Bacteriophage Latent-Period Evolution as a Response to Resource Availability

Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models—in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy—can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (~10⁸ cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (10⁷ cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 10⁸ cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 10⁵ cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.     

Optimizing bacteriophage plaque fecundity

Bacteriophages (phages), the viruses of bacteria, form visible lesions within bacterial lawns (called plaques), which are employed ubiquitously in phage isolation and characterization. Plaques also can serve as models for phage population growth within environments that display significant spatial structure, e.g. soils, sediments, animal mucosal tissue, etc. Furthermore, phages growing within plaques, in experimental evolution studies, may become adapted to novel conditions, may be selected for faster expansion, or may evolve toward producing more virions per plaque. Here, we examine the evolution of the latter, greater plaque fecundity, considering especially tradeoffs between phage latent period and phage burst size. This evolution is interesting because genetically lengthening latent periods, as seen with phage lysis-timing mutants, should increase phage burst sizes, as more time is available for phage-progeny maturation during infection. Genetically shortening latent periods, however, is a means toward producing larger phage plaques since phage virions then can spend more time diffusing rather than infecting. With these larger plaques more bacteria become phage infected, resulting in more phage bursts. Given this conflict between latent period's impact on per-plaque burst number versus per-infection burst size, and based on analysis of existing models of plaque expansion, we provide two assertions. First, latent periods that optimize plaque fecundity are longer (e.g. at least two-fold longer) than latent periods that optimize plaque size (or that optimize phage population growth within broth). Second, if increases in burst size can contribute to plaque size (i.e. larger plaques with larger bursts), then latent-period optima that maximize plaque fecundity should be longer still. As a part of our analysis, we provide a means for predicting latent-period optima-for maximizing either plaque size or plaque fecundity-which is based on knowledge of only phage eclipse period and the relative contribution of phage burst size versus latent period toward plaque size.     

Experimental Examination of Bacteriophage Latent-Period Evolution as a Response to Bacterial Availability

For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (≥~10⁷ bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ~70 to 80% of that of the wild type—which is nearly as short as the RB69 eclipse period—and which has a corresponding burst size that is ~30% of that of the wild type. We show that at higher host densities (≥~10⁷ bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (≤~10⁶ bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.     

Selection for bacteriophage latent period length by bacterial density: A theoretical examination

In bacteriophage (phage), rapid and efficient intracellular progeny production is of obvious benefit. A short latent period is not. All else being equal, a longer latent period utilizes host cell resources more completely. Using established parameters of phage growth, a simulation of three successive phage lysis cycles is presented. I have found that high, but not low, host cell densities can select for short phage latent periods. This results from phage with short latent periods more rapidly establishing multiple parallel infections at high host cell concentrations, whereas phage with long latent periods are restricted to growth within a single cell over the same period. This implies that phage with short latent periods habitually grow in environments that are rich in host cells.     

Population Dynamics of a Salmonella Lytic Phage and Its Host: Implications of the Host Bacterial Growth Rate in Modelling

The prevalence and impact of bacteriophages in the ecology of bacterial communities coupled with their ability to control pathogens turn essential to understand and predict the dynamics between phage and bacteria populations. To achieve this knowledge it is essential to develop mathematical models able to explain and simulate the population dynamics of phage and bacteria. We have developed an unstructured mathematical model using delay-differential equations to predict the interactions between a broad-host-range Salmonella phage and its pathogenic host. The model takes into consideration the main biological parameters that rule phage-bacteria interactions likewise the adsorption rate, latent period, burst size, bacterial growth rate, and substrate uptake rate, among others. The experimental validation of the model was performed with data from phage-interaction studies in a 5 L bioreactor. The key and innovative aspect of the model was the introduction of variations in the latent period and adsorption rate values that are considered as constants in previous developed models. By modelling the latent period as a normal distribution of values and the adsorption rate as a function of the bacterial growth rate it was possible to accurately predict the behaviour of the phage-bacteria population. The model was shown to predict simulated data with a good agreement with the experimental observations and explains how a lytic phage and its host bacteria are able to coexist.     

The evolution of phage lysis timing

Summary The effect of host quantity and host quality on the evolution of phage lysis timing is analysed using marginal value theorem of optimal foraging theory. Both factors have been shown to strongly influence the latent period. A high host density selects for short latent period, which is the same result as previous investigators have found. A good host quality also promotes a short latent period. However, elasticity analysis shows that these two factors exert their influences under different sets of conditions. When host density is low, the host density is more important in determining the length of latent period, whereas when host density is high, the host quality is more important.     

Fitness benefits of low infectivity in a spatially structured population of bacteriophages

For a parasite evolving in a spatially structured environment, an evolutionarily advantageous strategy may be to reduce its transmission rate or infectivity. We demonstrate this empirically using bacteriophage (phage) from an evolution experiment where spatial structure was maintained over 550 phage generations on agar plates. We found that a single substitution in the major capsid protein led to slower adsorption of phage to host cells with no change in lysis time or burst size. Plaques formed by phage isolates containing this mutation were not only larger but also contained more phage per unit area. Using a spatially explicit, individual-based model, we showed that when there is a trade-off between adsorption and diffusion (i.e. less ‘sticky’ phage diffuse further), slow adsorption can maximize plaque size, plaque density and overall productivity. These findings suggest that less infective pathogens may have an advantage in spatially structured populations, even when well-mixed models predict that they will not.     

Spatial Structure,Transmission Modes and the Evolution of Viral Exploitation Strategies

Spatial structure and local migration are predicted to promote the evolution of less aggressive host exploitation strategies in horizontally transmitted pathogens. Here we explore the effect of spatial structure on the evolution of pathogens that can use both horizontal and vertical routes of transmission. First, we analyse theoretically how vertical transmission can alter evolutionary trajectories and confirm that space can impede the spread of virulent pathogens. Second, we test this prediction using the latent phage λ which transmits horizontally and vertically in Escherichia coli populations. We show that the latent phage λ wins competition against the virulent mutant λcI857 in spatially structured epidemics, but loses when spatial structure is eroded. The vertical transmission of phage λ immunizes its local host pool against superinfection and prevents the spread of the virulent λcI857. This effect breaks down when mixing facilitates horizontal transmission to uninfected hosts. We thus confirm the importance of spatial structure for the evolutionary maintenance of prudent infection strategies in latent viruses.     

Characterization of group H streptococcal temperate bacteriophage phi 227.

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.     

Replication of Coliphage M-13 I. Effects on Host Cells After Synchronized Infection

Techniques have been described for synchronization of bacteriophage M-13 infection of host cells. The latent period in infected cells was 10 min, and no appreciable number of intracellular phage was observed. Phage production proceeded in three phases after release of the starvation block: an initial rapid exponential rate of progeny phage release without cell lysis, a period of rate transition accompanying the resumption of host cell division, and a second, slower exponential rate of phage production which paralleled the rate of host cell division. The size of infected cells was not affected by infection, but the generation time was increased by 25%. Starved infected cells exhibited a much longer lag in attaining an exponential rate of growth upon the addition of nutrients than did an uninfected control culture.     

Isolation and characterization of a lipopolysaccharide-specific bacteriophage of Pseudomonas aeruginosa

Phage H22 was isolated from sewage using Pseudomonas aeruginosa NCTC 8505 (serotype 0:3) as the host. Although not O-specific, this phage was found to have lipopolysaccharide (LPS) as a receptor. The broad host-range and lack of O-specificity of the phage suggested that its receptor site was in the core region of the LPS. Phage H22 had a Bradley type A structure. It was unaffected by chloroform and diethyl ether, and was stable between pH 5 and 8 and in the temperature range 0 to 60 degrees C. The adsorption rate constant was 14.6 X 10(-9) ml min-1. The phage had a latent period of 43 min, with a rise time of 18 min and a burst size of 6. The adsorption of phage to whole cells and LPS occurred over a broad pH range. Maximum adsorption occurred at 50 degrees C and pH 7.5 in the presence of 0.001 M Ca2+.     

Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant‐bacteria‐phage system*

The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context‐dependent nature of host‐parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high‐nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage‐selected resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage‐resistant mutants (evolved in vitro) did not realize a fitness benefit over phage‐sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed in vitro. Thus, this context‐dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.     

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy

Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

The isolation and characterization of a temperate phage, Y46/(E2), from Erwinia herbicola Y40.

A temperate phage was induced from exponential phase cells of Erwinia herbicola Y46 by treatment with mitomycin C. The phage was purified by single plaque isolation, and produced in bulk by successive cultivation in young cultures of E. herbicola Y 178. Phages were concentrated from culture filtrates by rate zonal centrifugation and resuspension in 0.02 M Tris buffer, pH 7.2, twice, yielding suspensions of about 5 times 10(11) PFU/ml. Purification was achieved by centrifugation in buffered sucrose solutions. The band at the 30/40% sucrose interface yielded intact particles having regular hexagonal heads and lonb contractile tails, with base plates. Fibers were not seen. The mean dimensions were head, 51 nm; neck length, 11 nm; overall tail length, extended, 98 nm and contracted, 75 nm; diameter of tail sheath, 24 nm. The phage was stable from pH 4.0 to 11.0, but unstable at pH 3.0, the response being independent of the suspending medium used. At pH 3.0, a survival curve having biphasic appearance was observed, which was not due to a mixed population of phages. Stability to heat was good up to 45 degrees C, above which a logarithmic decline with temperature increase occurred. The average inactivation rate constant at 50 degrees C and pH 6.8 was 0.15 min-1. Adsorption to E. herbicola Y 178 cells exhibited first-order kinetics, the adsorption rate constant being 2.5 times 10(-10) ml/min. One-step growth-curve experiments indicated a burst size of 35-40, and a minimum latent period of 80 min. Probit analysis gave a mean latent period of 140 min (SD 25). The phage caused lysis of only E. herbicola strains Y178 and Y186.     

Optimality models of phage life history and parallels in disease evolution

Optimality models constitute one of the simplest approaches to understanding phenotypic evolution. Yet they have shortcomings that are not easily evaluated in most organisms. Most importantly, the genetic basis of phenotype evolution is almost never understood, and phenotypic selection experiments are rarely possible. Both limitations can be overcome with bacteriophages. However, phages have such elementary life histories that few phenotypes seem appropriate for optimality approaches. Here we develop optimality models of two phage life history traits, lysis time and host range. The lysis time models show that the optimum is less sensitive to differences in host density than suggested by earlier analytical work. Host range evolution is approached from the perspective of whether the virus should avoid particular hosts, and the results match optimal foraging theory: there is an optimal "diet" in which host types are either strictly included or excluded, depending on their infection qualities. Experimental tests of both models are feasible, and phages provide concrete illustrations of many ways that optimality models can guide understanding and explanation. Phage genetic systems already support the perspective that lysis time and host range can evolve readily and evolve without greatly affecting other traits, one of the main tenets of optimality theory. The models can be extended to more general properties of infection, such as the evolution of virulence and tissue tropism.     

Biological characterization of the lytic cycle of actinophage φA7 in Streptomyces antibioticus

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.     

多耐药鲍曼不动杆菌噬茵体IME—AB6的分离、生物特性及保存方法研究

目的：分离并鉴定一株多耐药鲍曼不动杆菌噬菌体,对其生物特性进行研究,为治疗多耐药鲍曼不动杆菌感染提供新的方法和实验依据。方法：以一株多耐药不动杆菌AB6为宿主菌,从医院废水中分离出噬菌体;用聚乙二醇6000对噬菌体进行浓缩和纯化,并就噬菌体的形态、一步生长曲线、裂解特性和不同保存条件对噬菌体活性的影响进行初步研究。结果：分离出一株噬菌体IME-AB6,电镜显示为肌尾噬菌体,其潜伏期为10 min,爆发期为40 min,爆发量160 pfu/cell;IME-AB6能迅速使菌液变清晰,温度灵敏性强,并且发现在4℃保存是一种方便高效的方法。结论：噬菌体IME-AB6是一株新的有独特特点的噬菌体,用聚乙二醇能很好地提高滴度,其潜伏期和爆发期短且杀菌效能强,性能稳定易保存,这对于噬菌体制剂的开发和耐药性鲍曼不动杆菌的防治具有潜在应用价值。     

Characteristics of Tφ3, a Bacteriophage for Bacillus stearothermophilus

A bacteriophage (Tphi3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60 C, the optimal growth temperature of the host. At 60 C, the phage had a latent period of 18 min and a burst size of about 200. The phage was comparatively thermostable in broth. The halflife of Tphi3 was 400 min at 60 C, 120 min at 65 C, 40 min at 70 C, and 12 min at 75 C. The activation energy for the heat inactivation of Tphi3 was 56,000 cal. The buoyant density of Tphi3 in a cesium chloride density gradient was 1.526 g/ml. Electron micrographs of Tphi3 indicate that the phage has a head that is 57 mmu long. The dimensions and shape of the head are compatible with those of a regular icosahedron. Each edge of the head is 29 mmu long. The tail of Tphi3 is 125 mmu long and 10 mmu wide. There are about 30 cross-striations that are spaced at 3.9-mmu intervals along the tail. Under the conditions investigated, Tphi3 adsorbed slowly to the host. Only 2.8% of the phage adsorbed in 10 min at 60 C, the normal incubation temperature that was used. Tphi3 was not infective to four other thermophilic strains or to two mesophilic strains of bacteria.