C-Terminal loss of RelA protein suggests as a potential cause of an infective endocarditis relapse with Enterococcus faecium

Infective endocarditis (IE) caused by Enterococcus spp represents the third most common cause of IE, with high rates of relapse compared with other bacteria species. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported infective endocarditis relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by Multi Locus Sequence Typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis with the aim of identifying potential genes involved in virulence and stringent response. Our results showed one missense mutation and one 13 nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the EI. These data represent the first clinical evidence of mutations in the stringent response related with E. faecium IE relapse.

肠球菌属（Enterococcus spp）引起的感染性心内膜炎（infective endocarditis, IE）是第三大常见的IE病因，相较于其他致病菌，其复发率更高。值得注意的是，此前仅报道过粪肠球菌（Enterococcus faecalis）引发的迟发性复发（复发间隔>6个月），而本文首次报道了1例屎肠球菌（Enterococcus faecium）在初始心内膜炎发作17个月（超过1年）后发生的迟发性复发感染性心内膜炎。首先，通过多位点序列分型（Multi Locus Sequence Typing, MLST），我们证实两株临床分离菌（EF646与EF641）属于同一序列型（ST117）。鉴于EF641能够在饥饿与抗生素处理条件下长期存活，我们开展了生物信息学分析，旨在鉴定与细菌毒力及严谨反应相关的潜在功能基因。研究结果显示，relA基因中存在1处错义突变与1处13核苷酸重复序列（位置1638-1650），该突变导致提前出现终止密码子，使得RelA酶的C端结构域缺失167个氨基酸。RelA介导细菌的严谨反应，调控警报因子四磷酸鸟苷（guanosine tetraphosphate, ppGpp）的合成水平。与EF646菌株相比，relA突变株（EF641）的生长能力更弱，且存在小菌落变异株，同时生物膜生成能力更强，但依据标准流程检测的抗菌药物敏感性无显著差异。我们认为上述所有表型变化与屎肠球菌的长期存活及感染性心内膜炎的迟发性复发密切相关。本研究首次提供了严谨反应相关基因突变与屎肠球菌感染性心内膜炎复发相关的临床证据。