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.

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).

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.

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.

Background/Objectives: Biofilms are a form of defense that enables bacteria to withstand antibiotic pressure and demonstrate antibiotic resistance. It is crucial to develop anti-biofilm strategies in order to combat chronic and persistent multidrug-resistant (MDR) infections. Methods: In this study, we developed 3D biofilms of single-, dual-, and poly-species MDR ESKAPE components, including the pathogens P. aeruginosa S. aureus and K. pneumoniae, in CF Mu3Gel. We evaluated the efficacy of using a phage, a di-hetero phage cocktail or a poly-hetero phage cocktail in combination with ciprofloxacin to eliminate mature biofilm biomass after 72 h or one week in a single treatment. Results: The phage components mostly exhibited synergistic behavior when combined with ciprofloxacin and with each other in di- and poly-hetero-cocktails. The reduction in 72-h dual- and poly-species biofilms was one log higher than that of one-week biofilms treated with the phage–antibiotic combination. The greatest reductions were observed in the 72-h single-species biofilm with combination therapy, at 1.4–3.0 log. Reductions of 2.16 and 1.6 log were observed in the dual-species P. aeruginosa and S. aureus biofilm and the poly-species biofilm, respectively. Conclusions: This study examined how a single application of phages or phage cocktails, either alone or in combination with ciprofloxacin, impacted established biofilm models, and how this affected the proportion of microcolonies of different species within each model. These insights will facilitate the development of strategies for multiple follow-up treatments, as well as the reordering of phages, phage cocktails, and combinations with antibiotics, to improve outcomes. The 3D biofilm models developed here could be used to screen phages or phage cocktails either on their own or alongside other therapies. This would facilitate the application of in vitro findings to real physiological settings.