Transcriptomic investigations of polymyxins and colistin/sulbactam combination against carbapenem-resistant Acinetobacter baumannii

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a Priority 1 (Critical) pathogen urgently requiring new antibiotics. Polymyxins are a last-line option against CRAB-associated infections. This transcriptomic study utilized a CRAB strain to investigate mechanisms of bacterial killing with polymyxin B, colistin, colistin B, and colistin/sulbactam combination therapy. After 4 h of 2 mg/L polymyxin monotherapy, all polymyxins exhibited common transcriptomic responses which primarily involved disruption to amino acid and fatty acid metabolism. Of the three monotherapies, polymyxin B induced the greatest number of differentially expressed genes (DEGs), including for genes involved with fatty acid metabolism. Gene disturbances with colistin and colistin B were highly similar (89 % common genes for colistin B), though effects on gene expression were generally lower (0-1.5-fold in most cases) with colistin B. Colistin alone (2 mg/L) or combined with sulbactam (64 mg/L) resulted in rapid membrane disruption as early as 1 h. Transcriptomic analysis of this combination revealed that the effects were driven by colistin, which included disturbances in fatty acid synthesis and catabolism, and inhibition of nutrient uptake. Combination therapy produced substantially higher fold changes in 72 % of DEGs shared with monotherapy, leading to substantially greater reductions in fatty acid biosynthesis and increases in biofilm, cell wall, and phospholipid synthesis. This indicates synergistic bacterial killing with the colistin/sulbactam combination results from a systematic increase in perturbation of many genes associated with bacterial metabolism. These mechanistic insights enhance our understanding of bacterial responses to polymyxin mono- and combination therapy and will assist to optimize polymyxin use in patients.

Keywords: Carbapenem-resistant Acinetobacter baumannii; Combination; Polymyxins; Sulbactam; Transcriptomics.

Graphical abstract

Fig. 1

The numbers of common and unique up- or down-regulated DEGs in A. baumannii 163560 following 4 h of treatment with polymyxin or sulbactam monotherapy (2 mg/L of colistin, colistin B, polymyxin B, or 64 mg/L sulbactam) or the combination of 2 mg/L colistin plus 64 mg/L sulbactam.

Fig. 2

COG category of (A) common up- and down-regulated DEGs with 2 mg/L polymyxin monotherapy (colistin, colistin B, or polymyxin B), and (B) unique DEGs with combination treatment (2 mg/L colistin plus 64 mg/L sulbactam). Data shown is following 4 h of antibiotic treatment.

Fig. 3

Fold changes of common DEGs associated with highly perturbed COG categories following 4 h of treatment with 2 mg/L polymyxin monotherapy (colistin, colistin B, or polymyxin B).

Fig. 4

Fold changes of unique DEGs associated with highly perturbed COG categories following 4 h of treatment with 2 mg/L polymyxin B monotherapy.

Fig. 5

Fold changes of common DEGs associated with highly perturbed COG categories following 4 h of treatment with 2 mg/L colistin monotherapy or 2 mg/L colistin combined with 64 mg/L sulbactam.

Fig. 6

Fold changes of unique DEGs associated with highly perturbed COG categories following 4 h of treatment with 2 mg/L colistin combined with 64 mg/L sulbactam.

Fig. 7

Scanning electron microscopy showing cell morphology of A. baumannii 163560 at 1 h and 4 h in the absence of antibiotic treatment (Control) and following exposure to 2 mg/L colistin and 32 mg/L sulbactam as mono- or combination therapy.

Fig. 8

The fluorescence intensity across 4 h of (A) N-phenyl-1-naphthylamine (NPN) and (B) propidium iodide (PI) following no antibiotic treatment (Control) and treatment with colistin and sulbactam mono- and combination therapy. * 0.05 < p < 0.01, * *0.01 < p < 0.001, * ** 0.001 < p < 0.0001, * ** *p < 0.0001.