Cocktail is an easy-to-use computer program for mathematical modelling of bacteriophage (phage) infection kinetics in a chemostat. The infection of bacteria by phages results in complicated dynamic processes as both have the ability to multiply and change during the course of an infection. There is a need for a simple way to visualise these processes, not least due to the increased interest in phage therapy. Cocktail is completely self-contained and runs on a Windows 64-bit operating system. By changing the publicly available source code, the program can be developed in the directions that users see fit. Cocktail’s models consist of coupled differential equations that describe the infection of a bacterium in a vessel by one or two (interfering) phages. In the models, the bacterial population can be controlled by sixteen parameters, for example, through different growth rates, phage resistance, metabolically inactive cells or biofilm formation. The phages can be controlled by eight parameters each, such as different adsorption rates or latency periods. As the models in Cocktail describe the infection kinetics of phages in vitro, the program is primarily intended to generate hypotheses, but the results can however be indicative in the application of phage therapy.

In this study, we looked at the population dynamics of a two phages-one host system using phages vB_EcoP_SU10 (SU10) and vB_EcoD_SU57 (SU57) and the bacteria Escherichia coli, strain ECOR57. Phage-specific growth curves were observed where infections by SU10 resulted in a moderate production of phages and infections by SU57 resulted in a fast and extensive production of phage progeny. Sequentially adding SU10 followed by SU57 did not produce a significant change in growth rates, whereas adding SU57 followed by SU10 resulted in a decrease in SU10 titer The efficiency of the plating assays showed that ECOR57 exhibited a resistance spectrum after infection by both the single and combined phages. Phage-resistant bacteria exhibited four different morphotypes (i.e., normal, slimy, edgy, and pointy). The normal and edgy morphotypes had a high frequency of developing resistance. Bacterial growth and biofilm assays indicated that the edgy and pointy morphotypes reached a stationary phase faster and produced more biofilm compared to the wild type. These findings suggest that the dynamic structure of phage–bacteria communities dictate resistance evolution and development. Understanding when and how resistances arise and phage(s)–hosts interactions could aid in the design of phage therapy treatments.

Escherichia coli phage SU10 belongs to the genus Kuravirus from the class Caudoviricetes of phages with short non-contractile tails. In contrast to other short-tailed phages, the tails of Kuraviruses elongate upon cell attachment. Here we show that the virion of SU10 has a prolate head, containing genome and ejection proteins, and a tail, which is formed of portal, adaptor, nozzle, and tail needle proteins and decorated with long and short fibers. The binding of the long tail fibers to the receptors in the outer bacterial membrane induces the straightening of nozzle proteins and rotation of short tail fibers. After the re-arrangement, the nozzle proteins and short tail fibers alternate to form a nozzle that extends the tail by 28 nm. Subsequently, the tail needle detaches from the nozzle proteins and five types of ejection proteins are released from the SU10 head. The nozzle with the putative extension formed by the ejection proteins enables the delivery of the SU10 genome into the bacterial cytoplasm. It is likely that this mechanism of genome delivery, involving the formation of the tail nozzle, is employed by all Kuraviruses.

Enterotoxigenic Escherichia coli (ETEC) strains are an important cause of bacterial diarrheal illness in humans and animals. Infections arising from ETEC could potentially be treated through the use of bacteriophage (phage) therapy, as phages encode for enzymes capable of bacterial cell lysis. vB_EcoP_SU7 was isolated from the Käppala wastewater treatment plant in Stockholm, Sweden, and propagated on an ETEC strain exhibiting the O:139 serovar. Transmission electron microscopy confirmed that vB_EcoP_SU7 belongs to the Podoviridae family and has the rare C3 morphotype of an elongated head. Bioinformatic analyses showed that the genome was 76,626 base pairs long and contained 35 genes with predicted functions. A total of 81 open reading frames encoding proteins with hypothetical function and two encoding proteins of no significant similarity were also found. A putative tRNA gene, which may aid in vB_EcoP_SU7's translation, was also identified. Phylogenetic analyses showed that compared to other Podoviridae, vB_EcoP_SU7 is a rare Kuravirus and is closely related to E. coli phages with the uncommon C3 morphotype, such as ECBP2, EK010, vB_EcoP_EcoN5, and vB_EcoP_SU10. Phage vB_EcoP_SU7 has a narrow host range, infecting 11 out of the 137 E. coli strains tested, a latency period of 30 min, a burst size of 12 PFU/cell, and an adsorption rate of 8.78 x 10(-9) mL/min five minutes post infection. With a limited host range and poor infection kinetics, it is unlikely that SU7 can be a standalone phage used for therapeutic purposes; rather, it must be used in combination with other phages for broad-spectrum therapeutic success.

The morphology, infection kinetics, genome sequence and phylogenetic characterization of the previously isolated bacteriophage vB_EcoD_SU57 are presented. The phage vB_EcoD_SU57 was isolated on Escherichia coli strain ECOR57 from the E. coli reference collection and was shown to produce four mm clear plaques with halos. Infection kinetics, as assessed by one-step growth analyses, suggest that vB_EcoD_SU57 is a virulent phage with an adsorption rate of 8.5 × 10^–10 mL × min^–1, a latency period of 14 min, and a burst size of 13 PFU per bacterium. Transmission electron microscopy confirmed vB_EcoD_SU57 to be a phage that used to be classified as a Siphoviridae phage. Bioinformatics analyses showed that the genome was 46,150 base pairs long, contained 29 genes with predicted protein functions, and 51 open reading frames encoding proteins with unknown function, many of which were gathered in clusters. A putative tRNA gene was also identified. Phylogenetic analyses showed that vB_EcoD_SU57 is a Braunvirinae phage of the newly formed Drexlerviridae family and closely related to T1-like E. coli phages vB_EcoS_ACG-M12 (Guelphvirus) and Rtp (Rtpvirus) as well as the unclassified phages vB_EcoS_CEB_EC3a and ECH1.

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.

Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.

Clinical trial results of phage treatment of bacterial infections show a low to moderate efficacy, and the variation in infection clearance between subjects within studies is often large. Phage therapy is complicated and introduces many additional components of variance as compared to antibiotic treatment. A large part of the variation is due to in vivo pharmacokinetics and pharmacodynamics being virtually unknown, but also to a lack of standardisation. This is a consequence of the great variation of phages, bacteria, and infections, which results in different experiments or trials being impossible to compare, and difficulties in estimating important parameter values in a quantitative and reproducible way. The limitations of phage therapy will have to be recognised and future research focussed on optimising infection clearance rates by e.g. selecting phages, bacteria, and target bacterial infections where the prospects of high efficacy can be anticipated, and by combining information from new mathematical modelling of in vivo pharmacokinetic and pharmacodynamic processes and quantitatively assessed experiments.

The resurgence of research into phage biology and therapy is, in part, due to the increasing need for novel agents to treat multidrug-resistant infections. Despite a long clinical history in Eastern Europe and initial success within the food industry, commercialized phage products have yet to enter other sectors. This relative lack of success is, in part, due to the inherent biological limitations of whole phages. These include (but are not limited to) reaching target sites at sufficiently high concentrations to establish an infection which produces enough progeny phages to reduce the bacterial population in a clinically meaningful manner and the limited host range of some phages. Conversely, parallels can be drawn between antimicrobial enzymes derived from phages and conventional antibiotics. In the current article the biological limitations of whole phage-based therapeutics and their derived antimicrobial enzymes will be discussed. In addition, the ability of more complex formulations to address these issues, in the context of medical and non-medical applications, will also be included.

The continuing threat of antimicrobial resistance presents a considerable challenge to researchers to develop novel strategies ensuring that bacterial infections remain treatable. Many plant extracts have been shown to have antibacterial properties and could potentially be combined with other antibacterial agents to create more effective formulations. In this study, the antibacterial activity of three plant extracts and virulent bacteriophages have been assessed as individual components and in combination. When assessed with a modified suspension test, these plant extracts also exhibit antiviral activity at bacterial inhibitory concentrations. Hence, to investigate any potential additive effects between the extracts and virulent phages, the extracts were tested at subantiviral concentrations. Phages alone and in combination with plant extracts significantly reduced (P < 0·05) the bacterial concentration compared to untreated and extract treated controls up to 6 h (2–3log₁₀), but this reduction did not extend to 24 h. In most cases, the phage and extract combinations did not significantly reduce bacterial content compared to phages alone. Additionally, there was little impact on the ability of the phages to reproduce within their bacterial hosts. To our knowledge, this study represents the first of its kind, in which antimicrobial plant extracts have been combined with virulent phages and has highlighted the necessity for plant extracts to be functionally characterized prior to the design of combinatorial therapies.

Significance and Impact of Study

This preliminary study provides insights into the potential combination of bacteriophages and antimicrobial plant bulk extracts to target bacterial pathogens. It is to our knowledge the first time in which virulent bacteriophages have been combined with antimicrobial plant extracts.