Evaluation of EDTA and Dipicolinic Acid in Broth Microdilution with Polymyxin B as a Phenotypic Test to Detect the mcr -1 Gene

Abstract

Polymyxins (colistin and polymyxin B) have recently regained significant importance as last-line drugs to treat infectious diseases due to multidrug-resistant gram-negative bacteria. However, resistance to polymyxins has increased, and the recognition of plasmid-mediated resistance (by the mcr gene) has led to an epidemiological concern. We aimed to evaluate the reduction of the polymyxin B minimum inhibitory concentration (MIC) in the presence of EDTA or dipicolinic acid (DPA) by using the broth microdilution (BMD) method for phenotypic screening of acquired polymyxin resistance mediated by the mcr-1 gene. Overall, 94 Enterobacterales (48 polymyxin-resistant and 46 polymyxin-susceptible) were evaluated: 47 mcr-1 positive (36 Escherichia coli, 2 Klebsiella pneumoniae, and 9 Salmonella spp.) and 47 mcr-1 negative (3 E. coli and 44 K. pneumoniae—27 isolates with MIC from ≤0.125 to 8 μg/mL and 20 isolates with MIC from 16 to 64 μg/mL). Results were categorized as positive when the chelator decreased the original BMD MIC by ≥2 logs. The majority (95.7%) of mcr-1 positive isolates displayed at least a 3 log dilution decrease in the MIC of polymyxin B with EDTA or DPA. The EDTA-based BMD assay detected 45 mcr-1-positive isolates, with only one false-positive among the mcr-1-negative isolates (sensitivity [SN], 95.7%; specificity [SP], 97.9%), whereas the DPA-based BMD assay detected 44 mcr-1-positive isolates (SN, 93.6%; SP, 95.7%), with two false-positive results. The accuracy of EDTA- and DPA-based BMD assays were 97% and 95%, respectively. The EDTA- and DPA-based assays were demonstrated to be reliable methods to detect mcr-1 positive isolates with excellent accuracy.