The study shows that some gut bacteria with the mcr-9 gene look sensitive to the antibiotic colistin in standard lab tests, but become highly resistant in other growth media. When these bacteria are exposed to colistin, they turn on the mcr-9 gene, which also helps them survive the human antimicrobial peptide LL‑37. A special agar can be used to spot these resistant bugs more easily.

Three isolates of the Enterobacter cloacae complex harboring <i>mcr-9</i>, a member of the colistin resistance <i>mcr</i> gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125&#x2009;to&#x2009;0.5&#x2009;&#x3bc;g/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&#x2009;to&#x2009;128&#x2009;&#x3bc;g/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 <i>mcr-9</i> expression in a dose-dependent manner when these <i>mcr-9</i>-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of <i>mcr-9</i>-positive isolates and Escherichia coli DH5&#x3b1; harboring <i>mcr-9</i> 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 DH5&#x3b1; harboring <i>mcr-9</i> when treated with colistin. Of 93 clinical isolates of <i>Enterobacteriaceae</i>, only the <i>mcr-9</i>-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These <i>mcr-9</i>-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3&#x2009;&#x3bc;g/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 <i>mcr-9</i>-positive isolates and that MCR9-JU agar is useful in screening for <i>Enterobacteriaceae</i> isolates harboring <i>mcr-9</i> and other colistin-resistant isolates.