The longitudinal expression of P. aeruginosa reference genes in infection-mimicking media

Quantitative reverse transcription PCR (RT-qPCR) is a popular and reliable tool for monitoring fluctuations in functional bacterial gene expression. A necessary step of the qRT-qPCR process is the use of a reference gene, which acts to distinguish between technical bias and true biological variation. Many reference genes have been defined for bacterial species; however, few studies have validated their stability across strain types and environmental test conditions. In this study of Pseudomonas aeruginosa, the expression consistency of seven commonly used reference genes (rpoD, proC, rpoS, 16S, algD, gyrA and ampC) was assessed in P. aeruginosa laboratory (PAO1) and clinical (LESB65) isolates grown in Lysogeny broth, synthetic cystic fibrosis (CF) media 2 (SCFM2) and CF lung media (CFLM) at various growth time points (2, 6, 24 and 72 h). The stability of the reference genes was then ranked using the RefFinder programme, and three differentially ranked (rpoS, 16S and ampC) were used to interpret the expression of a Pseudomonas virulence-related gene (exoS). The results showed that 16S was the only reference gene that was quantifiably expressed by both P. aeruginosa strains grown in all media types at all growth times. Furthermore, analysing the expression of exoS with different reference genes significantly influenced the calculated expression of exoS in SCFM2 and CFLM. This study has identified a suitable reference gene for RT-qPCR with P. aeruginosa grown in complex respiratory-mimicking media. The results presented here also highlight the importance of validating reference gene expression under the chosen experimental conditions and increase our understanding of how pathogen biology can fluctuate across diverse conditions. Such knowledge is paramount for the development of novel therapeutics, including antimicrobials and anti-virulence agents.