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Using the Evolution Machine to determine the mechanism of a novel Phage-Induced High-Cell-Density (PIHCD) phenotype

Periodic Reporting for period 1 - EvoMachine-Phage (Using the Evolution Machine to determine the mechanism of a novel Phage-Induced High-Cell-Density (PIHCD) phenotype)

Reporting period: 2017-04-01 to 2019-03-31

Bacteriophages (phages) are the major predators of bacteria on this planet and outnumber their prokaryotic prey by an order of magnitude. Phages are intimately involved in a range of biological processes from carbon cycling to the maintenance of human health. One rising global threat to human health is the emergence of bacteria that are resistant to all known antibiotics. For example, a recent study estimated 33,000 people die each in year in Europe due to infection by multi-drug resistant bacteria making the burden of these infections comparable to the combined impact of HIV/AIDs, Tuberculosis, and Influenza. The ability for phages to specifically target and kill bacteria combined with the desperate need for new antibiotics has led to a renaissance in the development of phage-based therapeutics. To develop the most effective strategies involving phage and phage-based products in the war against multi-drug resistant bacteria a better understanding of basic phage biology is required. Phages require a bacterial host to propagate and are therefore locked in a constant arms race with their hosts to evade detection while the host develops their own counterattack strategies. The goal of this project was to better understand this molecular arms race by determining the mechanism of a phage-induced high bacterial growth phenotype, described for the first time here. The phenotype was discovered by combining a mixture of naturally-occurring phages taken directly from a local compost heap in Paris. The first step was to determine the phage(s) responsible for the high growth phenotype.
Our approach to investigate this newly-described phenotype was to isolate and grow phages with their hosts in thousands of small sub-microliter droplets using a newly developed millifluidic device created by MilliDrop instruments, a spinoff biotech company founded at the institute where the work was performed (ESPCI Paris). The MilliDrop Analyzer provides excellent bacterial growth conditions by keeping bacterial cells in constant motion. In addition the MilliDrop analyzer provides the opportunity to select specific droplets of interest that can
In total fifty phages were isolated from compost over a three-month period with multiple phages exhibiting the high growth phenotype first observed in the initial mixture of naturally occurring phages. The underlying mechanism responsible for the high growth phenotype remains under investigation. These results are currently being prepared for publication.
This study involved phages that were initially isolated using traditional plaque-based methods that were subsequently grown in droplets. The plaque-based method requires approximately 48 hr to isolate phages from an environmental sample. For phage-therapeutic purposes timing can be critical as antibiotic-resistant infections can spread rapidly, therefore, any improvement in the speed of phage isolation could improve treatment outcome. An expansion of this study could be exploring the possibility of isolating phages using droplets directly from environmental samples, with the goal of decreasing the total time required for phage isolation in clinical situations. In addition, droplets could be utilized to rapidly test the virulence of specific phage isolates against bacterial strains of interest. Both of these approaches could serve as tools in the battle against multi-drug resistant bacteria.