Micro-flow cell washing technique combined with single-cell Raman spectroscopy for rapid and automatic antimicrobial susceptibility test of pathogen in urine.

Facing the rapid spread of antimicrobial resistance, methods based on single-cell Raman spectroscopy have proven their advances in reducing the turn-around time (TAT) of antimicrobial susceptibility tests (AST). However, the Raman-based methods are still hindered by the prolonged centrifugal cell washing procedure, which may require complex labor operation and induce high mechanical stress, resulting in a pretreatment time of over 1 h as well as a high cell-loss probability. In this study, we developed a micro-flow cell washing device and corresponding Raman-compatible washing chips, which were able to automatically remove the impurities in the samples, retain the bacterial cell and perform Raman spectra acquisition in situ. Results of washing the 5- and 10-μm polymethyl methacrylate (PMMA) microspheres showed that the novel technique achieved a successful removal of 99 % impurity and an 80 % particle retention rate after 6 to 10 cycles of washing. The micro-flow cell washing technique could complete the pretreatment for urine samples in a 96-well plate within 10 min, only taking 15 % of the handling time required by centrifugation. The AST profiles of urine sample spiked with E. coli 25922, E. faecalis 29212, and S. aureus 29213 obtained by the proposed Raman-based approach were found to be 100 % consistent with the results from broth micro-dilution while reducing the TAT to 3 h from several days which is required by the latter. Our study has demonstrated the micro-flow cell washing technique is a reliable, fast and compatible approach to replace centrifuge washing for sample pretreatment of Raman-AST and could be readily applied in clinical scenarios.