Bacteriophage therapy and infective endocarditis - is it realistic?

Infective endocarditis (IE) is a severe infection of the inner heart. Even with current standard treatment, the mean in-hospital mortality is as high as 15-20%, and 1-year mortality is up to 40% for left-sided IE. Importantly, IE mortality rates have not changed substantially over the past 30 years, and the incidence of IE is rising. The treatment is challenging due to the bacterial biofilm mode of growth inside the heart valve vegetations, resulting in antibiotic tolerance. Achieving sufficient antibiotic anti-biofilm concentrations in the biofilms of the heart valve vegetations is problematic, even with high-dose and long-term antibiotic therapy. The increasing prevalence of IE caused by antibiotic-resistant bacteria adds to the challenge. Therefore, adjunctive antibiotic-potentiating drug candidates and strategies are increasingly being investigated. Bacteriophage therapy is a reemerging antibacterial treatment strategy for difficult-to-treat infections, mainly biofilm-associated and caused by multidrug-resistant bacteria. However, significant knowledge gaps regarding the safety and efficacy of phage therapy impede more widespread implementation in clinical practice. Hopefully, future preclinical and clinical testing will reveal whether it is a viable treatment. The objective of the present review is to assess whether bacteriophage therapy is a realistic treatment for IE.

More evolvable bacteriophages better suppress their host.

The number of multidrug-resistant strains of bacteria is increasing rapidly, while the number of new antibiotic discoveries has stagnated. This trend has caused a surge in interest in bacteriophages as anti-bacterial therapeutics, in part because there is near limitless diversity of phages to harness. While this diversity provides an opportunity, it also creates the dilemma of having to decide which criteria to use to select phages. Here we test whether a phage's ability to coevolve with its host (evolvability) should be considered and how this property compares to two previously proposed criteria: fast reproduction and thermostability. To do this, we compared the suppressiveness of three phages that vary by a single amino acid yet differ in these traits such that each strain maximized two of three characteristics. Our studies revealed that both evolvability and reproductive rate are independently important. The phage most able to suppress bacterial populations was the strain with high evolvability and reproductive rate, yet this phage was unstable. Phages varied due to differences in the types of resistance evolved against them and their ability to counteract resistance. When conditions were shifted to exaggerate the importance of thermostability, one of the stable phages was most suppressive in the short-term, but not over the long-term. Our results demonstrate the utility of biological therapeutics' capacities to evolve and adjust in action to resolve complications like resistance evolution. Furthermore, evolvability is a property that can be engineered into phage therapeutics to enhance their effectiveness.

fENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes.

BACKGROUND: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. RESULTS: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. CONCLUSIONS: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.

Phage therapy combined with Gum Karaya injectable hydrogels for treatment of methicillin-resistant Staphylococcus aureus deep wound infection in a porcine model.

Skin and soft tissue infections (SSTIs) represent a significant healthcare challenge, particularly in the context of increasing antibiotic resistance. This study investigates the efficacy of a novel therapeutic approach combining bacteriophage (phage) therapy with a gum Karaya (GK)-based hydrogel delivery system in a porcine model of deep staphylococcal SSTIs. The study exploits the lytic activity and safety of the Staphylococcus phage 812K1/420 of the Kayvirus genus, which is active against methicillin-resistant Staphylococcus aureus (MRSA). The GK injectable hydrogels and hydrogel films, developed by our research group, serve as effective, non-toxic, and easy-to-apply delivery systems, supporting moist wound healing and re-epithelialization. In the porcine model, the combined treatment showed asynergistic effect, leading to a significant reduction in bacterial load (2.5 log CFU/gram of tissue) within one week. Local signs of inflammation were significantly reduced by day 8, with clear evidence of re-epithelialization and wound contraction. Importantly, no adverse effects of the GK-based delivery system were observed throughout the study. The results highlight the potential of this innovative therapeutic approach to effectively treat deep staphylococcal SSTIs, providing a promising avenue for further research and clinical application in the field of infections caused by antibiotic-resistant bacteria.

Biofilm in Endodontic Infection and its Advanced Therapeutic Options - An Updated Review.

Pulpal and periapical pathosis are biofilm-induced infections. Understanding the complex nature of endodontic biofilm would help to create a new disinfection strategy to eliminate the microorganism from the root canal system. The intricate canal structure creates challenges for proper disinfection, necessitating the need to understand the biofilm structure, composition, and mechanism within the biofilm community. This paper describes the endodontic biofilm structure, formation of biofilm, and advanced therapeutic options for combating the biofilm community within the root canal system.

Characterization of Salmonella phage of the genus Kayfunavirus isolated from sewage infecting clinical strains of Salmonella enterica.

The emergence of multi-drug resistance in Salmonella, causing food-borne infections, is a significant issue. With over 2,600 serovars in in Salmonella sp., it is crucial to identify specific solutions for each serovar. Phage therapy serves as an alternate treatment option. In this study, vB_SalP_792 phage was obtained from sewage, forming plaques in eight out of 13 tested clinical S. enterica isolates. Transmission electron microscopy (TEM) examination revealed a T7-like morphotype. The phage was characterized by its stability, life cycle, antibiofilm, and lytic ability in food sources. The phage remains stable throughout a range of temperatures (-20 to 70°C), pH levels (3-11), and in chloroform and ether. It also exhibited lytic activity within a range of MOIs from 0.0001 to 100. The life cycle revealed that 95% of the phages attached to their host within 3 min, followed by a 5-min latent period, resulting in a 50 PFU/cell burst size. The vB_SalP_792 phage genome has a dsDNA with a length of 37,281 bp and a GC content of 51%. There are 42 coding sequences (CDS), with 24 having putative functions and no resistance or virulence-related genes. The vB_SalP_792 phage significantly reduced the bacterial load in the established biofilms and also in egg whites. Thus, vB_SalP_792 phage can serve as an effective biocontrol agent for preventing Salmonella infections in food, and its potent lytic activity against the clinical isolates of S. enterica, sets out vB_SalP_792 phage as a successful candidate for future in vivo studies and therapeutical application against drug-resistant Salmonella infections.

Discovery and characterisation of new phage targeting uropathogenic Escherichia coli.

Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering.

Biological Traits and Comprehensive Genomic Analysis of Novel Enterococcus faecalis Bacteriophage EFP6.

Enterococcus faecalis is a prevalent opportunistic pathogen associated with chicken embryonic and neonatal chick mortality, posing a significant challenge in poultry farming. In the current study, E. faecalis strain EF6, isolated from a recent hatchery outbreak, served as the host bacterium for the isolation of a novel phage EFP6, capable of lysing E. faecalis. Transmission electron microscopy revealed a hexagonal head and a short tail, classifying EFP6 as a member of the Autographiviridae family. EFP6 showed sensitivity to ultraviolet radiation and resistance to chloroform. The lytic cycle duration of EFP6 was determined to be 50 min, highlighting its efficacy in host eradication. With an optimal multiplicity of infection of 0.001, EFP6 exhibited a narrow lysis spectrum and strong specificity towards host strains. Additionally, EFP6 demonstrated optimal growth conditions at 40 °C and pH 8.0. Whole genome sequencing unveiled a genome length of 18,147 bp, characterized by a GC concentration of 33.21% and comprising 25 open reading frames. Comparative genomic assessment underscored its collinearity with related phages, notably devoid of lysogenic genes, thus ensuring genetic stability. This in-depth characterization forms the basis for understanding the biological attributes of EFP6 and its potential utilization in phage therapy, offering promising prospects for mitigating E. faecalis-associated poultry infections.

Characterization of an Escherichia coli phage Tequatrovirus YZ2 and its application in bacterial wound infection.

The increasing prevalence of drug-resistant Escherichia coli (E. coli) resulting from the excessive utilization of antibiotics necessitates the immediate exploration of alternative approaches to counteract pathogenic E. coli. Phages, with their unique antibacterial mechanisms, are considered promising candidates for treating bacterial infections. Herein, we isolated a lytic Escherichia phage Tequatrovirus YZ2 (phage YZ2), which belongs to the genus Tequatrovirus. The genome of phage YZ2 consists of 168,356 base pairs with a G + C content of 35.34% and 269 putative open reading frames (ORFs). Of these, 146 ORFs have been annotated as functional proteins associated with nucleotide metabolism, structure, transcription, DNA replication, translation, and lysis. In the mouse model of a skin wound infected by E. coli, phage YZ2 therapy significantly promoted the wound healing. Furthermore, histopathological analysis revealed reductions in IL-1ß and TNF-α and increased VEGF levels, indicating the potential of phages as effective antimicrobial agents against E. coli infection.

A Novel Bacteriophage Infecting Multi-Drug- and Extended-Drug-Resistant Pseudomonas aeruginosa Strains.

The prevalence of carbapenem-resistant P. aeruginosa has dramatically increased over the last decade, and antibiotics alone are not enough to eradicate infections caused by this opportunistic pathogen. Phage therapy is a fresh treatment that can be administered under compassionate use, particularly against chronic cases. However, it is necessary to thoroughly characterize the virus before therapeutic application. Our work describes the discovery of the novel sequenced bacteriophage, vB_PaeP-F1Pa, containing an integrase, performs a phylogenetical analysis, describes its stability at a physiological pH and temperature, latent period (40 min), and burst size (394 ± 166 particles per bacterial cell), and demonstrates its ability to infect MDR and XDR P. aeruginosa strains. Moreover, this novel bacteriophage was able to inhibit the growth of bacteria inside preformed biofilms. The present study offers a road map to analyze essential areas for successful phage therapy against MDR and XDR P. aeruginosa infections, and shows that a phage containing an integrase is also able to show good in vitro results, indicating that it is very important to perform a genomic analysis before any clinical use, in order to prevent adverse effects in patients.

Exploring the historical roots, advantages and efficacy of phage therapy in plant diseases management.

In the drug-resistance era, phage therapy has received considerable attention from worldwide researchers. Phage therapy has been given much attention in public health but is rarely applied to control plant diseases. Herein, we discuss phage therapy as a biocontrol approach against several plant diseases. The emergence of antibiotic resistance in agriculturally important pathogenic bacteria and the toxic nature of different synthetic compounds used to control microbes has driven researchers to rethink the century-old strategy of phage therapy''. Compared to other treatment strategies, phage therapy offers remarkable advantages such as high specificity, less chances of drug resistance, non-harmful nature, and benefit to soil microbial flora. The optimizations and protective formulations of phages are significant accomplishments; however, steps towards a better understanding of the physiologic characteristics of phages need to be preceded to commercialize their use. The future of phage therapy in the context of plant disease management is promising and could play a significant role in sustainable agriculture. Ongoing research will likely affirm the safety of phage therapy, ensuring that it does not harm non-target organisms, including beneficial soil microbes. Phage therapy could become vital in addressing global food security challenges, particularly in regions heavily impacted by plant bacterial diseases. Efforts to create formulations that enhance the stability and shelf-life of phages will be crucial, especially for their use in varied environmental conditions.

Pharmacokinetics and Biodistribution of Phages and their Current Applications in Antimicrobial Therapy.

Antimicrobial resistance remains a critical global health concern, necessitating the investigation of alternative therapeutic approaches. With the diminished efficacy of conventional small molecule drugs due to the emergence of highly resilient bacterial strains, there is growing interest in the potential for alternative therapeutic modalities. As naturally occurring viruses of bacteria, bacteriophage (or phage) are being re-envisioned as a platform to engineer properties that can be tailored to target specific bacterial strains and employ diverse antibacterial mechanisms. However, limited understanding of key pharmacological properties of phage is a major challenge to translating its use from preclinical to clinical settings. Here, we review modern advancements in phage-based antimicrobial therapy and discuss the in vivo pharmacokinetics and biodistribution of phage, addressing critical challenges in their application that must be overcome for successful clinical implementation.

Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

The zebrafish embryo model: unveiling its potential for investigating phage therapy against methicillin-resistant Staphylococcus aureus infection.

Staphylococcus aureus is a pathogenic bacterium responsible for a broad spectrum of infections, including cutaneous, respiratory, osteoarticular, and systemic infections. It poses a significant clinical challenge due to its ability to develop antibiotic resistance. This resistance limits therapeutic options, increases the risk of severe complications, and underscores the urgent need for new strategies to address this threat, including the investigation of treatments complementary to antibiotics. The evaluation of novel antimicrobial agents often employs animal models, with the zebrafish embryo model being particularly interesting for studying host-pathogen interactions, establishing itself as a crucial tool in this field. For the first time, this study presents a zebrafish embryo model for the in vivo assessment of bacteriophage efficacy against S. aureus infection. A localized infection was induced by microinjecting either methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). Subsequent treatments involved administering either bacteriophage, vancomycin (the reference antibiotic for MRSA), or a combination of both via the same route to explore potential synergistic effects. Our findings indicate that the bacteriophage was as effective as vancomycin in enhancing survival rates, whether used alone or in combination. Moreover, bacteriophage treatment appears to be even more effective in reducing the bacterial load in S. aureus-infected embryos post-treatment than the antibiotic. Our study validates the use of the zebrafish embryo model and highlights its potential as a valuable tool in assessing bacteriophage efficacy treatments in vivo.

Neutralizing antibodies after nebulized phage therapy in cystic fibrosis patients.

BACKGROUND: Cystic fibrosis (CF) patients are prone to recurrent multi-drug-resistant (MDR) bacterial lung infections. Under this scenario, phage therapy has been proposed as a promising tool. However, the limited number of reported cases hampers the understanding of clinical outcomes. Anti-phage immune responses have often been overlooked and only described following invasive routes of administration. METHODS: Three monophage treatments against Staphylococcus aureus and/or Pseudomonas aeruginosa lung infections were conducted in cystic fibrosis patients. In-house phage preparations were nebulized over 10 days with standard-of-care antibiotics. Clinical indicators, bacterial counts, phage and antibiotic susceptibility, phage detection, and immune responses were monitored. FINDINGS: Bacterial load was reduced by 3-6 log in two of the treatments. No adverse events were described. Phages remained in sputum up to 33 days after completion of the treatment. In all cases, phage-neutralizing antibodies were detected in serum from 10 to 42 days post treatment, with this being the first report of anti-phage antibodies after nebulized therapy. CONCLUSIONS: Nebulized phage therapy reduced bacterial load, improving quality of life even without bacterial eradication. The emergence of antibodies emphasizes the importance of long-term monitoring to better understand clinical outcomes. These findings encourage the use of personalized monophage therapies in contrast to ready-to-use cocktails, which might induce undesirable antibody generation. FUNDING: This study was supported by the Spanish Ministry of Science, Innovation and Universities; Generalitat Valenciana; and a crowdfunding in collaboration with the Spanish Cystic Fibrosis Foundation.

Does Phage Therapy Need a Pan-Phage?

The emergence of multidrug-resistant bacteria is undoubtedly one of the most serious global health threats. One response to this threat that has been gaining momentum over the past decade is 'phage therapy'. According to this, lytic bacteriophages are used for the treatment of bacterial infections, either alone or in combination with antimicrobial agents. However, to ensure the efficacy and broad applicability of phage therapy, several challenges must be overcome. These challenges encompass the development of methods and strategies for the host range manipulation and bypass of the resistance mechanisms developed by pathogenic bacteria, as has been the case since the advent of antibiotics. As our knowledge and understanding of the interactions between phages and their hosts evolves, the key issue is to define the host range for each application. In this article, we discuss the factors that affect host range and how this determines the classification of phages into different categories of action. For each host range group, recent representative examples are provided, together with suggestions on how the different groups can be used to combat certain types of bacterial infections. The available methodologies for host range expansion, either through sequential adaptation to a new pathogen or through genetic engineering techniques, are also reviewed.

Optimizing bacteriophage treatment of resistant Pseudomonas.

Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of administering phages in clinical settings, preferred over monophage treatment because of their ability to eliminate multiple bacterial strains and reduce resistance formation. In our study, we compare monophage applications and phage cocktails to our chosen method of phage sequential treatments. To do so, we isolated four novel bacteriophages capable of infecting Pseudomonas alcaligenes T3, a close relative of P. aeruginosa, and characterized them using sequencing and transmission electron microscopy. While investigating monophage treatments, we observed that different phage concentrations had a strong impact on the timing and amount of resistance formation. When using phage cocktails, we observed that P. alcaligenes were capable of forming resistance in the same timespan it took them to become resistant to single phages. We isolated mutants resistant to each single phage as well as mutants exposed to phage cocktails, resulting in bacteria resistant to all four phages at once. Sequencing these mutants showed that different treatments yielded unique single nucleotide polymorphism mutation patterns. In order to combat resistance formation, we added phages one by one in intervals of 24 h, thus managing to delay resistance development and keeping bacterial growth significantly lower compared to phage cocktails.IMPORTANCEWHO declared antimicrobial resistance a top threat to global health; while antibiotics have stood at the forefront in the fight against bacterial infection, the increasing number of multidrug-resistant bacteria highlights a need to branch out in order to address the threat of antimicrobial resistance. Bacteriophages, viruses solely infecting bacteria, could present a solution due to their abundance, versatility, and adaptability. For this study, we isolated new phages infecting a fast-mutating Pseudomonas alcaligenes strain capable of forming resistance within 30 h. By using a sequential treatment approach of adding one phage after another, we were able to curb bacterial growth significantly more compared to state-of-the-art phage cocktails.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Phage K exposure generates phage-neutralizing activity in a rabbit model-humoral receptiveness may impact efficacy of phage therapy.

Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~1010±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 (P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual's "humoral receptiveness" to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo.