Isolation and characterization of lytic Shigella bacteriophages with rapid in vitro and ex vivo bactericidal activity

Introduction The emergence of shigellosis caused by Shigella spp. poses a significant threat to global health, and the prevalence of multidrug-resistant (MDR) strains necessitates the development of effective antimicrobial therapies. Bacteriophages present a promising approach for both prevention and treatment of MDR infections. Methods Ten lytic bacteriophages targeting Shigella flexneri 2457T were isolated from wastewater using the double-layer agar method. Phage morphology, lytic activity spectrum, latent period, burst size, bacterial killing efficiency, and efficacy under ex vivo microbiota conditions were evaluated. Transmission electron microscopy, one-step growth assays, bacterial killing assays, anaerobic fecal slurry co-culture experiments, and whole-genome sequencing of the representative phage PSW32 were performed. Results Transmission electron microscopy revealed that these phages possessed icosahedral heads and contractile tails, consistent with myovirus-like morphology commonly observed among tailed bacteriophages within the class Caudoviricetes . Most phages exhibited broad lytic activity against S. flexneri, S. sonnei , and S. dysenteriae , with limited activity against S. boydii in spot assay. Among them, phages PSW32, PSW37, PSW38, and PSW40 exhibited lytic activity against the largest number of tested Shigella strains under the spot assay conditions. One-step growth assays revealed latent periods of 30-70 minutes and burst sizes of 40-267 PFU/cell, indicating efficient host infection activity. At multiplicities of infection (MOIs) ranging from 10 to 0.001, all phages efficiently suppressed the growth of S. flexneri . Based on their lytic kinetics and host range, PSW32 and PSW40 were further evaluated in an ex vivo microbiota model using anaerobic fecal slurry co-cultures. PSW32 reduced CFUs by 88% at 2 h and decreased bacterial levels to below the limit of detection (LOD) by 3 h. PSW40 reduced CFUs by 69% at 2 h and reduced bacterial levels to below the limit of detection (LOD) by 3 h. Genomic analysis of PSW32 identified a T4-like lytic architecture with no detectable genes linked to antimicrobial resistance or virulence, supporting its potential applicability for further evaluation. Discussion These newly isolated lytic phages exhibited rapid bacterial activity under both in vitro and ex vivo microbiota-associated conditions and broad lytic activity against multiple Shigella species, Collectively, the findings support their potential for further investigation as phage-based strategies for controlling Shigella infections and contamination.