Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types
Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types
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DOI：
10.1128/jcm.42.6.2609-2617.2004
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发表时间：
2004-06-01
影响因子：
9.4
通讯作者：
Pons, JL
中科院分区：
文献类型：
--
作者：
Lemee, L;Dhalluin, A;Pons, JL
采用多位点序列分型（MLST）方法研究了72株艰难梭菌分离株的遗传关系和群体结构，这些分离株来自不同的寄主、地理来源、PCR核糖型和产毒型（由PCR靶向tcdA和tcdB基因确定）。MLST通过对7个管家基因（aroE, ddl, dutA, tpi, recA， gmk和sod，4）的DNA序列分析进行。等位基因数量从5个（dutA和ddl）到11个（recA）不等。等位基因图谱允许定义34种不同的序列类型（STs）。这些STs与地理来源缺乏相关性，但与产毒类型有很好的相关性。由成对遗传距离矩阵生成的树状图显示，动物分离株与人类分离株没有形成不同的谱系，在产毒的人类分离株群体中没有高毒谱系（从假膜性结肠炎和抗生素相关性腹泻中恢复的分离株没有聚集在不同的谱系中）。然而，A(-) B+变体分离株具有相同的ST，在研究群体中作为不同的谱系出现，表明单一的进化起源。通过等位基因多态性分析进一步研究了群体结构。基于复合序列分析生成的树状图显示一个同质群体与三个不同的谱系相关，其中一个仅限于a (-) B+变体分离株。根据位点间连锁不平衡（Ia）的估计，艰难梭菌表现出克隆群体结构。克隆复合体内的等位基因分析表明，点突变产生新等位基因的频率比重组交换高8倍，由独立的内护位点产生的树形图的一致性证实了该物种的突变进化。
A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sod,4). The number of alleles ranged from five (dutA and ddl) to eleven (recA). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirullent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages). However, A(-) B+ variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A(-) B+ variant isolates. C. difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.