Treatment sequences of a phage and colistin combination trigger different evolution pathways of a multidrug-resistant Acinetobacter baumannii yielding distinct treatment outcomes

• The treatment sequences of phage and colistin combination treatment yielded distinct difference in killing efficiency and evolution pathways of Acinetobacter baumannii • Pre-incubation of one antibacterial agent modified the surface morphology of bacteria and changed the interplays between bacteria with the subsequently added agent. • Only concomitant use of phage and colistin could effectively suppress the emergence of phage-resistant phenotypes. Phage and antibiotic synergy (PAS) are often adopted clinically to address shortcomings of phage monotherapy. While treatment order has shown to significantly influence the extent of synergy, the underlying mechanisms remain poorly understood, particularly in the context of planktonic bacterial populations. Building on a previously identified PAS combination, IME-AB2 phage and colistin, demonstrating sequential effects, we conducted a systematic investigation to elucidate mechanisms driving the phenotypic outcomes of three treatment orders (phage-first, simultaneous and antibiotic-first) against a resistant Acinetobacter baumannii . Our findings revealed phage pretreatment effectively decapsulated bacteria to promote colistin binding and bacterial killing, while colistin pretreatment altered bacterial morphology and intracellular machinery, reducing phage reproduction and hence the PAS level. For simultaneous treatment, colistin also reduced the phage reproduction rate, but phage-mediated bacterial decapsulation promoted colistin binding and outer membrane disruption, leading to significant PAS. Noteworthy, only the simultaneous treatment capable of suppressing resistance evolvement. Genome sequencing analysis revealed that colistin-pretreated bacteria modified LPS/outer membrane structure via regulating the QseB/C signal transduction system to promote resistance. On the other hand, phage-pretreated bacteria acquired mutation in gene clusters related to polysaccharide synthesis, impairing phage adsorption at the cost of weakening biofilm formation ability. Overall, this extended mechanistic study provided important insights into the impacts of treatment orders on PAS and resistance dynamics, suggesting the need for more fundamental studies to realize the full clinical potential of the PAS strategy.