Directed Evolution to Sodium Benzoate
Preservatives play an essential role when maintaining the quality of home and personal care (HPC) products. However, consumer demand and regulatory constraints have forced manufacturers to move away from some traditional preservation strategies that use isothiozolinones, formaldehyde releasers, and parabens esters. This has resulted in the need to rely on preservatives such as sodium benzoate to preserve HPC products, an approach that can be less efficacious. Pseudomonas aeruginosa is the most common contaminant within HPC products and it is problematic due to its nature as an antibiotic resistant opportunistic pathogen. This has caused concerns among manufacturers about the robustness of new strategies compared to traditional ones and how this impacts the adaptation of contaminants such as P. aeruginosa.  Directed evolution was used to understand how P. aeruginosa adapts when exposed to moderate levels of sodium benzoate in tryptone soy broth (TSB). Biofilms were established on stainless steel beads and successively transferred in two static levels of sodium benzoate, 0.15% (w/v) and 0.31% (w/v) sodium benzoate in TSB, referred to as NL and NH respectively, a non-treated control was run in parallel (P). Twelve endpoint isolates were selected to characterise both genotypically and phenotypically to further understand the role that sodium benzoate exposure plays in P. aeruginosa adaptation in this biofilm model. Most isolates selected (11/12) showed a slight increase of 2x the ancestral Minimum Inhibitory Concentration (MIC), however, this was irrespective of treatment group. Isolates were also tested for changes in attachment phenotypes using crystal violet assays. This revealed that isolates passaged in sodium benzoate performed worse in TSB than the ancestor or non-treated group to a high degree of significance (All comparisons, p = 0.0001). Conversely, all three groups showed higher significant attachment in 0.15% sodium benzoate versus the ancestor (NL: p = 0.0001, NH: p = 0.0001, P: p = 0.001). Whole-genome sequencing revealed several mutations associated with adaptation to biofilm lifestyle and parallel evolution in several genes, namely, wspA, wspF, and dipA as well as parallelism in putative function associated with cyclic-di-GMP regulation.  Our study is the first to show the types of evolutionary strategies that P. aeruginosa adopts in the presence of sodium benzoate preservative stress. This study highlights the importance of biofilms under these conditions and that the majority of adaptations that P. aeruginosa underwent were related to its lifestyle within biofilm populations. Studies such as this one contributes new findings to inform the HPC industry of new, robust preservation strategies and to limit risk factors that affect consumers and manufacturers in this shifting landscape.