Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9

Three isolates of the Enterobacter cloacae complex harboring , a member of the colistin resistance gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 μg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 μg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced expression in a dose-dependent manner when these -positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of -positive isolates and Escherichia coli DH5α harboring with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring when treated with colistin. Of 93 clinical isolates of , only the -positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These -positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 μg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect -positive isolates and that MCR9-JU agar is useful in screening for isolates harboring and other colistin-resistant isolates.