Kozáková J, Honskus M, Okonji Z
Epidemiol Mikrobiol Imunol. 2020 Summer;69(3):134-141.
In order to improve the surveillance of invasive pneumococcal disease (IPD), the National Reference Laboratory (NRL) for Streptococcal Infections implemented whole genome sequencing (WGS) of Streptococcus pneumoniae. This article reports the first WGS data on S. pneumoniae isolates in the Czech Republic.
Thirty-five isolates of S. pneumoniae from IPD recovered in 2017-2019 were selected for WGS. These were serotypes 4, 8, 9V, 19A, and 22F, which were determined by the Quellung reaction in combination with endpoint multiplex PCR (mPCR). Multilocus sequence typing (MLST) is routinely used for more detailed analysis termed sequence typing. The selected isolates were analysed by WGS on the Illumina MiSeq platform. The sequences obtained were processed using the Velvet de novo Assembler software. The assembled genomes were uploaded into the PubMLST database, using the BIGSdb platform, and then scanned automatically and molecularly characterized. The isolates were compared at three resolution levels: seven MLST genes, 53 ribosomal genes (rMLST), and 1420 genes (all loci). The all loci scheme covers MLST genes, ribosomal genes, and core genome MLST genes (cgMLST). These are all currently defined genes of S. pneumoniae available in the PubMLST database. Distance matrices based on the number and variability of all loci analysed were generated automatically using the Genome Comparator tool. Phylogenetic networks were created and edited with the SplitsTree4 package, using the NeighborNet algorithm. The final graphics were edited with the Inkscape software.
Based on an overall view of the phylogenetic networks, it can be concluded that the genetic lines within each of S. pneumoniae serotypes 4, 8, 9V, 19A, and 22F are highly unrelated, to the same extent as if the isolates were of different serotypes. S. pneumoniae isolates of the same serotype, whether or not of the same sequence type, can be described, based on the results, as a non-homogeneous group with a number of unrelated genetic clusters that share genes assigning them to a specific serotype. WGS has also shown its discriminatory power, allowing the assignment of isolates of the same serotype and sequence type to different genetic clusters.
Of the methods used so far in the Czech Republic, WGS allows the most detailed characterization of S. pneumoniae isolates. It is highly desirable to integrate it in the molecular surveillance of IPD in the Czech Republic, similarly to other countries in Europe and in the world.
为了加强侵袭性肺炎球菌病（IPD）监测，链球菌感染国家参考实验室（NRL）开展了肺炎链球菌全基因组测序（WGS）。本文报告了捷克共和国肺炎链球菌分离株的首批WGS数据。
选取2017 - 2019年从IPD中分离出的35株肺炎链球菌进行WGS。这些菌株为4、8、9V、19A和22F血清型，通过荚膜肿胀反应结合终点多重PCR（mPCR）进行鉴定。多位点序列分型（MLST）通常用于更详细的分析，即序列分型。所选分离株在Illumina MiSeq平台上进行WGS分析。获得的序列使用Velvet从头组装软件进行处理。组装后的基因组使用BIGSdb平台上传至PubMLST数据库，然后自动扫描并进行分子特征分析。分离株在三个分辨率水平上进行比较：七个MLST基因、53个核糖体基因（rMLST）和1420个基因（所有位点）。所有位点方案涵盖MLST基因、核糖体基因和核心基因组MLST基因（cgMLST）。这些都是PubMLST数据库中目前已定义的肺炎链球菌基因。使用Genome Comparator工具自动生成基于所分析所有位点数量和变异性的距离矩阵。使用NeighborNet算法，通过SplitsTree4软件包创建和编辑系统发育网络。最终图形使用Inkscape软件进行编辑。
基于系统发育网络的整体观察，可以得出结论，肺炎链球菌4、8、9V、19A和22F血清型中的每一种血清型内的遗传谱系高度不相关，就如同分离株属于不同血清型一样。相同血清型的肺炎链球菌分离株，无论是否为相同序列类型，根据结果可描述为一个非均匀群体，具有多个不相关的遗传簇，这些遗传簇共享将它们归为特定血清型的基因。WGS还显示出其鉴别能力，能够将相同血清型和序列类型的分离株归为不同的遗传簇。
在捷克共和国目前使用的方法中，WGS能够对肺炎链球菌分离株进行最详细的特征分析。与欧洲和世界其他国家类似，将其纳入捷克共和国IPD的分子监测非常有必要。
