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.

Bacteriophages and antibiotic interactions in clinical practice: what we have learned so far.

Bacteriophages (phages) may be used as an alternative to antibiotic therapy for combating infections caused by multidrug-resistant bacteria. In the last decades, there have been studies concerning the use of phages and antibiotics separately or in combination both in animal models as well as in humans. The phenomenon of phage-antibiotic synergy, in which antibiotics may induce the production of phages by bacterial hosts has been observed. The potential mechanisms of phage and antibiotic synergy was presented in this paper. Studies of a biofilm model showed that a combination of phages with antibiotics may increase removal of bacteria and sequential treatment, consisting of phage administration followed by an antibiotic, was most effective in eliminating biofilms. In vivo studies predominantly show the phenomenon of phage and antibiotic synergy. A few studies also describe antagonism or indifference between phages and antibiotics. Recent papers regarding the application of phages and antibiotics in patients with severe bacterial infections show the effectiveness of simultaneous treatment with both antimicrobials on the clinical outcome.

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.

Phage therapy: Current status and perspectives.

The spread of antimicrobial resistant bacterial pathogens combined with the lack of new drug classes in the antibiotic pipeline causes a resurgence of the use of bacterial viruses (phages) to treat bacterial infections (phage therapy [PT]). There has been a substantial increase in patients subjected to this experimental therapy and emergence of new PT centers in Europe and the United States paralleled by one clinical trial completed in accord with good medical practice (GMP) requirements and a few others underway. What is more, evidence has been accumulating to suggest that phages can also exert anti-inflammatory and immunomodulatory action which opens new pathways for the development of novel targets for PT. Here we present the status quo of the PT, recent regulatory, and clinical developments as well as new perspectives for its wider application in clinical medicine.

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.

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

BACKGROUND: Members 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. METHODS: Electron 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 CONCLUSIONS: Of 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.

Biological Properties of 12 Newly Isolated Acinetobacter baumannii-Specific Bacteriophages.

Infections with the opportunistic Gram-negative bacterium Acinetobacter baumannii pose a serious threat today, which is aggravated by the growing problem of multi-drug resistance among bacteria, caused by the overuse of antibiotics. Treatment of infections caused by antibiotic-resistant A. baumannii strains with the use of phage therapy is not only a promising alternative, but sometimes the only option. Therefore, phages specific for clinical multi-drug resistant A. baumannii were searched for in environmental, municipal, and hospital wastewater samples collected from different locations in Poland. The conducted research allowed us to determine the biological properties and morphology of the tested phages. As a result of our research, 12 phages specific for A. baumannii, 11 of which turned out to be temperate and only one lytic, were isolated. Their lytic spectra ranged from 11 to 75%. The plaques formed by most phages were small and transparent, while one of them formed relatively large plaques with a clearly marked 'halo' effect. Based on Transmission Electron Microscopy (TEM), most of our phages have been classified as siphoviruses (only one phage was classified as a podovirus). All phages have icosahedral capsid symmetry, and 11 of them have a long tail. Optimal multiplicity of infections (MOIs) and the adsorption rate were also determined. MOI values varied depending on the phage-from 0.001 to 10. Based on similarities to known bacteriophages, our A. baumannii-specific phages have been proposed to belong to the Beijerinckvirinae and Junivirinae subfamilies. This study provides an additional tool in the fight against this important pathogen and may boost the interest in phage therapy as an alternative and supplement to the current antibiotics.

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.

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.

Isolation and characterisation of KP34--a novel φKMV-like bacteriophage for Klebsiella pneumoniae.

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum ß-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with "phiKMV-like viruses". Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.

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.

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.

Potential for Phages in the Treatment of Bacterial Sexually Transmitted Infections.

Bacterial sexually transmitted infections (BSTIs) are becoming increasingly significant with the approach of a post-antibiotic era. While treatment options dwindle, the transmission of many notable BSTIs, including Neisseria gonorrhoeae, Chlamydia trachomatis, and Treponema pallidum, continues to increase. Bacteriophage therapy has been utilized in Poland, Russia and Georgia in the treatment of bacterial illnesses, but not in the treatment of bacterial sexually transmitted infections. With the ever-increasing likelihood of antibiotic resistance prevailing and the continuous transmission of BSTIs, alternative treatments must be explored. This paper discusses the potentiality and practicality of phage therapy to treat BSTIs, including Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Streptococcus agalactiae, Haemophilus ducreyi, Calymmatobacterium granulomatis, Mycoplasma genitalium, Ureaplasma parvum, Ureaplasma urealyticum, Shigella flexneri and Shigella sonnei. The challenges associated with the potential for phage in treatments vary for each bacterial sexually transmitted infection. Phage availability, bacterial structure and bacterial growth may impact the potential success of future phage treatments. Additional research is needed before BSTIs can be successfully clinically treated with phage therapy or phage-derived enzymes.

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.

Prophylactic effect of bacteriophages on mice subjected to chemotherapy-induced immunosuppression and bone marrow transplant upon infection with Staphylococcus aureus.

Due to the increased resistance of bacteria to antibiotics, phage therapy may be an alternative to treat or prevent suppurative infections in immunocompromised patients. The authors' recent studies indicated that such an approach is particularly beneficial in immunosuppressed mice. A5/L bacteriophages, specific for the Staphylococcus aureus strain L, were tested for their ability to protect CBA mice subjected to myeloablative (busulfan) and immunosuppressive (cyclophosphamide) conditioning followed by a syngeneic bone marrow transplantation (BMT) and infected with a sublethal or lethal dose of bacteria. The application of phages to immunocompromised mice given BMT led to a significant (>90%) reduction in bacterial load in the spleen and liver. Moreover, 72% of such mice attained long-term survival versus 8.2% survival of mice not treated with phages. Analysis of leukocyte number and blood cell type composition revealed that phage application increased the leukocyte numbers and neutrophil content in the circulating blood. Moreover, phage application led to an increased content of the myelocytic cell lineage in the bone marrow. The protective effects of phages in immunosuppressed mice are both direct (bacteriolytic) and indirect (by stimulation of myelopoiesis). The results suggest a potential benefit of phage therapy in immunocompromised patients subjected to bone marrow transplant procedures.

Bacteriophages and Lysins in Biofilm Control.

To formulate the optimal strategy of combatting bacterial biofilms, in this review we update current knowledge on the growing problem of biofilm formation and its resistance to antibiotics which has spurred the search for new strategies to deal with this complication. Based on recent findings, the role of bacteriophages in the prevention and elimination of biofilm-related infections has been emphasized. In vitro, ex vivo and in vivo biofilm treatment models with single bacteriophages or phage cocktails have been compared. A combined use of bacteriophages with antibiotics in vitro or in vivo confirms earlier reports of the synergistic effect of these agents in improving biofilm removal. Furthermore, studies on the application of phage-derived lysins in vitro, ex vivo or in vivo against biofilm-related infections are encouraging. The strategy of combined use of phage and antibiotics seems to be different from using lysins and antibiotics. These findings suggest that phages and lysins alone or in combination with antibiotics may be an efficient weapon against biofilm formation in vivo and ex vivo, which could be useful in formulating novel strategies to combat bacterial infections. Those findings proved to be relevant in the prevention and destruction of biofilms occurring during urinary tract infections, orthopedic implant-related infections, periodontal and peri-implant infections. In conclusion, it appears that most efficient strategy of eliminating biofilms involves phages or lysins in combination with antibiotics, but the optimal scheme of their administration requires further studies.

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 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.