Combination of Phage vB5647 and Chlorhexidine Gluconate: A Strategy to Combat Chlorhexidine-Insusceptible Acinetobacter Nosocomialis and Its Biofilms

Abstract Acinetobacter nosocomialis strains with reduced chlorhexidine gluconate susceptibility and robust biofilm capacity severely hinder clinical disinfection. We isolated lytic phage vB5647 targeting these isolates and characterized its standalone and combined antibacterial and antibiofilm activity with chlorhexidine gluconate. vB5647, a contractiletailed myoviral virulent phage free of virulence or resistance determinants, lysed 94.44% of tested Acinetobacter nosocomialis. Chlorhexidine gluconate alone produced concentration-dependent bacteriostasis yet permitted regrowth, whereas phage alone yielded transient elimination followed by efficacy attenuation. Combination treatment suppressed nascent biofilm formation but but cleared mature biofilms variably. Scanning electron microscopy in the silicon-wafer biofilm model revealed a nonmonotonic relationship with phage titer: low- and medium-titer phage with chlorhexidine gluconate eliminated abundant bacteria and induced cellular shrinkage and debris, whereas ultrahigh titer (108 PFU/mL) weakened this synergy. Extracellular polymeric substance quantification showed that high-titer phage plus chlorhexidine gluconate induced excessive extracellular polymeric substance production, whereas extracellular DNA accumulation and bacteriophage-insensitive mutant enrichment did not drive the reduced efficacy. In the BM6274 urinary catheter model, 64 μg/mL chlorhexidine gluconate with 108 PFU/mL phage gave the greatest surface biofilm reduction within 2 h, partially maintained at 24 h, suggesting the optimal phage titer may be substratum-dependent. However, this model used a single strain and material, so generalizability remains to be established. Overall, vB5647 in combination with chlorhexidine gluconate showed prominent synergy against reduced-susceptibility Acinetobacter nosocomialis, restraining planktonic regrowth and reducing BM6274 catheter-segment biofilms, and represents a promising candidate for device-related biofilm control.