Universiti Putra Malaysia, Faculty of Medicine and Health Sciences, Department of Medical Microbiology, Serdang, Selangor, Malaysia.
Universiti Putra Malaysia, Faculty of Biomedical Science, Department of Medical Microbiology, Serdang, Selangor, Malaysia.
Med J Malaysia. 2021 Mar;76(2):164-170.
Streptococcus pyogenes has a variety of virulence factors and the predominant invasive strains differ according to specific emm types and geographical orientation. Although emm typing is commonly used as the gold standard method for the molecular characterisation, multilocus sequence typing (MLST) has become an important tool for comparing the genetic profiles globally. This study aimed to screen selected virulence genes from invasive and non-invasive clinical samples and to characterise the molecular epidemiology by emm typing and MLST methods.
A total of 42 S. pyogenes isolates from invasive and non-invasive samples collected from two different tertiary hospitals were investigated for the distribution of virulence factors and their molecular epidemiology by emm and multilocus sequence typing methods. Detection of five virulence genes (speA, speB, speJ, ssa and sdaB) was performed using multiplex polymerase chain reaction (PCR) using the standard primers and established protocol. Phylogenetic tree branches were constructed from sequence analysis utilised by neighbour joining method generated from seven housekeeping genes using MEGA X software.
Multiplex PCR analysis revealed that sdaB/speF (78.6%) and speB (61.9%) were the predominant virulence genes. Regardless of the type of invasiveness, diverse distribution of emm types/subtypes was noted which comprised of 27 different emm types/subtypes. The predominant emm types/subtypes were emm63 and emm18 with each gene accounted for 11.8% whereas 12% for each gene was noted for emm28, emm97.4 and emm91. The MLST revealed that the main sequence type (ST) in invasive samples was ST402 (17.7%) while ST473 and ST318 (12% for each ST) were the major types in non-invasive samples. Out of 18 virulotypes, Virulotype A (five genes, 55.6%) and Virulotype B (two genes, 27.8%) were the major virulotypes found in this study. Phylogenetic analysis indicated the presence of seven different clusters of S. pyogenes. Interestingly, Cluster VI showed that selected emm/ST types such as emm71/ST318 (n=2), emm70.1/ST318 (n=1), emm44/ST31 (n=1) and emm18/ST442 (n=1) have clustered within a common group (Virulotype A) for both hospitals studied.
The present study showed that group A streptococcci (GAS) are genetically diverse and possess virulence genes regardless of their invasiveness. Majority of the GAS exhibited no restricted pattern of virulotypes except for a few distinct clusters. Therefore, it can be concluded that virulotyping is partially useful for characterising a heterogeneous population of GAS in hospitals.
化脓链球菌具有多种毒力因子，主要侵袭菌株根据特定的 emm 型和地理位置而有所不同。虽然 emm 分型通常被用作分子特征的金标准方法，但多位点序列分型（MLST）已成为比较全球遗传特征的重要工具。本研究旨在从侵袭性和非侵袭性临床样本中筛选选定的毒力基因，并通过 emm 分型和 MLST 方法对分子流行病学进行特征描述。
对来自两家不同的三级医院的侵袭性和非侵袭性样本中总共 42 株化脓链球菌分离株进行了毒力因子分布及其分子流行病学的研究。使用标准引物和既定方案，通过多重聚合酶链反应（PCR）检测五种毒力基因（speA、speB、speJ、ssa 和 sdaB）。使用 MEGA X 软件从七个管家基因的序列分析中构建的邻接法生成的分支构建系统发育树。
多重 PCR 分析显示 sdaB/speF（78.6%）和 speB（61.9%）是主要的毒力基因。无论侵袭性类型如何，均发现 emm 型/亚型分布广泛，包括 27 种不同的 emm 型/亚型。主要的 emm 型/亚型为 emm63 和 emm18，每种基因占 11.8%，而 emm28、emm97.4 和 emm91 每种基因占 12%。MLST 显示侵袭性样本中的主要序列型（ST）为 ST402（17.7%），而非侵袭性样本中的主要类型为 ST473 和 ST318（每种 ST 占 12%）。在 18 种毒力型中，毒力型 A（5 个基因，55.6%）和毒力型 B（2 个基因，27.8%）是本研究中发现的主要毒力型。系统发育分析表明化脓链球菌存在七个不同的聚类。有趣的是，聚类 VI 表明，所选 emm/ST 型（如 emm71/ST318（n=2）、emm70.1/ST318（n=1）、emm44/ST31（n=1）和 emm18/ST442（n=1））已在两所研究医院中聚集在一个共同的组（毒力型 A）中。
本研究表明，A 组链球菌（GAS）具有遗传多样性，并具有毒力基因，无论其侵袭性如何。除了少数几个明显的聚类外，大多数 GAS 表现出无限制的毒力型模式。因此，可以得出结论，毒力分型对于描述医院中 GAS 的异质群体部分有用。
