Table_2_Ribotype Classification of Clostridioides difficile Isolates Is Not Predictive of the Amino Acid Sequence Diversity of the Toxin Virulence Factors TcdA and TcdB.doc

Clostridioides (Clostridium) difficile is the most commonly recognized cause of infectious diarrhea in healthcare settings. Currently there is no vaccine to prevent initial or recurrent C. difficile infection (CDI). Two large clostridial toxins, TcdA and TcdB, are the primary virulence factors for CDI. Immunological approaches to prevent CDI include antibody-mediated neutralization of the cytotoxicity of these toxins. An understanding of the sequence diversity of the two toxins expressed by disease causing isolates is critical for the interpretation of the immune response to the toxins. In this study, we determined the whole genome sequence (WGS) of 478 C. difficile isolates collected in 12 countries between 2004 and 2018 to probe toxin variant diversity. A total of 44 unique TcdA variants and 37 unique TcdB variants were identified. The amino acid sequence conservation among the TcdA variants (≥98%) is considerably greater than among the TcdB variants (as low as 86.1%), suggesting that different selection pressures may have contributed to the evolution of the two toxins. Phylogenomic analysis of the WGS data demonstrate that isolates grouped together based on ribotype or MLST code for multiple different toxin variants. These findings illustrate the importance of determining not only the ribotype but also the toxin sequence when evaluating strain coverage using vaccine strategies that target these virulence factors. We recommend that toxin variant type and sequence type (ST), be used together with ribotype data to provide a more comprehensive strain classification scheme for C. difficile surveillance during vaccine development objectives.

艰难梭菌（Clostridioides difficile，旧称Clostridium difficile）是医疗环境中感染性腹泻最常识别的病原体。目前尚无疫苗可用于预防初次或复发性艰难梭菌感染（CDI）。TcdA和TcdB两种大型梭状芽孢杆菌毒素是CDI的主要致病因素。预防CDI的免疫学方法包括通过抗体介导的中和这些毒素的细胞毒性。理解疾病致病菌株所表达的两种毒素的序列多样性对于解读毒素的免疫反应至关重要。在本研究中，我们确定了2004年至2018年间在12个国家收集的478株艰难梭菌的全基因组序列（WGS），以探究毒素变异多样性。共鉴定出44种独特的TcdA变异体和37种独特的TcdB变异体。TcdA变异体之间的氨基酸序列保守性（≥98%）远大于TcdB变异体（最低可达86.1%），这表明不同的选择压力可能对两种毒素的进化产生了贡献。全基因组序列数据的系统发育分析表明，基于菌落型或多位点序列分型（MLST）代码，不同毒素变异体的菌株被归为一组。这些发现说明了在评估疫苗策略的菌株覆盖范围时，不仅需要确定菌落型，还需要确定毒素序列的重要性。我们建议将毒素变异体类型和序列类型（ST）与菌落型数据结合使用，以提供一个更全面的菌株分类方案，用于艰难梭菌的监测和疫苗开发目标。