Transcriptomic analysis of the food spoilers Pseudomonas fluorescens reveals the antibiofilm of carvacrol by interference with intracellular signaling processes

Abstract Pseudomonas fluorescens as an important food spoilage organism, has strong biofilm forming-ability to resist common biocides, increasing persistence and contamination in the cold fresh food chain. This bacterial biofilm organization is regulated by luxI/luxR mediated quorum sensing (QS) signaling. Carvacrol with antimicrobial properties is well documented, however, the mechanisms of its antibiofilm activity targeting QS are still not well known. The aim of this study was to explore the inhibition of carvacrol on biofilm formation and spoilage potential of two P. fluorescens PFuk4 and PF07 strains, and their luxI mutants (ΔluxI). The minimum inhibitory concentration (MIC) of carvacrol against planktonic wild type (WT) strains and the corresponding ΔluxI was 1.6 and 1.4 mM, respectively, and maximum sublethal concentration (MSC) was 0.8 mM. The acyl-homoserine-lactones (AHLs) C4-HSL and C6-HSL, as major autoinducer molecules, were greatly decreased in PFuk4 and PF07 treated with 0.4 mM carvacrol, while those in the ΔluxI were almost abolished. Biofilm formation and exopolysaccharide production of the P. fluorescens WT and ΔluxI strains were significantly inhibited by carvacrol at the MSC, as well as the observed sparer and thinner biofilm structures. Bacterial cell swimming and swarming motilities were both decreased by carvacrol without affecting cell viability. Protease activity secreted by P. fluorescens was significantly reduced treated by MSC of carvacrol, in consistent with the repression of sensory attributes, especially in the WT strains. Transcriptomics profiling revealed that exposure to 0.4 mM carvacrol dramatically altered gene expression in P. fluorescens PF07 biofilm, as a total of 402 differentially expressed genes (DEGs) were upregulated and 308 DEGs were downregulated, compared with the untreated biofilms. Those DECs were mainly involved in pathways of ribosome, bacterial chemotaxis, citrate cycle. Furthermore, carvacrol also altered the transcription of P. fluorescens genes associated with flagellar assembly, chemotaxis, exopolysaccharide synthesis, signaling pathways of LuxR mediated QS and c-di-GMP, and amino acid degradation, which may contribute to biofilm development and spoilage potential. In addition, carvacrol was found to competitively bind with LuxR predicted protein to interrupt the interaction with C4-HSL, supported by molecular docking. Our results provide novel insights into the regulatory signaling networks of antibiofilm formation in P. fluorescens affected by carvacrol.