Molecular and Structural Insights of carO Gene Variations in Carbapenem-Resistant Acinetobacter baumannii

Background/Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a WHO critical-priority pathogen, and variations in the outer membrane porin CarO are one mechanism implicated in carbapenem resistance. No prior study has characterized carO mutation patterns among A. baumannii in Tak, Thailand. This study aimed to characterize carO mutation patterns in clinical isolates, evaluate their association with CRAB, reconstruct phylogeny, and predict the structural consequences of CarO representatives relative to a crystallographic reference structure (PDB 4RL9). Seventy-seven A. baumannii isolates were recovered from Mae Sot Hospital, and re-confirmed by PCR based on the presence of bla_OXA-51-like gene. Results: The carO gene was sequenced and mutations compared between CRAB and carbapenem-susceptible (CSAB) isolates. Of the 77 isolates, 70 yielded successfully carO sequence data by next-generation sequencing (NGS), including 64 CRAB (91.43%) and 6 CSAB isolates. Two overlapping mutation clusters were significantly associated among CRAB isolates (73.44% and 75.00%; p = 0.0008 and p = 0.0006) and were absent from all 6 CSAB isolates (8.57%). Phylogenetic analysis resolved 3 clinical lineages, with variant III predominating. AlphaFold2 models predicted structural divergence among CarO variants with the single variant IV isolate showing the most extensive predicted remodeling of the CarO barrel. Conclusions:carO mutation was strongly associated with carbapenem resistance in this Thai cohort. However, these findings do not establish a direct causal role of carO mutation in carbapenem resistance. Predicted structures were consistent with potential alterations of porin function. These findings provide a regional characterization of carO-mediated resistance and support further functional validation of CarO as a resistance marker.