Multiple evolutionary pathways lead to vancomycin resistance in Clostridioides difficile

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years vancomycin resistance has emerged as a serious problem in several Gram positive pathogens, but high level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by severe fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified two distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity trains. Moreover, our data provide a mutational roadmap to inform genomic surveillance.

艰难梭菌（Clostridioides difficile）是一种重要的人类致病菌，目前可用的治疗方案极为有限，主要依赖糖肽类抗生素万古霉素（vancomycin）。近年来，万古霉素耐药性已在多种革兰氏阳性病原菌中成为严峻问题，但尚未见艰难梭菌高水平耐药的相关报道，目前尚不清楚这是否源于该物种自身的耐药进化限制。本研究证实，在逐步强化的选择压力下，艰难梭菌可快速进化出万古霉素耐药性，但该过程伴随严重的适合度代价与多效性权衡，包括预期会严重影响传播能力的芽孢形成缺陷。研究团队鉴定出两种独立的耐药进化通路，二者均被预测可改变肽聚糖末端D-丙氨酸-D-丙氨酸（muropeptide terminal D-Ala-D-Ala）——万古霉素的主要作用靶点。其中一种通路涉及一种此前未被表征的D,D-羧肽酶（D,D-carboxypeptidase），其表达受专属双组分信号转导系统（two-component signal transduction system）调控。本研究结果表明，尽管艰难梭菌能够进化出高水平万古霉素耐药性，但由于其对关键致病性状存在多效性影响，该耐药表型在临床中可能难以出现。此外，本研究数据为基因组监测提供了突变路线图。