Decoding the hidden resistome: Single-cell Raman detection of viable but non-culturable antibiotic-resistant bacteria...

Viable but non-culturable (VBNC) antibiotic-resistant bacteria (ARB) represent an important hidden reservoir of antimicrobial resistance (AMR) in aquatic environments, yet conventional methods based on membrane integrity or culturability may underestimate metabolically active non-culturable bacteria. Here, we developed a combined single-cell Raman spectroscopy (SCRS) and stable isotope probing (SIP) approach to detect Raman-defined active tetracycline-resistant bacteria (Tc-ARB) based on single-cell metabolic activity. In pure-culture experiments, the initial proportion of VBNC-like cells before simulated light exposure was low across methods (0.3-0.7%), and SCRS showed greater sensitivity to low-activity cells during light-induced stress. During light exposure, SCRS showed higher sensitivity to cells with low metabolic activity. After 80 min of light induction, only about 5% of cells were completely inactivated, whereas most retained low but detectable metabolic activity under non-culturable conditions. In environmental samples from 17 sites in the Songliao River Basin, the average concentration of culturable Tc-ARB was 2.15 × 10² CFU/mL. Bland-Altman analysis showed that FCM yielded an approximately 0.37% higher Tc-ARB proportion than SCRS, likely due to abiotic-particle interference. Raman-D₂O screening further revealed spatial heterogeneity in active Tc-ARB. These results suggest that culture-based surveillance alone may underestimate metabolically active ARB fractions in aquatic environments.