Ecological antagonism constrains receptor-diverse phage cocktail efficacy against multidrug-resistant Enterobacter...

Multidrug-resistant (MDR) Enterobacter hormaechei, a member of the Enterobacter cloacae complex, is an important cause of healthcare-associated infections with limited therapeutic options. This study aimed to investigate phage receptor usage and inter-phage interactions and to establish a rational strategy for optimizing therapeutic phage cocktails.

Five strictly lytic phages active against MDR E. hormaechei were isolated and characterized. Transposon sequencing (Tn-seq) and targeted gene knockouts were used to identify bacterial structures involved in phage infection. The antibacterial effects of individual phages and their combinations were evaluated using bacterial growth inhibition and biofilm assays. An optimized phage cocktail was further assessed against clinical isolates, in food matrices, and in a murine bacteremia model.

The five phages utilized diverse bacterial surface structures, including lipopolysaccharide core oligosaccharides, O-antigen, OmpA, and fimbriae, with one phage exhibiting simultaneous dependence on multiple receptors. Despite this receptor diversity, certain phage combinations exhibited antagonistic interactions. Although ΦEHO4B broadened host coverage, its inclusion reduced the inhibitory activity of complex phage cocktails, an effect not evident in pairwise combinations. Guided by receptor profiling and interaction screening, a four-phage cocktail (EHO-cocktail4) was developed, showing activity against 86.4% of clinical isolates and effectively suppressing planktonic growth and biofilm formation in vitro. In food matrices, the cocktail retained antibacterial activity. In a murine bacteremia model, phage treatment achieved 100% survival, outperforming polymyxin B therapy.

Receptor diversity alone does not guarantee optimal phage cocktail efficacy. Inter-phage ecological interactions should also be considered in cocktail design. These findings provide a mechanistic and experimental basis for rational phage cocktail optimization against MDR E. hormaechei and support further development of phage-based therapeutic strategies.