Interactions between fish isolates Pseudomonas fluorescens and Staphylococcus aureus in dual-species biofilms and sensitivity to carvacrol.

Biofilm formation is a frequent source of contamination of food products, which results in significant economic losses through microbial spoilage and poses serious health concerns. Little is known about the fate of Staphylococcus aureus in the dual-species biofilms with Pseudomonas fluorescens an important spoiler commonly found in aquatic products. This study evaluates the interactions between mono- or dual-species biofilms formed by P. fluorescens and S. aureus, as well as the sensitivity of the two tested strains to carvacrol. The biofilm cell population, expolysaccharide production, biofilm structures of P. fluorescens as mono- and dual-species with S. aureus at ratios of 1:1 and 1:0.01 were investigated with different concentrations of carvacrol (0, 0.4, 0.8 and 1.6 mM) in fish juice at 30 °C. The results show that the biofilm cell population of S. aureus in the dual-species was significantly lower (p < 0.05) than that in the mono-species, compared to no difference for P. fluorescens. In the co-culture the dominance of P. fluorescens inhibited the growing population of S. aureus in both planktonic and biofilm cells, however, two strains were stimulated to produce the large expolysaccharides and coaggregation, forming the complex spatial multibiofilm structures. The large increase in the dual-species biofilms was positively correlated with high quorum sensing autoinductor-2 (AI-2), and exogenous 4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor, DPD), rather than C4-HSL, greatly stimulated the dual-species biofilm formation. In addition, carvacrol significantly reduced the tested biofilms and expolysaccharide secretion without affecting cell viability in a concentration-dependent manner, especially for S. aureus. Furthermore, the two strains as the dual-species biofilms exhibited lower sensitivity to carvacrol than the mono-culture, regardless of the level of inoculum of S. aureus, which was consistent with the decrease of AI-2 activity. The present study highlights that the interactions between P. fluorescens and S. aureus in dual-species biofilms promoted the large production of expolysaccharides and complex biofilm structures modulated by AI-2 signal, which results in the community-level resistance to carvacrol.