Different Phenotypes of Mature Biofilm in Flavobacterium psychrophilum Share a Potential for Virulence That Differs from Planktonic State

Abstract

<i>Flavobacterium psychrophilum</i> is the etiological agent of bacterial coldwater disease and the rainbow trout fry syndrome in salmonid aquaculture worldwide. However, there have been few studies into the capacity of <i>F. psychrophilum</i> to form biofilms and how these cellular accretions differ from planktonic cells or how they affect potential virulence. We evaluated the biofilm formation by three Chilean isolates of <i>F. psychrophilum</i> (LM-02-Fp, LM-06-Fp, and LM-13-Fp) and two non-Chilean strains (JIP02/86 and NCMB1947^T), and compared biofilm and planktonic states to obtain insights into expression differences of virulence- and biofilm-related genes (VBRGs). Our findings are based on scanning confocal laser microscopy (SCLM) and LIVE/DEAD staining, enzymatic reactions, reverse transcription-quantitative PCR (RT-qPCR) of genes encoding putative virulence factors, and transcriptomes (RNA-Seq). The LM-02-Fp and NCMB1947^T strains were the strongest and weakest biofilm producers, respectively. The strong-biofilm producer showed different physiological cell states distributed in different layers of mature biofilms, whereas the NCMB1947^T biofilms consisted of cells arranged in a monolayer. WGA-binding exopolysaccharides would be the main components of their corresponding extracellular matrices. Transcriptomes of <i>F. psychrophilum</i> NCMB1947^T and LM-02-Fp were clustered by state (biofilm vs. planktonic) rather than by strain, indicating important state-dependent differences in gene expression. Analysis of differentially expressed genes between states identified putative VBRGs involved in polysaccharide biosynthesis, lateral gene transfer, membrane transport (e.g., for drugs and Fe³⁺), sensory mechanisms, and adhesion, and indicated that about 60-100% of VBRGs involved in these processes was significantly upregulated in the biofilm state. Conversely, upregulated motility-related genes in the biofilm state were not identified, whereas a lower fraction of proteolysis-related genes (33%) was upregulated in biofilms. In summary, <i>F. psychrophilum</i> strains that produce different biofilm phenotypes show global transcriptional activity in the mature biofilm state that differs significantly from their planktonic counterparts. Also, different biofilm phenotypes share a genetic potential for virulence that is transcriptionally enhanced with respect to free-living cells. Our results suggest that the <i>F. psychrophilum</i> biofilm lifestyle acts as a reservoir for a given set of putative virulence factors, and recommend a deeper understanding of which could help prevent recurring infections in salmonid farms.