Freeze-Drying of Encapsulated Bacteriophage T4 to Obtain Shelf-Stable Dry Preparations for Oral Application.

Therapeutic application of bacterial viruses (phage therapy) has in recent years been rediscovered by many scientists, as a method which may potentially replace conventional antibacterial strategies. However, one of the main problems related to phage application is the stability of bacterial viruses. Though many techniques have been used to sustain phage activity, novel tools are needed to allow long-term phage storage and application in versatile forms. In this study, we combined two well-known methods for bacteriophage immobilization. First, encapsulated phages were obtained by means of extrusion-ionic gelation, and then alginate microspheres were dried using the lyophilization process (freeze-drying). To overcome the risk of phage instability upon dehydration, the microspheres were prepared with the addition of 0.3 M mannitol. Bacteriophage-loaded microspheres were stored at room temperature for 30 days and subsequently exposed to simulated gastric fluid (SGF). The survival of encapsulated phages after drying was significantly higher in the presence of mannitol. The highest number of viable bacteriophages exceeding 4.8 log10 pfu/mL in SGF were recovered from encapsulated and freeze-dried microspheres, while phages in lyophilized lysate were completely inactivated. Although the method requires optimization, it may be a promising approach for the immobilization of bacteriophages in terms of practical application.

High value-added products derived from crude glycerol via microbial fermentation using Yarrowia clade yeast.

BACKGROUND: Contemporary biotechnology focuses on many problems related to the functioning of developed societies. Many of these problems are related to health, especially with the rapidly rising numbers of people suffering from civilization diseases, such as obesity or diabetes. One factor contributing to the development of these diseases is the high consumption of sucrose. A very promising substitute for this sugar has emerged: the polyhydroxy alcohols, characterized by low caloric value and sufficient sweetness to replace table sugar in food production. RESULTS: In the current study, yeast belonging to the Yarrowia clade were tested for erythritol, mannitol and arabitol production using crude glycerol from the biodiesel and soap industries as carbon sources. Out of the 13 tested species, Yarrowia divulgata and Candida oslonensis turned out to be particularly efficient polyol producers. Both species produced large amounts of these compounds from both soap-derived glycerol (59.8-62.7 g dm-3) and biodiesel-derived glycerol (76.8-79.5 g dm-3). However, it is equally important that the protein and lipid content of the biomass (around 30% protein and 12% lipid) obtained after the processes is high enough to use this yeast in the production of animal feed. CONCLUSIONS: The use of waste glycerol for the production of polyols as well as utilization of the biomass obtained after the process for the production of feed are part of the development of modern waste-free technologies.

Bacteriophage amplification - A comparison of selected methods.

The bactericidal properties of bacteriophages have been used almost since the moment of the discovery of bacterial viruses. In the light of the rapidly growing number of antibiotic-resistant bacteria, phage therapy is considered one of the most promising alternatives to classical treatment. Phage amplification is one of the most common procedures of working with phages, and high-titer preparations are beneficial at the experimental stage of studies as well as in practice. The objective of this study was to compare five commonly applied methods of phage amplification: (i) pooled plaques method, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage.

The Efficacy of Isolated Bacteriophages from Pig Farms against ESBL/AmpC-Producing Escherichia coli from Pig and Turkey Farms.

Extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases are plasmid (but also chromosomally) encoded enzymes found in Enterobacteriaceae, determining resistance to a variety of important antibiotics including penicillins, cephalosporins, and monobactams. In recent decades, the prevalence of ESBL/AmpC-producing bacteria has increased rapidly across the world. Here, we evaluate the potential use of bacteriophages in terms of a reduction of antibiotic-resistant bacteria in healthy animals. The aim of our studies was to isolate bacteriophages capable of destroying ESBL/AmpC-producing Escherichia coli isolated from livestock habitats. The efficacy of isolated phages against ESBL/AmpC E. coli strains varies, but creation of a phage cocktail with broad activity spectrum is possible. This may indicate that the role of phages may not be limited to phage therapy, but bacterial viruses may also be applied against spread of bacteria with antibiotic resistance genes in the environment. We also addressed the hypothesis, that phages, effective for therapeutic purposes may be isolated from distant places and even from different environments other than the actual location of the targeted bacteria. This may be beneficial for practical purposes, as the construction of effective phage preparations does not require access to disease outbreaks.

Influence of bacteriophage preparations on migration of HL-60 leukemia cells in vitro.

BACKGROUND: Bacteriophage therapy is considered one of the most attractive alternatives to antibiotic treatment, which may be significant due to the rising number of antibiotic-resistant bacterial strains. Patients with cancer frequently suffer bacterial infections resulting from immunosuppression caused by anticancer treatment; thus they constitute a considerable group of patients subjected to phage therapy. In this study, we investigated the influence of bacteriophages on the migration of human leukemia (HL-60) cells. Results of these studies provide data regarding phage treatment of patients with cancer, especially with this type of leukemia. MATERIALS AND METHODS: The influence of phage preparation on migration of HL-60 leukemia cells was evaluated with BD Bioscience Migration Chambers. RESULTS: Bacteriophages have no influence on migration of HL-60 cells. The only phage preparation which stimulated migration of HL-60 cells was Staph.liz, specific to S. aureus, however, the molecular basis of these interactions cannot be currently explained. CONCLUSION: Results of our studies may be in line with previous data indicating that phage therapy is safe for patients with cancer.

Influence of bacteriophage preparations on intracellular killing of bacteria by human phagocytes in vitro.

Bacteriophages are viruses that infect bacteria. It was shown that bacteriophage therapy is an effective method of combating bacterial infections, including infections caused by antibiotic-resistant bacterial strains. One of the main obstacles to widespread use of phage preparations is limited knowledge regarding the influence of bacteriophages on human organisms. In our study, we evaluated whether application of phage preparations impair bactericidal activities of human phagocytes (granulocytes and monocytes). In our study, we used preparations of phages T2 and T4 specific to Escherichia coli and A3 phage specific to Staphylococcus aureus. We found that bacteriophage preparations do not influence intracellular killing of bacteria by human phagocytes. The effect is irrespective of phage preparation type (lysate, purified phage preparation), phage titer of the preparation, and whether bacteria phagocytosed by phagocyte cells are sensitive or insensitive to phage (bacteriophages homologous and heterologous to bacteria). Although the results of our study are preliminary, they support previous data indicating safety of therapeutic application of phages.

Bacteriophages and cancer.

Bacteriophages can be used effectively to cure bacterial infections. They are known to be active against bacteria but inactive against eukaryotic cells. Nevertheless, novel observations suggest that phages are not neutral for higher organisms. They can affect physiological and immunological processes which may be crucial to their expected positive effects in therapies. Bacteriophages are a very differentiated group of viruses and at least some of them can influence cancer processes. Phages may also affect the immunological system. In general, they activate the immunological response, for example cytokine secretion. They can also switch the tumor microenvironment to one advantageous for anticancer treatment. On the other hand, bacteriophages are used as a platform for foreign peptides that may induce anticancer effects. As bacterial debris can interfere with bacteriophage activity, phage purification is significant for the final effect of a phage preparation. In this review, results of the influence of bacteriophages on cancer processes are presented which have implications for the perspective application of phage therapy in patients with cancer and the general understanding of the role of bacteriophages in the human organism.

The effects of staphylococcal bacteriophage lysates on cancer cells in vitro.

Bacteriophages constitute a serious alternative to antibiotic therapy of bacterial infections. They are also extremely numerous entities: phages can be found in almost all places on Earth and are constantly present in human and animal bodies. Observations of the effect of therapeutic staphylococcal phages and their bacterial hosts on melanoma migration in vitro are reported in this article. Together with bacteriophage preparations, disrupted Staphylococci (host strains) were investigated to compare the effects of bacteria with those of bacteriophages. Migration was decreased by all the investigated preparations in various ways and this was rather due to the activity of the bacterial components. Importantly, none of the investigated bacteriophage or bacterial preparations induced an increase in the migration activity of melanoma cells, which is important from the perspective of the therapeutic use of phage lysates. The possible presence of staphylococcal enterotoxins in the therapeutic bacteriophage preparations was also verified. All the studied therapeutic bacteriophage preparations were negative for the Staphylococcal enterotoxins A, B, C, D, and E (i.e., the enterotoxin content was less than 0.2-0.5 ng/ml).

Bacteriophage interactions with phagocytes and their potential significance in experimental therapy.

Bacteriophages are among the most numerous creatures on earth and they are omnipresent. They are thus in constant natural contact with humans and animals. However, the clinical and technological use of bacteriophages has also become more frequent, which is why all aspects of phage-mammal interactions need to be explored. Bacteriophages are able to interact with mammalian phagocytes. They may inhibit the phagocytosis of bacteria, but they may also undergo phagocytosis themselves. The ability of bacteriophages to reduce reactive oxygen species production by polymorphonuclear leukocytes in the presence of bacteria or their endotoxins was also confirmed. Studies show that the high immunogenicity of bacteriophages may also be employed in anti-tumor treatment. The present knowledge of phage interactions with cellular components of the mammalian immune system is sparse and insufficient, especially considering the increasing interest in the application of these viruses in human life. We believe that continuation of such research is indispensable.

The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration.

BACKGROUND: The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes. RESULTS: In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed. CONCLUSION: We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.