Synergistic effects of halo-plaque forming phage cocktails against polymicrobial Acinetobacter baumannii and Staphylococcus aureus

ABSTRACT Antimicrobial resistance is a growing global health crisis, with polymicrobial infections caused by Acinetobacter baumannii and Staphylococcus aureus posing a significant healthcare challenge due to their synergistic virulence, multidrug resistance, high incidence, and association with severe outcomes. Accordingly, urgent alternative treatments are needed, with bacteriophage or phage therapy showing considerable promise. Here, we isolated six phage types based on plaque morphology, including small clear plaques (SCP; phiAR002 and phiAR004), medium clear plaques (MCP; phiAR010 and phiAR011), and hazy-halo plaques (HHP; phiAR014 and phiAR015). These phages demonstrate the ability to lyse a broad spectrum of several carbapenem-resistant A. baumannii strains collected from secondary and tertiary care hospitals in Thailand, highlighting their suitability for therapeutic applications. To broaden host range and enhance bactericidal activity, phage cocktails comprising the halo-forming phage (phiAR014) in combination with either SCP (phiAR002) or MCP (phiAR010), which are genetically and mechanistically distinct, were formulated and demonstrated synergistic effects, suppressing bacterial growth for up to 36 h. These relatively small myophages, SCP and MCP, possess larger genomes (around 165–188 kbp) compared with HHP (around 106 kbp), exhibit a longer latent period (30 min versus 20 min), and produce a greater number of progenies, yielding 678–882 PFU per infected cell compared with 255 PFU per infected cell for HHP. To demonstrate their therapeutic potential, the phage cocktails were further assessed in co-cultures of A. baumannii and S. aureus under both planktonic and biofilm conditions. Cocktails combining HHP with SCP or MCP exhibited significant synergistic antibacterial activity at MOIs of 10 and 100, with effects evident within 10 h and sustained for 24 h. Relative to individual phages and uninfected controls, the cocktails achieved greater bacterial reduction, decreasing A. baumannii by approximately 2 log₁₀ CFU/mL compared with single phages (and ~5 log₁₀ versus controls) and S. aureus by ~2 log₁₀ CFU/mL. Moreover, the cocktails significantly enhanced biofilm inhibition and eradication compared to single-phage treatments, demonstrating synergistic interactions. These findings indicate that halo phages represent promising therapeutic candidates with potent antibacterial and anti-biofilm activity against polymicrobial infections. IMPORTANCE Phages are promising therapeutic agents due to their host specificity and ability to penetrate biofilms via structural and enzymatic mechanisms. The halo-plaque phenotype, indicative of depolymerase activity, facilitates the degradation of extracellular polysaccharides and cell wall components, thereby enhancing infectivity and biofilm penetration. Based on phenotypic and genomic characterization, halo-forming phage cocktails were developed and demonstrated effective suppression of polymicrobial growth and disruption of established biofilms in A. baumannii and S. aureus co-culture models. These cocktails exhibited synergistic inhibition of planktonic growth, biofilm formation, and biofilm eradication. Phage replication and lysis of A. baumannii may promote the release of putative depolymerases and/or cell wall hydrolases that could potentially contribute to matrix degradation; however, further investigation is required to elucidate the precise mechanisms and enzymatic functions involved. Together, these findings would pave the way for advancing halo-forming phages and support the targeted engineering of tail fiber or spike proteins for therapeutic applications in multidrug-resistant polymicrobial infections.