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

Abstract

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. Escherichia coli (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 E. coli and its persisters were assessed by SCRS, and their metabolic activities were labeled and measured with D2O-based single-cell Raman spectroscopy (D2O-Ramanometry). Notable differences in the intensity of Raman bands related to major cellular components and metabolites were observed between E. coli and its ampicillin-treated persisters. Based on their distinct Raman spectra, E. coli and its persister cells were classified into different projective zones through the principal component analysis and t-distributed stochastic neighbor embedding. According to the D2O absorption rate, E. coli persisters exhibited higher metabolic activities than those of untreated E. coli. Importantly, after the termination of ampicillin exposure, these persister cells showed a temporal pattern of D2O intake that was distinct from non-persister cells. To our knowledge, this is the first report on identifying E. coli persisters and assessing their metabolic activities through the integrated SCRS and D2O-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.

Robust Spontaneous Raman Flow Cytometry for Single‐Cell Metabolic Phenome Profiling via pDEP‐DLD‐RFC

“…[ 5 , 24 ] The results produced via pDEP‐DLD‐RFC here are consistent with our past observations of cellular response after Amp treatment (i.e., the sharp decrease in intensity of nucleic acid‐related bands, while no change or an increase of lipid‐related bands, which is perhaps linked to the Amp associated inhibition of cell wall/membrane synthesis). [ 5 , 25 ] Previously, the acquisition of fs‐SCRS from drug‐exposed or D 2 O‐fed bacterial cells was typically conducted at a low throughput (≈3–5 cells min −1 ) using static cells on a substrate (e.g., CaF 2 ). [ 5 , 24 ] An automated flow‐based Raman platform reported a similarly low throughput of 3.3–8.3 cells min −1 , due to the inability of optical tweezers to efficiently trap fast‐moving cells.…”

Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing

“…The sample was prepared as previously described [ 34 , 35 ]. Briefly, cells were collected by centrifuging at 8000× g for 1 min and then washed three times using Milli-Q water.…”

Recent Advances in Bacterial Persistence Mechanisms

“…Considering these challenges, a variety of enrichment methods have been applied to study the gene expression of the persistent phenotype [ 86 ]. The antibiotic lysis of sensitive bacteria is a conventional way of obtaining the remaining persister subpopulation [ 87 , 88 , 89 ]. Similarly, exposure to a stress environment, such as extreme temperature or nutrient restriction, and waiting for the aging of normal bacteria can induce persister survival [ 90 ].…”

“…Chuan Wang et al developed single-cell Raman spectroscopy based on D2O to evaluate the biochemical properties of E. coli and its persisters [ 88 ]. Persister formation was induced by ampicillin of a high concentration.…”