ABSTRACTAntimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi-, extensive, and pandrug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., inEscherichia coli) is a common approach, so far, no standard methods for heterologous expression and characterization ofmcrgenes exist.E. coliB-strains, designed for optimum protein expression, are frequently utilized. Here, we report that fourE. coliB-strains are intrinsically resistant to colistin (MIC 8–16 μg/mL). The three tested B-strains that encode T7 RNA polymerase show growth defects when transformed with empty ormcr-expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects.E. coliSHuffle T7 express carrying empty pET17b also skips wells in colistin MIC assays in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in eachpmrAandpmrBin all fourE. coliB-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude thatE. coliB-strains are not appropriate heterologous expression hosts for identification and characterization ofmcrgenes.IMPORTANCEGiven the rise in multidrug, extensive drug, and pandrug resistance in bacteria and the increasing use of colistin to treat human infections, occurrence ofmcrgenes threatens human health, and characterization of these resistance genes becomes more important. We show that three commonly used heterologous expression strains are intrinsically resistant to colistin. This is important because these strains have previously been used to characterize and identify new mobile colistin resistance (mcr) genes. We also show that expression plasmids (i.e., pET17b) without inserts cause cell viability defects when carried by B-strains with T7 RNA polymerase and grown in the presence of IPTG. Our findings are important as they will facilitate improved selection of heterologous strains and plasmid combinations for characterizing AMR genes, which will be particularly important with a shift to Culture-independent diagnostic tests where bacterial isolates become increasingly less available for characterization.