Marlena Kłak

The influence of external factors on bacteriophages—review

The ability of bacteriophages to survive under unfavorable conditions is highly diversified. We summarize the influence of different external physical and chemical factors, such as temperature, acidity, and ions, on phage persistence. The relationships between a phage’s morphology and its survival abilities suggested by some authors are also discussed. A better understanding of the complex problem of phage sensitivity to external factors may be useful not only for those interested in pharmaceutical and agricultural applications of bacteriophages, but also for others working with phages.

Clinical aspects of phage therapy.

Phage therapy (PT) is a unique method of treatment of bacterial infections using bacteriophages (phages)-viruses that specifically kill bacteria, including their antibiotic-resistant strains. Over the last decade a marked increase in interest in the therapeutic use of phages has been observed, which has resulted from a substantial rise in the prevalence of antibiotic resistance of bacteria, coupled with an inadequate number of new antibiotics. The first, and so far the only, center of PT in the European Union is the Phage Therapy Unit (PTU) established at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland in 2005. This center continues the rich tradition of PT in Poland, which dates from the early 1920s. The main objective of this chapter is to present a detailed retrospective analysis of the results of PT of 153 patients with a wide range of infections resistant to antibiotic therapy admitted for treatment at the PTU between January 2008 and December 2010. Analysis includes the evaluation of both the efficacy and the safety of PT. In general, data suggest that PT can provide good clinical results in a significant cohort of patients with otherwise untreatable chronic bacterial infections and is essentially well tolerated. In addition, the whole complex procedure employed to obtain and characterize therapeutic phage preparations, as well as ethical aspects of PT, is discussed.

Phage as a modulator of immune responses: practical implications for phage therapy.

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

Phage as a Modulator of Immune Responses

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.

The perspectives of the application of phage therapy in chronic bacterial prostatitis

Chronic bacterial prostatitis (CBP) is a long-lasting and crippling disease that strongly impacts the patients quality of life. The diagnosis of CBP is difficult and the treatment regimens are not always successful. Poor penetration of antibiotics to the prostate tissue, the drug resistance of uropathogens, the adverse events associated with antibiotic treatment, the persistence of prostatic calculi, and biofilm formation in the prostate gland are factors that contribute toward decreasing the cure rate of CBP. The phenomenon of increasing antibiotic resistance, which has also been called a clinical super-challenge, has revived interest in therapy using bacterial viruses (bacteriophages or phages). Because of their mechanism of action, which is completely different from those of all antibiotics, phages are effective even against multidrug-resistant bacteria. Here, we describe the current perspectives on the possible application of phage therapy (PT) in treating CBP. The advantages of therapeutic phages, including their interactions with bacterial biofilm, as well as the safety of PT are discussed.

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.

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.

Possible Use of Bacteriophages Active against Bacillus anthracis and Other B. cereus Group Members in the Face of a Bioterrorism Threat

Anthrax is an infectious fatal disease with epidemic potential. Nowadays, bioterrorism using Bacillus anthracis is a real possibility, and thus society needs an effective weapon to neutralize this threat. The pathogen may be easily transmitted to human populations. It is easy to store, transport, and disseminate and may survive for many decades. Recent data strongly support the effectiveness of bacteriophage in treating bacterial diseases. Moreover, it is clear that bacteriophages should be considered a potential incapacitative agent against bioterrorism using bacteria belonging to B. cereus group, especially B. anthracis. Therefore, we have reviewed the possibility of using bacteriophages active against Bacillus anthracis and other species of the B. cereus group in the face of a bioterrorism threat.

Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between “microbiological disappearance” and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.