Efficacy of a bacteriophage cocktail targeting pathogenic bacteria associated with gastrointestinal dysfunction

Abstract

The use of bacteriophage viruses that infect and kill bacteria is increasingly explored as an alternative to antibiotics for treatment of infections caused by opportunistic pathogens. Examples of opportunistic pathogens that disrupt the intestinal barrier and contribute to microbial imbalance in the gastrointestinal tract include Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens. This study evaluated the in vitro effectiveness of a bacteriophage cocktail composed of three host-specific phages: Lilla1 targeting Klebsiella pneumoniae, PAE1 targeting Pseudomonas aeruginosa and Smarc1 targeting Serratia marcescens. Bacterial growth was monitored by measuring optical density at 600 nm, and phage replication was quantified by plaque-forming unit assays. Each phage exhibited strong host specificity and produced significant reduction of growth of the corresponding bacterium while increasing in concentration during incubation. When applied as a cocktail, these three phages produced stronger and more sustained suppression of bacterial growth than single-phage treatments, both in individual cultures and in mixed cultures containing all three pathogens. In mixed cultures, continuous inhibition of bacterial growth persisted throughout a 24-hour incubation, and concentrations of Lilla1, PAE1 and Smarc1 increased significantly, indicating active replication and enhanced efficacy in combination. In single bacterial growth assays, bacterial populations that were initially suppressed showed regrowth after extended incubation. Regrowth manifested as a renewed increase in optical density several hours after initial decline and is consistent with selection of phage-resistant subpopulations, potentially arising from modification of surface receptors, phase variation or activation of adaptive defence systems. The observed biphasic response highlights the need for measures to limit microbial resistance to phage treatment, including expanded phage cocktails, adjusted dosing strategies and combined phage-antibiotic regimens. The results demonstrate that a three-phage cocktail can simultaneously inhibit multiple bacterial pathogens associated with gastrointestinal dysfunction and provide a basis for further in vivo evaluation and therapeutic development.