Phenotypic Detection of Plasmid-Mediated Colistin Resistance in Enterobacteriaceae

Abstract

The aim of this work was to evaluate an easy-to-perform assay based upon inhibition of mobile colistin resistance (MCR) activity by EDTA. We included 92 nonrelated isolates of Enterobacteriaceae (74 Escherichia coli, 17 Klebsiella pneumoniae, and 1 Serratia marcescens). Our proposed method is based on a modification of the colistin agar-spot screening test (CAST), a plate containing 3 μg/ml colistin, by adding an extra plate of colistin agar-spot supplemented with EDTA (eCAST). Bacterial growth was evaluated after 24 h of incubation at 35°C. All the colistin-resistant isolates showed development on the CAST plates. Colistin-resistant K. pneumoniae without mcr-1 and S. marcescens also grew on the eCAST plates. In contrast, colistin-resistant MCR-producing E. coli was not able to grow in eCAST plates. The combined CAST/eCAST test could provide a simple and easy-to-perform method to differentiate MCR-producing Enterobacteriaceae from those in which colistin resistance is mediated by chromosomal mechanisms.

Keywords: EDTA; Enterobacteriaceae; MCR; colistin.

FIG 1

Differential growth in the combined CAST/eCAST test. Colistin-resistant isolates showed growth in the colistin agar-spot screening test (CAST) (plate A: Mueller-Hinton agar with 3 μg/ml COL). Of these isolates, only MCR producers did not grow in 1 mM EDTA colistin agar-spot screening test (eCAST) (plate B: Mueller-Hinton agar with 3 μg/ml COL + 1 mM EDTA). In contrast, mcr-negative strains harboring other resistance mechanisms also grew in these plates. A control plate (plate C: Mueller-Hinton agar with 1 mM EDTA) was used as a growth control for each isolate. Sections 1 to 10, mcr-positive COL-resistant isolates; 11 to 16, mcr-negative COL-resistant isolates; 17 to 21, mcr-negative COL-susceptible isolates.