Pharmacokinetic and Pharmacodynamic Obstacles for Phage Therapy From the Perspective of Clinical Practice.

Bacteriophages present unique features that enable targeted killing of bacteria, including strains resistant to many antibiotics. However, phage pharmacokinetics and pharmacodynamics constitute much more complex and challenging aspects for researchers than those attributable to antibiotics. This is because phages are not just chemical substances, but also biological nanostructures built of different proteins and genetic material that replicate within their bacterial hosts and may induce immune responses acting as simple antigens. Here, we present a few examples of how primary general assumptions on phage pharmacokinetics and pharmacodynamics are verified by current preclinical and clinical observations, leading to conclusions that may not be obvious at first but are of significant value for the final success of phage therapy in humans.

CTCF is a DNA-tension-dependent barrier to cohesin-mediated loop extrusion.

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4-7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin's loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

Characterization and Comparative Genomic Analysis of Three Virulent E. coli Bacteriophages with the Potential to Reduce Antibiotic-Resistant Bacteria in the Environment.

The emerging global crisis of antibiotic resistance demands new alternative antibacterial solutions. Although bacteriophages have been used to combat bacterial infections for over a century, a dramatic boost in phage studies has recently been observed. In the development of modern phage applications, a scientific rationale is strongly required and newly isolated phages need to be examined in detail. In this study, we present the full characterization of bacteriophages BF9, BF15, and BF17, with lytic activity against extended-spectrum ß-lactamases (ESBLs)- and AmpC ß-lactamases (AmpC)-producing Escherichia coli, the prevalence of which has increased significantly in livestock in recent decades, representing a great hazard to food safety and a public health risk. Comparative genomic and phylogenetic analysis indicated that BF9, BF15, and BF17 represent the genera Dhillonvirus, Tequatrovirus, and Asteriusvirus, respectively. All three phages significantly reduced in vitro growth of their bacterial host and retained the ability to lyse bacteria after preincubation at wide ranges of temperature (-20-40 °C) and pH (5-9). The results described herein indicate the lytic nature of BF9, BF15, and BF17, which, along with the absence of genes encoding toxins and bacterial virulence factors, represents an undoubted asset in terms of future phage application.

Characteristics of Environmental Klebsiella pneumoniae and Klebsiella oxytoca Bacteriophages and Their Therapeutic Applications.

In recent years, multidrug-resistant (MDR) strains of Klebsiella pneumoniae have spread globally, being responsible for the occurrence and severity of nosocomial infections. The NDM-1-kp, VIM-1 carbapenemase-producing isolates as well as extended-spectrum beta lactamase-producing (ESBL) isolates along with Klebsiella oxytoca strains have become emerging pathogens. Due to the growing problem of antibiotic resistance, bacteriophage therapy may be a potential alternative to combat such multidrug-resistant Klebsiella strains. Here, we present the results of a long-term study on the isolation and biology of bacteriophages active against K. pneumoniae, as well as K. oxytoca strains. We evaluated biological properties, morphology, host specificity, lytic spectrum and sensitivity of these phages to chemical agents along with their life cycle parameters such as adsorption, latent period, and burst size. Phages designated by us, vB_KpnM-52N (Kpn52N) and VB_KpnM-53N (Kpn53N), demonstrated relatively broad lytic spectra among tested Klebsiella strains, high burst size, adsorption rates and stability, which makes them promising candidates for therapeutic purposes. We also examined selected Klebsiella phages from our historical collection. Notably, one phage isolated nearly 60 years ago was successfully used in purulent cerebrospinal meningitis in a new-born and has maintained lytic activity to this day. Genomic sequences of selected phages were determined and analyzed. The phages of the sequenced genomes belong to the Slopekvirus and Jiaodavirus genus, a group of phages related to T4 at the family level. They share several features of T4 making them suitable for antibacterial therapies: the obligatorily lytic lifestyle, a lack of homologs of known virulence or antibiotic resistance genes, and a battery of enzymes degrading host DNA at infection.

How Interest in Phages Has Bloomed into a Leading Medical Research Activity in Poland.

Poland has a leading position in phage therapy, as reflected by the number of patients treated and relevant publications in quality journals. The Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences was established by Ludwik Hirszfeld, a prominent microbiologist and serologist who also initiated studies on phages and pioneered the activities that set into motion phage therapy at the Institute. To achieve this goal, Hirszfeld had to overcome many difficulties in a post-war Poland. He died a month before the official start of the Institute's activity and was not able to witness the advancement of the Institute bearing his name. However, his hard work and dedication have been recently rewarded. In a recent evaluation of scientific performance, the Institute received the highest ranking in medical sciences among all universities and research institutions in Poland. One could consider it a posthumous tribute to the memory of L. Hirszfeld, being well-deserved on the grounds of the Institute's achievements (especially in the field of phage therapy) as well as his life and work.

Zygotic genome activation by the totipotency pioneer factor Nr5a2.

Life begins with a switch in genetic control from the maternal to the embryonic genome during zygotic genome activation (ZGA). Despite its importance, the essential regulators of ZGA remain largely unknown in mammals. On the basis of de novo motif searches, we identified the orphan nuclear receptor Nr5a2 as a key activator of major ZGA in mouse two-cell embryos. Nr5a2 is required for progression beyond the two-cell stage. It binds to its motif within SINE B1/Alu retrotransposable elements found in cis-regulatory regions of ZGA genes. Chemical inhibition suggests that 72% of ZGA genes are regulated by Nr5a2 and potentially other orphan nuclear receptors. Nr5a2 promotes chromatin accessibility during ZGA and binds nucleosomal DNA in vitro. We conclude that Nr5a2 is an essential pioneer factor that regulates ZGA.

Do Anti-Phage Antibodies Persist after Phage Therapy? A Preliminary Report.

Phages are immunogenic and may evoke an immune response following their administration. Consequently, patients undergoing phage therapy (PT) produce phage-neutralizing serum antibodies. The clinical significance of this phenomenon for the success or failure of the therapy is currently unclear. Interestingly, even a strong anti-phage humoral response does not exclude the success of PT. On the other hand, it cannot be ruled out that phage-antibody complexes may be trapped in tissues and organs causing injury and late complications of PT. Therefore, patients should be monitored for the presence of serum antibodies and therapy discontinued if their level is high. Our preliminary data suggest that the kinetics of the disappearance of those antibodies may vary from patient to patient and in some cases may take more than a year.

A Thorough Synthesis of Phage Therapy Unit Activity in Poland-Its History, Milestones and International Recognition.

The year 2020 marked 15 years of the Phage Therapy Unit in Poland, the inception of which took place just one year after Poland's accession to the European Union (2004). At first sight, it is hard to find any connection between these two events, but in fact joining the European Union entailed the need to adapt the regulatory provisions concerning experimental treatment in humans to those that were in force in the European Union. These changes were a solid foundation for the first phage therapy center in the European Union to start its activity. As the number of centers conducting phage therapy in Europe and in the world constantly and rapidly grows, we want to grasp the opportunity to take a closer look at the over 15-year operation of our site by analyzing its origins, legal aspects at the local and international levels and the impressive number and diversity of cases that have been investigated and treated during this time. This article is a continuation of our work published in 2020 summarizing a 100-year history of the development of phage research in Poland.

Bronislawa Fejgin (1883-1943): Forgotten Important Contributor to International Microbiology and Phage Therapy.

Bronislawa Brandla Fejgin was a Polish-born Jewish female physician. Among Fejgin's numerous articles in the field of microbiology, her later work was almost entirely devoted to phage research. Although not equally famous as the phage pioneers from Western Europe, F.W. Twort and F. d'Herelle, Fejgin's contribution to phage research deserves proper recognition. Her studies on phages resulted in the publication of numerous original scientific reports. These articles, published mostly in French, constitute an important source of information and expertise on early attempts towards therapeutic use of phages in humans. The interwar period marks the most intense years in Bronislawa Fejgin's research activity, brutally interrupted by her death in the Warsaw Ghetto in 1943. Her microbiology contributions have not been analyzed so far. Thus, the aim of this article is to fill the existing gap in the history of microbiology and phage therapy.

Anti-biofilm activity of bacteriophages and lysins in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is an otolaryngological disease with a recalcitrant nature, predominantly due to antibiotic resistant bacteria and the biofilm formation. The intracellular residency of Staphylococcus aureus bacteria was observed in CRS. The overall prevalence of CRS is estimated between 5-15% in the human population, and biofilms were formed in sinuses in 40-80% of cases. The bacterial species S. aureus and Pseudomonas aeruginosa are known to form difficult to treat biofilms in CRS. Bacteriophages (phages) or lysins can be alternatives to antibiotics in the biofilm treatment. The application of a P. aeruginosa phage cocktail ex vivo decreased biofilm biomass of bacterial isolates from the sinuses of CRS patients by a median of 70%. Further, animal studies performed on a sheep sinusitis model demonstrated significant reduction in S. aureus and P. aeruginosa biofilm biomass by phage cocktails while maintaining safe prolonged topical application (up to 20 days). Staphylococcal lysin P128 used at a concentration of ≥12.5 µg/ml in vitro against the biofilm of methicillin sensitive S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) isolates from the sinuses of CRS patients demonstrated a significant reduction of the biofilm (up to 95.5%). Staphylococcal lysin CHAP(k) applied in vivo in mice nasal infection caused a significant 2 log reduction of S. aureus suggesting its potential use against bacteria in nasal mucosa. Furthermore, a beneficial effect of phage therapy in the treatment of chronic sinusitis in humans was observed. Here, we summarize the recent, quite scarce data regarding phage application in chronic rhinosinusitis and look further into this phenomenon. Keywords: bacteriophages; biofilm; chronic rhinosinusitis; lysins; phage therapy.

Low Immunogenicity of Intravesical Phage Therapy for Urogenitary Tract Infections.

Patients with chronic urinary and urogenital multidrug resistant bacterial infections received phage therapy (PT) using intravesical or intravesical and intravaginal phage administration. A single course of PT did not induce significant serum antibody responses against administered phage. Whilst the second cycle of PT caused a significant increase in antibody levels, they nevertheless remained quite low. These data combined with good therapy results achieved in some patients suggest that this mode of PT may be an efficient means of therapy for urogenital infections and a reliable model for a clinical trial of PT.

The Presence of Bacteriophages in the Human Body: Good, Bad or Neutral?

The presence of bacteriophages (phages) in the human body may impact bacterial microbiota and modulate immunity. The role of phages in human microbiome studies and diseases is poorly understood. However, the correlation between a greater abundance of phages in the gut in ulcerative colitis and diabetes has been suggested. Furthermore, most phages found at different sites in the human body are temperate, so their therapeutic effects and their potential beneficial effects remain unclear. Hence, far, no correlation has been observed between the presence of widespread crAssphage in the human population and human health and diseases. Here, we emphasize the beneficial effects of phage transfer in fecal microbiota transplantation (FMT) in Clostridioides difficile infection. The safety of phage use in gastrointestinal disorders has been demonstrated in clinical studies. The significance of phages in the FMT as well as in gastrointestinal disorders remains to be established. An explanation of the multifaceted role of endogenous phages for the development of phage therapy is required.

Phage Prevalence in the Human Urinary Tract-Current Knowledge and Therapeutic Implications.

Recent metagenomic analyses imply an immense abundance of phages in the human body. Samples collected from different sites (lungs, skin, oral cavity, intestines, ascitic fluid, and urine) reveal a generally greater number of phage particles than that of eukaryotic viruses. The presence of phages in those tissues and fluids reflects the paths they must overcome in the human body, but may also relate to the health statuses of individuals. Besides shaping bacterial metabolism and community structure, the role of phages circulating in body fluids has not been fully understood yet. The lack of relevant reports is especially visible with regard to the human urobiome. Certainly, phage presence and the role they have to fulfill in the human urinary tract raises questions on potential therapeutic connotations. Urinary tract infections (UTIs) are among the most common bacterial infections in humans and their treatment poses a difficult therapeutic dilemma. Despite effective antibiotic therapy, these infections tend to recur. In this review, we summarized the recent data on phage presence in the human urinary tract and its possible implications for health and disease.

Phages as a Cohesive Prophylactic and Therapeutic Approach in Aquaculture Systems.

Facing antibiotic resistance has provoked a continuously growing focus on phage therapy. Although the greatest emphasis has always been placed on phage treatment in humans, behind phage application lies a complex approach that can be usefully adopted by the food industry, from hatcheries and croplands to ready-to-eat products. Such diverse businesses require an efficient method for combating highly pathogenic bacteria since antibiotic resistance concerns every aspect of human life. Despite the vast abundance of phages on Earth, the aquatic environment has been considered their most natural habitat. Water favors multidirectional Brownian motion and increases the possibility of contact between phage particles and their bacterial hosts. As the global production of aquatic organisms has rapidly grown over the past decades, phage treatment of bacterial infections seems to be an obvious and promising solution in this market sector. Pathogenic bacteria, such as Aeromonas and Vibrio, have already proved to be responsible for mass mortalities in aquatic systems, resulting in economic losses. The main objective of this work is to summarize, from a scientific and industry perspective, the recent data regarding phage application in the form of targeted probiotics and therapeutic agents in aquaculture niches.

Phage Therapy in Poland - a Centennial Journey to the First Ethically Approved Treatment Facility in Europe.

Although phage discovery is an unquestionable merit of the English bacteriologist Frederick W. Twort and the Canadian-French microbiologist Félix d'Hérelle, who both discovered phages over 100 years ago, the Polish history of phage studies also dates back to those years. In contrast to the Western world, developing phage treatment in Poland has never been abandoned despite the country's tense history marked by the Second World War (WWII) and the communism era. Today, Poland takes a prominent and remarkable place in the phage research area. Furthermore, established in 2005, the Phage Therapy Unit at the Hirszfeld Institute of Immunology and Experimental Therapy in Wroclaw, the first such center within European borders, has quickly become a model for other centers in the world facing the issue of widespread antibiotic resistance. This article constitutes an attempt to fill the gap in the scientific literature by providing a comprehensive summary of the long tradition of phage research in Poland.

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

Prospects of Phage Application in the Treatment of Acne Caused by Propionibacterium acnes.

Propionibacterium acnes is associated with purulent skin infections, and it poses a global problem for both patients and doctors. Acne vulgaris (acne) remains a problem due to its chronic character and difficulty of treatment, as well as its large impact on patients' quality of life. Due to the chronic course of the disease, treatment is long lasting, and often ineffective. Currently there are data regarding isolation of P. acnes phages, and there have been numerous studies on phage killing of P. acnes, but no data are available on phage application specifically in acne treatment. In this review, we have summarized the current knowledge on the phages active against P. acnes described so far and their potential application in the treatment of acne associated with P. acnes. The treatment of acne with phages may be important in order to reduce the overuse of antibiotics, which are currently the main acne treatment. However, more detailed studies are first needed to understand phage functioning in the skin microbiome and the possibility to use phages to combat P. acnes.

Antiphage activity of sera during phage therapy in relation to its outcome.

AIM: The aim was to study the association between the phage neutralization of patients' sera and the clinical outcome of phage therapy (PT). PATIENTS: About 62 patients with various bacterial infections receiving PT as well as 30 healthy volunteers were studied. MATERIALS & METHODS: Antiphage activity of sera (AAS) was examined using the phage neutralization test of different types of phages before and during PT in relation to the route of phage administration and correlated with the results of PT. RESULTS & CONCLUSION: The analysis of the association between AAS level and clinical results indicated that the level of AAS is not correlated with the outcome of PT.