Figure 3.

Modified rapid polymyxin NP test (mRPNP). Wells A1-A6 were filled with colistin-free RPNP solution. Wells B1 to B6 were filled with RPNP solution + colistin sulfate. Wells C1 to C6 were filled with colistin-free NP + EDTA. Wells D1 to D6 were added with RPNP solution + colistin sulfate + EDTA. Wells in column 1 were filled with 0.85% NaCl (negative sterility control), whereas Columns 2 to 7 represent the mRPNP test performed for E. coli ATCC 25922, P. mirabilis ATCC 25933, colistin-resistant mcr-1-5 negative K. pneumoniae, colistin-resistant mcr-1-5 negative E. coli strain, and mcr-1-positive E. coli NCTC 13846, respectively. The plates were incubated at 35 ± 2°C under aerobic conditions for 4 h, and visual changes in the color of the wells were monitored each hour. In wells, B1 to B6, a color change from orange to yellow was considered positive to colistin resistance, whereas the mRPNP test was considered positive to MCR-1 phosphoethanolamine transferase production when the colistin-containing solution supplemented with EDTA (wells D1 to D6) remained orange (i.e. absence of glucose metabolization); this shows that growth of the colistin-resistant E. coli (mcr-1-positive) in the well containing colistin solution (well D6) was inhibited by EDTA. Since mcr-1 translates to PEtN transferase, which is a zinc enzyme, exposure to chelators like EDTA could reduce colistin resistance in mcr-1-producing strains.