Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis.

Tuberculosis (TB) remains a major global health concern, with drug-resistant strains posing a significant challenge to effective treatment. Bacteriophage (phage) therapy has emerged as a potential alternative to combat antibiotic resistance. In this study, we investigated the efficacy of widely used mycobacteriophages (D29, TM4, DS6A) against Mycobacterium tuberculosis (M. tuberculosis) under pathophysiological conditions associated with TB, such as low pH and hypoxia. We found that even at low multiplicity of infection (MOI), mycobacteriophages effectively infected M. tuberculosis, got rapidly amplified, and lysed M. tuberculosis, demonstrating their potential as therapeutic agents. Furthermore, we observed a novel phage tolerance mechanism with bacteria forming aggregates after several days of phage treatment. These aggregates were enriched with biofilm components and metabolically active bacteria. However, no phage tolerance was observed upon treatment with the three-phage mixture, highlighting the dynamic interplay between phages and bacteria and emphasizing the importance of phage cocktails. We also observed that phages were effective in lysing bacteria even under low pH and low oxygen concentrations as well as antibiotic-resistant bacteria. Our results provide key insights into phage infection of slow-growing bacteria and suggest that mycobacteriophages can effectively eliminate M. tuberculosis in complex pathophysiological environments like hypoxia and acidic pH. These results can aid in developing targeted phage-based therapies to combat antibiotic-resistant mycobacterial infections.

Proportion of patients with prosthetic joint infection eligible for adjuvant phage therapy: a French single-centre retrospective study.

BACKGROUND: Bone and joint infections represent a major public health issue due to their increasing prevalence, their functional prognosis and their cost to society. Phage therapy has valuable anti-biofilm properties against prosthetic joint infections (PJI). The aim of this study was to establish the proportion of patients eligible for phage therapy and to assess their clinical outcome judged against all patients presenting with PJI. METHOD: . Patients admitted for periprosthetic joint infection (PJI) at a French general hospital between 2015 and 2019 were retrospectively included. Eligibility for phage therapy was determined based on French recommendations, with polymicrobial infections serving as exclusion criteria. Patients were categorized into two groups: those eligible and those ineligible for phage therapy. We analyzed their characteristics and outcomes, including severe adverse events, duration of intravenous antibiotic therapy, length of hospitalization, and relapse rates. RESULTS: . In this study, 96 patients with PJI were considered in multidisciplinary medical meetings. Of these, 44% patients (42/96) were eligible for additional phage therapy. This group of patients had a longer duration of intravenous therapy (17 days vs. 10 days, p = 0.02), more severe adverse events (11 vs. 3, p = 0.08) and had a longer hospital stay (43 days vs. 18 days, p < 0.01). CONCLUSION: . A large number of patients met eligibility criteria for phage therapy and treatment and follow-up is more complex. A larger epidemiological study would more accurately describe the prognosis of eligible patients.

Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens.

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens.

Insights into the Preparation of and Evaluation of the Bactericidal Effects of Phage-Based Hydrogels.

The rise of antibiotic-resistant strains demands new alternatives in antibacterial treatment. Bacteriophages, with their precise host specificity and ability to target and eliminate bacteria safely, present a valuable option. Meanwhile, hydrogels, known for their excellent biodegradability and biocompatibility, serve as ideal carriers for bacteriophages. The combination of bacteriophages and hydrogels ensures heightened phage activity, concentration, controlled release, and strong antibacterial properties, making it a promising avenue for antibacterial treatment. This article provides a comprehensive review of different crosslinking methods for phage hydrogels, focusing on their application in treating infections caused by various drug-resistant bacteria and highlighting their effective antibacterial properties and controlled release capabilities.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Optimization of bacteriophage therapy for difficult-to-treat musculoskeletal infections: a bench-to-bedside perspective.

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.

Immunomodulation in Non-traditional Therapies for Methicillin-resistant Staphylococcus aureus (MRSA) Management.

The rise of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge in clinical settings due to its ability to evade conventional antibiotic treatments. This overview explores the potential of immunomodulatory strategies as alternative therapeutic approaches to combat MRSA infections. Traditional antibiotics are becoming less effective, necessitating innovative solutions that harness the body's immune system to enhance pathogen clearance. Recent advancements in immunotherapy, including the use of antimicrobial peptides, phage therapy, and mechanisms of immune cells, demonstrate promise in enhancing the body's ability to clear MRSA infections. However, the exact interactions between these therapies and immunomodulation are not fully understood, underscoring the need for further research. Hence, this review aims to provide a broad overview of the current understanding of non-traditional therapeutics and their impact on immune responses, which could lead to more effective MRSA treatment strategies. Additionally, combining immunomodulatory agents with existing antibiotics may improve outcomes, particularly for immunocompromised patients or those with chronic infections. As the landscape of antibiotic resistance evolves, the development of effective immunotherapeutic strategies could play a vital role in managing MRSA infections and reducing reliance on traditional antibiotics. Future research must focus on optimizing these approaches and validating their efficacy in diverse clinical populations to address the urgent need for effective MRSA management strategies.

Isolation and characterization of two novel bacteriophages against carbapenem-resistant Klebsiella pneumoniae.

The increase of antibiotic-resistant bacteria has become a global health emergency and the need to explore alternative therapeutic options arises. Phage therapy uses bacteriophages to target specific bacterial strains. Phages are highly specific and can target resistant bacteria. Currently, research in this regard is focused on ensuring reliability and safety to bring this tool into clinical practice. The first step is to conduct comprehensive preclinical research. In this work, we present two novel bacteriophages vB_Kpn_F13 and vB_Kpn_F14 isolated against clinical carbapenem-resistant Klebsiella pneumoniae strains obtained from hospital sewage. Multiple studies in vitro were conducted, such as sequencing, electron microscopy, stability, host range infectivity, planktonic effect and biofilm inhibition in order to discover their ability to be used against carbapenem-resistant K. pneumoniae pathogens causing difficult-to-treat infections.

Fighting fire with fire: using infectious agents to treat persistent infection.

Infectious diseases lead to significant morbidity and mortality. Often, resolution of the acute stage of the disease leads to microbial persistence, resulting in chronic debilitating disease. Management of persistent infections frequently requires lifelong therapy with antimicrobial agents. These infections could be chronic viral infections like HIV, hepatitis B or chronic bacterial persistent infections like prosthetic joint infections caused by multi-drug resistant organisms. Bacteriophages have been designed specifically to target recalcitrant bacterial infections, such as prosthetic joint infections with varying success. In this review, we describe the historic evolution of scenarios and risks associated with innovative therapy using infectious agents to treat other persistent infections.

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The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae.

One of the most common bacteria that cause nosocomial infections is Klebsiella pneumonia (K. pneumoniae), especially in patients who are very sick and admitted to the intensive care unit (ICU). The frequency of multi-drug-resistant Klebsiella pneumoniae (MDRKP) has dramatically increased worldwide in recent decades, posing an urgent threat to public health. The Western world's bacteriophage (phage) studies have been revitalized due to the increasing reports of antimicrobial resistance and the restricted development and discovery of new antibiotics. These factors have also spurred innovation in other scientific domains. The primary agent in phage treatment is an obligately lytic organism (called bacteriophage) that kills the corresponding bacterial host while sparing human cells and lessening the broader effects of antibiotic usage on commensal bacteria. Phage treatment is developing quickly, leading to many clinical studies and instances of life-saving medicinal use. In addition, phage treatment has a few immunological adverse effects and consequences in addition to its usefulness. Since K. pneumoniae antibiotic resistance has made treating multidrug-resistant (MDR) infections challenging, phage therapy (PT) has emerged as a novel therapeutic strategy. The effectiveness of phages has also been investigated in K. pneumoniae biofilms and animal infection models. Compared with antibiotics, PT exhibits numerous advantages, including a particular lysis spectrum, co-evolution with bacteria to avoid the emergence of phage resistance, and a higher abundance and diversity of phage resources than found in antibiotics. Moreover, phages are eliminated in the absence of a host bacterium, which makes them the only therapeutic agent that self-regulates at the sites of infection. Therefore, it is essential to pay attention to the role of PT in treating these infections. This study summarizes the state of knowledge on Klebsiella spp. phages and provides an outlook on the development of phage-based treatments that target K. pneumoniae in clinical trials.

Antimicrobial resistance: use of phage therapy in the management of resistant infections.

The emergence and increase in antimicrobial resistance (AMR) is now widely recognized as a major public health challenge. Traditional antimicrobial drugs are becoming increasingly ineffective, while the development of new antibiotics is waning. As a result, alternative treatments for infections are garnering increased interest. Among these alternatives, bacteriophages, also known as phages, are gaining renewed attention and are reported to offer a promising solution to alleviate the burden of bacterial infections. This review discusses the current successes of phage therapy (PT) against multidrug-resistant organisms (MDROs), such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter spp. The review also compares the efficacy of PT with that of chemical antibiotics, reporting on its benefits and limitations, while highlighting its impact on the human gut microbiome and immune system. Despite its potential, phage therapy is reported to face challenges such as the narrow antibacterial range, the complexity of developing phage cocktails, and the need for precise dosing and duration protocols. Nevertheless, continued research, improved regulatory frameworks, and increased public awareness are essential to realize its full potential and integration into standard medical practice, paving the way for innovative treatments that can effectively manage infections in an era of rising antimicrobial resistance.

Advanced Vibrational Spectroscopy and Bacteriophages Team Up: Dynamic Synergy for Medical and Environmental Applications.

Bacteriophages are emerging as a promising alternative in combating antibiotic-resistant bacteria amidst the escalating global antimicrobial resistance crisis. Recently, there has been a notable resurgence of interest in phages, prompting extensive research into their therapeutic potential. Beyond conventional microbiology and virology techniques, such as genomics and proteomics, novel phenotypic and chemical characterization methods are being explored. Among these, there is a growing interest in vibrational spectroscopy, especially in advanced modalities such as surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), and atomic force microscopy-infrared spectroscopy (AFM-IR), which offer improved sensitivity and spatial resolution. This review explores the spectrum of uses of vibrational spectroscopy for bacteriophages, including its role in diagnostics, biosensing, phage detection, assistance in phage-based therapy, and advancing basic research.

Phage Therapy: An Alternative Approach to Combating Multidrug-Resistant Bacterial Infections in Cystic Fibrosis.

Patients with cystic fibrosis (CF) are prone to developing life-threatening lung infections with a variety of pathogens that are difficult to eradicate, such as Burkholderia cepacia complex (Bcc), Hemophilus influenzae, Mycobacterium abscessus (Mab), Pseudomonas aeruginosa, and Staphylococcus aureus. These infections still remain an important issue, despite the therapy for CF having considerably improved in recent years. Moreover, prolonged exposure to antibiotics in combination favors the development and spread of multi-resistant bacteria; thus, the development of alternative strategies is crucial to counter antimicrobial resistance. In this context, phage therapy, i.e., the use of phages, viruses that specifically infect bacteria, has become a promising strategy. In this review, we aim to address the current status of phage therapy in the management of multidrug-resistant infections, from compassionate use cases to ongoing clinical trials, as well as the challenges this approach presents in the particular context of CF patients.

Role of bacteriophages in shaping gut microbial community.

Phages are the most diversified and dominant members of the gut virobiota. They play a crucial role in shaping the structure and function of the gut microbial community and consequently the health of humans and animals. Phages are found mainly in the mucus, from where they can translocate to the intestinal organs and act as a modulator of gut microbiota. Understanding the vital role of phages in regulating the composition of intestinal microbiota and influencing human and animal health is an emerging area of research. The relevance of phages in the gut ecosystem is supported by substantial evidence, but the importance of phages in shaping the gut microbiota remains unclear. Although information regarding general phage ecology and development has accumulated, detailed knowledge on phage-gut microbe and phage-human interactions is lacking, and the information on the effects of phage therapy in humans remains ambiguous. In this review, we systematically assess the existing data on the structure and ecology of phages in the human and animal gut environments, their development, possible interaction, and subsequent impact on the gut ecosystem dynamics. We discuss the potential mechanisms of prophage activation and the subsequent modulation of gut bacteria. We also review the link between phages and the immune system to collect evidence on the effect of phages on shaping the gut microbial composition. Our review will improve understanding on the influence of phages in regulating the gut microbiota and the immune system and facilitate the development of phage-based therapies for maintaining a healthy and balanced gut microbiota.

A game of resistance: War between bacteria and phages and how phage cocktails can be the solution.

While phages hold promise as an antibiotic alternative, they encounter significant challenges in combating bacterial infections, primarily due to the emergence of phage-resistant bacteria. Bacterial defence mechanisms like superinfection exclusion, CRISPR, and restriction-modification systems can hinder phage effectiveness. Innovative strategies, such as combining different phages into cocktails, have been explored to address these challenges. This review delves into these defence mechanisms and their impact at each stage of the infection cycle, their challenges, and the strategies phages have developed to counteract them. Additionally, we examine the role of phage cocktails in the evolving landscape of antibacterial treatments and discuss recent studies that highlight the effectiveness of diverse phage cocktails in targeting essential bacterial receptors and combating resistant strains.

Combination of bacteriophages and vancomycin in a co-delivery hydrogel for localized treatment of fracture-related infections.

Fracture-related infections (FRIs), particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA), are challenging to treat. This study designed and evaluated a hydrogel loaded with a cocktail of bacteriophages and vancomycin (1.2 mg/mL). The co-delivery hydrogel showed 99.72% reduction in MRSA biofilm in vitro. The hydrogel released 54% of phages and 82% of vancomycin within 72 h and maintained activity for eight days, in vivo the co-delivery hydrogel with systemic antibiotic significantly reduced bacterial load by 0.99 log10 CFU compared to controls, with active phages detected in tissues at euthanasia (2 × 103 PFU/mL). No phage resistance was detected in the phage treatment groups, and serum neutralization resulted in only a 20% reduction in phage count. In this work, we show that a phage-antibiotic co-delivery system via CMC hydrogel is a promising adjunct to systemic antibiotic therapy for MRSA-induced FRI, highlighting its potential for localized, sustained delivery and improved treatment outcomes.

Characterization of a novel phage against multidrug-resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant challenge in global healthcare, underscoring the urgency for innovative therapeutic approaches. Phage therapy emerges as a promising strategy amidst rising antibiotic resistance, emphasizing the crucial need to identify and characterize effective phage resources for clinical use. In this study, we introduce a novel lytic phage, RCIP0100, distinguished by its classification into the Chaoyangvirus genus and Fjlabviridae family based on International Committee on Taxonomy of Viruses (ICTV) criteria due to low genetic similarity to known phage families. Our findings demonstrate that RCIP0100 exhibits broad lytic activity against 15 out of 27 tested MDR-KP strains, including diverse profiles such as carbapenem-resistant K. pneumoniae (CR-KP). This positions phage RCIP0100 as a promising candidate for phage therapy. Strains resistant to RCIP0100 also showed increased susceptibility to various antibiotics, implying the potential for synergistic use of RCIP0100 and antibiotics as a strategic countermeasure against MDR-KP.

PhagoScreener: A novel phagogram platform based on a capillary-wave microbioreactor.

Due to the overuse of antibiotics, the number of multidrug-resistant pathogen bacteria is rising in recent years posing a serious threat to human health. One promising alternative for treatment is the application of phage therapy using highly selective bacteriophages. Because of their selectivity, individual screens called phagograms for each patient are required to select phages from a phage library. Phagograms are mostly performed via bacterial cultivation on double layer agar plates and phage addition causing bacterial lysis. However, these assays are work-intensive and have a low ability for parallelization and automation. Hence, highly parallelizable and automatable microbioreactors in the lowest microliter scale could offer an economic solution increasing the throughput of phagograms. This paper demonstrates the applicability of a novel capillary-wave microbioreactor (cwMBR) to perform phagograms. Due to its small volume of only 7 µL and the open-droplet design, it can be easily automated and parallelized in future. Furthermore, the ability of online biomass measurement makes the cwMBR a perfect phagogram platform in the future. Herein, phagograms with E. coli and different concentrations of the phages MM02 and EASG3 were performed as proof of concept for phagograms in the cwMBR. Thereby, the cwMBR was able to measure differences in lysis kinetics of different phages. Furthermore, the phagograms were compared to those in conventional microtiter plate readers revealing the cwMBR as ideal alternative for phagograms as it combines favorable mixing conditions and a phage repellent hydrophilic glass surface with online biomass measurement in an open-droplet design for future parallelization and automation.

Phytopathological management through bacteriophages: enhancing food security amidst climate change.

The increasing global population and climate change pose significant challenges to agriculture, particularly in managing plant diseases caused by phytopathogens. Traditional methods, including chemical pesticides and antibiotics, have become less effective due to pathogen resistance and environmental concerns. Phage therapy emerges as a promising alternative, offering a sustainable and precise approach to controlling plant bacterial diseases without harming beneficial soil microorganisms. This review explores the potential of bacteriophages as biocontrol agents, highlighting their specificity, rapid multiplication, and minimal environmental impact. We discuss the historical context, current applications, and prospects of phage therapy in agriculture, emphasizing its role in enhancing crop yield and quality. Additionally, the paper examines the integration of phage therapy with modern agricultural practices and the development phage cocktails and genetically engineered phages to combat resistant pathogens. The findings suggest that phage therapy could revolutionize phytopathological management, contributing to global food security and sustainable agricultural practices. ONE-SENTENCE SUMMARY: The burden of plant diseases and phage-based phytopathological treatment.

Rationalisation of the purification process for a phage active pharmaceutical ingredient.

The resurgence of phage therapy, once abandoned in the early 20th century in part due to issues related to the purification process and stability, is spurred by the global threat of antibiotic resistance. Engineering advances have enabled more precise separation unit operations, improving overall purification efficiency. The present review discusses the physicochemical properties of impurities commonly found in a phage lysate, e.g., contaminants, phage-related impurities, and propagation-related impurities. Differences in phages and bacterial impurities properties are leveraged to elaborate a four-step phage purification process: clarification, capture and concentration, subsequent purification and polishing. Ultimately, a framework for rationalising the development of a purification process is proposed, considering three operational characteristics, i.e., scalability, transferability to various phages and duration. This guide facilitates the preselection of a sequence of unit operations, which can then be confronted with the expected impurities to validate the theoretical capacity of the process to purify the phage lysate.