(499) Determining the molecular underpinnings of virulence enhancing drug resistance mutations in Pseudomonas aeruginosa.

Background: During chronic cystic fibrosis (CF) lung infections, Pseudomonas aeruginosa mutates and becomes antibiotic resistant. Recently, we showed that aztreonam selects for several P. aeruginosa mutations, including deletion of the mexEF-oprN efflux pump (1). Unexpectedly, the ΔmexEF mutant was more lethal in mice and expressed rhamnolipid virulence genes at higher levels than wild-type (WT) (2). This is concerning because the absence of mexE gene expression has been demonstated in P. aeruginosa clinical isolates (3), suggesting that these strains may also exhibit hypervirulent phenotypes. In this study, we test the hypothesis that the ΔmexEF mutant is more invasive than WT and elucidate the molecular mechanisms underlying the mutant hypervirulence.

Methods: In vivo mouse intratracheal infections and in vitro models with murine macrophages and epithelial cells were used to test virulence of WT and ΔmexEF strains. Bacterial burdens were quantified by colony forming units. Bacterial and host gene expression was measured with qRT-PCR and RNA-seq. Fluorescence and dye-based assays were used to quantify virulence and host antimicrobial factors.

Results: In vivo, compared to WT, the ΔmexEF showed significantly higher bacterial burdens in the lungs and increased dissemination to the liver.In vitro, infections of macrophages revealed higher numbers of intracellular mutant bacteria than WT, despite similar levels of phagocytosis and reactive oxygen species (ROS) produced in response to both strains. This suggested that the ΔmexEF

strain may be more resistant to oxidative stress than WT. Exposing epithelial cells to conditioned medium (CM) from infected macrophages led to increased bacterial killing relative to CM exposure from naïve macrophages. However, the ΔmexEF strain also survived epithelial cell infection better than WT, which we linked to increased ROS tolerance of the mutant. Finally, we find that virulence factors including rhamnolipids, pyoverdine, alginate, and elastase were all elevated in the ΔmexEF relative to WT.

Conclusions: Our study shows that mutations that arise in P. aeruginosa due to antibiotic exposure can confer a dual advantage: 1. allowing bacteria to thrive during antibiotic treatment, and 2. enhancing virulence and host adaptation mechanisms which allow bacteria to grow more effectively and disseminate to secondary sites of infection.

Acknowledgements:

References: 1. Jorth P, McLean K, Ratjen A, Secor PR, Bautista GE, Ravishankar S, et al. Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa. mBio. 8(5):e00517-17.

2. Vaillancourt M, Limsuwannarot SP, Bresee C, Poopalarajah R, Jorth P. Pseudomonas aeruginosa mexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence. Antibiotics. 2021 Oct;10(10):1164.

3. Tomás M, Doumith M, Warner M, Turton JF, Beceiro A, Bou G, et al. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother. 2010 May;54(5):2219–24.