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Abstract：
Polymyxins [colistin and polymyxin B (PMB)] comprise an important class of natural product lipopeptide antibiotics used to treat multidrug-resistant Gram-negative bacterial infections. These positively charged lipopeptides interact with lipopolysaccharide (LPS) located in the outer membrane and disrupt the permeability barrier, leading to increased uptake and bacterial cell death. Many bacteria counter polymyxins by upregulating genes involved in the biosynthesis and transfer of amine-containing moieties to increase positively charged residues on LPS. Although 4-deoxy-l-aminoarabinose (Ara4N) and phosphoethanolamine (PEtN) are highly conserved LPS modifications in Escherichia coli, different lineages exhibit variable PMB susceptibilities and frequencies of resistance for reasons that are poorly understood. Herein, we describe a mechanism prevalent in E. coli B strains that depends on specific insertion sequence 1 (IS1) elements that flank genes involved in the biosynthesis and transfer of Ara4N to LPS. Spontaneous and transient chromosomal amplifications mediated by IS1 raise the frequency of PMB resistance by 10- to 100-fold in comparison to strains where a single IS1 element located 90 kb away from the end of the arn operon has been deleted. Amplification involving IS1 becomes the dominant resistance mechanism in the absence of PEtN modification. Isolates with amplified arn operons gradually lose their PMB-resistant phenotype with passaging, consistent with classical PMB heteroresistance behavior. Analysis of the whole genome transcriptome profile showed altered expression of genes residing both within and outside of the duplicated chromosomal segment, suggesting complex phenotypes including PMB resistance can result from tandem amplification events.IMPORTANCEPhenotypic variation in susceptibility and the emergence of resistant subpopulations are major challenges to the clinical use of polymyxins. While a large database of genes and alleles that can confer polymyxin resistance has been compiled, this report demonstrates that the chromosomal insertion sequence (IS) content and distribution warrant consideration as well. Amplification of large chromosomal segments containing the arn operon by IS1 increases the Ara4N content of the lipopolysaccharide layer in Escherichia coli B lineages using a mechanism that is orthogonal to transcriptional upregulation through two-component regulatory systems. Altogether, our work highlights the importance of IS elements in modulating gene expression and generating diverse subpopulations that can contribute to phenotypic polymyxin B heteroresistance.
摘要：
多粘菌素[粘菌素和多粘菌素B（PMB）]包含一类重要的天然产物脂肽抗生素，用于治疗多药耐药的革兰氏阴性菌感染。这些带正电荷的脂肽与位于外膜的脂多糖（LPS）相互作用，破坏通透性屏障，导致摄取增加和细菌细胞死亡。许多细菌通过上调参与含胺部分的生物合成和转移的基因以增加LPS上的带正电荷的残基来对抗多粘菌素。尽管4-脱氧-1-氨基阿拉伯糖（Ara4N）和磷酸乙醇胺（PEtN）是大肠杆菌中高度保守的LPS修饰，由于鲜为人知的原因，不同的谱系表现出可变的PMB敏感性和抗性频率。在这里，我们描述了大肠杆菌B菌株中普遍存在的机制，该机制取决于特定的插入序列1（IS1）元件，该元件侧翼参与Ara4N的生物合成和向LPS转移的基因。与缺失了位于arn操纵子末端90kb的单个IS1元件的菌株相比，由IS1介导的自发和瞬时染色体扩增使PMB抗性的频率提高了10至100倍。在没有PEtN修饰的情况下，涉及IS1的扩增成为主要的抗性机制。具有扩增的arn操纵子的分离株随着传代逐渐失去其PMB抗性表型，与经典的PMB异质抗性行为一致。全基因组转录组分析显示，重复染色体片段内外的基因表达发生了变化，提示包括PMB抗性在内的复杂表型可由串联扩增事件引起。易感性的表型变异和耐药亚群的出现是多粘菌素临床应用的主要挑战。虽然已经编制了一个可以赋予多粘菌素抗性的基因和等位基因的大型数据库，本报告表明,染色体插入序列(IS)的含量和分布也值得考虑.通过IS1扩增包含arn操纵子的大染色体片段，使用与通过双组分调节系统进行转录上调正交的机制，增加了大肠杆菌B谱系中脂多糖层的Ara4N含量。总之,我们的工作强调了IS元件在调节基因表达和产生可能导致表型多粘菌素B异质耐药的不同亚群中的重要性.
