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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.

BACKGROUND: Ticks are the most important pathogen vectors in Europe. They are known to be influenced by environmental factors, but these links are usually studied at specific temporal or spatial scales. Focusing on Ixodes ricinus in Belgium, we attempt to bridge the gap between current “single-sided” studies that focus on temporal or spatial variation only. Here, spatial and temporal patterns of ticks are modelled together. METHODS: A multi-level analysis of the Ixodes ricinus patterns in Belgium was performed. Joint effects of weather, habitat quality and hunting on field sampled tick abundance were examined at two levels, namely, sampling level, which is associated with temporal dynamics, and site level, which is related to spatial dynamics. Independent variables were collected from standard weather station records, game management data and remote sensing-based land cover data. RESULTS: At sampling level, only a marginally significant effect of daily relative humidity and temperature on the abundance of questing nymphs was identified. Average wind speed of seven days prior to the sampling day was found important to both questing nymphs and adults. At site level, a group of landscape-level forest fragmentation indices were highlighted for both questing nymph and adult abundance, including the nearest-neighbour distance, the shape and the aggregation level of forest patches. No cross-level effects or spatial autocorrelation were found. CONCLUSIONS: Nymphal and adult ticks responded differently to environmental variables at different spatial and temporal scales. Our results can advise spatio-temporal extents of environment data collection for continuing empirical investigations and potential parameters for biological tick models.

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.

In the present study, we evaluated the effect of spray-drying formulations and operating parameters of a laboratory-scale spray-dryer on the characteristics of spray-dried powders containing two Pseudomonas aeruginosa bacteriophages exhibiting different morphotypes: a podovirus (LUZ19) and a myovirus (14-1). We optimized the production process for bacteriophage-loaded powders, with an emphasis on long-term storage under ICH (international conference on harmonization) conditions. D-trehalose-/L-isoleucine-containing bacteriophage mixtures were spray-dried from aqueous solutions using a Büchi Mini Spray-dryer B-290 (Flawil, Switzerland). A response surface methodology was used for the optimization of the spray-drying process, with the following as-evaluated parameters: Inlet temperature, spray gas flow rate, and the D-trehalose/L-isoleucine ratio. The dried powders were characterized in terms of yield, residual moisture content, and bacteriophage lytic activity. L-isoleucine has demonstrated a positive impact on the activity of LUZ19, but a negative impact on 14-1. We observed a negligible impact of the inlet temperature and a positive correlation of the spray gas flow rate with bacteriophage activity. After optimization, we were able to obtain dry powder preparations of both bacteriophages, which were stable for a minimum of one year under different ICH storage conditions (up to and including 40 °C and 75% relative humidity).

Background: Bacteriophages (phages) are viruses that infect and kill bacteria. The antimicrobial resistance crisis has driven renewed interest in phage therapy, including the use of phages to treat chronic orthopaedic infections. Methods: Here, we present the results of the first three orthopaedic patients treated with phage therapy in the United Kingdom. Results: The first patient was treated in May 2023 and received phages active against Staphylococcus aureus. At nine months follow-up, the patient’s wound remained healed, the C-reactive protein normal and the patient was walking independently. The second patient received phages active against Klebsiella pneumoniae and S. aureus; the infection remained unresolved. The third patient received phages active against Staphylococcus epidermidis; at six months follow-up, the patient was free of infection. Endotoxin was considered at least partially responsible for mild self-limiting adverse effects in two cases. Conclusions: These promising results hint at the potential for phage therapy to transform the care of chronic orthopaedic infections.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compromises the ability of military forces to fulfill missions. At the beginning of May 2020, 22 out of 70 Belgian soldiers deployed to a military education and training center in Maradi, Niger, developed mild COVID-19 compatible symptoms. Immediately upon their return to Belgium, and two weeks later, all seventy soldiers were tested for SARS-CoV-2 RNA (RT-qPCR) and antibodies (two immunoassays). Nine soldiers had at least one positive COVID-19 diagnostic test result. Five of them exhibited COVID-19 symptoms (mainly anosmia, ageusia, and fever), while four were asymptomatic. In four soldiers, SARS-CoV-2 viral load was detected and the genomes were sequenced. Conventional and genomic epidemiological data suggest that these genomes have an African most recent common ancestor and that the Belgian military service men were infected through contact with locals. The medical military command implemented testing of all Belgian soldiers for SARS-CoV-2 viral load and antibodies, two to three days before their departure on a mission abroad or on the high seas, and for specific missions immediately upon their return in Belgium. Some military operational settings (e.g., training camps in austere environments and ships) were also equipped with mobile infectious disease (COVID-19) testing capacity.

IntroductionAs antimicrobial resistance continues to rise and the development of new antimicrobials lags, we face an urgent threat where routine infections could become life-threatening. Armed conflicts, like the war on Ukraine, intensify this crisis by accelerating the spread of resistant organisms across regions and borders. Phage therapy or the use of bacteriophages (phages), the viruses of bacteria, as a therapeutic has reemerged as a viable treatment for difficult to treat multidrug resistant (MDR) infections. Our Belgian consortium, led primarily by the Queen Astrid Military Hospital (QAMH), has been dedicated to treating patients, culminating in a landmark publication that documents 100 consecutive cases of phage therapy.1 Phage Support Ukraine (Phage SP UKR) is a collaborative effort led by QAMH in partnership with our group at KU Leuven to provide phages targeting circulating pathogens and enable a military hospital in Kyiv to effectively deliver personalized phage therapies.MethodsAs part of this initiative, we received from this hospital in Kyiv bacterial isolates belonging to a number of species, but decided to focus here on Klebsiella pneumoniae, the most concerning pathogen with regard to prevalence, virulence and antibiotic resistance. Strains were sequenced using both Illumina (short-read) and Nanopore (long-read) technologies to characterize their genomic content, including antibiotic resistance and virulence factors. Antibiograms were also conducted in accordance with standard protocols. Strains were assessed for hypervirulence using Galleria mellonella and a mouse model of pneumonia. Phages were isolated from Ukrainian wastewater, sequenced and characterized for therapeutic effectiveness.ResultsAmong these K. pneumoniae isolates we identified MDR sequence types (ST)39, 307 and 395 and one predominating clonal group—CG10146 first detected in Moscow, Russia—carrying a hybrid virulence and antibiotic resistance plasmid encoding the carbapenemase NDM-1 and aerobactin. When tested in virulence assays, only one isolate (ST395) was hypervirulent in a G. mellonella though not in a mouse model of infection. Several phages were isolated that could lyse these K. pneumoniae strains, including those isolated from soldiers displaced in Belgium, Germany, and Latvia. Bacterial resistance was observed during in vitro testing; however, some phage-resistant isolates had mutations in virulence factors, including one that lost its hybrid plasmid, resulting in re-sensitivity to antibiotics. As we continue to receive isolates for surveillance, new strains emerging from Ukraine this year have been identified as pan-drug resistant (PDR). However, they have also been found to be susceptible to the phages isolated to date.ConclusionThese phages show high potential as a therapeutic against MDR and PDR strains of K. pneumoniae. As part of this initiative, QAMH will provide these phages as well as equipment for the group in Kyiv to work with these phages and the training needed to implement phage therapy in Ukraine. Strategies like Phage SP UKR are essential to prevent the selection, persistence, and global spread of these drug-resistant clones.ReferencePirnay JP, Djebara S, Steurs G, Griselain J, Cochez C, De Soir S, et al. Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study. Nat Microbiol. 2024;9(6):1434–53.Abstract A24 Figure 1Phage SP UKR. A schematic outlining the goals of this project, which include: 1. Pathogen characterization, 2. Phage isolation and characterization, and 3. Delivery of phages to Ukraine[Image Omitted. See PDF.]DisclosuresThe authors declare no conflict of interest.

More than a year after the first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of the 2019 coronavirus disease (COVID-19) in China, the emergence and spread of genomic variants of this virus through travel raise concerns regarding the introduction of lineages in previously unaffected regions, requiring adequate containment strategies. Concomitantly, such introductions fuel worries about a possible increase in transmissibility and disease severity, as well as a possible decrease in vaccine efficacy. Military personnel are frequently deployed on missions around the world. As part of a COVID-19 risk mitigation strategy, Belgian Armed Forces that engaged in missions and operations abroad were screened (7683 RT-qPCR tests), pre- and post-mission, for the presence of SARS-CoV-2, including the identification of viral lineages. Nine distinct viral genotypes were identified in soldiers returning from operations in Niger, the Democratic Republic of the Congo, Afghanistan, and Mali. The SARS-CoV-2 variants belonged to major clades 19B, 20A, and 20B (Nextstrain nomenclature), and included “variant of interest” B.1.525, “variant under monitoring” A.27, as well as lineages B.1.214, B.1, B.1.1.254, and A (pangolin nomenclature), some of which are internationally monitored due to the specific mutations they harbor. Through contact tracing and phylogenetic analysis, we show that isolation and testing policies implemented by the Belgian military command appear to have been successful in containing the influx and transmission of these distinct SARS-CoV-2 variants into military and civilian populations.

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 . Analysis of 100 consecutive personalized bacteriophage therapy cases supports the use of personalized and sometimes pre-adapted, bacteriophage preparations, often in combination with antibiotics.

In the present study, we evaluated the effect of spray-drying formulations and operating parameters of a laboratory-scale spray-dryer on the characteristics of spray-dried powders containing two bacteriophages exhibiting different morphotypes: a podovirus (LUZ19) and a myovirus (14-1). We optimized the production process for bacteriophage-loaded powders, with an emphasis on long-term storage under ICH (international conference on harmonization) conditions. D-trehalose-/L-isoleucine-containing bacteriophage mixtures were spray-dried from aqueous solutions using a Büchi Mini Spray-dryer B-290 (Flawil, Switzerland). A response surface methodology was used for the optimization of the spray-drying process, with the following as-evaluated parameters: Inlet temperature, spray gas flow rate, and the D-trehalose/L-isoleucine ratio. The dried powders were characterized in terms of yield, residual moisture content, and bacteriophage lytic activity. L-isoleucine has demonstrated a positive impact on the activity of LUZ19, but a negative impact on 14-1. We observed a negligible impact of the inlet temperature and a positive correlation of the spray gas flow rate with bacteriophage activity. After optimization, we were able to obtain dry powder preparations of both bacteriophages, which were stable for a minimum of one year under different ICH storage conditions (up to and including 40 °C and 75% relative humidity).