Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii

Abstract

The increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii. Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.

Keywords: Acinetobacter baumannii; RNAseq; TraDIS; colistin resistance; multi-drug resistance; whole-genome sequencing.

Fig. 1.

Transcriptional changes in in vitro generated colistin-resistant A. baumannii. Differentially expressed genes common to all colistin-resistant cultures (C1 and C2) grown in the presence of colistin (64 or 128 mg l⁻¹) or without selection compared to the day 0 BAL062 culture (WT). The heat map, obtained using values from DESeq2 analysis, excludes genes that were differentially expressed in the passaged BAL062 culture that was not exposed to any colistin. Blue colours indicate genes with higher expression relative to the comparator, red colours indicate lower expression.

Fig. 2.

Inferred recombination in clinical isolates of colistin-resistant A. baumannii. These data were visualized using Phandango (https://github.com/jameshadfield/phandango/). (a) A maximum-likelihood tree of the clinical colistin-resistant organisms (BAL505, BAL543 and BAL719) and colistin-sensitive A. baumannii GC strains [42]. (b) Schematic representation of the reference A. baumannii genome. (c) Recombination regions are indicated as coloured blocks relative to the taxa involved, red blocks indicate predicted recombination that occurred on an internal branch, blue blocks are only present in one isolate (I) and the genomic region affected (II). (d) The line graph summarizes the data in (c).