Locus CauMT1 Provides a Higher-Resolution Alternative to Ribosomal Gene Sequencing for Initial Candida auris Genotyping.
Locus CauMT1 Provides a Higher-Resolution Alternative to Ribosomal Gene Sequencing for Initial Candida auris Genotyping.
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Locus CauMT1 为初始耳念珠菌基因分型提供了核糖体基因测序的更高分辨率替代方案。
DOI：
10.1128/jcm.01039-20
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发表时间：
2020
影响因子：
9.4
通讯作者：
Edlind,Tom
中科院分区：
文献类型：
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作者：
Katiyar,Santosh;Edlind,Tom
在严重急性呼吸系统综合征冠状病毒2 （SARS-CoV-2）之前，纽约市是另一种最近出现的病原体——耳念珠菌的中心。Zhu等人最近在该杂志上描述了2016年至2018年与这种机会性酵母相关的大规模爆发的实验室分析(1)，并附有Wickes的评论(2)。本研究和其他研究（3-6）的数据强烈表明，人类和环境表面的定植促进了金黄色葡萄球菌的爆发。疫情的发现和管理需要进行流行病学调查；为此，基因分型至关重要。全基因组测序提供最大的分离物分辨率；然而，由于成本和技术复杂性，它在临床实验室的常规使用被排除在外。Zhu等人描述了使用廉价的PCR和核糖体基因D1/D2和内部转录间隔（ITS）位点的Sanger测序进行金黄色葡萄球菌基因分型。虽然这些基因座只在进化支水平上区分分离株，但数据显示南亚进化支I占优势；少数为东亚进化支II，南非进化支III、南美进化支IV和伊朗进化支V未检出。对于D1/D2-ITS基因分型，Zhu等人采用了两次单独的扩增；另外，威克斯建议将这些位点组合成一个1200 - 1500 bp的扩增子，并用两个引物进行测序。这两个基因座都需要完全解析这五个进化支。在这里，我们提出了一个单一的替代位点，CauMT1，它的序列分析可以解析所有进化支，并进一步解析优势进化支i中的多个等位基因。短串联重复序列（STRs）由于DNA复制过程中的滑动而表现出相对较高的插入/删除率。利用这一点，de Groot等人(7)开发了一种基于C. auris长度的分型系统，并对444种不同的分离株进行了评估。在12个选择的str中，M3-1a表现出更大的多态性，具有13 ~ 76个3-bp重复序列，多样性指数为0.82（其余为0.58 ~ 0.70）。虽然该STR系统可解析40个基因型，但对12个PCR产物和毛细管电泳的要求再次阻碍了其在临床实验室的常规使用。如所附评论(8)所述，基于长度的数据也不便于携带，使日常和实验室间的比较复杂化。为了探索含有M3-1a重复位点的序列分型，我们首先比较了NCBI数据库中21株耳球菌的基因组序列。该分析证实了重复长度多态性，但也揭示了侧翼区域内存在多个单核苷酸多态性（SNPs）（见补充材料中的图S1）。引物（见图1图例）被设计用于扩增和测序重复加上约700bp的侧翼序列。如前所述(9)，用从FDA-CDC抗生素耐药性分离库（10）和8个分离株中提取的10个代表性金黄色葡萄球菌分离株的基因组dna进行了测试
Prior to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), New York City was the epicenter for another recently emerged pathogen, Candida auris. The laboratory analysis of a large 2016 to 2018 outbreak associated with this opportunistic yeast was recently described in this journal by Zhu et al.(1), with commentary by Wickes (2). The data from this study and others (3–6) strongly suggest that C. auris outbreaks are facilitated by colonization of both human and environmental surfaces. Outbreak detection and management require epidemiological investigation; for this, genotyping is critical. Whole-genome sequencing provides maximal isolate resolution; however, its routine use in clinical labs is precluded by cost and technical complexity. Zhu et al. describe the use of inexpensive PCR and Sanger sequencing of ribosomal gene D1/D2 and internal transcribed spacer (ITS) loci for C. auris genotyping. Although these loci resolved isolates only to the clade level, the data were informative in showing a preponderance of South Asia clade I; a minority were East Asia clade II, while South Africa clade III, South America clade IV, and Iran clade V were not detected. For D1/D2-ITS genotyping, Zhu et al. employed two separate amplifications; alternatively, Wickes suggests combining the loci into one 1,200-to 1,500-bp amplicon and sequencing with two primers. Both loci are required to fully resolve the five clades. Here, we suggest a single, alternative locus, CauMT1, for which sequence analysis resolves all clades and furthermore resolves multiple alleles within the predominant clade I.Short tandem repeats (STRs) exhibit relatively high rates of insertion/deletion due to slippage during DNA replication. Exploiting this, de Groot et al.(7) developed a C. auris length-based typing system and evaluated it with 444 diverse isolates. Of the 12 selected STRs, M3-1a exhibited significantly greater polymorphism, with 13 to 76 copies of a 3-bp repeat and a diversity index of 0.82 (compared to 0.58 to 0.70 for the remainder). While this STR system resolved 40 genotypes, the requirement for 12 PCR products and capillary electrophoresis again precludes its routine use in clinical labs. Length-based data are also less portable, complicating day-to-day and lab-to-lab comparisons, as noted in the accompanying commentary (8). To explore sequence-based typing of the M3-1a repeat-containing locus, we first compared the genome sequences of 21 C. auris strains available in NCBI databases. This analysis confirmed repeat length polymorphism but also revealed multiple single nucleotide polymorphisms (SNPs) within flanking regions (see Fig. S1 in the supplemental material). Primers (see the Fig. 1 legend) were designed to amplify and sequence the repeat plus ca. 700bp of flanking sequence. These were tested as previously described (9) with genomic DNAs prepared from 10 representative C. auris isolates from the FDA-CDC Antibiotic Resistance Isolate Bank (10) and 8 isolates