New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

多重耐药肺炎克雷伯菌噬菌体KP002的生物学特性及基因组学研究

以上海某些医院临床分离到的多重耐药肺炎克雷伯菌为宿主菌,从不同环境的污水中分离获得1株肺炎克雷伯菌噬菌体KP002。电子显微镜显示其为有尾噬菌体,头部直径约70nm,尾长约80nm,尾宽约20nm。对其生物学特性进行研究,结果显示此株噬菌体在pH 3~9及4~50℃的环境中具有较高活性;6min吸附率达95%以上;潜伏期为10min,爆发期为50min;裂解量为172pfu/cell。结果表明,该噬菌体对pH值和温度适应范围较宽。对其全基因组进行测序分析,结果显示其基因组为环状双链DNA,全长47 173bp,GC含量为48%。本研究筛选获得1株对pH值和温度适应范围较宽的耐药肺炎克雷伯菌烈性噬菌体KP002,为建立耐药肺炎克雷伯菌的噬菌体库以用于治疗临床多重耐药菌感染提供了新的思路。     

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.     

Bacteriophage adsorption rate and optimal lysis time

The first step of bacteriophage (phage) infection is the attachment of the phage virion onto a susceptible host cell. This adsorption process is usually described by mass-action kinetics, which implicitly assume an equal influence of host density and adsorption rate on the adsorption process. Therefore, an environment with high host density can be considered as equivalent to a phage endowed with a high adsorption rate, and vice versa. On the basis of this assumption, the effect of adsorption rate on the evolution of phage optimal lysis time can be reinterpreted from previous optimality models on the evolution of optimal lysis time. That is, phage strains with a higher adsorption rate would have a shorter optimal lysis time and vice versa. Isogenic phage lambda-strains with different combinations of six different lysis times (ranging from 29.3 to 68 min), two adsorption rates (9.9 x 10(-9) and 1.3 x 10(-9) phage(-1) cell(-1) ml(-1) min(-1)), and two markers (resulting in "blue" or "white" plaques) were constructed. Various pairwise competitions among these strains were conducted to test the model prediction. As predicted by the reinterpreted model, the results showed that the optimal lysis time is shorter for phage strains with a high adsorption rate and vice versa. Competition between high- and low-adsorption strains also showed that, under current conditions and phenotype configurations, the adsorption rate has a much larger impact on phage relative fitness than the lysis time.     

Population dynamics ofAzospirillum brasilense and its bacteriophage in soil

Summary This study examined the ecology and interaction ofAzospirillum brasilense and its bacteriophage in soil. Four Chernozemic soils from Canada, a Latosol and three Podzolic soils from Brazil were assayed for phage. Only the Latosol containedA. brasilense phage. None of the soils contained phage for otherA. brasilense orA. lipoferum strains tested. Recovery of phage from soil depended on the growth of indigenous or added host cells. A phage isolated from the Latosol had a hexagonal head of 100 nm and a tail of 200 nm. This phage was morphologically distinct from previously described Azospirillum phage and its host range was limited toA. brasilense strains 29145 and 29711.Survival and recovery of phage added to phage-free soil was dependent on the phage, the initial phage population, the presence of host cells and nutrients, and the soil. Phage persisted in soils at undetectable levels for at least seven weeks, but were still able to interact with multiplying host cells and exhibit a 1000-fold increase in number. Phage required a host cell population of at least 100–1000 per g of soil in order to multiply. The phage burst detected under these conditions increased as the cell to phage ratio increased. Long term incubation studies showed that the activity of phage in soil closely followed the activity of host cells and thus both were manipulated by appropriate amendments to soil.     

Atomic Force Microscopy Analysis of the Acinetobacter baumannii Bacteriophage AP22 Lytic Cycle

Background

Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution.

Methodology/Principal Findings

In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C.

Conclusions/Significance

High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.     

Characteristics of φT, the Temperate Bacteriophage Carried by Bacillus megaterium 899a

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.     

Bacteriophage X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.     

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 (approximately 10(8) cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (10(7) 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 10(8) 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 10(5) 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.     

Adsorption of temperate phages of Lactobacillus delbrueckii strains and phage resistance linked to their cell diversity

Aims: The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage‐resistant derivatives with interesting technological properties. Methods and Results: The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca²⁺ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage‐resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Conclusions: Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage‐resistant derivatives of their host strains were obtained. Significance and Impact of the Study: Some phage‐resistant derivatives isolated exhibited good technological properties with the prospective to be used at industrial level.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Inhibition of lactic streptococcus bacteriophage by crystal violet and other agents

Ninety-nine selected compounds and eleven antibiotic-producing organisms were tested for antiphage activity and host toxicity. A paper disc-agar diffusion method was used for primary screening and quantitative methods were employed for confirmatory investigation. Most of the agents tested, although previously reported as inhibitory to one or more other virus-host systems, did not selectively prevent multiplication of lactic streptococcus bacteriophage. Several compounds which prevented mass lysis were extremely toxic to host bacteria. Crystal violet suppressed growth of two phage strains at a level (1.0 x 10(-7)M) which permitted normal growth of the host cells. Failure of crystal violet to prevent multiplication of many phage strains suggested possible variations in the multiplication mechanisms of different strains of virus. Virustatic levels of crystal violet did not destroy unadsorbed virus, reduce adsorption, or prevent invasion; increase of virus was reduced in one-step growth experiments; mass lysis was prevented or delayed in long time experiments. Addition and removal of crystal violet at various intervals during the latent period resulted in virus yields directly related to the portion of the latent period during which no dye was present. Duration of the latent period was unaffected. Single burst experiments indicated that the yield of plaque-forming particles per infected bacterium was reduced; the proportion of infected bacteria giving rise to active progeny did not appear to be influenced to a significant degree. Crystal violet apparently interferes with intracellular multiplication of the virus, possibly by combination of the dye with phage DNA or fractions thereof at some critical stage in the incorporation of DNA into the virus particle.     

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.     

Diffusion of bacteriophages through artificial biofilm models

The simple two-chamber diffusion method was improved to study the diffusion properties of bacteriophage (phage) T4 through a model biofilm agarose gel membrane (AGM) embedded with dead host Escherichia coli K12 cells. The apparent diffusion coefficient (D(app) ) of phage T4 was calculated to be 2.4 × 10(-12) m(2) /s in 0.5% AGM, which was lower than the coefficient of 4.2 × 10(-12) m(2) /s in 0.5% AGM without host cells. The phage adsorption process by dead host cells slowed the apparent phage diffusion. The Langmuir adsorption equation was used to simulate phage adsorption under different multiplicity of infections (MOIs); the maximum adsorbed phage MOI was calculated to be 417 PFU/CFU, and the Langmuir adsorption constant K(L) was 6.9 × 10(-4) CFU/PFU. To evaluate the effects of phage proliferation on diffusion, a simple syringe-based biofilm model was developed. The phage was added into this homogenous biofilm model when the host cells were in an exponential growth phase, and the apparent diffusion coefficient was greatly enhanced. We concluded that D(app) of phages through biofilms could be distinctly affected by phage adsorption and proliferation, and that the idea of D(app) and these methods can be used to study diffusion properties through real biofilms.     

Lactococcal Bacteriophages Require a Host Cell Wall Carbohydrate and a Plasma Membrane Protein for Adsorption and Ejection of DNA

The mechanism of the initial steps of bacteriophage infection in Lactococcus lactis subsp. lactis C2 was investigated by using phages c2, ml3, kh, l, h, 5, and 13. All seven phages adsorbed to the same sites on the host cell wall that are composed, in part, of rhamnose. This was suggested by rhamnose inhibition of phage adsorption to cells, competition between phage c2 and the other phages for adsorption to cells, and rhamnose inhibition of lysis of phage-inoculated cultures. The adsorption to the cell wall was found to be reversible upon dilution of the cell wall-adsorbed phage. In a reaction step that apparently follows adsorption to the cell wall, all seven phages adsorbed to a host membrane protein named PIP. This was indicated by the inability of all seven phages to infect a strain selected for resistance to phage c2 and known to have a defective PIP protein. All seven phages were inactivated in vitro by membranes from wild-type cells but not by membranes from the PIP-defective, phage c2-resistant strain. The mechanism of membrane inactivation was an irreversible adsorption of the phage to PIP, as indicated by adsorption of [³⁵S] methionine-labeled phage c2 to purified membranes from phage-sensitive cells but not to membranes from the resistant strain, elimination of adsorption by pretreatment of the membranes with proteinase K, and lack of dissociation of ³⁵S from the membranes upon dilution. Following membrane adsorption, ejection of phage DNA occurred rapidly at 30°C but not at 4°C. These results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.     

Growth of Male-Specific Bacteriophage in Pasteurella Harboring F-Genotes Derived from Escherichia coli

When either the F' lac or the F'Cm plasmid was transferred from Escherichia coli into Pasteurella pseudotuberculosis, the P. pseudotuberculosis (F') strains isolated formed plaques with both ribonucleic acid (RNA)-containing and deoxyribonucleic acid-containing male-specific phages. In contrast, strains of P. pestis harboring E. coli (F') plasmids did not form plaques with male-specific phages, although such strains permitted limited multiplication of phage MS2. The adsorption and burst size of MS2 were approximately the same in both species of Pasteurella, but the per cent of adsorbed MS2 that produced infective centers was much lower in P. pestis than it was in P. pseudotuberculosis. By use of a sib-selection technique of P. pestis (F') cells, we isolated a single clone that could form MS2 plaques. (32)P-labeled MS2 adsorbed equally to and its RNA penetrated equally into both the typical MS2-nonpermissive P. pestis cells and the MS2-permissive P. pestis cells. No host modification occurred after growth of MS2 in Pasteurella. Our data suggest that typical strains of P. pestis inhibit the intracellular development of phage MS2.     

Interaction of the PhiHSIC virus with its host: lysogeny or pseudolysogeny?

The marine phage PhiHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at -80( degrees ) C and were lost. When HSIC was reinfected with phage PhiHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10(11) ml(-1)) in the absence of inoculating phage and yet reached culture densities of nearly 10(9) ml(-1). Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the PhiHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that PhiHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.     

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. Methods and Results: Phage–host interactions between eight EPS‐producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (φYsca, φ3, φ5, φ6, φ8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular‐producing strains were more sensitive to phage attacks than the noncapsular‐producing strains. Streptococcus thermophilus CRL1190 (capsular‐producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular‐negative, EPS low‐producing mutant of strain CRL1190 was reduced, especially for φYcsa and φ8. Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. Significance and Impact of the Study: Capsular‐producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.