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.

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 .

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.

In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success.

Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications.

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective.

As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a "passive" and or "active" treatment. Beside these methods, several techniques, based on "real-time" growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology.

A Design of Experiment Approach to Optimize Spray-Dried Powders Containing Pseudomonas aeruginosaPodoviridae and Myoviridae Bacteriophages.

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).