Gram-negative pathogens represent an urgent threat due to their intrinsic and acquired antibiotic resistance. Many recent drug candidates display prominent antimicrobial activity against Gram-positive bacteria being inefficient against Gram-negative pathogens. Ultrahigh-throughput, microfluidics-based screening techniques represent a new paradigm for deep profiling of antibacterial activity and antibiotic discovery. A key stage of this technology is based on single-cell cocultivation of microbiome biodiversity together with reporter fluorescent pathogen in emulsion, followed by the selection of reporter-free droplets using fluorescence-activated cell sorting. Here, a panel of reporter strains of Gram-negative bacteria Escherichia coli was developed to provide live biosensors for precise monitoring of antimicrobial activity. We optimized cell morphology, fluorescent protein, and selected the most efficient promoters for stable, homogeneous, high-level production of green fluorescent protein (GFP) in E. coli. Two alternative strategies based on highly efficient constitutive promoter pJ23119 or T7 promoter leakage enabled sensitive fluorescent detection of bacterial growth and killing. The developed live biosensors were applied for isolating potent E. coli-killing Paenibacillus polymyxa P4 strain by the ultrahigh-throughput screening of soil microbiome. The multi-omics approach revealed antibiotic colistin (polymyxin E) and its biosynthetic gene cluster, mediating antibiotic activity. Live biosensors may be efficiently implemented for antibiotic/probiotic discovery, environmental monitoring, and synthetic biology.

Over the past decades, the problem of bacterial resistance to most antibiotics has become a serious threat to patients’ survival. Nevertheless, antibiotics of a novel class have not been approved since the 1980s. The development of antibiotic potentiators is an appealing alternative to the challenging process of searching for new antimicrobials. Production of H₂S—one of the leading defense mechanisms crucial for bacterial survival—can be influenced by the inhibition of relevant enzymes: bacterial cystathionine γ-lyase (bCSE), bacterial cystathionine β-synthase (bCBS), or 3-mercaptopyruvate sulfurtransferase (MST). The first one makes the main contribution to H₂S generation. Herein, we present data on the synthesis, in silico analyses, and enzymatic and microbiological assays of novel bCSE inhibitors. Combined molecular docking and molecular dynamics analyses revealed a novel binding mode of these ligands to bCSE. Lead compound 2a manifested strong potentiating activity when applied in combination with some commonly used antibiotics against multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The compound was found to have favorable in vitro absorption, distribution, metabolism, excretion, and toxicity parameters. The high effectiveness and safety of compound 2a makes it a promising candidate for enhancing the activity of antibiotics against high-priority pathogens.

Obesity is a significant metabolic disorder associated with excessive fat accumulation and insulin resistance. In this study, we explored a gene therapy approach to treat obesity in agouti mice using adeno-associated viruses (AAVs) carrying PRDM16, FoxP4, or Follistatin (FST) genes, which are known to promote the browning of white adipose tissue. Mice treated with AAVs encoding PRDM16, FoxP4, or FST genes showed a reduction in body weight (10–14%) within the first three weeks after administration, compared to the control groups. A lipidomic analysis of the adipose tissue revealed a dramatic reduction in triacylglycerol (TAG) species with low carbon numbers (40–54 acyl carbons) in treated mice.