Analysis of Regulatory Mechanism of AcrB and CpxR on Colistin Susceptibility Based on Transcriptome and Metabolome of Salmonella Typhimurium.

With the increasing and inappropriate use of colistin, the emerging colistin-resistant isolates have been frequently reported during the last few decades. Therefore, new potential targets and adjuvants to reverse colistin resistance are urgently needed. Our previous study has confirmed a marked increase of colistin susceptibility (16-fold compared to the wild-type Salmonella strain) of cpxR overexpression strain JSΔacrBΔcpxR::kan/pcpxR (simplified as JSΔΔ/pR). To searching for potential new drug targets, the transcriptome and metabolome analysis were carried out in this study. We found that the more susceptible strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels. The virulence-related genes and colistin resistance-related genes (CRRGs) were significantly downregulated in JSΔΔ/pR. There were significant accumulation of citrate, α-ketoglutaric acid, and agmatine sulfate in JSΔΔ/pR, and exogenous supplement of them could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. Additionally, we also demonstrated that AcrB and CpxR could target the ATP and reactive oxygen species (ROS) generation, but not proton motive force (PMF) production pathway to potentiate antibacterial activity of colistin. Collectively, these findings have revealed several previously unknown mechanisms contributing to increased colistin susceptibility and identified potential targets and adjuvants for potentiating colistin treatment of Salmonella infections. IMPORTANCE Emergence of multidrug-resistant (MDR) Gram-negative (G-) bacteria have led to the reconsideration of colistin as the last-resort therapeutic option for health care-associated infections. Finding new drug targets and strategies against the spread of MDR G- bacteria are global challenges for the life sciences community and public health. In this paper, we demonstrated the more susceptibility strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels and revealed several previously unknown regulatory mechanisms of AcrB and CpxR on the colistin susceptibility. Importantly, we found that exogenous supplement of citrate, α-ketoglutaric acid, and agmatine sulfate could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. These results provide a theoretical basis for finding potential new drug targets and adjuvants.

Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella.

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.