Lateral transduction is inherent to the life cycle of the archetypical Salmonella phage P22

Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with early prophage excision followed by DNA replication and packaging (ERP). This temporal program is considered universal for P22-like temperate phages, though there is no direct evidence to support the timing and sequence of these events. Here we report that the long-standing ERP program is an observation of the experimentally favored Salmonella phage P22 tsc229 heat-inducible mutant, and that wild-type P22 actually follows the replication-packaging-excision (RPE) program. We find that P22 tsc229 excises early after induction, but P22 delays excision to just before it is detrimental to phage production. This allows P22 to engage in lateral transduction. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation.

A Rapid Method for Performing a Multivariate Optimization of Phage Production Using the RCCD Approach

Bacteriophages can be used in various applications, from the classical approach as substitutes for antibiotics (phage therapy) to new biotechnological uses, i.e., as a protein delivery vehicle, a diagnostic tool for specific strains of bacteria (phage typing), or environmental bioremediation. The demand for bacteriophage production increases daily, and studies that improve these production processes are necessary. This study evaluated the production of a T4-like bacteriophage vB_EcoM-UFV09 (an E. coli-infecting phage with high potential for reducing environmental biofilms) in seven types of culture media (Luria–Bertani broth and the M9 minimal medium with six different carbon sources) employing four cultivation variables (temperature, incubation time, agitation, and multiplicity of infection). For this purpose, the rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for starting to scale up the production process. We used the RCCD to set up the experimental design by combining the cultivation parameters in a specific and systematic way. Despite the high number of conditions evaluated, the results showed that when specific conditions were utilized, viral production was effective even when using a minimal medium, such as M9/glucose, which is less expensive and can significantly reduce costs during large-scale phage production.

The secret life (cycle) of temperate bacteriophages

Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with prophage excision followed by DNA replication and packaging (ERP) – a temporal program that is considered universal for most temperate phages. Here we report that the long-standing ERP program is an artefact of the experimentally favoured Salmonella phage P22 tsc229 heat-inducible mutant, and that wildtype P22 actually follows a replication-packaging-excision (RPE) program. We found that unlike P22 tsc229, P22 delayed excision to just before it was detrimental to phage production. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation.One Sentence SummaryGenetic analyses propose a new life cycle for temperate bacteriophages.

Chronic release of tailless phage particles from Lactococcus lactis

Lactococcus lactis strains residing in the microbial community of a complex dairy starter culture named 'Ur' are hosts to prophages belonging to the family Siphoviridae. L. lactis strains (TIFN1 to TIFN7) showed detectable spontaneous phage production and release (109-1010 phage particles/mL) and up to 10-fold increases upon prophage induction, while in both cases we observed no obvious cell lysis, typically described for the lytic life cycle of Siphoviridae phages. Intrigued by this phenomenon, we investigated the host-phage interaction using strain TIFN1 (harboring prophage proPhi1) as a representative. We confirmed that during the massive phage release, all bacterial cells remain viable. Further, by monitoring phage replication in vivo, using a green fluorescence protein reporter combined with flow cytometry, we demonstrated that the majority of the bacterial population (over 80%) is actively producing phage particles when induced with mitomycin C. The released tailless phage particles were found to be engulfed in lipid membranes, as evidenced by electron microscopy and lipid staining combined with chemical lipid analysis. Based on the collective observations, we propose a model of phage-host interaction in L. lactis TIFN1, where the phage particles are engulfed in membranes upon release, thereby leaving the producing host intact. Moreover, we discuss possible mechanisms of chronic, or non-lytic release of LAB Siphoviridae phages and its impact on the bacterial host.

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology

Background Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria–bacteriophage co-cultures, are required. Results In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage–bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase). Conclusions Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Phage Therapy

Phage therapy refers to the use of bacteriophages (phages - bacterial viruses) as therapeutic agents against infectious bacterial diseases. This therapeutic approach emerged in the beginning of the 20th century but was progressively replaced by the use of antibiotics in most parts of the world after the second world war. More recently however, the alarming rise of multidrug-resistant bacteria and the consequent need for antibiotic alternatives has renewed interest in phages as antimicrobial agents. Several scientific, technological and regulatory advances have supported the credibility of a second revolution in phage therapy. Nevertheless, phage therapy still faces many challenges that include: i) the need to increase phage collections from reference phage banks; ii) the development of efficient phage screening methods for the fast identification of the therapeutic phage(s); iii) the establishment of efficient phage therapy strategies to tackle infectious biofilms; iv) the validation of feasible phage production protocols that assure quality and safety of phage preparations; and (v) the guarantee of stability of phage preparations during manufacturing, storage and transport. Moreover, current maladapted regulatory structures represent a significant hurdle for potential commercialization of phage therapeutics. This article describes the past and current status of phage therapy and presents the most recent advances in this domain.

Strategy for Mass Production of Lytic Staphylococcus Aureus Bacteriophage pSa-3: Contribution of Multiplicity of Infection

BackgroundAntibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures are required.ResultsIn the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13 fold increase).ConclusionsTherefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, which this combinatorial strategy could decrease phage production time. Further, we suggest that response surface methodology has the potential for optimizing mass production of lytic bacteriophages.

A phage satellite tunes inducing phage gene expression using a domesticated endonuclease to balance inhibition and virion hijacking

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1’s capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.

The Impact of Pf Bacteriophages on the Fitness of Pseudomonas aeruginosa; A Mathematical Modeling Approach

Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work by ourselves and others has implicated Pf bacteriophages, non-lytic filamentous viruses produced by Pa, in the chronicity and severity of Pa infections. Pf phages act as structural elements in Pa biofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf+ bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf- strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model predicts that Pf+ strains of Pa can only outcompete Pf- strains if the benefits of phage production falls solely onto Pf+ strains and not onto the overall bacterial community in the lung. Further, phage production only leads to a net positive gain in fitness at antibiotic concentrations slightly above the minimum inhibitory concentration (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production), but which are not lethal for Pf+ strains. As a result, our model predicts that frequent administration of intermediate doses of antibiotics with low decay rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+ Pa infections.ImportanceFilamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains of Pa in different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages and may facilitate improved treatment strategies for combating antibiotic tolerance.