Université PSL

New ANR for Andrew Griffiths and coworkers

New ANR for Andrew Griffiths and coworkers
The "microFlu4AMR" project submitted by Andrew Griffiths (LBC - CBI) is the winner of the "ANTIBIORESISTANCE: UNDERSTAND, INNOVATE, ACT" call for projects within the framework of the "Investissements & Avenir". This project is in partnership with Paul Rainey's, LGE team, INRAE, UBFC and Deinove.

Soil is a formidable reservoir of microbial biodiversity and existing antibiotics are principally secondary metabolites produced by soil microorganisms, or semisynthetic derivatives thereof. In nature, these compounds serve as weapons in the competition between genotypes, and their evolution and dissemination are intimately coupled with the evolution and dissemination of antibiotic resistance. This project aims i) to investigate the functional diversity (genes and metabolic pathways) of soil microbial communities involved in antibiotic production, and corresponding resistomes, ii) to develop a better understanding of ecological and evolutionary processes in the soil communities involved in antibiotic production, and as a consequence, to their associated resistomes, iii) to develop a better understanding of the role and mechanisms of horizontal and vertical gene transfer in the evolution and dissemination of antibiotics and antibiotic resistance, and iv) to discover new natural classes of antibiotics, in particular more pathogen-specific antibiotics that reduce the risk of emergence of adapted resistomes.

To this end, we will analyze soil samples, selected to cover the largest microbial diversity possible. The multiplicity of genetic diversity associated with antibiotic production and antibiotic resistance will be functionally characterized using i) targeted and global metagenomic approaches and ii) ultrahigh-throughput phenotypic and genotypic screening and single-cell whole genome sequencing using a novel droplet-based microfluidic platform. This will give unprecedented insight into the diversity of, and interplay between, antibiotic and resistance mechanisms, and potentially allow the discovery of novel antibiotics and resistance mechanisms.
Furthermore, we will use the same soil samples, and the same analytical systems, in combination with a powerful new laboratory evolution system, to i) exploit the ability of natural selfish genetic elements (SGEs) to amplify and disseminate antibiotic and resistance genes, ii) study mechanisms to
control of the emergence and dissemination of resistance, iii) observe the emergence of new antibiotic and resistance genes and clusters due to recombination events, and iv) use laboratory evolution in highly diverse microbial communities to evolve novel antibiotics. Using the droplet-based microfluidic system, which we will develop during the project, single microorganisms will be compartmentalized in droplets and antibiotic production will be detected using specifically developed reporter bacteria, allowing fluorescence-activated sorting of the droplets at rates of up to 1,000 per second, and screening of ~1 million microorganisms per hour. This innovative technology will increase both screening throughput (about 100-fold) and sensitivity compared to conventional microbiological screens. The same system will also be applied to screen for antibiotic resistance, in this case screening in the presence of antibiotic, and using a fluorogenic assay to quantify live bacteria in the drops. Furthermore, it will allow screening of species that are usually considered as non-culturable. To this end, we will develop a system to allow single-cell whole genome sequencing (WGS) of thousands of sorted bacteria to directly identify biosynthetic gene clusters producing new antibiotic compounds or comprising mechanisms of antibiotic resistance.
Sorted cultivatable bacteria will be grown to validate antibiotic activity on selected pathogens analysed by mass-spectrometry to identify active molecules and analysed by NGS. Gene clusters potentially encoding novel antibiotics from these bacteria, or from sorted non-cultivatable bacteria
will be transferred into a bacterial chassis for production and further characterisation, and potential development into novel therapeutics.