Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2–16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs > 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAβN) to yield MIC values of 8–16 µg/mL and 2–4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.

中文翻译：

---

Robenidine 类似物 NCL259 和 NCL265 对革兰氏阴性病原体的体外活性

多重耐药 (MDR) 革兰氏阴性病原体，尤其是鲍曼不动杆菌、铜绿假单胞菌、大肠杆菌和肠杆菌spp.，被世界卫生组织确认为迫切需要药物开发的最关键的优先病原体。在这项研究中，测试了罗贝尼定类似物 NCL259 和 NCL265 对关键人类和动物革兰氏阴性临床分离株和参考菌株的体外抗菌活性。NCL259 和 NCL265 对这些革兰氏阴性优先病原体表现出适度的抗菌活性，NCL265 始终更加活跃，实现了 2–16 µg/mL 范围内的较低最低抑菌浓度 (MIC)。当与亚抑制浓度的多粘菌素 B 结合使用以透化外膜时，NCL259 和 NCL265 对测试的参考菌株产生协同或相加活性，将 NCL259 的 MIC 和 NCL265 的 MIC 降低 8 至 256 倍4 至 256 倍。克雷伯氏菌属 分离株（三个）对 NCL259 和 NCL265 均有耐药性，MIC > 256 µg/mL。在存在外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺 (PAβN) 的情况下，这种阻力完全逆转，NCL259 和 NCL256 的 MIC 值分别为 8–16 µg/mL 和 2–4 µg/mL。当 NCL259 和 NCL265 针对野生型大肠杆菌进行测试时分离株 BW 25113 及其同基因多药外排泵亚基 AcrB 缺失突变体 (ΔAcrB)，与野生型亲本相比，两种化合物对突变体 ΔAcrB 分离株的 MIC 降低了 16 倍，表明 AcrAB-来自肠杆菌的 TolC 外排泵赋予对这些罗贝尼定类似物的抗性。体外细胞毒性测试显示，与亲本罗贝尼定相比，NCL259 和 NCL265 对一系列人类细胞系具有更高水平的毒性，因此排除了它们作为针对革兰氏阴性病原体的新型抗生素的进一步开发。