Antibiotic resistance is arguably the biggest current threat to global health. An increasing number of infections are becoming harder or almost impossible to treat, carrying high morbidity, mortality, and financial cost. The therapeutic use of bacteriophages, viruses that infect and kill bacteria, is well suited to be part of the multidimensional strategies to combat antibiotic resistance. Although phage therapy was first implemented almost a century ago, it was brought to a standstill after the successful introduction of antibiotics. Now, with the rise of antibiotic resistance, phage therapy is experiencing a well-deserved rebirth. Among the admittedly vast literature recently published on this topic, this review aims to provide a forward-looking perspective on phage therapy and its role in modern society. We cover the key points of the antibiotic resistance crisis and then explain the biological and evolutionary principles that support the use of phages, their interaction with the immune system, and a comparison with antibiotic therapy. By going through up-to-date reports and, whenever possible, human clinical trials, we examine the versatility of phage therapy. We discuss conventional approaches as well as novel strategies, including the use of phage-antibiotic combinations, phage-derived enzymes, exploitation of phage resistance mechanisms, and phage bioengineering. Finally, we discuss the benefits of phage therapy beyond the clinical perspective, including opportunities for scientific outreach and effective education, interdisciplinary collaboration, cultural and economic growth, and even innovative use of social media, making the case that phage therapy is more than just an alternative to antibiotics.

Phage Therapy in the Postantibiotic Era

Bacteriophage (phage) therapy, i.e., the use of viruses that infect bacteria as antimicrobial agents, is a promising alternative to conventional antibiotics. Indeed, resistance to antibiotics has become a major public health problem after decades of extensive usage. However, one of the main questions regarding phage therapy is the possible rapid emergence of phage-resistant bacterial variants, which could impede favourable treatment outcomes. Experimental data has shown that phage-resistant variants occurred in up to 80% of studies targeting the intestinal milieu and 50% of studies using sepsis models. Phage-resistant variants have also been observed in human studies, as described in three out of four clinical trials that recorded the emergence of phage resistance. On the other hand, recent animal studies suggest that bacterial mutations that confer phage-resistance may result in fitness costs in the resistant bacterium, which, in turn, could benefit the host. Thus, phage resistance should not be underestimated and efforts should be made to develop methodologies for monitoring and preventing it. Moreover, understanding and taking advantage of the resistance-induced fitness costs in bacterial pathogens is a potentially promising avenue.

https://serval.unil.ch/resource/serval:BIB_6572B8A55D64.P001/REF.pdf

Resistance Development to Bacteriophages Occurring during Bacteriophage Therapy

In contrast to planktonic cells, bacteria imbedded biofilms are notoriously refractory to treatment by antibiotics or bacteriophage (phage) used alone. Given that the mechanisms of killing differ profoundly between drugs and phages, an obvious question is whether killing is improved by combining antibiotic and phage therapy. However, this question has only recently begun to be explored. Here, in vitro biofilm populations of Pseudomonas aeruginosa PA14 were treated singly and with combinations of two phages and bactericidal antibiotics of five classes. By themselves, phages and drugs commonly had only modest effects in killing the bacteria. However some phage-drug combinations reduced bacterial densities to well below that of the best single treatment; in some cases, bacterial densities were reduced even below the level expected if both agents killed independently of each other (synergy). Furthermore, there was a profound order effect in some cases: treatment with phages before drugs achieved maximum killing. Combined treatment was particularly effective in killing in Pseudomonas biofilms grown on layers of cultured epithelial cells. Phages were also capable of limiting the extent to which minority populations of bacteria resistant to the treating antibiotic ascend. The potential of combined antibiotic and phage treatment of biofilm infections is discussed as a realistic way to evaluate and establish the use of bacteriophage for the treatment of humans.

/pdf/synergy-and-order-effects-of-antibiotics-and-phages-in-osajwifiba.pdf

Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms.

https://www.cell.com/trends/microbiology/pdf/S0966-842X(15)00302-9.pdf

Evolutionary Rationale for Phages as Complements of Antibiotics

Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options.

The evolution of antibiotic resistance in bacteria is a global concern and the use of bacteriophages alone or in combined therapies is attracting increasing attention as an alternative. Evolutionary theory predicts that the probability of bacterial resistance to both phages and antibiotics will be lower than to either separately, due for example to fitness costs or to trade-offs between phage resistance mechanisms and bacterial growth. In this study, we assess the population impacts of either individual or combined treatments of a bacteriophage and streptomycin on the nosocomial pathogen Pseudomonas aeruginosa. We show that combining phage and antibiotics substantially increases bacterial control compared to either separately, and that there is a specific time delay in antibiotic introduction independent of antibiotic dose, that minimizes both bacterial density and resistance to either antibiotics or phage. These results have implications for optimal combined therapeutic approaches.

/pdf/a-window-of-opportunity-to-control-the-bacterial-pathogen-2mn45xu7tp.pdf

A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining antibiotics and phages.

Natural microbial communities perform many functions that are crucial for human well-being. Yet we have very little control over them, and we do not know how to optimize their functioning. One idea is to breed microbial communities as we breed dogs: by comparing a set of microbiomes and allowing the best-performing ones to generate new communities, and so on. Although this idea seems simple, designing such a selection experiment brings with it many decisions with surprising outcomes. Xie and colleagues developed a computational model that reveals this complexity and shows how different experimental design decisions can impact the success of such an experiment.

/pdf/artificially-selecting-microbial-communities-if-we-can-breed-1etpezx6sd.pdf

Artificially selecting microbial communities: If we can breed dogs, why not microbiomes?

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsbl.2016.0064

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Understanding adaptation to complex environments requires information about how exposure to one selection pressure affects adaptation to others. For bacteria, antibiotics and viral parasites (phages) are two of the most common selection pressures and are both relevant for treatment of bacterial infections: increasing antibiotic resistance is generating significant interest in using phages in addition or as an alternative to antibiotics. However, we lack knowledge of how exposure to antibiotics affects bacterial responses to phages. Specifically, it is unclear how the negative effects of antibiotics on bacterial population growth combine with any possible mutagenic effects or physiological responses to influence adaptation to other stressors such as phages, and how this net effect varies with antibiotic concentration. Here, we experimentally addressed the effect of pre-exposure to a wide range of antibiotic concentrations on bacterial responses to phages. Across 10 antibiotics, we found a strong association between their effects on bacterial population size and subsequent population growth in the presence of phages (which in these conditions indicates phage-resistance evolution). We detected some evidence of mutagenesis among populations treated with fluoroquinolones and β-lactams at sublethal doses, but these effects were small and not consistent across phage treatments. These results show that, although stressors such as antibiotics can boost adaptation to other stressors at low concentrations, these effects are weak compared to the effect of reduced population growth at inhibitory concentrations, which in our experiments strongly reduced the likelihood of subsequent phage-resistance evolution.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jeb.13220

Effects of prior exposure to antibiotics on bacterial adaptation to phages.

There is increasing interest in artificially selecting or breeding microbial communities, but experiments have reported modest success and it remains unclear how to best design such a selection experiment. Here, we develop computational models to simulate two previously known selection methods and compare them to a new “disassembly” method that we have developed. Our method relies on repeatedly competing different communities of known species combinations against one another, and sometimes changing the species combinations. Our approach significantly outperformed previous methods that could not maintain enough between-community diversity for selection to act on. Instead, the disassembly method allowed many species combinations to be explored throughout a single selection experiment. Nevertheless, selection at the community level in our simulations did not counteract selection at the individual level. Species in our model can mutate, and we found that they evolved to invest less into community function and more into growth. Increased growth compensated for reduced investment, however, and overall community performance was barely affected by within-species evolution. Our work provides important insights that will help design community selection experiments.

/pdf/novel-artificial-selection-method-improves-function-of-14hcn2ug.pdf

Flor I. Arias-Sánchez

Papers

A window of opportunity to control the bacterial pathogen Pseudomonas aeruginosa combining antibiotics and phages.

Artificially selecting microbial communities: If we can breed dogs, why not microbiomes?

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Effects of prior exposure to antibiotics on bacterial adaptation to phages.

Novel artificial selection method improves function of simulated microbial communities