Antibiotic-induced cellular enlargement drives phage synergy and resensitizes carbapenem-resistant Acinetobacter baumannii

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) represents a critical clinical challenge, as phage monotherapy is frequently compromised by the rapid emergence of bacterial resistance. This study characterizes phiAR008, a newly isolated Hadassahvirus phage with therapeutic potential against clinical CRAB isolates, and evaluates its synergistic efficacy alongside five antibiotic classes to develop robust combination therapies. Although phiAR008 monotherapy initially reduced bacterial growth, rapid bacterial regrowth occurred. In contrast, combination treatment with subinhibitory concentrations of meropenem (MEM) or ciprofloxacin (CIP) produced complete bacterial clearance in vitro and significantly enhanced antibacterial activity compared with either treatment alone. MEM and CIP induced distinct morphological changes, including spheroid enlargement and filamentation, respectively, resulting in increased bacterial cell surface area. These morphological alterations were associated with accelerated phage adsorption and increased intracellular phage accumulation during the early stages of infection, whereas the final phage yield remained unchanged, suggesting that antibiotic-induced cell enlargement primarily enhances early infection kinetics rather than phage productivity. In contrast, antibiotics that did not substantially alter bacterial morphology, including colistin, rifampin, and chloramphenicol, exhibited limited synergy with phiAR008. In a Galleria mellonella infection model, phiAR008-MEM and phiAR008-CIP combination therapy significantly improved larval survival compared with phage or antibiotic monotherapy. Phage-resistant isolates recovered following combination treatment also exhibited reduced virulence and altered antibiotic susceptibility. Although the genetic and mechanistic basis of these phenotypic changes remains to be determined, our findings indicate that antibiotic-induced bacterial morphological changes may contribute to phage-antibiotic synergy by enhancing early phage-host interactions. These results support the rational optimization of phage-antibiotic combinations as a promising therapeutic strategy for refractory CRAB infections. IMPORTANCE Phage-antibiotic synergy offers a promising strategy to overcome the therapeutic limitations of phage monotherapy against CRAB. In this study, phiAR008 showed limited standalone killing because bacterial regrowth and phage resistance emerged rapidly. However, when combined with specific antibiotics, especially meropenem and ciprofloxacin, phiAR008 produced strong synergistic antibacterial activity, leading to rapid and complete bacterial eradication in vitro and improved survival in vivo . Importantly, synergy was not universal across antibiotic classes, indicating that the choice of partner drug is critical. Our findings further show that synergy is mechanistically linked to antibiotic-induced changes in bacterial morphology that enhance phage adsorption rather than phage production. These results provide practical guidance for selecting phage-antibiotic pairs and support phage-antibiotic synergy as a mechanistically informed approach for treating multidrug-resistant A. baumannii infections.