Single-cell Raman spectroscopy identifies <i>E. coli</i> persisters and their functionalities

Abstract

Objectives: Microbial persisters are the featured tiny sub-population of microorganisms highly tolerant to multiple antimicrobials. Currently, studies on persisters remain challenging owing to technical limitations. Here, we explored the chemical composition and metabolism of Escherichia coli persisters, via D2O-based Single-Cell Raman Spectroscopy (SCRS). Methods: E. coli (ATCC 25922) cells were cultured to mid-log and stationary phases, and then treated with fresh Lysogeny broth (LB) containing ampicillin (100 μg/mL) for 4 h. The remaining cells, deemed as E. coli ‘persisters’, were further incubated in LB with 100% D2O and ampicillin (100 μg/mL) for another 4 h. In parallel, the persister cells were resuscitated in LB (100% D2O) without antibiotics for 0, 1, 2, 3 and 4 h, respectively. Single-cell Raman spectra (500 ~ 3200 cm-1) were acquired for all samples using a WiRE5.3 (Renishaw) confocal micro-Raman system. All data were analyzed by R (version 3.4.3) via customized scripts. Results: Principal component analysis showed that two types of E. coli persisters and normal E. coli can be distinguished in two-dimensional space based on their Raman spectra. Load matrix analysis revealed that there were notable differences in chemical composition distribution between the two types of E. coli persisters. According to the D2O absorption rate, E. coli persisters exhibited higher metabolic activities than the untreated E. coli at the same period. Of note, the metabolic activity of Type II E. coli persisters was consistently higher than Type I persisters. Conclusions: This study identifies for the first time the Types I and II E. coli persisters and assesses their metabolic activities by a D2O-based Single-Cell Raman Spectroscopy. These findings can enhance our understanding on the phenotypes and functionalities of microbial persister cells. Further investigations can be extended to oral/periodontal pathogens for precisely disclosing their pathogenicity and underlying mechanisms in the etiopathogenesis of oral/periodontal diseases.