The Virulence Index: A Metric for Quantitative Analysis of Phage Virulence.

Background: One of the main challenges in developing phage therapy and manufacturing phage products is the reliable evaluation of their efficacy, performance, and quality. Since phage virulence is intrinsically difficult to fully capture, researchers have turned to rapid but partially inadequate methods for its evaluation. Materials and Methods: This study demonstrates a standardized quantitative method to assess phage virulence based on three parameters: the virulence index (VP )-quantifying the virulence of a phage against a host, the local virulence (vi )-assessing killing potential at given multiplicities of infection (MOIs), and MV50 -the MOI at which the phage achieves 50% of its maximum theoretical virulence. This was shown through comparative analysis of the virulence of phages T4, T5, and T7. Results: Under the conditions tested, phage T7 displayed the highest virulence, followed by phage T4 and, finally, by phage T5. The impact of parameters such as temperature and medium composition on virulence was shown for each phage. The use of the method to evaluate the virulence of combinations of phages-for example, for cocktail formulation-is also shown with phages T5 and T7. Conclusions: The method presented provides a platform for high-throughput quantitative assessment of phage virulence and quality control of phage products. It can also be applied to phage screening, evaluation of phage strains, phage mutants, infection conditions and/or the susceptibility of host strains, and the formulation of phage cocktails.

Modeling tailed bacteriophage adsorption: Insight into mechanisms.

The process of a bacteriophage attaching to its host cell is a combination of physical diffusion, biochemical surface interactions, and reaction-induced conformational changes in receptor proteins. Local variations in the physico-chemical properties of the medium, the phage׳s mode of action, and the physiology of the host cell also all influence adsorption kinetics. These characteristics can affect a specific phage׳s binding capabilities and the susceptibility of the host cell to phage attack. Despite the complexity of this process, describing adsorption kinetics of a population of bacteriophages binding to a culture of cells has been accomplished with relatively simple equations governed by the laws of mass-action. Many permutations and modifications to the basic set of reactions have been suggested through the years. While no single solution emerges as a universal answer, this review provides the fundamentals of current phage adsorption modeling and will guide researchers in the selection of valid, appropriate models.

A simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

Impact of the cell life-cycle on bacteriophage T4 infection.

Synchronized Escherichia coli cultures were infected with bacteriophage T4 at discrete points in the cell growth cycle. The cell cycle had a significant impact on the outcome of infection. Cell burst size was smallest for newly formed cells and increased dramatically as these progressed in the cell cycle. The largest burst sizes were achieved when infecting cells immediately prior to cell division. When cells were infected during cell division, the burst size was reduced back to its initial value. Interestingly, lysis time was longest for young cells, reached a minimum at the same point that burst size reached its maximum value, and then increased at the commencement of cell division. Consequently, phage productivity in cells about to undergo cell division was almost three times greater than the productivity of young, newly formed cells. The availability of intracellular resources is believed to be the major driving force behind phage productivity during infection. Indeed, intracellular RNA contents at the time of infection were found to correlate strongly with phage productivity. There was no significant relationship between cell DNA levels and phage productivity. Finally, burst size experiments suggested that the cell cycle also influenced the likelihood of a phage to undergo productive infection.

Evidence that the heterogeneity of a T4 population is the result of heritable traits.

Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments (residual fraction). This residual fraction generally constitutes a minority of phages that exhibit significantly slower adsorption kinetics than the main phage stock (main fraction). While this phenomenon is likely the result of evolutionary driving forces, the present study demonstrates that the residual fraction is not always the result of phenotypic variations within a single genotype, as is generally thought. Experiments with phage T4 showed that two subgroups with distinct adsorption traits that were passed on to their progeny could be isolated from the original phage stock. Sequencing of genes involved in adsorption revealed two point mutations in gene 37 of residual fraction isolates, which resulted in modifications to the long tail-fiber, the organelle of attachment and host cell recognition. Adsorption studies consistently showed that T4 phage stocks amplified from residual fraction isolates had significantly lower adsorption efficiencies than those amplified from main fractions. The conducted experiments provide convincing evidence that the observed heterogeneity in T4 adsorption behavior is the result of conserved mutations to the phage genome and is not exclusively the result of phenotypic variations within the population. While it is believed high mutation rates exist to hasten phage adaptation, this study shows that this bet hedging strategy can also, in the short term, inadvertently handicap the phage's adsorption capabilities to a given host under normal infection conditions, resulting in the residual fraction observed in adsorption experiments.

Self-cycling operation increases productivity of recombinant protein in Escherichia coli.

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express ß-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.

Bacteriophage adsorption efficiency and its effect on amplification.

Existing models for bacteriophage adsorption are modified with the addition of a new term, adsorption efficiency, and applied to a T4-Escherichia coli system. The adsorption efficiency is the fraction of phage that adsorbs irreversibly to the host. Adsorption kinetics were modeled using the adsorption rate constant(k) and the adsorption efficiency(epsilon). Experimental data demonstrated that the adsorption rate constant depends strongly on the condition of the host while the adsorption efficiency is a property of the bacteriophage population. The adsorption efficiency exhibited a marked dependence on the concentration of L-tryptophan. The system was used to study the effect of adsorption kinetics on bacteriophage amplification. Increasing adsorption efficiency had an effect similar to increasing the initial multiplicity of infection; the number of phages produced during amplification decreased. Optimizing the adsorption efficiency by manipulating the L-tryptophan concentration yielded a 14-fold increase in the number of phages produced.