Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

The global rise of multi-drug resistant bacteria has resulted in the notion that an “antibiotic apocalypse” is fast approaching. This has led to a number of well publicized calls for global funding initiatives to develop new antibacterial agents. The long clinical history of phage therapy in Eastern Europe, combined with more recent in vitro and in vivo success, demonstrates the potential for whole phage or phage based antibacterial agents. To date, no whole phage or phage derived products are approved for human therapeutic use in the EU or USA. There are at least three reasons for this: (i) phages possess different biological, physical and pharmacological properties compared to conventional antibiotics. Phages need to replicate in order to achieve a viable antibacterial effect, resulting in complex pharmacodynamics / pharmacokinetics. (ii) The specificity of individual phages requires multiple phages to treat single species infections, often as part of complex cocktails. (iii) The current approval process for antibacterial agents has evolved with the development of chemically based drugs at its core, and is not suitable for phages. Due to similarities with conventional antibiotics, phage derived products such as endolysins are suitable for approval under current processes as biological therapeutic proteins. These criteria render the approval of phages for clinical use theoretically possible but not economically viable. In this review, pitfalls of the current approval process will be discussed for whole phage and phage derived products, in addition to the utilization of alternative approval pathways including adaptive licensing and “Right to try” legislation.

Stunting, a severe and multigenerational growth impairment, globally affects 22% of children under the age of 5 years. Stunted children have altered gut bacterial communities with higher proportions of Proteobacteria, a phylum with several known human pathogens. Despite the links between an altered gut microbiota and stunting, the role of bacteriophages, highly abundant bacterial viruses, is unknown. Here, we describe the gut bacterial and bacteriophage communities of Bangladeshi stunted children younger than 38 months. We show that these children harbor distinct gut bacteriophages relative to their non-stunted counterparts. In vitro, these gut bacteriophages are infectious and can regulate bacterial abundance and composition in an age-specific manner, highlighting their possible role in the pathophysiology of child stunting. Specifically, Proteobacteria from non-stunted children increased in the presence of phages from younger stunted children, suggesting that phages could contribute to the bacterial community changes observed in child stunting.

In this review we provide an overview of current challenges and advances in bacteriophage research within the growing field of viromics. In particular, we discuss, from a human virome study perspective, the current and emerging technologies available, their limitations in terms of de novo discoveries, and possible solutions to overcome present experimental and computational biases associated with low abundance of viral DNA or RNA. We summarize recent breakthroughs in metagenomics assembling tools and single-cell analysis, which have the potential to increase our understanding of phage biology, diversity, and interactions with both the microbial community and the human body. We expect that these recent and future advances in the field of viromics will have a strong impact on how we develop phage-based therapeutic approaches.