A Lytic Bacteriophage Cocktail Attenuates Carbapenem-Resistant Klebsiella pneumoniae-Acinetobacter baumannii Mixed-Species Biofilms and Reshapes Transcriptional Responses.

Mixed-species biofilms formed by carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Acinetobacter baumannii can complicate the clinical management of device-associated infections; however, phage-based interventions targeting these communities remain insufficiently characterized. We assessed the antibiofilm activity of a lytic HZJ31 + HZY2308 phage cocktail against KPZ2-AB48 mixed-species biofilms. Compared with the corresponding single-species cultures, cocultures showed greater crystal violet-stained biomass, higher metabolic activity as measured by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, and microscopic surface coverage. Phage-cocktail exposure during biofilm development or after biofilm formation reduced biomass, XTT reduction, culturable bacterial counts, and scanning electron microscopy-derived surface coverage, with similar changes observed on central venous catheter surfaces. In Galleria mellonella infection models, cocktail treatment increased survival and was associated with less extensive histopathological lesions and lower Kirschner wire-associated bacterial burdens. RNA sequencing and quantitative reverse transcription polymerase chain reaction identified treatment-associated transcriptional changes involving metabolic pathways, autoinducer-2 transport, cell-envelope functions, efflux systems, and stress responses. These findings support further preclinical investigation of phage cocktails targeting multidrug-resistant mixed-species biofilms.