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

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 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 -1 gene. Overall, 94 (48 polymyxin-resistant and 46 polymyxin-susceptible) were evaluated: 47 -1 positive (36 , 2 , and 9 spp.) and 47 -1 negative (3 and 44 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 -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 -1-positive isolates, with only one false-positive among the -1-negative isolates (sensitivity [SN], 95.7%; specificity [SP], 97.9%), whereas the DPA-based BMD assay detected 44 -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 -1 positive isolates with excellent accuracy.