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Insights into the role of phages on the bacterial resistome

Periodic Reporting for period 2 - ROPHARE (Insights into the role of phages on the bacterial resistome)

Reporting period: 2018-12-26 to 2019-12-25

Antibiotics have revolutionized the treatment of infectious diseases. Unfortunately, antimicrobial resistance (AMR) is today a global problem that requires an integral solution. AMR is responsible for an estimated 25,000 deaths per year in the EU and 700,000 worldwide. A report commissioned by the UK government in 2016 predicts that by 2050, AMR infections will kill 10 million people across the world, representing more than the current toll from cancer. One of the most promising alternatives to antibiotics is phage therapy – i.e. the use of viruses (bacteriophages) to specifically infect and kill bacteria. From an ecological perspective, (bacterio)phages are the most abundant and ubiquitous organisms on Earth, and play important roles in microbial physiology, population structure, dynamics and ecosystem functioning. Phages have the advantage to be extremely strain-specific and do not have a major impact on the commensal flora.

The appearance and increase of AMR observed throughout the world cannot be explained only by the modern and growing use of antibiotics, as it implies a complex interaction of microbial communities, antibiotics and AMR genes from different ecosystems. In addition, horizontal gene transfer is the most relevant form of AMR propagation and is mediated by elements capable of transferring genetic information from one bacterium to another. The bacterial modulator properties of phages make them suitable vehicles for the mobilization of genes between microorganisms from different ecosystems.

The general objective of this project is to understand the role of phages in the emergence, abundance and dissemination of AMR genes in human-impacted environments. Ultimately, a major exploitation goal consists of the recovery and characterization of novel phages with potential for the development of alternative antimicrobial therapies.

We have unveiled an unprecedented amount of novel phages through culture-independent (agricultural soil) and culture-dependent (staphylococcal phages from wastewater) approaches, with complex and extended phage-bacterial networks. Our huge data set substantially increases the amount of currently available viral data, and provides insights into the yet largely undescribed environmental viral sequence space. We are further characterizing relevant phages for (i) their ability to mobilize AMR genes and (ii) their properties to be exploited for the development of novel antimicrobial strategies.
(1) We have analyzed the bacterial and viral communities in the experimental field soil under study. For this, several workflows have been developed and optimized. This has enabled us to (i) detected thousands of novel soil viral genomes, which are being characterized; (ii) reconstruct novel soil bacterial genomes; and (ii) establishment of host-virus interactions. These workflows and resultant data are enabling us to deep on the bacterial soil ecology and microbial composition of agricultural soils.

(2) A number of bioinformatics tools have been formatted to perform a specialized annotation against AMRGs, metal and biocide resistance genes. Ongoing analyses have enabled us to detect a high number of novel potential AMRGs with a low percentage of identity to reference genes in the bacterial fraction. These genes will be cloned and expressed for functionality. Fine manual curation of viral contigs has resulted vital to prevent false positive AMRG in the viral fraction.

(3) We have optimized the procedure for the extraction of phage dsDNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. We have shown a remarkable enhanced extraction of the soil phage community. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

(4) We have discovered a great diversity of potentially novel staphylococcal phages from wastewater samples. Based on host-range assay with 60 diverse staphylococcal strains, we have categorized 100 different phages and half of them are being whole-genome sequenced. Importantly, many of these phages have a broad host-range, being able to infect clinically relevant multidrug resistant strains. Out of 125 strains tested, our phages can together infect 62 different strains belonging to 27 different species. Notably, out of 10’884 infections, we have obtained 1’164 different phage-bacteria interactions plus 552 lysis zones. Our primary data also reveal that S. xylosus and S. aureus can transfer a small clindamycin resistance plasmid to clinically relevant species. Our work shows the immense unexplored staphylococcal phage-bacterial network.

(5) In-depth analysis of the genome of an environment-associated multidrug resistant methicillin resistant Staphylococcus sciuri strain has been performed. A novel functionally active thrimethoprim resistance gene in a novel multiresistant mosaic plasmid, which contains additional adaptive genes has been characterized. Additional novel resistance genomic islands and three novel prophages apparently functional have been identified and analysed. Further characterization of these prophages, as well as its ability to transfer the “cohabitant” resistance plasmids is ongoing to unveil the role of carrying phages on the spread of concomitant AMRGs and resistance plasmids.
• We have performed the first comparative metagenomic analysis of the phage and bacterial communities of environmental biomes with different anthropogenic impact. We have discovered an unprecedented viral and bacterial diversity and revealed that fertilization practices do modify microbial composition. This is essential for a better understanding of the role of viruses in the ecological functioning and evolution of microorganisms. With the upcoming functional viromic approaches, we will appraise the role of environmental phages in the emergence and dissemination of AMR genes between biomes.

• This project provides data on AMR gene biodiversity and dynamics of man-managed systems, which can be applied for designing application guidelines based on: (i) potential emerging bacterial/phage located AMR genes in human related biomes, (ii) potential antimicrobial agents that may be most suitable for restraining AMR spread and (iii) possible future clinical treatment failures, (iv) Antibiotics and antibiotic combinations could be used to prevent the horizontal gene transfer of AMR determinants. Such guidelines will define the conditions under which antibiotics are used in animal or human medicine.

• This project helps answer needs raised by policy stakeholders on risk mitigation strategies: i) policy impact for long-term decisions on animal waste uses in soil systems, ii) policy impact of results for immediate decision-making regarding the management of wastewater, or the use of specific agents on farming, iii) provide early notice to the competent agencies on potential reservoirs or new AMR genes in the environment.

• We will exploit our results obtained from the relevant staphylococcal phages discovered for the potential development of promising new antimicrobial strategies, as phage-therapy or the use of phage particles to obtain combined clinical therapies to fight against multidrug resistant bacteria or the development of biocontrol agent for food contaminants.
overview of the project postulate
graphical view of one of the main project hypothesis
overview of one of the main goals