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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)