Phage Lysates Research Articles

Protective Efficiency of Pasteurella multocida A:1 Bacteriophage lysate Vaccine against Homologous and Heterologous Challenge in Poultry

Fowl cholera (FC) caused by serotypes of Pasteurella multocida includes A:1, A:3, A:4 is a highly fatal septicemic disease. Preliminary trials of P. multocida A:1 bacteriophage lysate vaccine against FC was evaluated. Lytic phage and P. multocida ratio was standardized to obtain stable lysate batches. Consequently, three batches of lytic phage preparation were produced; estimation of protein and carbohydrate content amongst batches did not shown any significant variation indicating same batches can be produced by standardized procedure. Protective response trials in poultry with P. multocida A:1, A:3, A:4 against plain lysate and alum adsorbed lysate (1% alum) on vaccinated group showed both homologous and heterologous protection compared with inactivated whole cell group provided only homologous protection. Assessment of antibody response towards P. multocida A:1, A:3, A:4 antigen evaluated by Indirect Haemagglutination test (IHA) reveals presence of protective antibody titer.

Effect of Carbon Sources on Stability and Competition of T7aiiA Phage Lysates

Abstract Biofouling is a pervasive challenge in industrial and medical settings with enormous economic and health impacts. Use of quorum-quenching phages is a potential solution in either combating the biofilm or inhibiting biofilm formation when an engineered phage is designed for a specific bacterial system. In the event of a real-life application of synthetic phage, it is necessary to consider the effect of environmental conditions on the synthetic phage. This study focused on both (1) stability of phage lysate under different types of carbon sources and (2) competition of the engineered phage in comparison to a wild-type (wt) phage. Optimal results were found (in plaque forming units [PFUs]) with glucose as a carbon source and were kept stable for almost 30 days. Results suggested that competition happens before 4 h. After that time, polymerase chain reaction results from PFU samples showed an increase in T7aiiA, which indicates that these conditions favored the phage replication of the engineered pha...

Some immunological studies on a new vaccine derived frombacteriophage lysate of Pasteurella multocida in mice

The study was undertaken to evaluate the humoral immune response in mice induced by a novel phage lysate vaccine against Hemorrhagic Septicemia developed from Pasteurella multocida grown under iron-restricted conditions and lysed by a bacteriophage. Two groups of mice were immunized with lysate vaccine (LV) and conventional killed HS vaccine (CV) and blood was collected at various days post – immunization (DPI). The serum protein concentration in lysate vaccinated mice (4.675±0.223) was significantly higher (p less than 0.05) than the conventional vaccinated mice (4.100±0.282) at 150 DPI. The serum globulin levels at 90 DPI and 180 DPI in LV mice (1.330±0.071 and 0.650±0.100) were significantly (p less than 0.01) higher than the CV mice (0.850±0.084 and 0.366±0.098). The serum IgG levels at 150 DPI and 180 DPI in LV (0.564±0.188 and 0.485±0121) mice were significantly higher (p less than 0.01) than the CV mice (0.178±0.039 and 0.121±0.026). Although our results in mice are promising, further studies involving bovines are needed.

In Vivo Studies on the Influence of Bacteriophage Preparations on the Autoimmune Inflammatory Process.

Phage preparations used for phage therapy may have not only direct antibacterial action but also immunomodulating effects mediated by phages themselves as well as by bacterial antigens. Therefore phage application in patients with immune disorders, and especially with autoimmune diseases, requires special attention. The aim of this study was to investigate the effect of phage lysates (staphylococcal phages A3/R, phi200, and MS-1 cocktail, enterococcal phage 15/P, Pseudomonas phage 119x, and E. coli T4 phage) as well as purified T4 phage on the course of murine collagen-induced arthritis (CIA), commonly used as an animal model of rheumatoid arthritis. Intraperitoneal application of phage lysates or purified T4 phage did not aggravate the course of autoimmune joint disease. Moreover, although endotoxins are known to potentiate CIA, the systemic administration of phage lysate of Pseudomonas aeruginosa, which contains debris of this Gram-negative bacillus, did not significantly influence CIA although the sonicate of the corresponding bacterial strain did. Interestingly, a purified T4 phage revealed some anti-inflammatory activity when applied under the therapeutic scheme. Our preliminary results do not suggest that phages may aggravate the symptoms of rheumatoid arthritis. In contrast T4 phage may even exert an immunosuppressive effect.

Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization

Brucellosis is an economically important zoonosis of worldwide significance. Earlier (Jain et al., 2015) we reported methodology for generation of phage lysate preparations against Brucella abortus S19 using brucellaphage ‘ϕLd’. In this study, using a fixed dose (Two mouse PD100) of lysates, the prophylactic efficacies of both plain and alum gel adjuvanted lysates were evaluated in guinea pig by direct virulent challenge and passive mouse protection test (PMPT). Strong humoral and cell mediated immune responses in guinea pigs and protection comparable to S19 vaccine was observed with low dose (1.0 μg protein and 120 μg carbohydrate adsorbed on 0.1% aluminium gel). Passive transfer of antibodies to mice using d 90 post immunization sera of guinea pig protected the animals against challenge. The study suggested the significance of humoral immunity in murine brucellosis. Further, the methodology can be explored to produce a new class of immunotherapeutic agents against bovine brucellosis.

Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) Phages From Sewage at a Tertiary Pediatric Hospital

Background: The methicillin-resistant Staphylococcus aureus (MRSA) strain is now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages are a potential alternative treatment for MRSA infections. Objectives: The objective of this study was the isolation and characterization of MRSA phages from the sewage at a tertiary pediatric hospital. Methods: Two MRSA strains were isolated from patient blood. The isolated strains were confirmed as MRSA through the conventional methods. Phages were then isolated from the sewage at the tertiary pediatric hospital. Lytic activity was determined with a spot test, while the titers of phage lysates were measured using the DLA technique. The morphology was assessed using electron microscopy, and the latent period time and burst size were determined. Results: Electron microscopy showed MRSA phages’ resemblance to members of the family Siphoviridae, serogroups A and F. They exhibited a latent period of 70 minutes and a relatively burst size of 2,400 plaque-forming units (PFU)/infected cell. The titer of the phages was determined to be 3 × 109 PFU/mL using the by DLA technique. Conclusions: In this study, two phages from the family Siphoviridae were isolated and characterized from sewage at a tertiary pediatric hospital; these phages specifically target MRSA. Their presence may be one reason for the transfer of antibiotic resistance or virulence factors, resulting in transduction and the conversion of nonvirulent strains of Staphylococcus to virulent strains. Considering that phages through can be transferred to wards in an air-born manner, it is better to create negative pressure in the sewage pipes at hospitals until the phages can no longer transfer to the wards via aerosols.

Selection of polyvalent bacteriophages infecting Salmonella enterica serovar Choleraesuis

Background: Ideally, bacteriophages of pathogenic bacterial hosts should be polyvalent to be able to replicate in an alternative nonpathogenic bacterium. Thus, accidental infection by the original host can be avoided when bacteriophage lysates are used in biocontrol protocols. Results: From 15 wastewater samples, collected at different sites in the V Region in Chile, we selected three bacteriophages (FC, FP, and FQ) capable of productively infecting Salmonella enterica serovar Choleraesuis. By transmission electron microscopy (TEM) observation, the bacteriophages were found to belong to the order Caudoviridae. Molecular analyses indicated that FC, FP, and FQ contained double-stranded DNA genomes, of sizes similar to bacteriophage P22, and distinct recognition sites for the restriction endonucleases HaeIII and HindIII. Assays of host range revealed that the bacteriophages were polyvalent and thus capable of infecting different strains of Escherichia coli and other serovars of Salmonella . Conclusion: We have isolated new bacteriophages of the serovar Choleraesuis with various potential applications in relation to this pathogenic bacterium.

Use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)® for the Control of Recurrent Pyoderma Eczema in Dogs with Atopic Dermatitis

Background: Recurrent staphylococcal infections are frequent in dogs with atopic dermatitis (AD). Many factors seem to contribute to making bacterial pyoderma refractory to treatment. Short-term systemic antibiotic therapy is effective for the treatment of acute symptoms, and may, along with pulsatile therapy, contribute to the long-term control of the disease. However, microbial resistance has become a growing and alarming problem. The aim of this study was to evaluate whether the use of Staphylococcus aureus Phage Lysate Staphage Lysate (SPL)®, can minimize the symptoms of recurrent pyoderma and increase the interval between acute atopic manifestations in dogs.Materials, Methods & Results: Thirteen dogs with a history of Canine Atopic Dematitis (CAD) and recurrent bacterial pyoderma received SPL at increasing intervals for 23 weeks. The contents of an intact pustule of each dog was collected and submitted to microbiological analysis. Systemic antibiotic therapy was established for the first 4-6 weeks of SPL protocol, based on the antibiotic sensitivity tests. The animals included in the study underwent a therapeutic protocol receiving shots of 0.5 mL of SPL subcutaneously (SC) twice a week for the first 12 weeks; 1.0 mL of SPL (SC) once a week for four weeks; 1.0 mL of SPL (SC) once every 15 days; 1.0 mL of SPL (SC) after a three-week interval from the last dose on week 20, until final observation at week 26, with no application. The animals underwent clinical examination every week and the evaluation of pruritus was used according Rybnicek et al. During the therapeutic protocol with SPL, a significant decline in the pruritus was observed in the treated dogs (P < 0.05). In week 1, the mean pruritus index was 7.33 on the Rybnicek scale; in weeks 12 and 23, the mean indices were 2.41 and 1.91. An effectiveness of 83.33% for the control of pruritus along with regression of the lesions was observed.Discussion: Before treatment, the selected animals presented worsening of the pruritus during the pyoderma eczema episodes (pruritic), resulting in the emergence of a vicious cycle where the pruritus induced the appearance of new lesions, requiring the use of antibiotics for a long period. During the therapeutic protocol with SPL, a significant decline in the pruritus was observed in the treated dogs. The control of pruritus associated with pyoderma eczema of the dogs in this study before the vaccination protocol with SPL was satisfactory when they were subjected to antibiotic therapy; however, after suspending therapy, the bacterial infections recurred, on average, after 2-4 weeks. On the other hand, with the use of SPL, the animals were recurrence-free until the end of the experimental protocol. This was attributed to the antibiotic therapy administered at the beginning of the protocol, as this led to a regression of the bacterial pyoderma and involution of the lesions. However, after suspending antibiotics, it was observed that, by the end of the study, 83.33% of the dogs still had a low level of pruritus, few or no lesions, which were considered acceptable to most owners. At this moment none of these patients needed to be subjected to antibiotic treatment. The sums of the scores for the dogs on weeks 1, 12, and 23 were 53.33, 4.41, and 3.5, respectively, indicating significant improvements of the lesions, showing that the proposed protocol with SPL was able to prevent new episodes of pyoderma.

Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544

The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage ‘ϕLd’ was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 108 CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 μg protein and 60 μg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.

Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 103 and 105 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 103-105 EU/109 PFU (plaque forming units) down to an average of 2.8 EU/109 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa).

Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.

Phage therapy as an approach to prevent Vibrio anguillarum infections in fish larvae production.

Fish larvae in aquaculture have high mortality rates due to pathogenic bacteria, especially the Vibrio species, and ineffective prophylactic strategies. Vaccination is not feasible in larvae and antibiotics have reduced efficacy against multidrug resistant bacteria. A novel approach to controlling Vibrio infections in aquaculture is needed. The potential of phage therapy to combat vibriosis in fish larvae production has not yet been examined. We describe the isolation and characterization of two bacteriophages capable of infecting pathogenic Vibrio and their application to prevent bacterial infection in fish larvae. Two groups of zebrafish larvae were infected with V. anguillarum (∼106 CFU mL−1) and one was later treated with a phage lysate (∼108 PFU mL−1). A third group was only added with phages. A fourth group received neither bacteria nor phages (fish control). Larvae mortality, after 72 h, in the infected and treated group was similar to normal levels and significantly lower than that of the infected but not treated group, indicating that phage treatment was effective. Thus, directly supplying phages to the culture water could be an effective and inexpensive approach toward reducing the negative impact of vibriosis in larviculture.

161P - Effect of E.Coli Phage Lysate Vaccinations on Cd4 + Cd25 + Foxp3+ T-Regulatory Cells and Cytokine Levels in Cancer Bearing Mice

ABSTRACT Aim: Regulatory T cells (Treg) play a key role in maintaining the balance of immune responses in human health and in disease. Generally, Tregs are considered as the most powerful inhibitors of antitumor immunity and the greatest barrier to successful immunotherapy and vaccinations. Modulation of Tregs can enhance the efficacy of cancer immunotherapy and has potential to result in tumor regression. The aim of this study was to stimulate antitumor innate immunity via suppression of Treg and activation of effector immune cells using E.coli phage lysate vaccinations. Methods: Specimen (spleen, blood serum) were obtained from control (with Ehrlich carcinoma) and E.coli phage lysate vaccinated (0,25 ml/day, with 5 day intervals, during 5 weeks) 20–25 g C57BL/6J mice. Number of CD4+CD25+FoxP3+ Treg as well as CD3+CD4+, CD3+CD8+, NK1.1 cells were assessed using FACS Array Bioanalyzer (BD FACSCalibur, USA) and WST-8 Cell Proliferation Assay Kit. Isolated cells were stained by fluorochrome conjugated antibodies (anti-CD3, Foxp3, NK1.1, anti-CD4, anti-CD8, anti-CD25 and isotype-matched controls). Two-color data analysis for CD4CD8, CD3CD4, CD4CD25, CD25Foxp3 staining was performed using dot plots with WinMDI Software. Cytokines: IL-12, TGF-b, IFN-γ were studied by ELISA according to manufactures protocol (RD BD Biosciences). Results: Investigations have shown that E.coli phage lysate vaccinations stimulate innate and further acquired immunity manifested by increase in NK1.1, CD3CD4, CD3CD8 cells and secretion of IL-12, IFN-γ with further decrease in TGF-b, CD4CD25, CD25Foxp3 compared to control. More than 5 vaccinations had no effect and further vaccinations decreased immune response. Our results indicate that although vaccinations enhance antitumor immunity and host defenses decreasing Tregs and stimulating CD3+CD4+, CD3+CD8+, NK1.1 effector immune cells, the successful treatment requires optimal regimen of vaccination. Conclusions: E.coli phage lysate vaccinations have in vivo antitumor immunomodulatory effect and can be considered as a new immunotherapeutic approach of cancers. Disclosure: All authors have declared no conflicts of interest.

Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

We investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 10(9) to 10(10) PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

Properties of Klebsiella phage P13 and associated exopolysaccharide depolymerase

The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide (EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL−1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL−1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60°C and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.

High efficiency generalized transduction in Escherichia coli O157:H7

Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes ( stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

Isolation and partial characterization of lytic phage against Salmonella Abortusequi

Aim: To use partially characterized phages for generation of phage lysates having potential to be developed as vaccines against Salmonella Abortusequi. Material and Methods: Phage/s isolation from several sewage samples was attempted against Salmonella Abortusequi strains procured from the National Salmonella Reference Centre, IVRI using enrichment and agar overlay technique. A concentrated stock of the phage was prepared after its purification. The lytic range, morphology and DNA size were determined. Results: Two lytic phages could be isolated and purified. The phage with wider lytic range was found to possess a hexagonal head with a very short tail. The DNA size was found to be 21kb. Conclusion: The investigation reports first isolation of a lytic phage against Salmonella Abortusequi. The phage can be used in future for development of phage lysate based immune therapeutic agent against equine abortions.

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.

Thermal treatment of the bacteriophage lysate of Klebsiella pneumoniae B5055 as a step for the purification of capsular depolymerase enzyme

The lytic bacteriophage which produces the hydrolase enzyme capable of depolymerizing exopolysaccharide of Klebsiella pneumoniae and of other bacteria was isolated earlier. In this study, a simple method of depolymerase purification from the phage lysate by dissociating the enzyme from the phage particle was developed. The bacteriophage showed a relatively smaller plaque size surrounded by a wide halo indicating a depolymerase action on the capsular polysaccharide of K. pneumoniae B5055. The depolymerase activity was associated predominantly with the phage particles. Different methods have been used by various researchers to dissociate the enzyme associated with phage particles either by exposure to chemicals or by altering the environmental conditions. In this study, the potential application of thermal treatment of the bacteriophage lysate was evaluated as a step for the purification of depolymerase in comparison to the mild acid treatment method of Rieger et al. (1975). The results showed that the relative thermal stability of phage depolymerase at 60°C for 30min was the basis for harvesting the enzyme leading to disintegration of all phage particles in the lysate. Both thermal and mild acid treatment resulted in comparable enzyme levels, however; mild acid treatment appeared to be cumbersome and cause chemical contamination.

Following Cell-fate in <em>E. coli</em> After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.