Gene expression analysis between planktonic and biofilm states of Flavobacterium columnare

Flavobacterium columnare, the causative agent of columnaris disease causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. F. columnare naturally occurs in a planktonic, free living state where it can survive for long periods of time, even in the absence of nutrients. In contrast, F. columnare also possesses the ability to form biofilms, broadly defined as surface bound microbial communities inhabiting an organic matrix composed of autogenously derived extracellular polymeric substances. The advantages of adopting this life stage are not completely clear for F. columnare, but biofilm formation could increase virulence by offering protection from desiccation, augment resistance to antimicrobials, improve nutrient acquisition, and protection against other bacteria. To examine gene expression between F. columnare planktonic cells and biofilms, we conducted a study where both phases were grown with and without stimulation and then sampled for RNA sequencing. Overall design: F. columnare isolate 94-081 was retrieved from frozen glycerol stocks and streaked onto the appropriate medium. After 24-48 h, bacterial colonies were dislodged from the agar using a sterile cotton swab and inoculated into FCGM. Bacterial broth cultures were incubated for 24 h at 28℃ prior to use in all growth assays. For planktonic and biofilm assays, an overnight broth culture of 94-081 was diluted (1:100) as needed into fresh FCGM broth into separate flasks with or without the addition of 20 µg/ml sterilized catfish mucus. After 48 h of growth at 28℃ the planktonic culture was removed from the flask prior to harvesting the biofilm. Experiments were performed in triplicate to represent biological replicates. A total of 12 samples were taken: three biological replicates, sampling two states, with two treatment groups (with or without mucus) for the isolate.