Single-cell Raman spectroscopy identifies persisters and reveals their enhanced metabolic activities.

Microbial persisters are the featured tiny sub-population of microorganisms that are highly tolerant to multiple antimicrobials. Currently, studies on persisters remain a considerable challenge owing to technical limitations. Here, we explored the application of single-cell Raman spectroscopy (SCRS) in the investigation of persisters. (ATCC 25922) cells were treated with a lethal dosage of ampicillin (100 μg/mL, 32 × MIC, 4 h) for the formation of persisters. The biochemical characters of and its persisters were assessed by SCRS, and their metabolic activities were labeled and measured with DO-based single-cell Raman spectroscopy (DO-Ramanometry). Notable differences in the intensity of Raman bands related to major cellular components and metabolites were observed between and its ampicillin-treated persisters. Based on their distinct Raman spectra, and its persister cells were classified into different projective zones through the principal component analysis and t-distributed stochastic neighbor embedding. According to the DO absorption rate, persisters exhibited higher metabolic activities than those of untreated . Importantly, after the termination of ampicillin exposure, these persister cells showed a temporal pattern of DO intake that was distinct from non-persister cells. To our knowledge, this is the first report on identifying persisters and assessing their metabolic activities through the integrated SCRS and DO-Ramanometry approach. These novel findings enhance our understanding of the phenotypes and functionalities of microbial persister cells. Further investigations could be extended to other pathogens by disclosing microbial pathogenicity mechanisms for developing novel therapeutic strategies and approaches.