Contribution of Novel Amino Acid Alterations in PmrA or PmrB to Colistin Resistance in <i>mcr</i> -Negative Escherichia coli Clinical Isolates, Including Major Multidrug-Resistant Lineages O25b:H4-ST131- <i>H</i> 30Rx and Non-x

Abstract

Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance (<i>mcr</i>) gene-negative colistin-resistant <i>Escherichia coli</i> clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-<i>H</i>30Rx (isolates SRE34 and SRE44; MIC for colistin = 16 mg/liter), non-x (SME296; MIC = 8 mg/liter), and O18-ST416 (SME222; MIC = 4 mg/liter). In this study, we investigated the colistin resistance mechanism and identified novel amino acid substitutions or deletions in the PmrAB two-component system that activates <i>eptA</i> (encoding a phosphoethanolamine transferase) and <i>arnT</i> (encoding an undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase) in all colistin-resistant isolates. SRE34 possessed deletion Δ27-45 (LISVFWLWHESTEQIQLFE) in PmrB, SRE44 possessed substitution L105P in PmrA, and both SME222 and SME296 included substitution G206D in PmrB. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that lipid A is modified with phosphoethanolamine in all four isolates. Deletion of <i>pmrAB</i> decreased colistin MICs to 0.5 mg/liter and lowered <i>eptA</i> and <i>arnT</i> expression. Chromosomal replacement of mutated <i>pmrA</i> or <i>pmrB</i> in colistin-susceptible O25b:H4-ST131 strain SME98 (colistin MIC = 0.5 mg/liter) increased the colistin MIC to that of the respective parent colistin-resistant isolate. In addition, SME98 mutants in which <i>pmrAB</i> was replaced with mutated <i>pmrAB</i> showed no significant differences in bacterial growth and competition culture from the parent strain, except for the mutant with L105P in PmrA, whose growth was significantly suppressed in the presence of the parent strain. In conclusion, some O25b:H4-ST131 strains appear to acquire colistin resistance via phosphoethanolamine modification of lipid A through amino acid changes in PmrAB, and the amino acid changes in PmrB do not influence bacterial growth.