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Inflammatory periodontal diseases associated with the accumulation of dental biofilm, such as gingivitis and periodontitis, are very common and pose clinical problems for clinicians and patients. Gingivitis is a mild form of gum disease and when treated quickly and properly is completely reversible. Periodontitis is an advanced and irreversible disease of the periodontium with periods of exacerbations, progressions and remission. Periodontitis is a chronic inflammatory condition that damages the tissues supporting the tooth in its socket, i.e., the gums, periodontal ligaments, root cementum and bone. Periodontal inflammation is most commonly triggered by bacteria present in excessive accumulations of dental plaque (biofilm) on tooth surfaces. This disease is driven by disproportionate host inflammatory immune responses induced by imbalance in the composition of oral bacteria and changes in their metabolic activities. This microbial dysbiosis favors the establishment of inflammatory conditions and ultimately results in the destruction of tooth-supporting tissues. Apart microbial shift and host inflammatory response, environmental factors and genetics are also important in etiology In addition to oral tissues destruction, periodontal diseases can also result in significant systemic complications. Conventional methods of periodontal disease treatment (improving oral hygiene, dental biofilm control, mechanical plaque removal, using local or systemic antimicrobial agents) are not fully effective. All this prompts the search for new methods of therapy. Advanced periodontitis with multiple abscesses is often treated with antibiotics, such as amoxicillin, tetracycline, doxycycline, minocycline, clindamycin, or combined therapy of amoxicillin with metronidazole. However, due to the growing problem of antibiotic resistance, treatment does not always achieve the desired therapeutic effect. This review summarizes pathogenesis, current approaches in treatment, limitations of therapy and the current state of research on the possibility of application of bacteriophages and predatory bacteria to combat bacteria responsible for periodontitis. We present the current landscape of potential applications for alternative therapies for periodontitis based on phages and bacteria, and highlight the gaps in existing knowledge that need to be addressed before clinical trials utilizing these therapeutic strategies can be seriously considered. [ABSTRACT FROM AUTHOR]

The studies on phage therapy have shown an overall protective effect of phages in bacterial infections, thus providing an optimistic outlook on the future benefits of phage-based technologies for treating bacterial diseases. However, the therapeutic effect is highly affected by the proper composition of phage cocktails. The rational approach to the design of bacteriophage cocktails, which is the subject of this study, allowed for development of an effective anti-mastitis solution, composed of virulent bacteriophages acting on Escherichia coli and Staphylococcus aureus. Based on the in-depth bioinformatic characterization of bacteriophages and their in vitro evaluation, the cocktail of five phages against E. coli and three against S. aureus strains was composed. Its testing in the milk model experiment revealed a reduction in the number of S. aureus of 45% and 30% for E. coli strains, and in the study of biofilm prevention, it demonstrated 99% inhibition of biofilm formation for all tested S. aureus strains and a minimum of 50% for 50% of E. coli strains. Such insights justify the need for rational design of cocktails for phage therapy and indicate the potential of the developed cocktail in the treatment of diseased animals, but this requires further investigations to evaluate its in vivo efficacy. [ABSTRACT FROM AUTHOR]

Although advances in contemporary medical care have broadened access to healthcare and extended the human life span, deaths resulting from antimicrobial-resistant pathogens continue to increase. This minireview summarizes the evidence that AI and machine learning, coupled with precision medicine and alternative therapies, such as repurposing non-antibiotic drugs and the use of bacteriophages, has promise to halt this advance.

Abstract Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics.

Given the increasing threat of antimicrobial resistance, scientists are urgently seeking adjunct antimicrobial strategies, such as phage therapy (PT). However, despite promising results for the treatment of musculoskeletal infections in our center, crucial knowledge gaps remain. Therefore, a prospective observational study (PHAGEFORCE) and a multidisciplinary approach was set up to achieve and optimize standardized treatment guidelines. At our center, PT is strictly controlled and monitored by a multidisciplinary taskforce. Each phage treatment follows the same pathway to ensure standardization and data quality. Within the PHAGEFORCE framework, we established a testing platform to gain insight in the safety and efficacy of PT, biodistribution, phage kinetics and the molecular interaction between phages and bacteria. The draining fluid is collected to determine the phage titer and bacterial load. In addition, all bacterial isolates are fully characterized by genome sequencing to monitor the emergence of phage resistance. We hereby present a standardized bench-to-bedside protocol to gain more insight in the kinetics and dynamics of PT for musculoskeletal infections. [ABSTRACT FROM AUTHOR]

Although advances in contemporary medical care have broadened access to healthcare and extended the human life span, deaths resulting from antimicrobial-resistant pathogens continue to increase. This minireview summarizes the evidence that AI and machine learning, coupled with precision medicine and alternative therapies, such as repurposing non-antibiotic drugs and the use of bacteriophages, has promise to halt this advance. [ABSTRACT FROM AUTHOR]

Bacterial diseases of the gastrointestinal (GI) tract continue to be a major worldwide cause of human morbidity and mortality. Among various enteric pathogens, Shigella spp. are some of the most common and deadly bacterial pathogens. They are responsible for ~125 million worldwide cases of shigellosis, and ~14,000 deaths annually, the majority in children under the age of 5 and occurring in developing countries. Preventing and treating shigellosis with conventional drugs (e.g., vaccines and antibiotics) has proven to be very difficult. Here, we assessed the safety and tolerability of ShigActive™, a lytic bacteriophage preparation targeting Shigella spp., in a randomized, placebo-controlled, double-blind Phase 1 clinical trial. Ten participants randomized 4:1 received ShigActive™ or placebo co-administered with sodium bicarbonate orally three times daily for 7 days. Solicited and unsolicited adverse events (AEs) were observed for 29 days. Fifty percent of the subjects receiving ShigActive™ reported mild GI-related symptoms, while one participant experienced moderate fatigue. No serious or medically attended AEs occurred through day 90. Additionally, no significant differences in GI-associated inflammatory mediators or fecal microbiome changes were observed between placebo- and ShigActive™-treated subjects, or from a participants' baseline value. The results of this first-in-human (FIH) randomized, controlled Phase 1 trial of ShigActive™ demonstrate that it is safe and well tolerated when orally administered with no significant differences compared to placebo controls. [ABSTRACT FROM AUTHOR]

The conventional treatment of bacterial infections with antibiotics is becoming increasingly ineffective due to the emergence of multidrug-resistant (MDR) pathogens. This literature review explores the potential of bacteriophages as an alternative or adjunctive therapy to antibiotics in combating MDR infections in Africa. This analysis focuses on current research regarding the integration of phage therapy into African healthcare, highlighting its challenges and opportunities. This review begins with the AMR crisis and the need for new treatments, then covers the history, mechanisms, benefits, and limitations of phage therapy. Key African studies are summarized, identifying major obstacles such as regulatory issues, infrastructure, and research standardization. Research efforts in West Africa that have made notable progress in bacteriophage research are highlighted. This review concludes with recommendations for policymakers, researchers, and healthcare professionals to enhance the development and use of phage therapy in Africa, aiming to reduce antibiotic resistance and improve patient outcomes. By addressing the identified challenges and leveraging the unique advantages of phages, there is potential to significantly mitigate the impact of antibiotic resistance and improve patient outcomes in Africa. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a food-borne pathogen, which is often isolated from various seafood products. In this study, two kinds of bacteriophages was isolated from the offshore sediments samples. The anti-phage mutant strain were obtained after seventeen rounds of co-culture of Vibrio parahaemolyticus and mixed bacteriophage, multigroup sequencing was carried out on spontaneous the anti-phage mutant strain and the wild-type strain. We used the Sanger sequencing to verify the accuracy of the mutation sites. Biolog GEN III MicroPlates were used to evaluate the metabolic capacity of wild-type strains and the anti-phage mutant strain. In this study, we found that with flaG gene (slight homology to N terminus of multiple flagellins) mutated, making the bacteriophage unable to absorb to the cell surface of the host. And, the growth competitiveness of the anti-phage mutant strain is lower than the wild-type strain. These results indicated that the fitness cost, including loss of the growth competitiveness, constitutes a barrier to the prevalence of these defense mechanisms. And the selection pressure on different anti-phage strategies depends on the trade-off between mortality imposed by bacteriophages and fitness cost of the defense strategy under the given environmental conditions. In conclusion, this study provides valuable insights into the phagehost interaction and phage resistance in Vibrio parahaemolyticus. Our study provided knowledge for the evolutionary adaption of bacteria against the bacteriophage, which could add more information to understand the phage resistance mechanism before applying in the industry. [ABSTRACT FROM AUTHOR]

Introduction: The present study aimed to optimize the doses and schedule of specific bacteriophage cocktails in freshwater fish infections as prophylactic and therapeutic measures. Methods: The three most active phages against Aeromonas hydrophila (A. hydrophila) (φAHBHU12, φAHBHU16, and φAHBHU19) were characterized phenotypically and genotypically. Intramuscular and water immersion routes were used to calculate the absolute lethal dose of A. hydrophila in Pangasius buchanani. Phage therapy was given simultaneously and after 6, 12, and 24 h of bacterial challenge through intramuscular and water immersion routes. Results: The prophylactic and early phage administration could save the fish. Furthermore, the dose of intramuscular 1.0 × 104 plaque-forming unit (PFU)/fish and water immersion 1.0 × 106 PFU mL-1 of the phage cocktail was optimal. Discussion: The efficacy of bacteriophage therapy as preventive or curative measures practical when administered simultaneously or early hours of A. hydrophila infection in aquaculture systems. Phage-based approaches may be used as an alternative to antibiotics in aquaculture to reduce antibiotic use as a part of the "One Health" approach. [ABSTRACT FROM AUTHOR]

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

The global health threat posed by carbapenem-resistant Klebsiella pneumoniae (CRKP) is exacerbated by the limited availability of effective treatments. Bacteriophages are promising alternatives to conventional antimicrobial agents. However, current phage databases are limited. Thus, identifying and characterizing new phages could provide biological options for the treatment of multi-drug resistant bacterial infections. Here, we report the characterization of a novel lytic phage, vB_KpnP_23, isolated from hospital sewage. This phage exhibited potent activity against carbapenemase-producing CRKP strains and was characterised by an icosahedral head, a retractable tail, and a genome comprising 40,987 base pairs, with a G + C content of 51 %. Capable of targeting and lysing nine different capsule types (K-types) of CRKP, including the clinically relevant ST11-K64, it demonstrated both high bacteriolytic efficiency and stability in various environmental contexts. Crucially, vB_KpnP_23 lacks virulence factors, antimicrobial resistance genes, or tRNA, aligning with the key criteria for therapeutic application. In vitro evaluation of phage-antibiotic combinations revealed a significant synergistic effect between vB_KpnP_23 and meropenem, levofloxacin, or amikacin. This synergy could lead to an 8-fold reduction in the minimum inhibitory concentration (MIC), suggesting that integrated treatments combining this phage with the aforementioned antibiotics may substantially enhance drug effectiveness. This approach not only extends the clinical utility of these antibiotics but also presents a strategic advance in combating antibiotic resistance. Specifically, it underscores the potential of phage-antibiotic combinations as a powerful tool in the treatment of infections caused by CRKP, offering a promising avenue to mitigate the public health challenges of antibiotic-resistant pathogens.

ABSTRACT Infections caused by multidrug resistant (MDR) pathogenic bacteria are a global health threat. Bacteriophages (“phage”) are increasingly used as alternative or last-resort therapeutics to treat patients infected by MDR bacteria. However, the therapeutic outcomes of phage therapy may be limited by the emergence of phage resistance during treatment and/or by physical constraints that impede phage–bacteria interactions in vivo. In this work, we evaluate the role of lung spatial structure on the efficacy of phage therapy for Pseudomonas aeruginosa infections. To do so, we developed a spatially structured metapopulation network model based on the geometry of the bronchial tree, including host innate immune responses and the emergence of phage-resistant bacterial mutants. We model the ecological interactions between bacteria, phage, and the host innate immune system at the airway (node) level. The model predicts the synergistic elimination of a P. aeruginosa infection due to the combined effects of phage and neutrophils, given the sufficient innate immune activity and efficient phage-induced lysis. The metapopulation model simulations also predict that MDR bacteria are cleared faster at distal nodes of the bronchial tree. Notably, image analysis of lung tissue time series from wild-type and lymphocyte-depleted mice revealed a concordant, statistically significant pattern: infection intensity cleared in the bottom before the top of the lungs. Overall, the combined use of simulations and image analysis of in vivo experiments further supports the use of phage therapy for treating acute lung infections caused by P. aeruginosa, while highlighting potential limits to therapy in a spatially structured environment given impaired innate immune responses and/or inefficient phage-induced lysis.IMPORTANCEPhage therapy is increasingly employed as a compassionate treatment for severe infections caused by multidrug-resistant (MDR) bacteria. However, the mixed outcomes observed in larger clinical studies highlight a gap in understanding when phage therapy succeeds or fails. Previous research from our team, using in vivo experiments and single-compartment mathematical models, demonstrated the synergistic clearance of acute P. aeruginosa pneumonia by phage and neutrophils despite the emergence of phage-resistant bacteria. In fact, the lung environment is highly structured, prompting the question of whether immunophage synergy explains the curative treatment of P. aeruginosa when incorporating realistic physical connectivity. To address this, we developed a metapopulation network model mimicking the lung branching structure to assess phage therapy efficacy for MDR P. aeruginosa pneumonia. The model predicts the synergistic elimination of P. aeruginosa by phage and neutrophils but emphasizes potential challenges in spatially structured environments, suggesting that higher innate immune levels may be required for successful bacterial clearance. Model simulations reveal a spatial pattern in pathogen clearance where P. aeruginosa are cleared faster at distal nodes of the bronchial tree than in primary nodes. Interestingly, image analysis of infected mice reveals a concordant and statistically significant pattern: infection intensity clears in the bottom before the top of the lungs. The combined use of modeling and image analysis supports the application of phage therapy for acute P. aeruginosa pneumonia while emphasizing potential challenges to curative success in spatially structured in vivo environments, including impaired innate immune responses and reduced phage efficacy.

The treatment of musculoskeletal infections (MSIs), including periprosthetic joint infection (PJI) and fracture-related infection (FRI), is often complicated by biofilm-related challenges necessitating multiple revision surgeries and incurring substantial costs. The emergence of antimicrobial resistance (AMR) adds to the complexity of the problem, leading to increased morbidity and healthcare expenses. There is an urgent need for novel antibacterial strategies, with the World Health Organization endorsing non-traditional approaches like bacteriophage (phage) therapy. Phage therapy, involving the targeted application of lytic potent phages, shows promise in the treatment of MSIs. Although historical clinical trials and recent case studies present significant milestones in the evolution of phage therapy over the past century, challenges persist, including variability in study designs, administration protocols and phage selection. Efforts to enhance treatment efficacy consist of personalized phage therapy and combination with antibiotics. Future perspectives entail addressing regulatory barriers, standardizing treatment protocols, and conducting high-quality clinical trials to establish phage therapy’s efficacy for the treatment of MSIs. Initiatives like the PHAGEFORCE study and the PHAGEinLYON Clinic programme aim to streamline phage therapy, facilitating personalized treatment approaches and systematic data collection to advance its clinical utility in these challenging infections.

Increasing antimicrobial resistance rates have revitalized bacteriophage (phage) research, the natural predators of bacteria discovered over 100 years ago. In order to use phages therapeutically, they should (1) preferably be lytic, (2) kill the bacterial host efficiently, and (3) be fully characterized to exclude side effects. Developing therapeutic phages takes a coordinated effort of multiple stakeholders. Herein, we review the state of the art in phage therapy, covering biological mechanisms, clinical applications, remaining challenges, and future directions involving naturally occurring and genetically modified or synthetic phages.

Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. We observed a repertoire of phage resistance mechanisms in K pneumoniae , such as disruption of phage binding (fhuA ::Tn and tonB ::Tn), extension of the phage latent period (mnmE ::Tn and rpoN ::Tn), and increased mutation frequency (mutS ::Tn and mutL ::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN ::Tn increased susceptibility to colistin while mutS ::Tn and mutL ::Tn increased resistance to rifampicin and colistin. Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations. [Display omitted] [ABSTRACT FROM AUTHOR]

Klebsiella spp. are causative agents of healthcare-associated infections in patients who are immunocompromised and use medical devices. The antibiotic resistance crisis has led to an increase in infections caused by these bacteria, which can develop into potentially life-threatening illnesses if not treated swiftly and effectively. Thus, new treatment options for Klebsiella are urgently required. Phage therapy can offer an alternative to ineffective antibiotic treatments for antibiotic-resistant bacteria infections. The aim of the present study was to produce a safe and effective phage cocktail treatment against Klebsiella pneumoniae and Klebsiella oxytoca, both in liquid in vitro culture and an in vivo Galleria mellonella infection model. The phage cocktail was significantly more effective at killing K. pneumoniae and K. oxytoca strains compared with monophage treatments. Preliminary phage cocktail safety was demonstrated through application in the in vivo G. mellonella model: where the phage cocktail induced no toxic side effects in G. mellonella. In addition, the phage cocktail significantly improved the survival of G. mellonella when administered as a prophylactic treatment, compared with controls. In conclusion, our phage cocktail was demonstrated to be safe and effective against Klebsiella spp. in the G. mellonella infection model. This provides a strong case for future treatment for Klebsiella infections, either as an alternative or adjunct to antibiotics. IMPORTANCE Klebsiella infections are a concern in individuals who are immunocompromised and are becoming increasingly difficult to treat with antibiotics due to their drug-resistant properties. Bacteriophage is one potential alternative therapy that could be used to tackle these infections. The present study describes the design of a non-toxic phage cocktail that improved the survival of Galleria mellonella infected with Klebsiella. This phage cocktail demonstrates potential for the safe and effective treatment of Klebsiella infections, as an adjunct or alternative to antibiotics. [ABSTRACT FROM AUTHOR]

In the advancement of Inflammatory Bowel Disease (IBD) treatment, existing therapeutic methods exhibit limitations; they do not offer a complete cure for IBD and can trigger adverse side effects. Consequently, the exploration of novel therapies and multifaceted treatment strategies provides patients with a broader range of options. Within the framework of IBD, gut microbiota plays a pivotal role in disease onset through diverse mechanisms. Bacteriophages, as natural microbial regulators, demonstrate remarkable specificity by accurately identifying and eliminating specific pathogens, thus holding therapeutic promise. Although clinical trials have affirmed the safety of phage therapy, its efficacy is prone to external influences during storage and transport, which may affect its infectivity and regulatory roles within the microbiota. Improving the stability and precise dosage control of bacteriophages--ensuring robustness in storage and transport, consistent dosing, and targeted delivery to infection sites --is crucial. This review thoroughly explores the latest developments in IBD treatment and its inherent challenges, focusing on the interaction between the microbiota and bacteriophages. It highlights bacteriophages' potential as microbiome modulators in IBD treatment, offering detailed insights into research on bacteriophage encapsulation and targeted delivery mechanisms. Particular attention is paid to the functionality of various carrier systems, especially regarding their protective properties and ability for colon-specific delivery. This review aims to provide a theoretical foundation for using bacteriophages as microbiome modulators in IBD treatment, paving the way for enhanced regulation of the intestinal microbiota. [ABSTRACT FROM AUTHOR]

Inflammatory periodontal diseases associated with the accumulation of dental biofilm, such as gingivitis and periodontitis, are very common and pose clinical problems for clinicians and patients. Gingivitis is a mild form of gum disease and when treated quickly and properly is completely reversible. Periodontitis is an advanced and irreversible disease of the periodontium with periods of exacerbations, progressions and remission. Periodontitis is a chronic inflammatory condition that damages the tissues supporting the tooth in its socket, i.e., the gums, periodontal ligaments, root cementum and bone. Periodontal inflammation is most commonly triggered by bacteria present in excessive accumulations of dental plaque (biofilm) on tooth surfaces. This disease is driven by disproportionate host inflammatory immune responses induced by imbalance in the composition of oral bacteria and changes in their metabolic activities. This microbial dysbiosis favors the establishment of inflammatory conditions and ultimately results in the destruction of tooth-supporting tissues. Apart microbial shift and host inflammatory response, environmental factors and genetics are also important in etiology In addition to oral tissues destruction, periodontal diseases can also result in significant systemic complications. Conventional methods of periodontal disease treatment (improving oral hygiene, dental biofilm control, mechanical plaque removal, using local or systemic antimicrobial agents) are not fully effective. All this prompts the search for new methods of therapy. Advanced periodontitis with multiple abscesses is often treated with antibiotics, such as amoxicillin, tetracycline, doxycycline, minocycline, clindamycin, or combined therapy of amoxicillin with metronidazole. However, due to the growing problem of antibiotic resistance, treatment does not always achieve the desired therapeutic effect. This review summarizes pathogenesis, current approaches in treatment, limitations of therapy and the current state of research on the possibility of application of bacteriophages and predatory bacteria to combat bacteria responsible for periodontitis. We present the current landscape of potential applications for alternative therapies for periodontitis based on phages and bacteria, and highlight the gaps in existing knowledge that need to be addressed before clinical trials utilizing these therapeutic strategies can be seriously considered.

Given the increasing threat of antimicrobial resistance, scientists are urgently seeking adjunct antimicrobial strategies, such as phage therapy (PT). However, despite promising results for the treatment of musculoskeletal infections in our center, crucial knowledge gaps remain. Therefore, a prospective observational study (PHAGEFORCE) and a multidisciplinary approach was set up to achieve and optimize standardized treatment guidelines. At our center, PT is strictly controlled and monitored by a multidisciplinary taskforce. Each phage treatment follows the same pathway to ensure standardization and data quality. Within the PHAGEFORCE framework, we established a testing platform to gain insight in the safety and efficacy of PT, biodistribution, phage kinetics and the molecular interaction between phages and bacteria. The draining fluid is collected to determine the phage titer and bacterial load. In addition, all bacterial isolates are fully characterized by genome sequencing to monitor the emergence of phage resistance. We hereby present a standardized bench-to-bedside protocol to gain more insight in the kinetics and dynamics of PT for musculoskeletal infections.

ABSTRACT: Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3–12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

Proteus mirabilis, a prevalent urinary tract pathogen and formidable biofilm producer, especially in Catheter-Associated Urinary Tract Infection, has seen a worrying rise in multidrug-resistant (MDR) strains. This upsurge calls for innovative approaches in infection control, beyond traditional antibiotics. Our research introduces bacteriophage (phage) therapy as a novel non-antibiotic strategy to combat these drug-resistant infections. We isolated P2-71, a lytic phage derived from canine feces, demonstrating potent activity against MDR P. mirabilis strains. P2-71 showcases a notably brief 10-minute latent period and a significant burst size of 228 particles per infected bacterium, ensuring rapid bacterial clearance. The phage maintains stability over a broad temperature range of 30-50°C and within a pH spectrum of 4-11, highlighting its resilience in various environmental conditions. Our host range assessment solidifies its potential against diverse MDR P. mirabilis strains. Through killing curve analysis, P2-71's effectiveness was validated at various MOI levels against P. mirabilis 37, highlighting its versatility. We extended our research to examine P2-71's stability and bactericidal kinetics in artificial urine, affirming its potential for clinical application. A detailed genomic analysis reveals P2-71's complex genetic makeup, including genes essential for morphogenesis, lysis, and DNA modification, which are crucial for its therapeutic action. This study not only furthers the understanding of phage therapy as a promising non-antibiotic antimicrobial but also underscores its critical role in combating emerging MDR infections in both veterinary and public health contexts. [ABSTRACT FROM AUTHOR]

Simple Summary: Aeromonas schubertii is one of the most significant opportunistic pathogen threats to humans, other mammals, and aquatic animals. Lytic bacteriophages have been recognized as effective alternatives to antibiotics for controlling bacterial infections. In this study, a lytic bacteriophage, which could effectively infect A. schubertii, was isolated for the first time. We report some of the characteristics of the phage, including its morphology, host range, storage stability, and structural proteins. Furthermore, in vivo experimental infections included intraperitoneal injection and immersion administration to evaluate the effects of phage therapy for A. schubertii infection. Our results suggest a potential use for phage SD04 in controlling this disease. Current experimental data indicate the value of using phage SD04 to prevent A. schubertii infection in snakehead. Aeromonas schubertii is a pathogen that severely affects aquatic animals, including the snakehead, Channa maculata. Lytic bacteriophages have been recognized as effective alternatives to antibiotics for controlling bacterial infections. However, there have been no reports of A. schubertii phages as far as we know. In this study, a lytic bacteriophage SD04, which could effectively infect A. schubertii, was isolated from pond water cultured with diseased snakehead. The SD04 phage formed small, round plaques on Petri dishes. Electron microscopy revealed a hexagonal head and a contractile tail. Based on its morphology, it may belong to the Myoviridae family. Two major protein bands with molecular weights of 50 and 38 kilodaltons were observed after the phage was subjected to SDS-PAGE. The phage showed a large average burst size, high specificity, and a broad host range. When stored at 4 °C, phage SD04 had high stability over 12 months and showed almost no variation within the first six months. All fish were healthy after both intraperitoneal injection and immersion administration of SD04, indicating the safety of the phage. After treatment with SD04, Channa maculata in both phage therapy groups and prevention groups showed high survival rates (i.e., 83.3 ± 3.3% and 100 ± 1.3%, respectively). Phage therapy inhibits bacterial growth in the liver, the target organ of the infected Channa maculat. The experimental results indicate the potential use of phage SD04 for preventing A. schubertii infection in Channa maculata. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the viability of four Staphylococcal bacteriophages when exposed to different concentrations of commonly used lavage solutions in the surgical treatment of prosthetic joint infections (PJI). Four tailed Staphylococcal bacteriophages and six different lavage solutions (chlorhexidine 4%, hydrogen peroxide 3%, acetic acid 3%, povidone iodine 10%, sodium hypochlorite 0.5%, and Vashe solution) at 100%, 1%, and 0.01% concentrations were used in this experiment. In addition, the temporal impact of exposing bacteriophages to these lavage solutions was also evaluated at 5‐min exposures and 24‐h exposures. The results show that the titers of the four bacteriophages were statistically significantly decreased for all lavage solutions (100% and 1%) at 5‐min exposures and 24‐h exposures. However, with 0.01% concentrations of the lavage solutions, only acetic acid caused a statistically significant decrease in bacteriophage titers compared to normal saline control. Our findings suggest that tailed Staphylococcal bacteriophages do not remain stable in high concentrations of the most commonly used lavage solutions. However, at very dilute concentrations the bacteriophages do remain viable. This has important clinical ramifications in that it shows when using bacteriophage therapy for PJI it is critical to thoroughly wash out any lavage solutions before the introduction of therapeutic bacteriophages especially when acetic acid is used. [ABSTRACT FROM AUTHOR]

Drug-resistant Staphylococcus aureus stands as a prominent pathogen in nosocomial and community-acquired infections, capable of inciting various infections at different sites in patients. This includes Staphylococcus aureus bacteremia (SaB), which exhibits a severe infection frequently associated with significant mortality rate of approximately 25%. In the absence of better alternative therapies, antibiotics is still the main approach for treating infections. However, excessive use of antibiotics has, in turn, led to an increase in antimicrobial resistance. Hence, it is imperative that new strategies are developed to control drug-resistant S. aureus infections. Bacteriophages are viruses with the ability to infect bacteria. Bacteriophages, were used to treat bacterial infections before the advent of antibiotics, but were subsequently replaced by antibiotics due to limited theoretical understanding and inefficient preparation processes at the time. Recently, phages have attracted the attention of many researchers again because of the serious problem of antibiotic resistance. This article provides a comprehensive overview of phage biology, animal models, diverse clinical case treatments, and clinical trials in the context of drug-resistant S. aureus phage therapy. It also assesses the strengths and limitations of phage therapy and outlines the future prospects and research directions. This review is expected to offer valuable insights for researchers engaged in phage-based treatments for drug-resistant S. aureus infections. [ABSTRACT FROM AUTHOR]

Stroke poses a critical global health challenge, leading to substantial morbidity and mortality. Existing treatments often miss vital timeframes and encounter limitations due to adverse effects, prompting the pursuit of innovative approaches to restore compromised brain function. This review explores the potential of filamentous phages in enhancing stroke recovery. Initially antimicrobial-centric, bacteriophage therapy has evolved into a regenerative solution. We explore the diverse role of filamentous phages in post-stroke neurological restoration, emphasizing their ability to integrate peptides into phage coat proteins, thereby facilitating recovery. Experimental evidence supports their efficacy in alleviating post-stroke complications, immune modulation, and tissue regeneration. However, rigorous clinical validation is essential to address challenges like dosing and administration routes. Additionally, genetic modification enhances their potential as injectable biomaterials for complex brain tissue issues. This review emphasizes innovative strategies and the capacity of filamentous phages to contribute to enhanced stroke recovery, as opposed to serving as standalone treatment, particularly in addressing stroke-induced brain tissue damage. [ABSTRACT FROM AUTHOR]

Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Currently there is no cure for JD and test-based culling has proved ineffective at preventing the spread. To isolate new mycobacteriophages (mbps) that can potentially be used to control JD transmission and infection on dairy farms, we optimized an isolation protocol by fecal spiking and the testing of different isolation solution compositions. Using this protocol, we successfully enhanced the yield of mbps from spiked fecal samples, elevating it from less than 1% to 59%. With this method, we isolated 14 mbps from 475 environmental samples collected from MAP-positive dairy farms, after in-sample enrichment with MAP and the fast-growing M. smegmatis. The sample sources included soil, manure pits, lactation barns, feces, milk, and drain water. After fingerprinting these mbps by restriction enzyme profiling, we concluded that 12 were distinct and novel. Further characterization of their host range revealed that eight were capable of lysing multiple MAP strains. We also studied the cross-resistance, lysogeny, the effect of pH and their antimycobacterial properties in milk replacer. Each novel mbp showed limited cross-resistance and prophage immunity and showed no reduction in the titer in a range of pHs after 4 h. The novel phages were also able to reduce the mycobacterial counts to zero after 8 h in milk replacer. In conclusion, these novel mbps could be considered to be used in the control strategies of JD on farms. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

ABSTRACTClostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%–92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry.IMPORTANCEClostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

The study aimed to assess the effect of inhaled bacteriophage therapy on oral mucosal immunity in children with acute tonsillitis. Materials and methods.We examined 212 patients aged 4 to 15 years old with acute tonsillitis and 110 age-matched apparently healthy children. Research methods: calculating the Neutrophil to lymphocyte ratio (NLR), saliva diagnostics — secretory immunoglobulin A (sIgA) and the pro-inflammatory cytokine (TNFα). Taking into account the scheme of the treatment, the patients were divided into mutually comparable groups: the first group included patients with acute tonsillitis who received the standard generally accepted treatment depending on the clinical form of the pathology, without using bacteriophage therapy — n = 107 (50.5%), the second group — patients receiving a course of bacteriophage therapy — n = 105 (49.95%), nebulizer bacteriophage therapy using liquid complex pyobacteriophage (PCL) (Microgen, Russia) from the first days of the disease along with standard treatment.Results.During bacteriophage therapy, on the 6th day of treatment, an increased sIgA level up to 97.2% was observed particularly in younger and adolescent patients up to 97.2% (p ≤ 0.05). At the same time, this parameter reached 75.8% and 81.6%, respectively (p ≤ 0.05), in patients who received only standard treatment. The following difference between the two study groups was observed: between patients in the younger age subgroup — 21.4%, in the older age subgroup -16.1% (p ≤ 0.05 relative to control group), which indicates a more effective drug-related effect in patients from the younger age group groups. Similar changes are observed while analyzing level of the pro-inflammatory cytokine (TNFα). Conclusion.The use of inhaled bacteriophage therapy in the combination treatment of children with acute tonsillitis helps to shorten the period of general and local clinical manifestations of the disease by 1.4-fold and improve mean local immunity from 5.7% up to 16.1% (p ≤ 0.05).

The studies on phage therapy have shown an overall protective effect of phages in bacterial infections, thus providing an optimistic outlook on the future benefits of phage-based technologies for treating bacterial diseases. However, the therapeutic effect is highly affected by the proper composition of phage cocktails. The rational approach to the design of bacteriophage cocktails, which is the subject of this study, allowed for development of an effective anti-mastitis solution, composed of virulent bacteriophages acting on Escherichia coli and Staphylococcus aureus. Based on the in-depth bioinformatic characterization of bacteriophages and their in vitro evaluation, the cocktail of five phages against E. coli and three against S. aureus strains was composed. Its testing in the milk model experiment revealed a reduction in the number of S. aureus of 45% and 30% for E. coli strains, and in the study of biofilm prevention, it demonstrated 99% inhibition of biofilm formation for all tested S. aureus strains and a minimum of 50% for 50% of E. coli strains. Such insights justify the need for rational design of cocktails for phage therapy and indicate the potential of the developed cocktail in the treatment of diseased animals, but this requires further investigations to evaluate its in vivo efficacy.

Bacterial diseases of the gastrointestinal (GI) tract continue to be a major worldwide cause of human morbidity and mortality. Among various enteric pathogens, Shigella spp. are some of the most common and deadly bacterial pathogens. They are responsible for ~125 million worldwide cases of shigellosis, and ~14,000 deaths annually, the majority in children under the age of 5 and occurring in developing countries. Preventing and treating shigellosis with conventional drugs (e.g., vaccines and antibiotics) has proven to be very difficult. Here, we assessed the safety and tolerability of ShigActive™, a lytic bacteriophage preparation targeting Shigella spp., in a randomized, placebo-controlled, double-blind Phase 1 clinical trial. Ten participants randomized 4:1 received ShigActive™ or placebo co-administered with sodium bicarbonate orally three times daily for 7 days. Solicited and unsolicited adverse events (AEs) were observed for 29 days. Fifty percent of the subjects receiving ShigActive™ reported mild GI-related symptoms, while one participant experienced moderate fatigue. No serious or medically attended AEs occurred through day 90. Additionally, no significant differences in GI-associated inflammatory mediators or fecal microbiome changes were observed between placebo- and ShigActive™-treated subjects, or from a participants’ baseline value. The results of this first-in-human (FIH) randomized, controlled Phase 1 trial of ShigActive™ demonstrate that it is safe and well tolerated when orally administered with no significant differences compared to placebo controls.

The conventional treatment of bacterial infections with antibiotics is becoming increasingly ineffective due to the emergence of multidrug-resistant (MDR) pathogens. This literature review explores the potential of bacteriophages as an alternative or adjunctive therapy to antibiotics in combating MDR infections in Africa. This analysis focuses on current research regarding the integration of phage therapy into African healthcare, highlighting its challenges and opportunities. This review begins with the AMR crisis and the need for new treatments, then covers the history, mechanisms, benefits, and limitations of phage therapy. Key African studies are summarized, identifying major obstacles such as regulatory issues, infrastructure, and research standardization. Research efforts in West Africa that have made notable progress in bacteriophage research are highlighted. This review concludes with recommendations for policymakers, researchers, and healthcare professionals to enhance the development and use of phage therapy in Africa, aiming to reduce antibiotic resistance and improve patient outcomes. By addressing the identified challenges and leveraging the unique advantages of phages, there is potential to significantly mitigate the impact of antibiotic resistance and improve patient outcomes in Africa.

Bacteriophages (phages) are viruses of bacteria and have been used as antibacterial agents now for over one-hundred years. The primary pharmacodynamics of therapeutic phages can be summed up as follows: phages at a certain concentration can reach bacteria at a certain rate, attach to bacteria that display appropriate receptors on their surfaces, infect, and (ideally) kill those now-adsorbed bacteria. Here, I consider the rate at which phages reach bacteria, during what can be dubbed as an ‘extracellular search’. This search is driven by diffusion and can be described by what is known as the phage adsorption rate constant. That constant in turn is thought to be derivable from knowledge of bacterial size, virion diffusion rates, and the likelihood of phage adsorption given this diffusion-driven encounter with a bacterium. Here, I consider only the role of bacterial size in encounter rates. In 1932, Schlesinger hypothesized that bacterial size can be described as a function of cell radius (R, or R1), as based on the non-phage-based theorizing of Smoluchowski (1917). The surface area of a cell—what is actually encountered—varies however instead as a function R2. Here, I both provide and review evidence indicating that Schlesinger’s assertion seems to have been correct.

Abstract Background Klebsiella aerogenes can cause ventilator-associated pneumonia by forming biofilms, and it is frequently associated with multidrug resistance. Phages are good antibiotic alternatives with unique advantages. There has been a lack of phage therapeutic explorations, kinetic studies, and interaction mechanism research targeting K. aerogenes. Methods Plaque assay, transmission electron microscopy and whole-genome sequencing were used to determine the biology, morphology, and genomic characteristics of the phage. A mouse pneumonia model was constructed by intratracheal/endobronchial delivery of K. aerogenes to assess the therapeutic effect of phage in vivo. Bioinformatics analysis and a prokaryotic protein expression system were used to predict and identify a novel capsule depolymerase. Confocal laser scanning microscopy, Galleria mellonella larvae infection models and other experiments were performed to clarify the function of the capsule depolymerase. Results A novel lytic phage (pK4-26) was isolated from hospital sewage. It was typical of the Podoviridae family and exhibited serotype specificity, high lytic activity, and high environmental adaptability. The whole genome is 40,234 bp in length and contains 49 coding domain sequences. Genomic data show that the phage does not carry antibiotic resistance, virulence, or lysogenic genes. The phage effectively lysed K. aerogenes in vivo, reducing mortality and alleviating pneumonia without promoting obvious side effects. A novel phage-derived depolymerase was predicted and proven to be able to digest the capsule, remove biofilms, reduce bacterial virulence, and sensitize the bacteria to serum killing. Conclusions The phage pK4-26 is a good antibiotic alternative and can effectively relieve pneumonia caused by multidrug-resistant K. aerogenes. It carries a depolymerase that removes biofilms, reduces virulence, and improves intrinsic immune sensitivity.

ABSTRACT Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient’s gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient’s antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections.IMPORTANCEPhage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

ABSTRACTMycobacterium abscessus is a nontuberculous mycobacteria that contributes to the decline and death of patients with lung diseases such as cystic fibrosis and other muco-obstructive airway diseases. M. abscessus is challenging to treat due to its extensive antibiotic resistance and ability to survive inside mammalian cells. An alternative to antibiotics is the therapeutic use of bacteriophages (phages). There are recent cases of phage therapy being used to treat M. abscessus infections in people under compassionate-use conditions. However, little is known about the ability of phages to kill bacteria, such as M. abscessus, which reside in an intracellular environment. Here, we used M. abscessus strains and phages from recent phage therapy cases to determine if phages can enter mammalian cells and if they can infect and kill intracellular M. abscessus. Using fluorescence microscopy, we demonstrate phage uptake by macrophages and lung epithelial cells, and we further demonstrate phage infection of intracellular M. abscessus with fluorescent reporter phages. Transmission electron microscopy was additionally used to image phage infection of intracellular M. abscessus. Together, these findings provide the first visualizations of phage-M. abscessus interactions in an intracellular environment. Finally, we show that phage treatment can significantly reduce the intracellular burden of M. abscessus in a manner that depends on both the specific phage and mammalian cell type involved. These results demonstrate the potential to use phage therapy to treat intracellular bacteria, specifically M. abscessus, while also highlighting the importance of prescreening phage therapy candidates for activity in an intracellular environment.IMPORTANCEAs we rapidly approach a post-antibiotic era, bacteriophage (phage) therapy may offer a solution for treating drug-resistant bacteria. Mycobacterium abscessus is an emerging, multidrug-resistant pathogen that causes disease in people with cystic fibrosis, chronic obstructive pulmonary disease, and other underlying lung diseases. M. abscessus can survive inside host cells, a niche that can limit access to antibiotics. As current treatment options for M. abscessus infections often fail, there is an urgent need for alternative therapies. Phage therapy is being used to treat M. abscessus infections as an option of last resort. However, little is known about the ability of phages to kill bacteria in the host environment and specifically in an intracellular environment. Here, we demonstrate the ability of phages to enter mammalian cells and to infect and kill intracellular M. abscessus. These findings support the use of phages to treat intracellular bacterial pathogens.

ABSTRACT In recent years, bacteriophages (or phages) have reentered the spotlight as alternative or adjuvant therapeutic agents to antibiotics. Their efficacy in more recalcitrant forms of infections like biofilms, frequently encountered in the orthopedic setting, remains less characterized. The present study aimed at evaluating the activity of phage-antibiotic combinations against Pseudomonas aeruginosa biofilms. A large collection of phages was de novo isolated from a wide variety of environmental sources. Three phages with a large host range against a.o. clinical orthopedic isolates, PSP2 (Yuavirus), PSP3 (Pbunavirus), and PSP30 (Bruynoghevirus), were selected for phage-antibiotic synergy assays on mature P. aeruginosa PAO1 biofilms. Phages were combined with ciprofloxacin, meropenem, and ceftazidime, all used as standard of care in bone-related infections. Significant reductions in both cell counts and biomass were observed for all phage-antibiotic combinations. The highest reduction in viable cell counts was observed for PSP3 and PSP2 in combination with ceftazidime [4.58 and 4.30 log10 Colony Forming Units (CFU)/well], followed by all phage combinations with ciprofloxacin (up to 3.56 log10 CFU/well). The highest reduction in biomass was obtained by combining PSP3 with ciprofloxacin (29.8%). Metabolic assays confirmed these findings with reductions in biofilm respiratory rate of up to 65%. Scanning electron microscopy imaging of PAO1 biofilms grown on titanium coupons and treated by ciprofloxacin and PSP30 confirmed the efficacy of the combined treatment with PSP30 and ciprofloxacin. This study highlights the synergetic effects of phage-antibiotic combinations on P. aeruginosa biofilms, thereby offering a promising approach to combat biofilm-associated infections. IMPORTANCE Biofilm-related infections are among the most difficult-to-treat infections in all fields of medicine due to their antibiotic tolerance and persistent character. In the field of orthopedics, these biofilms often lead to therapeutic failure of medical implantable devices and urgently need novel treatment strategies. This forthcoming article aims to explore the dynamic interplay between newly isolated bacteriophages and routinely used antibiotics and clearly indicates synergetic patterns when used as a dual treatment modality. Biofilms were drastically more reduced when both active agents were combined, thereby providing additional evidence that phage-antibiotic combinations lead to synergism and could potentially improve clinical outcome for affected patients.

In the age of progressive antimicrobial resistance and increased difficulty combating infections in immunocompromised hosts, there has been renewed interest in the use of nontraditional therapeutics for infections. Herein, we review the use of investigational non‐pharmaceutical anti‐infective agents targeting fungal, bacterial, and viral infections in patients with hematologic malignancies, focusing on those receiving hematopoietic cell transplantation or cellular therapies. We discuss immune checkpoint inhibitors, granulocyte transfusions, bone marrow colony‐stimulating factors, bacteriophages, fecal microbiota transplantation, and virus specific T‐cell therapy. Although there is promising early experience with many of these treatments, further studies will be required to define their optimal role in the therapeutic armamentarium against infections in immunocompromised hosts. [ABSTRACT FROM AUTHOR]

The Burkholderia cepacia complex (Bcc) causes life-threatening respiratory tract infections in persons with cystic fibrosis (CF). In CF patients, end-stage pulmonary disease often requires lung transplantation, and pre-transplant colonization with antibiotic-resistant Burkholderia is predictive of poor post-transplant outcomes. To address this issue, phage therapy has been proposed as a treatment for these infections. However, the majority of characterized Bcc phages are temperate and are therefore difficult to use as therapeutics, and the few obligately lytic phages that have been isolated have limited host ranges. To overcome these limitations, we have produced a virulent, broad-host range derivative of the temperate Burkholderia cenocepacia phage Milagro. Phage Milagro is a 39.1-kb temperate myophage related to phage KL3 and the paradigm coliphage P2. This phage showed a phenotype of spontaneous autoplaquing on lawns of Milagro lysogens, and these autoplaques were found to be produced by virulent mutants of the parental phage Milagro. Mutations associated with virulence were identified as single base changes, insertions or deletions in the phage lysogeny control region that define potential operator sites required for lysogen maintenance. To improve phage host range, the C-terminal domain of the Milagro tail fiber was replaced with the receptor-binding domain of the broad-host range tailocin (high molecular weight bacteriocin) BceTMilo. A spontaneous virulent mutant of this engineered phage, designated Milagro vir gp20:Milo, exhibited an expanded host range over the parental phage and is able to infect multiple Bcc species including B. cenocepacia, Burkholderia multivorans, Burkholderia gladioli, Burkholderia dolosa, and Burkholderia vietnamensis. [ABSTRACT FROM AUTHOR]

Microbial resistance to antibiotics poses a tremendous challenge. Bacteriophages may provide a useful alternative or adjunct to traditional antibiotics. To be used in therapy, bacteriophages need to be purified from endotoxins and tested for their effects on human immune cells. Interleukin-1 Receptor Associated Kinase-3 (IRAK3) is a negative regulator of inflammation and may play a role in the modulation of immune signalling upon bacteriophage exposure to immune cells. This study aimed to investigate the immune effects of crude and purified bacteriophage FNU1, a bacteriophage that targets the oral pathobiont Fusobacterium nucleatum, on wildtype and IRAK3 knockout THP-1 monocytic cell lines. The IRAK3 knockout cell line was also used to develop a novel endotoxin detection assay. Exposure to crude FNU1 increased the production of pro-inflammatory cytokines (Tumour necrosis factor – alpha (TNF-α) and Interleukin 6 (IL-6)) compared to purified FNU1 in wildtype and IRAK3 knockout THP-1 monocytes. In the IRAK3 knockout THP-1 cells, exposure to crude FNU1 induced a higher immune response than the wildtype monocytes, supporting the suggestion that the inhibitory protein IRAK3 regulates reactions to endotoxins and impurities in bacteriophage preparations. Finally, the novel endotoxin detection assay generated here provides a robust and accurate method for determining endotoxin concentrations. [ABSTRACT FROM AUTHOR]

Given the high frequency of patients presenting with urinary tract infections (UTIs) and the ensuing high degree of antibiotic prescription, UTI is a critical point of intervention for non-antibiotic treatments to curb the further development of antimicrobial resistance and provide risk-appropriate care for patients. To highlight several select non-antibiotic therapies for the treatment of uncomplicated UTI and relevant indications (prevention and complicated UTI) from recent literature. PubMed, Google Scholar, and clinicaltrials.gov were searched for clinical trials published in the English language corresponding to non-antibiotic treatments for UTI. The focus of this narrative review centres on a limited number of non-antibiotic therapies for the treatment of UTI based on (a) herbal extracts or (b) antibacterial strategies (e.g. bacteriophage therapy and D-mannose). The experience of treatment with non-steroidal anti-inflammatory drugs is also used to fuel discussion on the risk of developing pyelonephritis without antibiotics—compared with the projected harms of continuing their widespread use. Non-antibiotic treatment strategies for UTI have shown varying results in clinical trials, and the current evidence does not yet indicate a clear, better alternative to antibiotics. However, the collective experience with non-antibiotic treatments suggests that there is a need to weigh the actual benefits/risks of unfettered, non–culture-confirmed antibiotic use in uncomplicated UTI. Given the different mechanisms of action of proposed alternatives, more in-depth knowledge on microbiological and pathophysiological factors influencing UTI susceptibility and prognostic indicators are highly needed to stratify patients most likely to benefit. The feasibility of alternatives in clinical practice should also be considered. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential healthcare applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (Chi) infects serovars of the pathogenic enterobacterium Salmonella enterica However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multi-gene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multi-gene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. This way, we identified motile mutants with varying degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multi-drug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and informs research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections.IMPORTANCEAntimicrobial resistance is a large concern in the healthcare field. With more multi-drug resistant bacterial pathogens emerging, other techniques for eliminating bacterial infections are being explored. Among these is phage therapy, where combinations of specific phages are used to treat infections. Generally, phages utilize cell appendages and surface receptors for the initial attachment to their host. Phages that are flagellotropic are of particular interest because flagella are often important in bacterial virulence, making resistance to attachment of these phages harder to achieve without reducing virulence. This study discovered the importance of a multi-drug efflux pump for the infection of Salmonella enterica by a flagellotropic phage. In theory, if a bacterial pathogen develops phage resistance by altering expression of the efflux pump then the pathogen would simultaneously become more susceptible to the antibiotic substrates of the pump. Thus, co-administering antibiotics and flagellotropic phage may be a particularly potent antibacterial therapy.