Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.This work was supported by the German Research Council (DFG) – FOR2251 (www.acinetobacter.de). JN was supported by the MagicBullet study. MagicBullet is a project funded by the European Union–Directorate General for Research and Innovation through the Seventh Framework Program for Research and Development [grant agreement 278232] and has been running since 1 January 2012 (duration, 48 months). CSN was supported by the Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) [process no. 88881.187573/2018-01]. IR was supported by the Department of Health, Generalitat de Catalunya [SLT002/16/00349]