Single-Cell Identification, Drug Susceptibility Test, and Whole-genome Sequencing of Helicobacter pylori Directly from Gastric Biopsy by Clinical Antimicrobial Susceptibility Test Ramanometry

Abstract

Background The battle against Helicobacter pylori (H. pylori) infections demands fast, reliable, and sensitive methods for pathogen identification (ID), antimicrobial susceptibility tests (ASTs) based on metabolic response, and genome-wide mutation profiling that reveals resistance mechanisms. Methods Here we introduce Clinical Antimicrobial Susceptibility Test Ramanometry for H. pylori (CAST-R-HP), and its validation with clinical samples. This method performs rapid ID, metabolism inhibition–based AST, and high-quality whole-genome sequencing for cells of targeted resistance phenotype, all at precisely 1-cell resolution and directly from biopsy samples. Results In CAST-R-HP, automated acquisition and machine learning of single-cell Raman spectra (SCRS) enable distinguishing individual H. pylori cells directly from a biopsy sample, with 98.5 ± 0.27% accuracy in ID. Moreover, by adding a 48- to72-h D2O feeding and drug exposure step prior to SCRS acquisition, CAST-R-HP reports AST for levofloxacin and clarithromycin with 100% accuracy, based on metabolic inhibition level. Furthermore, CAST-R-HP supports rapid sorting, low-bias DNA amplification, and full genome sequencing of single H. pylori cells with the SCRS defined, targeted drug-susceptibility phenotype, via Raman-activated gravity-driven cell encapsulation and sequencing. The genome-wide mutation map (maximum 99.70% coverage), at precisely 1-cell resolution, not only elucidates the drug-susceptibility phenotypes but also unveils their underlying molecular mechanisms. Conclusion The culture independency, shorter turnaround time, high resolution, and comprehensive information output suggest that CAST-R-HP is a powerful tool for diagnosing and treating H. pylori infections.

Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature

“…of 13.66% for 30-cell pools [63]). Together with our recent demonstration of deriving one-cell high-coverage genomes after RACS, such as 99.48% from urine [11], 99.70% from stomach [10] and 92.62% from soil [32], our findings further support the high genomic data quality and robustness of scRACS-Seq for various kinds of microbiome samples.…”

“…1). (i) At the SCRS-based functional-cell detection step, a SCRS which is an intrinsic biochemical fingerprint of a cell can identify or classify the cell based on a wide range of metabolic functions [6][7][8] or via its taxonomy (e.g., for microalgae [9] or for bacteria [10]); in particular, the shifts of specific Raman bands in SCRS can quantitatively measure its substrate-intake activity, when substrates with stable isotopes (e.g. 13 C, 15 N, 2 H, 18 O) are assimilated by the cell [6,8].…”

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

“…Next, the transcriptome of E. coli persisters can be compared to those of the non-persister cells, on the basis of Raman-activated cell sorting and sequencing technologies (RACS-Seq) (He et al, 2019 ). We have recently demonstrated that such links of metabolic phenotype of targeting microbes and high-coverage genome sequences can be established at the resolution of the precisely single bacterial cell directly from human or soil microbiota (Xu et al, 2020 ; Jing et al, 2021 ; Liu et al, 2022 ), using Raman-activated gravity-driven single-cell encapsulation and sequencing (RAGE-Seq). Such efforts could promise to unveil the transcriptomic or epigenetic mechanisms that underpin microbial persistence in this model system.…”

Single cell metabolic phenome and genome via the ramanome technology platform: Precision medicine of infectious diseases at the ultimate precision?

“…On the basis of ramanome and MIC‐MA concepts, the (MP‐G) n data type, and the instrument series of CAST‐R, RACS‐seq, and FlowRACS, a comprehensive SCIVVS workflow to diagnose and treat infectious diseases has been demonstrated by clinical applications. For example, we established the CAST‐R‐HP approach that accomplishes single‐cell ID, AST, and high‐coverage whole‐genome sequencing of H. pylori directly from a gastric biopsy [38]. This approach consists of three steps: (a) treatment of clinical gastric biopsy specimens in medium with 50% D 2 O, 1% IsoVitale X™, Dent supplement, and a particular antibiotic, and then microaerobic incubation for 2–3 days for drug exposure without the need to isolate H. pylori ; (b) acquisition of SCRS for D 2 O‐treated, antibiotic‐challenged cells directly from the biopsy sample for rapid ID and AST of H. pylori single cells; (c) sorting of individual cells whose SCRS identifies H. pylori or the AST phenotype into microdroplets in a RAGE chip for a precisely one cell reaction of multiple displacement amplification and then shotgun sequencing, which produces a precisely one cell assembled genome with high coverage (>95%) to support genotype‐based validation of drug susceptibility, identification of new drug resistance mutations, and full genome‐based tracking of pathogens at one cell resolution [38].…”