Microcin MccI47 reduces Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic with no effect on resident microbiota

The gastrointestinal (GI) tract acts a reservoir for multi-drug resistant pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other CR Enterobacteriaceae, which often lead to severe extraintestinal infections and lethal outcomes in vulnerable populations1. To combat this, selective decolonization of these bacteria from the GI tract has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals2. Towards this goal, we first purify the previously uncharacterized bacteriocin, microcin I47 (MccI47), and demonstrate its potent inhibition of multiple Enterobacteriaceae strains in vitro at concentrations resembling those of commonly prescribed antibiotics. We then engineer the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to constitutively express MccI47-producing genes from a stably retained multicopy plasmid, while rendering provisions for inducible gene expression and plasmid selection unnecessary. We then explore the in vivo applicability of the MccI47-producing EcN in a murine model of CR K. pneumoniae GI colonization and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae GI colonization after seven days of daily oral live biotherapeutic administration with no significant disruption of the resident microbiota.

胃肠道（gastrointestinal tract，缩写GI）是多重耐药病原体的储存库，尤以碳青霉烯类耐药（carbapenem-resistant, CR）肺炎克雷伯菌（Klebsiella pneumoniae）及其他碳青霉烯类耐药肠杆菌科（CR Enterobacteriaceae）为代表，这类病原体常导致易感人群发生严重肠外感染，甚至引发致命结局¹。为应对这一临床挑战，已有研究提出将这些细菌从胃肠道中选择性去定植作为全新策略，以阻断其向身体其他部位传播，并减少其向易感个体的扩散²。为此，我们首先纯化了此前未被表征的细菌素——微菌素I47（microcin I47, MccI47），并证实其在体外可有效抑制多种肠杆菌科菌株，其活性浓度与临床常用处方抗生素相当。随后，我们对益生菌大肠杆菌Nissle 1917（Escherichia coli Nissle 1917, EcN）进行基因工程改造，使其从稳定保留的多拷贝质粒上组成型表达合成MccI47的相关基因，同时无需额外设置诱导型基因表达及质粒筛选的相关条件。我们进一步在碳青霉烯类耐药肺炎克雷伯菌胃肠道定植的小鼠模型中，探究了表达MccI47的EcN的体内应用可行性；结果显示，每日口服该活菌生物制剂7天后，CR K. pneumoniae的胃肠道定植水平出现了显著的MccI47依赖性降低，且未对宿主常驻微生物群造成明显干扰。