Revisiting colistin susceptibility testing: will adding calcium to Mueller–Hinton agar improve the detection of colistin resistance?

The aim was to investigate whether adding calcium to Mueller–Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories. An isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca2+ in concentrations from 2.5 to 40 mM was added to the Mueller–Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes. Results were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca2+ to Mueller–Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca2+ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca2+ did not improve separation between susceptible and resistant populations. The results from this study show that addition of Ca2+ to the Mueller–Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.