Transcriptomic Basis of Serum Resistance and Virulence Related Traits in XDR P. aeruginosa Evolved Under Antibiotic Pressure in a Morbidostat Device

Colistin is a last resort antibiotic against the critical status pathogen Pseudomonas aeruginosa. Virulence and related traits such as biofilm formation and serum resistance after exposure to sub-inhibitory levels of colistin have been underexplored. We cultivated P. aeruginosa in a semi-automated morbidostat device with colistin, metronidazole and a combination of the two antibiotics for 21 days, and completed RNA-Seq to uncover the transcriptional changes over time. Strains became resistant to colistin within this time period. Colistin-resistant strains show significantly increased biofilm formation: the cell density in biofilm increases under exposure to colistin, while the addition of metronidazole can remove this effect. After seven days of colistin exposure, strains develop an ability to grow in serum , suggesting that colistin drives bacterial modifications conferring a protective effect from serum complement factors. Of note, strains exposed to colistin showed a decrease in virulence, when measured using the Galleria mellonella infection model. These phenotypic changes were characterised by a series of differential gene expression changes, particularly those related to LPS modifications, spermidine synthesis (via speH and speE) and the major stress response regulator rpoS. Our results suggest a clinically important bacterial evolution under sub-lethal antibiotic concentration leading to potential for significant changes in the clinical course of infection.