Transcriptomics Reveals How Minocycline-Colistin Synergy Overcomes Antibiotic Resistance in Multidrug-Resistant Klebsiella pneumoniae

Abstract

Multidrug-resistant Gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using transcriptome sequencing (RNA-Seq), we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline.

Keywords: RNA-Seq; antibiotic resistance; antimicrobial activity; antimicrobial agents; antimicrobial combinations; antimicrobial synergy; transcriptomics.

FIG 1

Chemical structure of colistin (polymyxin E) (top) and polymyxin B nonapeptide (PMBN) (bottom). Note the absence of the fatty acid tail in PMBN.

FIG 2

Diagram illustrating the mechanism of action of colistin. (a) A section of Gram-negative cell wall is shown (a). When a colistin-susceptible cell is exposed to colistin, permeabilization of the outer membrane, disruption of the cytoplasmic membrane, and cell lysis occur. Red colistin molecules illustrate binding to the lipid A component of LPS. Gray colistin molecules indicate additional proposed activities of colistin, including insertion of the fatty acid tail into the membrane interior and activity at the cytoplasmic membrane (b). When a colistin-resistant cell is exposed to colistin, permeabilization of the outer membrane still occurs, allowing increased entry of minocycline. However, disruption of the cytoplasmic membrane and cell lysis do not occur (c).

FIG 3

Time-kill curves of MGH-149 and BIDMC-32 treated with minocycline (MIN) and colistin (CST). Results represent the mean and standard deviation of 3 biological replicates. At 2 h, DNA was extracted from a culture set up under identical conditions in parallel with each replicate. “Low” and “high” refer to the low and high drug concentrations referenced in the text.

FIG 4

Principal-component analysis of the gene expression profiles the for antibiotic-treated and untreated control K. pneumoniae. (A) PCA of MGH-149; (B) PCA of BIDMC-32. “Low” and “high” refer to the low and high drug concentrations referenced in the text.

FIG 5

Shared upregulated and downregulated genes between colistin (high) treatment, minocycline (high) treatment and the combination of colistin and minocycline in MGH-149. (A) Upregulated and (B) downregulated differentially expressed genes in each treatment group in K. pneumoniae MGH-149 after treatment with colistin (purple), minocycline (blue), and the combination of colistin and minocycline (yellow). All comparisons are to the untreated control.

FIG 6

Shared upregulated and downregulated genes between colistin (high) treatment, minocycline (high) treatment, and the combination of colistin and minocycline in BIDMC-32. (A) Upregulated and (B) downregulated differentially expressed genes in each treatment group in K. pneumoniae BIDMC-32 after treatment with colistin (purple), minocycline (blue), and the combination of colistin and minocycline (yellow). All comparisons are to the untreated control.

FIG 7

Differential gene expression in the arnBCADTEF operon and other genes involved in polymyxin resistance. (A) Schematic of the PhoPQ and PmrAB two-component systems and how they control expression of the arn operon. Activation of arnBCADTEF mediated through the PmrD relay of PmrAB signaling, as well as direct activation by PhoP have been described (35, 37). (B) Heatmap of gene expression of the pathway in MGH-149 using normalized CPMs showing clustering of biological replicates. Expression was scaled using Z-score normalization prior to clustering, with red representing high expression and blue representing low expression. “Low” and “high” refer to the low and high drug concentrations referenced in the text.

FIG 8

Principal-component analysis of the gene expression profiles for antibiotic-treated and untreated control K. pneumoniae strain BIDMC-32. “Low” and “high” refer to the low and high drug concentrations referenced in the text. PMBN does not inhibit bacterial growth at any concentration tested (it does not have an MIC) and therefore was tested both alone and in combination with minocycline at its FIC (“low”).