Marzanna Łusiak-Szelachowska

Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae

BackgroundMembers of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.MethodsElectron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.Results and conclusionsOf the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Bacteriophage Procurement for Therapeutic Purposes

Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented.

Phages and immunomodulation

In the past years, the microbiome and its role in the pathophysiology of diseases have gained great interest. The progress of our knowledge in this field opens completely novel prospects for treating disorders, including those which are most challenging to medicine today. Of special interest are studies on the interactions of the microbiome with the immune system. Only recently has the presence of bacteriophages in the microbiome been highlighted, and their potential role in maintaining normal immunity has gained increasing attention. We summarize the available data pointing to the potential impact of phages in maintaining immunological homeostasis.

Bacteriophages in the gastrointestinal tract and their implications

The gut microbiota plays an essential role in health and disease of humans. Bacteriophages are the most abundant members of the gut virobiota and display great diversity. Phages can translocate through the mucosa to lymph and internal organs and play a role as regulators of the bacterial population in the gut. Increasing abundance of phages in the gut mucosa may reduce colonization by bacteria. Moreover, phages may have an immunomodulatory role in the immune response in the human gut. The role of phages in inflammatory bowel disease (IBD) remains unknown. Phages may take part in the development of IBD, but there are also data suggesting the protective role of phages in the gut of patients with IBD. Furthermore, recent data suggest that phages may mediate the beneficial effects of fecal microbiota transplantation (FMT). Therefore, evidence is accumulating to highlight the protective immunomodulating activity of the gut phages.

Bacteriophages targeting intestinal epithelial cells: a potential novel form of immunotherapy

In addition to their established role as a physical barrier to invading pathogens and other harmful agents, intestinal epithelial cells (IEC) are actively involved in local immune reactions. In the past years, evidence has accumulated suggesting the role of IEC in the immunopathology of intestinal inflammatory disorders (IBD). Recent advances in research on bacteriophages strongly suggest that—in addition to their established antibacterial activity—they have immunomodulating properties that are potentially useful in the clinic. We suggest that these immunomodulating phage activities targeting IEC may open novel treatment perspectives in disorders of the alimentary tract, particularly IBD.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wrocław, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics

Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes—contained in granules—that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy. The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy.

The Potential of Phage Therapy in Sepsis

Sepsis remains a difficult clinical challenge, since our understanding of its immunopathology is incomplete and no efficacious treatment currently exists. Its earlier stage results from an uncontrolled inflammatory response to bacteria while in the later stage disturbed immune response with immunodeficiency syndrome develops. More than a hundred of clinical trials have not provided an efficient therapy which could ascertain an improvement or cure. Recent advancements in immunobiology of bacterial viruses (phages) indicate that in addition to their well-known antibacterial action phages have potent immunomodulating properties. Those data along with preliminary observations in experimental animals and the clinic strongly suggest that clinical trials on the efficacy of phages in sepsis are urgently needed.

Phage therapy in allergic disorders

Allergic disorders pose a growing challenge to medicine and our society. Therefore, novel approaches to prevention and therapy are needed. Recent progress in studies on bacterial viruses (phages) has provided new data indicating that they have significant immunomodulating activities. We show how those activities could be translated into beneficial effects in allergic disorders and present initial clinical data that support this hope. Impact statement Allergic disorders pose a growing challenge to medicine and our society, so new approaches to prevention and therapy are urgently needed. Our article summarizes progress that has been recently made and presents a shift in our understanding of the immunobiological significance of bacterial viruses (phages). Currently, phages may be considered not only as mere “bacteria eaters” but also as regulators of immunity. The new understanding of phages as important factors in maintenance of immune homeostasis opens completely new perspectives for their use in controlling aberrant immune responses. It is likely that this new knowledge could be translated into novel means of immunotherapy of allergic disorders.

Phage Therapy: What Have We Learned?

In this article we explain how current events in the field of phage therapy may positively influence its future development. We discuss the shift in position of the authorities, academia, media, non-governmental organizations, regulatory agencies, patients, and doctors which could enable further advances in the research and application of the therapy. In addition, we discuss methods to obtain optimal phage preparations and suggest the potential of novel applications of phage therapy extending beyond its anti-bacterial action.