From planktonic to biofilm states: single-cell transcriptomics reveals metabolic reprogramming and cellular heterogeneity in Acinetobacter baumannii.

Acinetobacter baumannii, a notorious nosocomial pathogen, exhibits enhanced antibiotic resistance through biofilm formation. However, a comprehensive understanding of the heterogeneity and regulatory dynamics underlying biofilm development at single-cell resolution is lacking. This study outlines the transcriptional landscape of A. baumannii biofilm formation at single-cell resolution and explores potential therapeutic targets. We monitored the dynamic formation process of biofilms using single-cell RNA sequencing (scRNA-seq) technology. Subsequently, we conducted characteristic genes, gene ontology (GO) enrichment, and pseudotemporal analysis on each identified cluster. In this study, scRNA-seq and pseudotemporal trajectory results showed the transition from planktonic to biofilm states in A. baumannii. Increased cellular heterogeneity was observed during biofilm maturation: planktonic subpopulations (AB_0 h) displayed a metabolic divergence between phenylacetate catabolism (paa genes) and the tricarboxylic acid cycle (acnD, atp genes), whereas the 12 h mixed population (M_12 h) exhibited co-upregulation of ribosomal (rpl, rps) and stress response genes (recA, uvrA), facilitating protein synthesis and environmental adaptation. Mature biofilm subpopulations (BF_48 h) activated iron acquisition (bauA, basD) and sulfur/nitrogen metabolism pathways (ssuC, purine degradation genes) under nutrient limitation, alongside DNA repair (uvrB, uvrC) and proteostasis mechanisms (clpB, clpX). Pseudotemporal analysis identified a critical branchpoint (Node 2) that marked the transition from the high-metabolism planktonic to the low-metabolism biofilm state, characterized by the downregulation of ribosomal (rpl, rps) and transporter (putP) genes. These findings characterize transcriptional programs associated with biofilm maturation and reveal subpopulation-specific metabolic features that may represent potential vulnerabilities warranting further investigation through targeted mutagenesis and functional assays.