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Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria

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-bas...

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Autores principales: Baranova, Margarita N., Babikova, Polina A., Kudzhaev, Arsen M., Mokrushina, Yuliana A., Belozerova, Olga A., Yunin, Maxim A., Kovalchuk, Sergey, Gabibov, Alexander G., Smirnov, Ivan V., Terekhov, Stanislav S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8532632/
https://www.ncbi.nlm.nih.gov/pubmed/34680742
http://dx.doi.org/10.3390/antibiotics10101161
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author Baranova, Margarita N.
Babikova, Polina A.
Kudzhaev, Arsen M.
Mokrushina, Yuliana A.
Belozerova, Olga A.
Yunin, Maxim A.
Kovalchuk, Sergey
Gabibov, Alexander G.
Smirnov, Ivan V.
Terekhov, Stanislav S.
author_facet Baranova, Margarita N.
Babikova, Polina A.
Kudzhaev, Arsen M.
Mokrushina, Yuliana A.
Belozerova, Olga A.
Yunin, Maxim A.
Kovalchuk, Sergey
Gabibov, Alexander G.
Smirnov, Ivan V.
Terekhov, Stanislav S.
author_sort Baranova, Margarita N.
collection PubMed
description 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.
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spelling pubmed-85326322021-10-23 Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria Baranova, Margarita N. Babikova, Polina A. Kudzhaev, Arsen M. Mokrushina, Yuliana A. Belozerova, Olga A. Yunin, Maxim A. Kovalchuk, Sergey Gabibov, Alexander G. Smirnov, Ivan V. Terekhov, Stanislav S. Antibiotics (Basel) Article 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. MDPI 2021-09-24 /pmc/articles/PMC8532632/ /pubmed/34680742 http://dx.doi.org/10.3390/antibiotics10101161 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Baranova, Margarita N.
Babikova, Polina A.
Kudzhaev, Arsen M.
Mokrushina, Yuliana A.
Belozerova, Olga A.
Yunin, Maxim A.
Kovalchuk, Sergey
Gabibov, Alexander G.
Smirnov, Ivan V.
Terekhov, Stanislav S.
Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title_full Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title_fullStr Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title_full_unstemmed Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title_short Live Biosensors for Ultrahigh-Throughput Screening of Antimicrobial Activity against Gram-Negative Bacteria
title_sort live biosensors for ultrahigh-throughput screening of antimicrobial activity against gram-negative bacteria
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8532632/
https://www.ncbi.nlm.nih.gov/pubmed/34680742
http://dx.doi.org/10.3390/antibiotics10101161
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