Phage therapy is increasingly put forward as a “new” potential tool in the fight against antibiotic resistant infections. During the “Centennial Celebration of Bacteriophage Research” conference in Tbilisi, Georgia on 26–29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.

Highlights d Acr-positive phages limit evolution of CRISPR resistance during clonal and mixed infections d Acr-positive phages provide benefits to Acr-negative phages present in the community d Strong Acr help Acr-negative phages to amplify on immunosuppressed CRISPR-resistant cells d Weaker Acr provides larger advantages during competition with Acr-negative phages

1 Palindromic Repeats; CRISPR-associated) adaptive immune systems to protect 2 themselves against their viruses (phages) 1 . To overcome resistance, phages have evolved 3 anti-CRISPR proteins (Acr), which naturally vary in their potency to suppress the host 4 immune system and avoid phage extinction 2,3,4,5 . However, these Acr-phages need to 5 cooperate in order to overcome CRISPR-based resistance 4,5 : while many initial 6 infections by Acr-phages are unsuccessful, they nonetheless lead to the production of 7 Acr proteins, which generate immunosuppressed cells that can be successfully exploited 8 by other Acr-phages in the population 4,5 . Here we test the prediction that phages 9 lacking acr genes (Acr-negative phages) may exploit this cooperative behaviour 6 . We 10 demonstrate that Acr-negative phages can indeed benefit from the presence of Acr-11 positive phages during pairwise competitions, but the extent of this exploitation depends 12 on the potency of the Acr protein. Specifically, "strong" Acr proteins are more 13 exploitable and benefit both phage types, whereas "weak" Acr proteins predominantly 14 benefit Acr-positive phages only and therefore provide a greater fitness advantage 15 during competition with Acr-negative phages. This work further helps to explain what 16 defines the strength of an Acr protein, how selection acts on different Acr types in a 17 phage community context, and how this can shape the dynamics of phage populations in 18 natural communities. 19 20To explore the hypothesis that cooperative behaviours of Acr-phages may be exploited by 21 Acr-negative phages in the environment, we first performed a theoretical analysis (see 22

The constant arms race between bacteria and their phages has resulted in a large diversity of bacterial defence systems, with many bacteria carrying several systems. In response, phages often carry counter-defence genes. If and how bacterial defence mechanisms interact to protect against phages with counter-defence genes remains unclear. Here, we report the existence of a novel defence system, coined MADS (Methylation Associated Defence System), which is located in a strongly conserved genomic defence hotspot in Pseudomonas aeruginosa and distributed across Gram-positive and Gram-negative bacteria. We find that the natural co-existence of MADS and a Type IE CRISPR-Cas adaptive immune system in the genome of P. aeruginosa SMC4386 provides synergistic levels of protection against phage DMS3, which carries an anti-CRISPR (acr) gene. Previous work has demonstrated that Acr-phages need to cooperate to overcome CRISPR immunity, with a first sacrificial phage causing host immunosuppression to enable successful secondary phage infections. Modelling and experiments show that the co-existence of MADS and CRISPR-Cas provides strong and durable protection against Acr-phages by disrupting their cooperation and limiting the spread of mutants that overcome MADS. These data reveal that combining bacterial defences can robustly neutralise phage with counter-defence genes, even if each defence on its own can be readily by-passed, which is key to understanding how selection acts on defence combinations and their coevolutionary consequences.

No abstract