Comparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapy

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 µm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

Active Immunization with Pasteurella multocida Lysate Elicits Antibody that Protects Rabbits against Virulent Pasteurella multocida and Protects Mice by Passive Immunization

Background: Pasteurellosis is an economically important disease of livestock worldwide and vaccination is effective mean to control the disease outbreaks. One of alternative method for vaccine preparation could be the use of lytic phages as bacterial inactivating agents and employing “phage lysate immunogen” as an immunizing agent. Therefore, the study was undertaken to explore the protective immune response of novel phage lysate vaccine against Pasteurella multocida. Methods: A phage lysate vaccine of Pasteurella multocida was prepared using isolated lytic phage and subjected to vaccine response study in rabbits. In this experiment, two mouse fixed-dose of lysate was used and the prophylactic efficacies of lysates were evaluated in rabbits by passive mouse protection test (PMPT), western blotting and direct virulent challenge.Result: Passive transfer of antibodies to mice using post-immunization sera of rabbits protected the animals against the challenge with A: 1 strain (75%) and B: 2 strain (50%). In western blotting, a total number of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The study suggested the significance increase in humoral immunity by phage lysate in murine Pasteurellosis.

Bacteriophages: A Potential New Therapeutic Alternate to Antibiotics to Treat Chronic Septic Wounds in Large Animals

Background: Overuse of antibiotics is a major problem in the treatment of chronic infections. The antibiotic treatment is frequently non-curative due to emergence of antibiotic resistance, thus alternative treatment is necessary. The natural ability of pathogens to develop resistance is not only a threat to animal health but also leads to accumulation of antibiotic resistance determinants or resistant bacteria livestock products. Phage therapy has been developed as an alternative therapeutic approach against antibiotic resistance microorganisms.Methods: In present study 150 samples of sewage were collected from livestock farm, NDVSU, Jabalpur (M.P.). Bacteriophages were isolated and characterized from the collected sewage samples. “Cocktail” of the recovered phage lysate isolates (ØVS1, ØVS5, ØVS9 and ØVS27) was prepared for assessment of therapeutic utility of S. aureus phages in chronic septic infections. Therapeutic trial was performed in large animals (cattle and buffalo) at livestock farm, Adhartal, NDVSU, Jabalpur (MP.). Pathogens isolated from chronic septic wounds were showing antibiotic resistance but the bacteria were sensitive to phage lysate. Phage ‘cocktail’ was applied topically over the chronic septic wounds as single dose. Follow up of the cases was done at regular intervals (0, 5 and 10 days) which included clinical examination of wound by appearance, status of discharge and formation of granulation tissue.Result: Six animals showed complete recovery (60%) out of 10 animals from wounds as predicted by progressive healing status of wounds till the formation of granulation tissue within 10 days after phage application. Thus, recovery was much higher than the conventional antibiotic therapy normally used for the treatment of chronic septic wound infections in animals.

BACTERIOPHAGE PREPARATION ENGINEERING FOR BIOCONTROL OF PSEUDOMONAS SYRINGAE IN CROP SCIENCE

Phytopathogenic bacteria Pseudomonas syringae cause diseases of many cultivated plants, causing tumor neoformation, rot, chlorosis, necrosis, etc . The advanced biological mean to control bacteriosis in crop science is bacteriophages. In this work full biological characteristic of 8 bacteriophages is shown, active according to Pseudomonas syringae. The studied phages formed similar negative colonies- clear, rounded, in diameter of 5-9 mm. Lytic actifity of phages Pseudomonas syringae by Appelman from 10-4 to 10-8; by Gratia from 1,0±0,1×106 to 2,0±0,1×109 (BFU/ml). Bacteriophages Ps.s-7 UlGAU, Ps.s-13 UlGAU and Ps.s-27 UlGAU did not change lytic activity when storing in fridge during 12 months. Lytic activity of phages Ps.s-1 UlGAU, Ps.s-8, Ps.s-15 UlGAU, Ps.s-30 UlGAU, Ps.s-77 UlGAU in the same conditions fell within 1-2 orders. Spectrum of lytic activity of phages varied from 21,4% (Ps.s-13 UlGAU) to 85,7% (Ps.s-7 UlGAU, Ps.s-27 UlGAU). The study of phage specificity on 15 species og heterologous cultures showed that phages are specific for Pseudomonas syringae. Phages are moderately stable to heating and lose activity during 30 –minute temperature effect above 62°С. The optimal way to relieve phage lysate from living cells of Pseudomonas syringae was thrichloromethane at a ratio of 10:1and temporal exposition 45 minutes. On the basis of obtained data we determined capacity of each bacteriophage for the use as biocontrol agent. For further research for the study of molecular genetic characteristics we selected advanced strains of phages Pseudomonas syringae - Ps.s-7 UlGAU и Ps.s-27 UlGAU.

Temperate Bacteriophages from ChronicPseudomonas aeruginosaLung Infections Show Disease-Specific Changes in Host Range and Modulate Antimicrobial Susceptibility

ABSTRACTTemperate bacteriophages are a common feature ofPseudomonas aeruginosagenomes, but their role in chronic lung infections is poorly understood. This study was designed to identify the diverse communities of mobileP. aeruginosaphages by employing novel metagenomic methods, to determine cross infectivity, and to demonstrate the influence of phage infection on antimicrobial susceptibility. Mixed temperate phage populations were chemically mobilized from individualP. aeruginosa, isolated from patients with cystic fibrosis (CF) or bronchiectasis (BR). The infectivity phenotype of each temperate phage lysate was evaluated by performing a cross-infection screen against all bacterial isolates and tested for associations with clinical variables. We utilized metagenomic sequencing data generated for each phage lysate and developed a novel bioinformatic approach allowing resolution of individual temperate phage genomes. Finally, we used a subset of the temperate phages to infectP. aeruginosaPAO1 and tested the resulting lysogens for their susceptibility to antibiotics. Here, we resolved 105 temperate phage genomes from 94 lysates that phylogenetically clustered into 8 groups. We observed disease-specific phage infectivity profiles and found that phages induced from bacteria isolated from more advanced disease infected broader ranges ofP. aeruginosaisolates. Importantly, when infecting PAO1in vitrowith 20 different phages, 8 influenced antimicrobial susceptibility. This study shows thatP. aeruginosaisolated from CF and BR patients harbors diverse communities of inducible phages, with hierarchical infectivity profiles that relate to the progression of the disease. Temperate phage infection altered the antimicrobial susceptibility of PAO1 at subinhibitory concentrations of antibiotics, suggesting they may be precursory to antimicrobial resistance.IMPORTANCEPseudomonas aeruginosais a key opportunistic respiratory pathogen in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. The genomes of these pathogens are enriched with mobile genetic elements including diverse temperate phages. While the temperate phages of the Liverpool epidemic strain have been shown to be active in the human lung and enhance fitness in a rat lung infection model, little is known about their mobilization more broadly acrossP. aeruginosain chronic respiratory infection. Using a novel metagenomic approach, we identified eight groups of temperate phages that were mobilized from 94 clinicalP. aeruginosaisolates. Temperate phages fromP. aeruginosaisolated from more advanced disease showed high infectivity rates across a wide range ofP. aeruginosagenotypes. Furthermore, we showed that multiple phages altered the susceptibility of PAO1 to antibiotics at subinhibitory concentrations.

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.