Detecting mcr-1 and mcr-3 in E. coli

E. coli strains collected from pig
farms were obtained from the Faculty of Veterinary Science, Mahidol
University. Six E. coli strains (ATCC
25922, V13-2LF2, MI907-3bLF, MI951-bLF/62, E2LF, and E5-2LF) were
used in this study. E. coli ATCC 25922
and V13-2LF2 were used as negative controls. The mcr-1–3 genes were detected by multiplex PCR^{42 (link)} (Figure S2). E. coli MI907-3bLF and E2LF are positive for mcr-1 and mcr-3, respectively, while E. coli MI951-bLF/62 and E5-2LF show coexistence
of mcr-1 and mcr-3. Colistin was
purchased from Sigma-Aldrich. Pyrazolone compounds were synthesized
as described previously.^{43 (link)} Susceptibility
testing was performed using the broth microdilution procedure according
to the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) recommendation. E. coli colonies
were selected from Brain Heart Infusion (BHI) agar and transferred
to BHI broth to grow bacteria to OD₆₀₀ = 0.08–0.1
(1 × 10⁸ CFU/mL) and were then transferred to Mueller-Hinton
broth for minimum inhibitory concentration (MIC) testing at a final
cell concentration of 5 × 10⁵ CFU/mL. In the case
of colistin, its final concentration was in the range of 0.0625 to
64 μg/mL in 2-fold dilutions. For synergism testing, pyrazolone
compounds were added at a final concentration of 8 μg/mL with
final colistin concentrations tested in the range of 0.5 to 16 μg/mL
in 2-fold dilutions. All bacterial strains were incubated at 35 ±
1 °C for 18 ± 2 h. The MIC values were detected by the resazurin
assay.^{44 (link)}