Evidence for high-frequency parallel evolution in virulent A. baumannii cultures

Acinetobacter baumannii, is a gram-negative opportunistic pathogen notorious for its antibiotic resistance and adaptability. These attributes present significant challenges in clinical infection management and failure to manage infection results in around 1,000,000 global deaths per year. A greater knowledge of the layers of regulation employed by such a versatile pathogen may yield an improved understanding of the factors important for A. baumannii survival in diverse conditions and then facilitate the development of countermeasures. This study initially began with the investigation of phenotypic changes in colony opacity under varying environmental conditions with experiments designed to probe aspects of virulence, motility, biofilm formation, and antibiotic resistance. Our initial data also suggest evidence for a phenotype driving mutation system which simultaneously occurred in multiple lineages at the same time. This genetic alteration was observed at higher than expected frequencies, seemingly providing a striking example of parallel evolution. Using proteomic profiling and PacBio sequencing, we characterized lineages of AB5075 which, following changes in culture conditions, grew into colonies of a split translucent/ opaque phenotype which was inherited by translucent and opaque progeny lineages. A genetic alteration in the capsule operon gene wzy, marked by an ISAba13 transposon insert, led to the downregulation of the wzy gene product. Translucent variants demonstrated denser sedimentation and reduced biofilm formation, whereas both opaque and translucent variants showed unexpectedly similar antibiotic resistance profiles, challenging the assumptions that capsule formation and antibiotic resistance are always linked. Our findings suggest that the adaptability and resistance mechanisms of A. baumannii are related but distinct, where capsule loss is part of a broader, adaptive strategy rather than a sole determinant of antibiotic susceptibility. These insights highlight the need for a nuanced therapeutic approach, considering the dynamic interplay of environmental cues, phenotypic changes, and genetic rearrangements. We believe that this study further opens a path for understanding the adaptability of A. baumannii and lays the groundwork for developing innovative therapeutic strategies against this resilient pathogen. Significance StatementThis study advances our understanding of the adaptability mechanisms of Acinetobacter baumannii, a pathogen notorious for its antibiotic resistance in clinical settings. We initially focused on the role of phenotype switches and identified genetic alterations in the Wzy operon that impact capsule production. Our studies led us to the surprising realization that we were observing multiple identical but independent evolution events. In addition, contrary to the traditional view that capsule variability directly correlates with antibiotic resistance, our findings revealed that susceptibility to aminoglycosides can occur independently of capsule loss, indicating capsule loss may be part of multiple survival strategies. These insights challenge existing paradigms and underscore the necessity for multiple therapeutic and preventative strategies that address the pathogens multifaceted adaptability for survival. Ultimately, this research contributes significantly to our knowledge of the biology of A. baumannii, paving the way for more effective management of infections caused by this formidable pathogen.