Fast bacterial growth reduces antibiotic accumulation and efficacy.
Fast bacterial growth reduces antibiotic accumulation and efficacy.
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DOI：
10.7554/elife.74062
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发表时间：
2022-06-07
期刊：
影响因子：
7.7
通讯作者：
Pagliara, Stefano
中科院分区：
文献类型：
--
作者：
Lapinska, Urszula;Voliotis, Margaritis;Lee, Ka Kiu;Campey, Adrian;Stone, M. Rhia L.;Tuck, Brandon;Phetsang, Wanida;Zhang, Bing;Tsaneva-Atanasova, Krasimira;Blaskovich, Mark A. T.;Pagliara, Stefano
关键词：
单个微生物细胞之间的表型变异在微生物病原体对药物治疗的抗性中起关键作用。然而，对抗生素积累中的细胞个体性知之甚少。在这里，我们假设，表型多样化可以由药物转运速率的基本细胞间差异驱动。为了验证这一假设，我们采用了基于微流体的单细胞显微镜、荧光抗生素探针库和数学建模。这种方法使我们能够快速识别表型变异，避免大肠杆菌，铜绿假单胞菌，新洋葱伯克霍尔德氏菌和金黄色葡萄球菌种群内的抗生素积累。至关重要的是，我们发现快速生长的表型变体避免了大环内酯的积累，并且在没有基因突变的情况下存活下来。这些发现与目前的共识相反，即细胞休眠和缓慢的代谢是细菌对抗生素存活的基础。我们的研究结果还表明，快速增长的变体显示显着更高的表达的核糖体启动子药物治疗前相比，缓慢增长的变体。无药物活性核糖体促进这些快速生长变体的基本细胞过程，包括可以减少大环内酯积累的外排。我们利用这一新知识通过化学操纵其外膜来根除显示低抗生素积累的变体，从而激发了克服当前抗生素治疗失败的新途径。细菌可以引起一系列疾病，从轻微的不适到致命的。事实上，全世界每年有五百万人死于细菌感染。抗生素可以杀死细菌或阻止它们的生长，但许多细菌物种现在能够逃避这些药物。要想有效，大多数抗生素首先需要进入细菌内部;在那里，它们积累，直到达到它们需要的浓度。通常，药物通过细菌外膜上的通道状结构（“孔”）进入细胞，这些结构控制分子进出细胞。当基因变化为微生物提供对抗抗生素的优势时，或者当微生物以较慢的速度进行生命所必需的生化反应时，通常会出现耐药性。相比之下，Pagliara等人决定研究基因相似的大肠杆菌细菌在生长速度上的差异如何对抗抗生素。这种药物靶向核糖体，核糖体是细胞中产生蛋白质的机器。一种技术组合被用来跟踪单个细胞，揭示了快速生长的变体更好地存活。仔细观察发现，快速生长的细菌有多余的核糖体，然后产生更多的孔，可以将抗生素泵出细胞。接下来，Pagliara等人将细菌暴露于抗生素和一种削弱细菌膜的化合物;这消除了快速生长变体所显示的优势。总的来说，这项工作使人们更好地了解抗生素耐药性的机制，这可能有助于为对抗有害细菌的新策略铺平道路。
Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures. Bacteria can cause an array of diseases ranging from mildly inconvenient to deadly. In fact, every year around the world, five million people succumb to a bacterial infection. Antibiotics can kill bacteria or stop their growth, but many bacterial species are now able to evade these drugs. To be efficient, most antibiotics first need to get inside a bacterium; there, they accumulate until they reach the concentration they need to act. Often, the drugs make their way through channel-like structures (‘pores’) studded through the external membranes of bacteria and which control the passage of molecules in and out of cells. Resistance usually emerges when genetic changes provide the microorganism with an advantage against antibiotics, or when the microorganism performs the biochemical reactions necessary for life at a slower pace. In contrast, Łapińska, Pagliara et al. decided to examine how genetically similar Escherichia coli bacteria which differed in their growth rate would fare against antibiotics. The drug targeted ribosomes, the machinery that produces proteins in a cell. A combination of techniques was used to follow individual cells, revealing that fast-growing variants better managed to survive. A closer look showed that bacteria which were growing quickly had a surplus of ribosomes, which then produced more pores that could pump the antibiotic out the cell. Next, Łapińska, Pagliara et al. exposed the bacteria to both the antibiotic and a compound that weakens bacterial membrane; this erased the advantage shown by the fast-growing variants. Overall, this work gives a finer understanding of the mechanisms that underlie antibiotic resistance, which could help pave the way to new strategies to combat harmful bacteria.