Murine thigh infection model.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

链丝菌素（streptothricin）天然产物混合物，又称诺尔丝菌素（nourseothricin），于20世纪40年代初被发现，因其优异的革兰氏阴性菌抗菌活性，在最初便引发了广泛关注。本研究明确了诺尔丝菌素及其主要组分——链丝菌素F（streptothricin F, S-F，含1个赖氨酸）与链丝菌素D（streptothricin D, S-D，含3个赖氨酸）——的抗菌谱，上述组分均经纯化至均一纯度，测试对象为高度耐药的碳青霉烯类耐药肠杆菌目细菌（carbapenem-resistant Enterobacterales, CRE）与鲍曼不动杆菌（Acinetobacter baumannii）。针对CRE，S-F与S-D的最低抑菌浓度50（minimum inhibitory concentration 50, MIC50）、最低抑菌浓度90（minimum inhibitory concentration 90, MIC90）分别为2、4 μM与0.25、0.5 μM。S-F与诺尔丝菌素展现出快速的杀菌活性。在体外翻译实验（in vitro translation assays）中，S-F与S-D对原核核糖体的选择性均约为真核核糖体的40倍。体内实验显示，与S-D相比，S-F需在高于其有效剂量10倍以上的给药量下才会引发迟发性肾毒性。在小鼠大腿感染模型（murine thigh model）中，S-F对泛耐药（pandrug-resistant）、表达NDM-1的肺炎克雷伯菌（Klebsiella pneumoniae）内华达菌株展现出显著的治疗效果，且几乎无毒性或毒性极低。冷冻电镜（Cryo-EM）对S-F与鲍曼不动杆菌70S核糖体（70S ribosome）结合复合物的表征显示，作为鸟苷类似物（guanine mimetic）的S-F链丝替丁（steptolidine）结构域与34号螺旋中的16S rRNA的C1054核苷碱基（以大肠杆菌Escherichia coli编号系统标注）形成大量氢键，同时S-F的氨基甲酰化古洛糖胺（gulosamine）结构域与A1196位点相互作用，这解释了在单核糖体RNA操纵子（rrn operon）大肠杆菌中，上述对应位点发生突变所介导的高水平耐药性。结构分析表明，S-F可靶向结合A解码位点（A-decoding site），这或可解释其致错码活性。基于其独特且极具潜力的抗菌活性，本研究认为链丝菌素骨架（streptothricin scaffold）可作为针对耐药革兰氏阴性病原菌的潜在治疗候选物，值得进一步开展临床前探索。