Contribution of Novel Amino Acid Alterations in PmrA or PmrB to Colistin Resistance in mcr -Negative Escherichia coli Clinical Isolates, Including Major Multidrug-Resistant Lineages O25b:H4-ST131- H 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 () gene-negative colistin-resistant clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-30Rx (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 (encoding a phosphoethanolamine transferase) and (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 decreased colistin MICs to 0.5 mg/liter and lowered and expression. Chromosomal replacement of mutated or 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 was replaced with mutated 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.

Non-clonal emergence of colistin resistance associated with mutations in the BasRS two-component system in Escherichia coli bloodstream isolates

“…Because chromosomal mutations in basRS , but not in other regulatory systems, have previously been suggested to cause colistin resistance in E. coli (24–28), we next aimed to establish the contribution of the basRS alleles in the colistin-resistant phenotype of these bloodstream isolates. Due to the multidrug-resistant nature of the clinical isolates (Figure 1C), we were unable to generate targeted mutations in these strains.…”

“…Indeed, Sato et al . also exclusively found phosphoethanolamine-modified lipid A in colistin-resistant clinical E. coli isolates (24). The reliance of Escherichia on the modification of lipid A by phosphoethanolamine to acquire colistin resistance, suggests that the inhibition of this class of enzymes by blocking the conserved catalytic site (31) could be a target for future drug development and opens the possibility of combination therapy with colistin and an inhibitor of phosphoethanolamine transferase (63).…”

“…In addition, phoPQ expression in E. coli is not only controlled by MgrB but also by the sRNA MicA, adding to the mechanisms controlling PhoPQ activation and making it less likely that the deletion or inactivation of mgrB can contribute to colistin resistance in E. coli (14, 23). This may explain why colistin resistance in clinical E. coli strains has only been linked to mutations in basRS (24–28), although experimental validation of the role of these mutations in colistin resistance is currently mostly lacking.…”

<p>Small Molecule Adjuvants Potentiate Colistin Activity and Attenuate Resistance Development in <em>Escherichia coli</em> by Affecting <em>pmr</em>AB System</p>

“…In other studies, different pmrB and pmrA mutations ( Table 7) responsible for colistin resistance in E. coli were observed. 48,53,58 In Col R E. coli O25 isolated from human clinical specimens in Japan, deletion (Δ27-45, LISVFWLWHESTEQIQLFE) in pmrB and substitution (L105P) in pmrA were reported. 53 In the same study, substitution (G206D) was observed in pmrB in Col R E. coli O25 and O18 isolates.…”

“…48,53,58 In Col R E. coli O25 isolated from human clinical specimens in Japan, deletion (Δ27-45, LISVFWLWHESTEQIQLFE) in pmrB and substitution (L105P) in pmrA were reported. 53 In the same study, substitution (G206D) was observed in pmrB in Col R E. coli O25 and O18 isolates. 53 These mutations have led to modification of lipid A with the addition of PEtN and upregulation of eptA (pmrC) and arnT expression and thereby resistance to colistin.…”