We report that a combination of tobramycin and spermidine can drive the emergence of SCVs. The SCVs were studied for altered phenotypic and genetic characteristics, providing insight into both drivers of and systems involved in these clinically relevant morphotypes.

Background. Pseudomonas aeruginosa is a common cause of lung infection in people with cystic fibrosis (CF). Adaptation of P. aeruginosa to the lungs is associated with the emergence of phenotypically distinct morphotypes, termed small colony variants (SCVs). In laboratory medium, lung adapted SCVs have been linked to decreased bacterial fitness, increased biofilm formation, and increased antibiotic tolerance. However, the evolution and drivers of SCV phenotypes in the CF lung environment are poorly described, as genetic pathways to phenotypic conversion are challenging to decipher, and reversion is frequently observed. Aim. We investigated the phenotypic and genetic characteristics of SCV isolates that arose in the combined presence of the polyamine spermidine and aminoglycoside tobramycin from a variant of a successful epidemic clone – Liverpool Epidemic Strain (LES)B65 – in which the two component sensor kinase gene pmrB had been deleted. Methods. P. aeruginosa LESB65 and LESB65?pmrB were used throughout the study. In vitro assays were used to determine growth rate, minimum inhibitory concentrations (MICs), and biofilm formation. Genomic characterisation was based on short-read sequencing data. Results. SCVs specifically arose in the combined presence of tobramycin and the polyamine spermidine. Phenotypic assays revealed reduced fitness in rich medium but enhanced antimicrobial tolerance and biofilm production under growth-limiting conditions, features which may be hallmarks for persistence of infection in the lung environment. Using whole genome sequencing, small indels in both intragenic and intergenic regions were detected. Several mutations within genes associated with R bodies and a prophage region were identified. Conclusion. These data provide new insight regarding the conditions under which P. aeruginosa SCVs can arise, and their phenotypic and genotypic characteristics. Antimicrobial tolerance and increased biofilm production by SCVs may help to establish or maintain chronic lung infection and the findings presented here suggest a potential role for R bodies in this process. Understanding the drivers of phenotypic and genetic variation in P. aeruginosa may provide new insights to limit adaptation during chronic lung infections.