Phenotypic and Proteomic Alterations in Pseudomonas aeruginosa ATCC 9027 Induced by Prolonged Sub-MIC Gentamicin Exposure

Gentamicin (Ge) plays a crucial role in treating infections caused by Pseudomonas aeruginosa. This study examined the effects of prolonged exposure of P. aeruginosa to sub-minimum inhibitory concentrations (sub-MIC) of Ge. P. aeruginosa ATCC 9027 (initial strain) was exposed to sub-MIC Ge for 14 days (Ge-E1), then subsequently cultured in an antibiotic-free environment for 10 days (Ge-E2). Comparative analyses on antibiotic susceptibility profiles, morphology, key virulence features, proteome and noted target gene expression of the initial strain, Ge-E1 and Ge-E2 were performed. Resultedly, the initial strain shifted from being susceptible to Ge with an MIC of 2 μg/mL to resistant in Ge-E1 and Ge-E2 with an MIC of 256 and 128 μg/mL, respectively. Cross-resistance to multiple antibiotic classes was observed. Morphologically, Ge‑E1 exhibited a marked reduction in cell size, whereas Ge-E2 largely returned to its initial size. Regarding virulence, Ge exposure markedly suppressed rhamnolipid secretion, pyocyanin and pyoverdine production. Swarming motility was notably reduced in Ge-E1 and not fully reverted in Ge-E2. Proteomic analysis identified 994 proteins, of which approximately 47% were differentially expressed proteins (DEPs). A predominance of down-regulated proteins, primarily proteins related to ribosome function, cellular and metabolic processes, was found in both Ge-E1 and Ge-E2. Particularly, noted proteomic changes included the upregulation of mvaT, a global transcriptional regulator, and the down-regulation of rpoB and rpsL, involved in transcription and ribosomal function, respectively, as well as oprD, a porin protein linked to membrane transport. Furthermore, RT-qPCR results confirmed the proteomic data and further revealed downregulation of rpoB, sdhA, phzM, and rpsL, while mvaT and rmpA were consistently upregulated in both Ge-E1 and Ge-E2. In conclusion, prolonged exposure to sub-MIC levels of Ge induced stable resistance, relatively reversible phenotypic alterations, and coordinated transcriptional and proteomic responses in P. aeruginosa.