Case report: Personalized triple phage-antibiotic combination therapy to rescue necrotizing fasciitis caused by Panton-Valentine leukocidin-producing MRSA in a 12-year-old boy.

Maximal standard-of-care (SOC) management could not stop the life-threatening progression of a necrotizing fasciitis induced by Panton-Valentine Leukocidin-producing Methicillin-Resistant Staphylococcus aureus (MRSA) in a 12-year-old boy. Multi-route phage therapy was initiated along with antibiotics against Staphylococcus aureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, eventually leading to full recovery with no reported adverse events.

Case report: Local bacteriophage therapy for fracture-related infection with polymicrobial multi-resistant bacteria: hydrogel application and postoperative phage analysis through metagenomic sequencing.

Fracture-related infections can be challenging, particularly with concomitant severe bone defects and multi-resistant microorganisms. We present a case of a 42-year-old patient with a fracture-related infection following a war injury from a gunshot, resulting in a 12-cm subtrochanteric segmental bone defect and the detection of four different multi-resistant Gram-negative bacteria. Due to antibiotic drug resistance, treatment with bacteriophages was considered. Phage susceptibility testing revealed the activity of a commercially available bacteriophage cocktail (Intesti bacteriophage, Eliava Institute, Tbilisi, Georgia). This phage cocktail was included in a modified two-stage Masquelet technique. During the first intervention, the bone was debrided and samples for microbiological and phage testing were harvested. The indwelling intramedullary rod was removed, and the bone defect was filled with a PMMA spacer loaded with colistin and the bone stabilized with a plate. During the second procedure, the PMMA spacer was removed and a silver-coated angular stable plate was implanted. The bone defect was filled with a fibular autograft and allograft cancellous bone chips. At the end of the procedure, the Intesti bacteriophage cocktail was injected into a DAC hydrogel and this bacteriophage hydrogel composite was then put onto the angular stable plate. Postoperatively the wound fluid was collected over 72 h, and high-throughput metagenomic sequencing was performed. This showed a time-dependent release of the bacteriophages in the wound fluid, with a relatively high concentration after 12 h, decreasing to DNA copies of 0 after 72 h. Furthermore, we have assessed the release of phages from DAC gel and the effect of DAC gel on the phages in vitro. The results showed a stable and rapid release of phages from the DAC gel (~1×103 PFU/mL). The clinical course of the patient showed no relapse of the infection with good bone consolidation of the bone defect after 1 year without the need for any surgical revision. To the best of our knowledge, this is the first case that shows the detection of bacteriophage DNA copies by high-throughput metagenomics sequencing in a patient with a complex fracture-related infection. Successful treatment of this case encourages further investigation of bacteriophage therapy in patients with complex bone and joint infections.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. METHODS: Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. RESULTS: At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. CONCLUSION: Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.

Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study.

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127-0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage-antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363 .

Use of the Naturally Occurring Bacteriophage Grouping Model for the Design of Potent Therapeutic Cocktails.

The specificity of phages and their ability to evolve and overcome bacterial resistance make them potentially useful as adjuncts in the treatment of antibiotic-resistant bacterial infections. The goal of this study was to mimic a natural grouping of phages of interest and to evaluate the nature of their proliferation dynamics with bacteria. We have, for the first time, transferred naturally occurring phage groups directly from their sources of isolation to in vitro and identified 13 P. aeruginosa and 11 K. pneumoniae phages of 18 different genera, whose host range was grouped as 1.2-17%, 28-48% and 60-87%, using a large collection of P. aeruginosa (n = 102) and K. pneumoniae (n = 155) strains carrying different virulence factors and phage binding receptors. We introduced the interpretation model curve for phage liquid culturing, which allows easy and quick analysis of bacterial and phage co-proliferation and growth of phage-resistant mutants (PRM) based on qualitative and partially quantitative evaluations. We assayed phage lytic activities both individually and in 14 different cocktails on planktonic bacterial cultures, including three resistotypes of P. aeruginosa (PAO1, PA14 and PA7) and seven K. pneumoniae strains of different capsular serotypes. Based on the results, the natural phage cocktails designed and tested in this study largely performed well and inhibited PRM growth either synergistically or in proto-cooperation. This study contributes to the knowledge of phage behavior in cocktails and the formulation of therapeutic phage preparations. The paper also provides a detailed description of the methods of working with phages.

Burn or trauma scoring: experience of the burn unit of the Queen Astrid Military Hospital during the terror attacks on 22 March 2016.

PURPOSE: On 22 March 2016, the burn unit (BU) of Queen Astrid Military Hospital assessed a surge in severely injured victims from terror attacks at the national airport and Maalbeek subway station according to the damage control resuscitation (DCR) and damage control surgery (DCS) principles. This study delves into its approach to identify a suitable triage scoring system and to determine if a BU can serve as buffer capacity for mass casualty incidents (MCIs). METHODS: The study reviewed retrospectively the origin of explosion, demographic data, sustained injuries, performed surgery, and length of stay of all admitted patients. Trauma scores (Injury Severity Score (ISS) and New Injury Severity Score (NISS)) and triage scores (Revised Trauma Score (RTS), New Trauma Score (NTS), and Trauma Score Injury Severity Score (TRISS)), were compared to burn mortality scores (Osler updated Baux Score and Tobiasen's Abbreviated Burn Severity Index (ABSI)). RESULTS: Of the 23 casualties admitted to the BU, the scores calculated on average 3.5 indications for a level 1 trauma center (ISS 4, NISS 6, RTS 0, T-NTS 4). Nevertheless, no deaths occurred during admission or the 1-year follow-up. CONCLUSION: MCIs create chaos and a high demand for care. Avoiding bottlenecks and adhering to the DCR/DCS principles are necessary to deliver the best care to the largest number of people. This study indicates that a BU can serve as buffer capacity for MCIs. Nevertheless, its integration into the medical resilience plan depends on accurate scoring, comprehensive care availability, and understanding of the DCR/DCS concept. NTS for triage seems the best fit for scoring polytrauma referrals to a BU during MCIs.

Improving phage therapy by evasion of phage resistance mechanisms.

Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.

Bacteriophages for the treatment of pseudomonas-infected vascular prosthesis.

We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a prosthetic aortic graft. As there are currently no Danish laboratories offering phages for clinical therapy, and this case, to our knowledge represents the first applied phage therapy in Denmark, the practical and regulatory aspects of offering this treatment option in Denmark is briefly reviewed along with the clinical case.

Exploiting phage-antibiotic synergies to disrupt Pseudomonas aeruginosa PAO1 biofilms in the context of orthopedic 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.

Bacteriophage Production in Compliance with Regulatory Requirements.

In this chapter, we discuss production requirements for therapeutic bacteriophage preparations. We review the current regulatory expectancies and focus on pragmatic production processes, implementing relevant controls to ensure the quality, safety, and efficacy of the final products. The information disclosed in this chapter can also serve as a basis for discussions with competent authorities regarding the implementation of expedited bacteriophage product development and licensing pathways, taking into account some peculiarities of bacteriophages (as compared to conventional medicines), such as their specificity for, and co-evolution with, their bacterial hosts. To maximize the potential of bacteriophages as natural controllers of bacterial populations, the implemented regulatory frameworks and manufacturing processes should not only cater to defined bacteriophage products. But, they should also facilitate personalized approaches in which bacteriophages are selected ad hoc and even trained to target the patient's infecting bacterial strain(s), whether or not in combination with other antimicrobials such as antibiotics.

Guidelines to Compose an Ideal Bacteriophage Cocktail.

Properly designed bacteriophage therapeutics are the cornerstone for a successful outcome of bacteriophage therapy. Here we present an overview of the different strategies and steps that can be taken to develop a bacteriophage cocktail that complies with relevant quality and safety requirements. It is based on empirical bacteriophage therapy knowledge from over a century of experience, more recently performed studies, and emerging technologies. We emphasize the selection of adequate bacteriophages and describe a modified Appelmans' method to improve the overall performance of therapeutic bacteriophages individually and collectively in the cocktail. We present two versions of the method, which differ from each other by the employed techniques to evaluate phage activity and synergy: photometric assessment of bacterial growth versus measurement of bacterial respiration via the Omnilog® system.

Magistral Phage Preparations: Is This the Model for Everyone?

Phage therapy is increasingly put forward as a promising additional tool to help curb the global antimicrobial resistance crisis. However, industrially manufactured phage medicinal products are currently not available on the European Union and United States markets. In addition, it is expected that the business purpose-driven phage products that are supposed to be marketed in the future would mainly target commercially viable bacterial species and clinical indications, using fixed phage cocktails. hospitals or phage therapy centers aiming to help all patients with difficult-to-treat infections urgently need adequate phage preparations. We believe that national solutions based on the magistral preparation of personalized (preadapted) phage products by hospital and academic facilities could bring an immediate solution and could complement future industrially manufactured products. Moreover, these unlicensed phage preparations are presumed to be more efficient and to elicit less bacterial phage resistance issues than fixed phage cocktails, claims that need to be scientifically substantiated as soon as possible. Just like Belgium, other (European) countries could develop a magistral phage preparation framework that would exist next to the conventional medicinal product development and licensing pathways. However, it is important that the current producers of personalized phage products are provided with pragmatic quality and safety assurance requirements, which are preferably standardized (at least at the European level), and are tiered based on benefit-risk assessments at the individual patient level. Pro bono phage therapy providers should be supported and not stopped by the imposition of industry standards such as Good Manufacturing Practice requirements. Keywords: antimicrobial resistance; antibiotic resistance; bacterial infection; bacteriophage therapy; magistral preparation.

Stability of magistral phage preparations before therapeutic application in patients with chronic rhinosinusitis, sepsis, pulmonary, and musculoskeletal infections.

IMPORTANCE: As antimicrobial resistance becomes more prevalent, the application of (bacterio)phage therapy as an alternative treatment for difficult-to-treat infections is (re)gaining popularity. Over the past decade, numerous promising case reports and series have been published demonstrating the therapeutic potential of phage therapy. However, important questions remain regarding the optimal treatment protocol and, unlike for medicinal products, there are currently no predefined quality standards for the stability of phage preparations. Phage titers can be influenced by several factors which could lead to reduced titers after preparation and storage and, ultimately, subtherapeutic applications. Determining the stability of different phages in different recipients according to the route of administration is therefore one of the first important steps in establishing a standardized protocol for phage therapy.

Phages and phage-borne enzymes as new antibacterial agents.

BACKGROUND: Persistent and resistant infections caused by bacteria are increasing in numbers and pose a treatment challenge to the medical community and public health. However, solutions with new agents that will enable effective treatment are lacking or delayed by complex development and authorizations. Bacteriophages are known as a possible solution for invasive infections for decades but were seldom used in the Western world. OBJECTIVES: To provide an overview of the current status and emerging use of bacteriophage therapy and phage-based products, as well as touch on the socioeconomic and regulatory issues surrounding their development. SOURCES: Peer-reviewed articles and authors' first-hand experience. CONTENT: Although phage therapy is making a comeback since its early discovery, there are many hurdles to its current use. The lack of appropriate standardized bacterial susceptibility testing; lack of a simple business model and authorization for the need of many phages to treat a single species infection; and the lack of knowledge on predictable outcome measures are just a few examples. In this review, we explore the possible routes for phage use, either based on local specialty centres or by industry; the current status of phage therapy, which is mainly based on single-centre or single-bacterial cohorts, and emerging clinical trials; local country-level frameworks for phage utilization even without full authorization; and the use of phage-derived products as alternatives to antibiotics. We also explore what may be the current indications based on the possible availability of phages. IMPLICATIONS: Although phages are emerging as a potential treatment for non-resolving and life-threatening infections, the models for their use and production still need to be defined by the medical community, regulatory bodies, and industry. Bacteriophages may have a great potential for infection treatment but many aspects still need to be defined before their routine use in the clinic.

Preanalytical variables influencing the interpretation and reporting of biological tests on blood samples of living and deceased donors for human body materials.

With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.

Phage Therapy for Diabetic Foot Infection: A Case Series.

PURPOSE: Infected diabetic foot ulcers can be difficult to treat and, despite appropriate antibiotic therapy, some diabetic foot infections (DFIs) require amputation. Bacteriophages (phages) are viruses that infect and kill bacteria. Phage therapy has been repeatedly used to successfully treat DFIs and other chronic wounds. METHODS: This article reports the provision of topical adjunctive anti-staphylococcal phage therapy to 10 patients with DFI at high risk of amputation at two UK hospitals as part of clinical care; tolerability and efficacy were clinically assessed. FINDINGS: The opinion of the experienced clinical teams caring for these patients was that 9 of the 10 patients appeared to benefit from adjunctive phage therapy. No adverse effects were reported by clinicians or patients. In 6 of 10 patients the clinical impression was that phage therapy facilitated clinical resolution of infection and limb salvage. Resolution of soft tissue infection was observed in a 7th patient but unresolved osteomyelitis required amputation. An 8th patient demonstrated eradication of Staphylococcus aureus from a polymicrobial infection and a 9th showed signs of clinical improvement before early cessation of phage therapy due to an unrelated event. One patient, with a weakly susceptible S aureus isolate, had no significant response. IMPLICATIONS: This report describes the largest application of phage therapy in the United Kingdom to date and the first application of phage therapy for DFI in the United Kingdom and offers subjective hints toward impressive tolerability and efficacy. Phage therapy has the potential to transform the prevention and treatment of DFIs.

Eudragit® FS Microparticles Containing Bacteriophages, Prepared by Spray-Drying for Oral Administration.

Phage therapy is recognized to be a promising alternative to fight antibiotic-resistant infections. In the quest for oral dosage forms containing bacteriophages, the utilization of colonic-release Eudragit® derivatives has shown potential in shielding bacteriophages from the challenges encountered within the gastrointestinal tract, such as fluctuating pH levels and the presence of digestive enzymes. Consequently, this study aimed to develop targeted oral delivery systems for bacteriophages, specifically focusing on colon delivery and employing Eudragit® FS30D as the excipient. The bacteriophage model used was LUZ19. An optimized formulation was established to not only preserve the activity of LUZ19 during the manufacturing process but also ensure its protection from highly acidic conditions. Flowability assessments were conducted for both capsule filling and tableting processes. Furthermore, the viability of the bacteriophages remained unaffected by the tableting process. Additionally, the release of LUZ19 from the developed system was evaluated using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) model. Finally, stability studies demonstrated that the powder remained stable for at least 6 months when stored at +5 °C.

Case report: Analysis of phage therapy failure in a patient with a Pseudomonas aeruginosa prosthetic vascular graft infection.

Clinical case of a patient with a Pseudomonas aeruginosa multidrug-resistant prosthetic vascular graft infection which was treated with a cocktail of phages (PT07, 14/01, and PNM) in combination with ceftazidime-avibactam (CZA). After the application of the phage treatment and in absence of antimicrobial therapy, a new P. aeruginosa bloodstream infection (BSI) with a septic residual limb metastasis occurred, now involving a wild-type strain being susceptible to ß-lactams and quinolones. Clinical strains were analyzed by microbiology and whole genome sequencing techniques. In relation with phage administration, the clinical isolates of P. aeruginosa before phage therapy (HE2011471) and post phage therapy (HE2105886) showed a clonal relationship but with important genomic changes which could be involved in the resistance to this therapy. Finally, phenotypic studies showed a decrease in Minimum Inhibitory Concentration (MIC) to ß-lactams and quinolones as well as an increase of the biofilm production and phage resistant mutants in the clinical isolate of P. aeruginosa post phage therapy.

Characterization of a Bacteriophage GEC_vB_Bfr_UZM3 Active against Bacteroides fragilis.

Bacteroides fragilis is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not susceptible to the common antibiotic regimes established for B. fragilis, have been more frequently reported. Bacteriophages (phages) were found to be a successful antibacterial alternative to antibiotic therapy in many cases of multidrug-resistant (MDR) bacterial infections. We have characterized the bacteriophage GEC_vB_Bfr_UZM3 (UZM3), which was used for the treatment of a patient with a chronic osteomyelitis caused by a B. fragilis mixed infection. Studied biological and morphological properties of UZM3 showed that it seems to represent a strictly lytic phage belonging to a siphovirus morphotype. It is characterized by high stability at body temperature and in pH environments for about 6 h. Whole genome sequencing analysis of the phage UZM3 showed that it does not harbor any known virulence genes and can be considered as a potential therapeutic phage to be used against B. fragilis infections.