Detection of mcr-1-mediated resistance to polymyxins in Enterobacterales using colistin disk chelator application

Objective. To evaluate the possibility of using the colistin disk chelator application (CDCA) method as simple and available screening tool for detection of mcr-1-mediated resistance to polymyxins in Enterobacterales. Materials and Methods. A total of 47 colistin-resistant Enterobacterales isolates obtained in 2014–2020 within multicenter MARATHON study were included in the experiment. Colistin susceptibility testing was performed using Mueller–Hinton broth microdilution method according to ISO 20776-1:2006. Interpretation of the results was performed according to EUCAST v.12.0 clinical breakpoints. MCR-genes were detected by multiplex real-time PCR. Phenotypic screening for mcr-expression was performed on Mueller–Hinton agar by application of dipicolinic acid in concentration of 1,000 mcg/disk in 10 µL volume per disk and 0.5 M solution of EDTA in 5 µL volume per disk. Chelating effect was registered by differences in zone of growth inhibition around colistin disks with and without chelator. Measurements were performed with the help of caliper in millimeters. Statistical data processing was carried out in accordance with guidelines for statistical analysis in medical researches using MS-Excel tool. Results. In 25 of 47 included in the experiment enterobacteria isolates mcr-genes were detected by molecular method. MCR-detection by CDCA method identified the average difference value of the zones of growth inhibition for colistin and its combination with EDTA and DPA as 4.1 mm and 3.7 mm respectively for mcr-positive isolates and 1.7 mm and 1.2 mm respectively for mcr-negative isolates. Statistical analysis estimated that a difference of ≥ 3 mm in zone of growth inhibition for combination of colistin with one of the chelating agents when compared to colistin only allows us to conclude that a studied isolated carries mcr-1-mediated resistance to polymyxins. In addition, sensitivity of the test was 96% and specificity was 91% if DPA is used, while EDTA showed only 88% sensitivity and 77% specificity. Conclusions. Proposed method appears as available technique for phenotypic screening of the Enterobacterales order for mcr-1-mediated resistance to polymyxins for practical laboratories in present conditions. The use of DPA is preferred because of better specificity and sensitivity rates.