As increasing numbers of colistin-resistant bacteria emerge, new therapies are urgently needed to treat infections caused by these pathogens. The discovery of new combination therapies is one important way to solve such problems. Here, we report that the antitumor drug PFK-158 and its analogs PFK-015 and 3PO can exert synergistic effects with colistin against colistin-resistant Enterobacteriaceae, including mcr-1-positive or high-level-colistin-resistant (HLCR) isolates, as shown by a checkerboard assay. The results of a time-kill assay revealed that colistin combined with PFK-158 continuously eliminated colistin-resistant Escherichia coli 13-43, Klebsiella pneumoniae H04, and Enterobacter cloacae D01 in 24 h. Images from scanning electron microscopy (SEM) at 5 h postinoculation confirmed the killing effect of the combination. Finally, in vivo treatment showed that PFK-158 had a better synergistic effect than its analogs. Compared to the corresponding rates after colistin monotherapy, the survival rates of systemically infected mice were significantly increased 30% or 60% when the mice received an intravenous injection of colistin in combination with 15 mg/kg of body weight PFK-158. These results have important implications for repurposing PFK-158 to combat colistin resistance.

MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.