New frontiers against antibiotic resistance: A Raman-based approach for rapid detection of bacterial susceptibility and biocide-induced antibiotic cross-tolerance

To overcome the widespread misuse of antibiotics and reduce the growing problem of multidrug resistance, rapid, sensitive and specific novel methods to determine bacterial antibiotic susceptibility are needed. This study presents a combined dielectrophoresis (DEP) - Raman Spectroscopy (RS) method to obtain direct, real-time measurements of the susceptibility of a suspension of planktonic bacteria without labelling or other time-consuming and intrusive sample preparation processes. Using an in-house constructed DEP-Raman device, we demonstrated the susceptibility of Escherichia coli MG1655 towards the second-generation fluoroquinolone antibiotic ciprofloxacin (CP) after only 1 h of treatment, by monitoring spectral changes in the chemical fingerprint of bacteria related to the mode of action of the drug. Spectral variance was modelled by multivariate techniques and a classification model was calculated to determine bacterial viability in the exponential growth phase at the minimum bactericidal concentration (MBC) over a 3 h time span. Further tests at a sub-minimum inhibitory concentration (MIC) and with a CP-resistant E. coli were carried out to validate the model. The method was then successfully applied for class prediction in a biocide cross-induced tolerance assay that involved pre-treating E. coli with triclosan (TCS), an antimicrobial used in many consumer products. High specificity and adequate sensitivity of the proposed method was thereby demonstrated. Simultaneously, standard microbiological assays based on cell viability, turbidity and fluorescence microscopy, were carried out as reference methods to confirm the observed Raman response.