Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland.

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood-a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.

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.

Means to Facilitate the Overcoming of Gastric Juice Barrier by a Therapeutic Staphylococcal Bacteriophage A5/80.

In this article we compare the efficacy of different pharmacological agents (ranitidine, and omeprazole) to support phage transit from stomach to distal portions of the gastrointestinal tract in rats. We show that a temporal modification of environment in the animal stomach may protect Twort-like therapeutic antistaphylococcal phage A5/80 (from bacteriophage collection of the Hirszfeld Institute of Immunology and Experimental Therapy PAS in Wroclaw, Poland) from the inactivation by gastric juice effectively enough to enable a significant fraction of orally administered A5/80 to pass to the intestine. Interestingly, we found that yogurt may be a relatively strong in enhancing phage transit. Given the immunomodulating activities of phages our data may suggest that phages and yogurt can act synergistically in mediating their probiotic activities and enhancing the effectiveness of oral phage therapy. We also demonstrate that orally applied phages of similar size, morphology, and sensitivity to acidic environment may differ in their translocation into the bloodstream. This was evident in mice in which a therapeutic staphylococcal phage A5/80 reached the blood upon oral administration combined with antacid agent whilst T4 phage was not detected even when applied in 103 times higher dose. Our findings also suggest that phage penetration from digestive tract to the blood may be species-specific.

Mammalian Host-Versus-Phage immune response determines phage fate in vivo.

Emerging bacterial antibiotic resistance draws attention to bacteriophages as a therapeutic alternative to treat bacterial infection. Examples of phage that combat bacteria abound. However, despite careful testing of antibacterial activity in vitro, failures nevertheless commonly occur. We investigated immunological response of phage antibacterial potency in vivo. Anti-phage activity of phagocytes, antibodies, and serum complement were identified by direct testing and by high-resolution fluorescent microscopy. We accommodated the experimental data into a mathematical model. We propose a universal schema of innate and adaptive immunity impact on phage pharmacokinetics, based on the results of our numerical simulations. We found that the mammalian-host response to infecting bacteria causes the concomitant removal of phage from the system. We propose the notion that this effect as an indirect pathway of phage inhibition by bacteria with significant relevance for the clinical outcome of phage therapy.

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes.

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

Biological activity of alpha-galactoside preparations from Lupinus angustifolius L. and Pisum sativum L. seeds.

Biological activity tests were performed on alpha-galactoside preparations obtained from Lupinus angustifolius L. cv. Mirela (alkaloid-rich) and Pisum sativum L. cv. Opal seeds. The studies included the following tests: acute toxicity, cytotoxic test, delayed type hypersensitivity (DTH), plaque-forming cell number (IgM-PFC), and influence on the growth of bifidobacteria and coliform presence in rat colon. Results of these studies showed that alpha-galactosides from lupin and pea seeds were essentially nontoxic. Their acute toxicity (LD(50)) in mice was >4000 mg kg(-1) of body weight. alpha-galactoside preparations were not cytotoxic for mouse thymocytes in vitro. The in vitro test shows that oligosaccharides from lupin and pea are utilized by selected beneficial colon bacterium strains. The in vivo experiment demonstrated that alpha-galactosides from legume significantly influenced the growth of bifidobacteria in rats colon. Simultaneously, the decrease of the coliform presence was observed. The chemical composition of the tested preparations had no significant effect on their biological activity.