New Locus for Candida glabrata Sequence-Based Strain Typing Provides Evidence for Nosocomial Transmission.
New Locus for Candida glabrata Sequence-Based Strain Typing Provides Evidence for Nosocomial Transmission.
复制标题
光滑念珠菌的新基因座基于序列的菌株分型为医院传播提供了证据。
DOI：
10.1128/jcm.02933-20
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发表时间：
2021
影响因子：
9.4
通讯作者：
Edlind,Tom
中科院分区：
文献类型：
--
作者：
Katiyar,Santosh;Edlind,Tom
在氟康唑时代，与保健相关的光滑念珠菌感染已经变得越来越常见，可能是内源性的或医院内的。有效的干预需要区分这些来源，这反过来又需要结合分离分型进行流行病学分析(1-4)。已经描述了多种方法来进行光滑毛囊线虫的分型，从基于长度的分析，包括微卫星和脉冲场凝胶电泳，到基于单核苷酸多态(SNP)的分析，包括多位点序列分型和全基因组测序(5，6)。然而，成本和技术复杂性的问题阻碍了这些方法在临床实验室的常规使用。多态基因座序列分型(PLST)通过对一个或两个具有高插入/缺失和SNPs频率的选定串联重复基因座进行常规的聚合酶链式反应和Sanger测序来解决这些问题。在我们之前的研究中，PLST基因座CgMT-J和CgMT-M将104个光面弯曲霉菌株分解为10个系统发育簇和20到24个等位基因(7)。尽管提供了相对温和的分辨率(Simpsonls多样性指数[DI]，0.92到0.94)，但CgMT-J/CgMT-M PLST重要地识别了一个标记为P的簇，其中包括来自同一医学中心接受造血干细胞移植的四个不同患者的多个分离物，这表明医院内传播(7)。然而，这一簇还包括不相关的菌株BWP，削弱了这一断言。另一个标记为N的CgMT-J/CgMT-M聚类群再次包括来自同一中心三个不同患者的分离株，但由于N聚类群至少包括7个不相关的菌株，因此无法得出关于它们之间的流行病学联系的结论。CgMT-J/CgMT-M是通过对当时仅有的两个光肩菌基因组序列进行生物信息学分析而鉴定的，这两个基因组序列分别属于近缘菌株CBS138和M202019(7)。由于目前的GenBank数据库包括另外15个菌株的序列，其中包括4对流行病学相关的菌株，因此有必要对新的PLST基因座进行生物信息学分析，以显示更高的菌株分辨率。串联重复序列(http://tandem.但是。在GenBank数据库的BLASTN筛选中，使用CBS138染色体序列中的EDU/)和500个侧翼核苷酸作为查询。该基因定位于Cgmt-C(DI，0.98)，代表C染色体上的一个基因间隔区(核苷酸514273~515201，GenBank登录号：CR380949)，它包括多个串联重复序列，并以插入/缺失和SNP的形式显示多态。GenBank序列的系统发育分析解决了13个CgMT-C基因座完整的菌株中的12个(图1)(其余4个菌株的序列在较长的重复序列中被分解，这是基因组测序技术的一个常见限制)。然而，流行病学上相关的菌株是聚集性的，例如，反复感染分离株1A/B和2A/B(8)。在实验室中，CgMT-C基因座可以直接从光肩星天牛菌落裂解物，包括那些显色制备的裂解物中直接扩增和测序(引物序列见图1，方法见参考文献7)。
Health care-associated infections with the opportunistic yeast Candida glabrata have become increasingly common in the fluconazole era and may be endogenous or nosocomial in origin. Effective intervention requires distinguishing between these sources, which in turn requires epidemiological analysis in conjunction with isolate typing (1-4). Multiple approaches to C. glabrata typing have been described, ranging from length-based analyses, including microsatellite and pulsed-field gel electrophoresis, to single nucleotide polymorphism (SNP)-based analyses, including multilocus sequence typing and whole-genome sequencing (5, 6). Issues of cost and technical complexity, however, have precluded the routine use of these approaches in clinical laboratories. Polymorphic locus sequence typing (PLST) addresses these issues by employing conventional PCR and Sanger sequencing of one or two selected tandem-repeat loci that exhibit high rates of both insertions/deletions and SNPs. In our previous study, PLST loci CgMT-J and CgMT-M resolved 104 C. glabrata isolates into 10 phylogenetic clusters and 20 to 24 alleles (7).Although providing relatively modest resolution (SimpsonLs diversity index [DI], 0.92 to 0.94), CgMT-J/CgMT-M PLST importantly identified one cluster, labeled P, that included multiple isolates from four different patients undergoing hematopoietic stem cell transplantation at the same medical center, suggesting nosocomial transmission (7). However, this cluster also included the unrelated strain BWP, weakening this assertion. An additional CgMT-J/CgMT-M cluster, labeled N, again included isolates from three different patients at a single center, but no conclusion could be drawn regarding their epidemiological connection because cluster N included at least 7 unrelated strains. CgMT-J/CgMT-M was identified by bioinformatic analysis of the only two C. glabrata genome sequences available at the time, for closely related strains CBS138 and M202019 (7). Because current GenBank databases include sequences for 15 additional strains, including 4 epidemiologically related pairs, bioinformatic analysis for new PLST loci exhibiting greater strain resolution was warranted. Tandem repeats (http://tandem. bu. edu/) within CBS138 chromosome sequences, plus 500 flanking nucleotides, were used as queries in BLASTN screens of GenBank databases. This identified locus CgMT-C (DI, 0.98), representing an intergenic region on chromosome C (nucleotides 514273 to 515201, GenBank accession no. CR380949), which includes multiple tandem repeats and displays polymorphism in the form of both insertions/deletions and SNPs. Phylogenetic analysis of GenBank sequences resolved 12 of 13 strains with intact CgMT-C loci (Fig. 1)(sequences for the 4 remaining strains were unassembled across the longer repeat, a not uncommon limitation of genome sequencing technologies). Epidemiologically related strains were, however, clustered, eg, recurrent infection isolates 1A/B and 2A/B (8). In the laboratory, the CgMT-C locus was readily amplified and sequenced (see Fig. 1 legend for primer sequences and reference 7 for methods) directly from C. glabrata colony lysates, including those prepared from chromogenic