumigatus

目的探讨烟曲霉菌临床分离株生物膜（BF）形成能力与基因型的关系,了解医院侵袭性烟曲霉菌感染的流行趋势,为医院烟曲霉菌及其BF相关感染的防治提供分子流行病学依据。方法收集2012年10月-2014年2月由医院各临床科室送检标本中分离的42株烟曲霉菌,96孔板法体外构建BF,结晶紫染色法测定BF形成能力,扫描电镜（SEM）观察并证实BF形成,对所有受试菌株进行随机扩增多态性DNA（RAPD）分型及聚类分析,比较菌株的同源性及不同基因型BF形成能力的差异。结果受试的42株烟曲霉菌中34株形成BF,占81.0%;RAPD方法可将全部受试菌株分为7个基因型：Ⅰ-Ⅶ,其中Ⅱ型17株占40.5%,为优势菌株;Ⅰ型2株、Ⅲ型7株、Ⅳ型7株、Ⅴ型2株、Ⅵ型6株、Ⅶ型1株;不同基因型菌株的BF形成能力不同（P〈0.05）,其中Ⅵ型BF形成能力较强,Ⅳ型较弱。结论大多数烟曲霉菌临床分离株具有BF形成能力,不同基因型的烟曲霉菌株BF形成能力存在差异,院内侵袭性烟曲霉菌感染主要由Ⅱ型克隆株水平传播引起;建立医院烟曲霉菌BF形成能力检测和RAPD基因分型体系,对预防控制烟曲霉菌感染具有重要意义。

OBJECTIVE To explore the relationship between biofilm（BF）forming ability and genotypes of clinically isolated Aspergillus fumigatus strains,so as to provide reference for controlling A.fumigatus BF-related nosocomial infections on molecular epidemiological level.METHODS In vitro BF models was established by 42 A.fumigatus isolates collected from the patients who were hospitalized from Oct.2012 to Feb.2014 by 96-well plates method and measured their BF forming ability,the BF formation confirmed was confirmed under scanning electron microscopy（SEM）.The genetic similarities of isolates were assessed by random amplified polymorphic DNA（RAPD）in combination with cluster analysis.The relationship between BF forming ability and genotypes was then explored.RESULTS Totally 34 clinical A.fumigatus isolates（81.0%）could form BF.By RAPD,42 isolates were divided into 7genotypes（Ⅰ-Ⅶ）,among which typeⅡ was the major prevalent genotype（40.5%）.Others included 2strains of type Ⅰ isolates,7strains of type Ⅲ,7strains of type Ⅳ,2strains of type Ⅴ,6strains of typeⅥ and 1strain of typeⅦ.There was significant difference between genotypes and the BF formation capacity（P〈0.05）.Type Ⅵ owned the strongest ability while Type Ⅳ performed the weakest.CONCLUSION Most of the A.fumigatusisolates possessed BF forming ability and their genotypes were of correlation.Isolates of TypeⅡwere proved to be the dominant strains which caused the nosocomial invasive aspergillosis.Establishing aperfect BF detecting and RAPD genotyping system based on the dynamic surveillance would play an important role in preventing and controlling nosocomial fungal infection caused by A.fumigatus.