Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use.

中文翻译：

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对大肠杆菌脂蛋白信号肽酶抑制剂的抗性的不稳定机制。

具有杀灭病原细菌的新作用机制的抗生素的临床开发具有挑战性，部分原因是不可避免地产生了抗药性。在临床前开发中被广泛忽视的潜在临床重要性现象是异抗性，这是一种经常不稳定的表型，其中细菌细胞的亚群相对于优势人群显示出降低的抗生素敏感性。在这里，我们描述了一种新的球霉素类似物G0790，具有针对大肠杆菌II型信号肽酶LspA的有效活性，并在临床尿路致病性大肠杆菌中发现了对G0790的两种新型耐药机制菌株CFT073。基于先前的发现，即主要的革兰氏阴性外膜脂蛋白Lpp的完全缺失会导致球霉素耐药，我们还发现，由基于插入的破坏调控元件介导的Lpp水平出乎意料的适度下降足以赋予G0790抗药性，增加对血清杀灭的敏感性。另外，我们描述了由lspA基因组扩增介导的异抗性表型导致LspA水平升高，足以克服培养物中G0790的抑制作用。这些基因组扩增非常不稳定，在不存在G0790的情况下进行了两次亚培养后就丢失了，这使含有扩增的抗性菌株极有可能被常规的抗菌药敏测试误分类为易感菌株。总而言之，我们的研究揭示了两种对大肠杆菌中LspA抑制剂的耐药性截然不同的机制，并强调了在开发临床用抗生素时考虑不稳定和异质表型的潜在影响的重要性。