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 (mcr) gene-negative colistin-resistant Escherichia coli clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-H30Rx (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 eptA (encoding a phosphoethanolamine transferase) and arnT (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 pmrAB decreased colistin MICs to 0.5 mg/liter and lowered eptA and arnT expression. Chromosomal replacement of mutated pmrA or pmrB 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 pmrAB was replaced with mutated pmrAB 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.

Keywords: Escherichia coli; PmrAB; antimicrobial resistance; bacterial infection; lipid A.

FIG 1

mRNA expression levels of eptA and arnT in colistin-resistant Escherichia coli clinical isolates. Values on the y axis are relative expression levels (fold change) normalized against levels in the colistin-susceptible ST131 strain SME98. * and **, significant differences from SME98 (P < 0.05 and P < 0.01, respectively).

FIG 2

Structures of lipid A from colistin-resistant E. coli isolates determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The molecular ion at m/z 1,796 was attributed to diphosphoryl hexaacyl lipid A. The molecular ion at m/z 1,839 was attributed to PEtN-modified monophosphoryl hexaacyl lipid A. The molecular ion at m/z 1,919 was attributed to PEtN-modified diphosphoryl hexaacyl lipid A (30).

FIG 3

mRNA expression levels of eptA and arnT in SME98 mutants with replacements with mutated pmrAB. Values on the y axis are relative expression levels (fold change) normalized against levels in SME98. The amino acid alterations of the mutated pmrAB are described in Table 2. * and **, significant differences from SME98 (P < 0.05 and P < 0.01, respectively).

FIG 4

Bacterial growth curves and bacterial competition assays of SME98 mutants with replacements with mutated pmrAB. (A) Bacterial growth curves of mutated pmrAB-replaced SME98 mutants. (B) Cocultivation of SME98 mutants with replacements with mutated pmrAB with the rifampin-resistant SME98 mutant (SME98RIF) as a competitor. **, significant difference from the competitor (P < 0.01).