The emergence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) was regarded as an emerging threat in clinical settings. Here, we investigated the prevalence of CRKP strains among inpatients in a new hospital over 1 year since its inception with various techniques, and carried out a WGS-based phylogenetic study to dissect the genomic background of these isolates. The genomes of three representative blaNDM-1-positive strains and the plasmids of four blaKPC-2-positive strains were selected for Nanopore long-read sequencing to resolve the complicated MDR structures. Thirty-five CRKP strains were identified from 193 K. pneumoniae isolates, among which 30 strains (85.7%) harbored blaKPC-2, whereas the remaining five strains (14.3%) were positive for blaNDM-1. The antimicrobial resistance profiles of blaNDM-1-positive isolates were narrower than that of blaKPC-2-positive isolates. Five isolates including two blaNDM-1-positive isolates and three blaKPC-2-positive strains could successfully transfer the carbapenem resistance phenotype by conjugation. All CRKP strains were categorized into six known multilocus sequence types, with ST11 being the most prevalent type. Phylogenetic analysis demonstrated that the clonal spread of ST11 blaKPC-2-positive isolates and local polyclonal spread of blaNDM-1-positive isolates have existed in the hospital. The blaNDM-1 gene was located on IncX3, IncFIB/IncHI1B, and IncHI5-like plasmids, of which IncFIB/IncHI1B plasmid has a novel structure. By contrast, all ST11 isolates shared the similar blaKPC-2-bearing plasmid backbone, and 11 of them possessed pLVPK-like plasmids. In addition, in silico virulome analysis, Galleria mellonella larvae infection assay, and siderophore secretion revealed the hypervirulence potential of most blaKPC-2-positive strains. Given that these isolates also had remarkable environmental adaptability, targeted measures should be implemented to prevent the grave consequences caused by hv-CRKP strains in nosocomial settings.
Abstract Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [ 125 I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.
Background and Purpose Metformin, a small molecule, antihyperglycaemic agent, is a well‐known activator of AMP‐activated protein kinase (AMPK) and protects against cardiac fibrosis. However, the underlying mechanisms remain elusive. TGFβ1 is a key cytokine mediating cardiac fibrosis. Here, we investigated the effects of metformin on TGFβ1 production induced by angiotensin II (AngII) and the underlying mechanisms. Experimental Approach Wild‐type and AMPKα2 −/− C57BL/6 mice were injected s.c. with metformin or saline and infused with AngII (3 mg·kg −1 ·day −1 ) for 7 days. Adult mouse cardiac fibroblasts (CFs) were isolated for in vitro experiments. Key Results In CFs, metformin inhibited AngII‐induced TGFβ1 expression via AMPK activation. Analysis using bioinformatics predicted a potential hepatocyte nuclear factor 4α (HNF4α)‐binding site in the promoter region of the Tgfb1 gene. Overexpressing HNF4α increased TGFβ1 expression in CFs. HNF4α siRNA attenuated AngII‐induced TGFβ1 production and cardiac fibrosis in vitro and in vivo . Metformin inhibited the AngII‐induced increases in HNF4α protein expression and binding to the Tgfb1 promoter in CFs. In vivo , metformin blocked the AngII‐induced increase in cardiac HNF4α protein levels in wild‐type mice but not in AMPKα2 −/− mice. Consequently, metformin inhibited AngII‐induced TGFβ1 production and cardiac fibrosis in wild‐type mice but not in AMPKα2 −/− mice. Conclusions and Implications HNF4α mediates AngII‐induced TGFβ1 transcription and cardiac fibrosis. Metformin inhibits AngII‐induced HNF4α expression via AMPK activation, thus decreasing TGFβ1 transcription and cardiac fibrosis. These findings reveal a novel antifibrotic mechanism of action of metformin and identify HNF4α as a new potential therapeutic target for cardiac fibrosis. Linked Articles This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc
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