Evaluation of rapid colistin susceptibility directly from positive blood cultures using a flow cytometry assay.

Accurate assessment of colistin susceptibility is crucial with the increasing number of multidrug-resistant Gram-negative bacteria and simultaneously increasing colistin resistance. Both EUCAST and CLSI recommend broth microdilution (BMD) to determine colistin susceptibility, however it is cumbersome and growth-dependent. In this study, a rapid flow cytometry method (FASTinovⓇ) to determine colistin susceptibility directly from positive blood cultures (BCs) was evaluated. BCs were spiked with 204 Gram-negative bacilli (137 Enterobacterales, 35 Pseudomonas spp. and 32 Acinetobacter baumannii) at a concentration of 2 × 103 cells/bottle, inoculated with human donor blood and incubated until flagged positive. As quality control strains, two susceptible (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and two resistant (colistin-resistant mcr-1 -positive E. coli NCTC 13846 and Serratia marcescens ATCC 14756) were used. Bacteria were extracted according to assay instructions and were incubated for 1 h at 37 °C with 2 and 4 mg/L colistin and a fluorescent dye, previously optimised. Cells were analysed on CytoFLEX (Beckman Coulter) and AccuriTM C6 Plus (BD Biosciences) flow cytometers. Colistin susceptibility results were automatically provided by BioFAST software (FASTinovⓇ) and compared with those obtained with standard BMD. Overall categorical agreement between this new flow cytometry method and BMD was 99.0%. No very major errors were detected as well as no discrepancies between both flow cytometers. Here we describe a rapid and accurate assay for colistin susceptibility directly from positive BCs with a turnaround time of 2 h versus 48 h required for BMD. This method represents an accurate alternative to standard BMD. [ABSTRACT FROM AUTHOR]