Abstract
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.
Highlights
Global use of antimicrobials provokes intensive antimicrobial resistance (AMR) selection
General Requirements for Live Biosensors Applied in Ultrahigh-Throughput Screening
Ultrahigh-throughput screening of antimicrobial activity is based on a single-cell encapsulation of microbiome representatives together with fluorescent protein-producing reporter bacteria followed by isolation of droplets with inhibited growth of reporter bacteria using fluorescence-activated cell sorting (FACS) (Figure 1)
Summary
Global use of antimicrobials provokes intensive antimicrobial resistance (AMR) selection. The overuse of antibiotics lays the foundations for further resistome propagation and multi-resistance evolution. Further deterioration in this field threatens the emergence of epidemics caused by multiresistant pathogens and their subsequent persistence in the population under selection pressure. New antibiotics targeting resistant Gram-negatives have been approved, but most of them belong to existing classes of antibiotics, and resistance to them has already emerged [6]. Gram-negative bacteria have an outer membrane, a protective and unique feature that distinguishes them from Gram-positive bacteria. This shield provides an efficient barrier for a vast variety of antimicrobials. Together with acquired resistance mechanisms, like mutations in chromosomal genes or mobile genetic elements carrying resistance genes, this provides a challenge to medication, often unresolvable [7]
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