Development of a Fluorescent In‐Situ Hybridisation (FISH) assay to detect vancomycinresistant Enterococcus faecalis in a novel biofilm model.

AimTo develop a FISH assay to detect the vanA gene of E. faecalis in a novel biofilm model.MethodsBiofilm model ‐ Vancomycin resistant E. faecalis (VRE) were grown in tryptone soy broth(TSB) to 2.5x109 CFU/ml. Cells were harvested, re‐suspended and diluted 1:200 in PBS, TSBor TSB (vancomycin 10μg/ml). VRE were inoculated into 25μl gene frames, and biofilmswere developed at 37oC for 24hrs.FISH ‐ Biofilms were washed with PBS and fixed in 97% ethanol for 5 mins. Fixed biofilmswere incubated with fluorescein ‐labelled FISH probes specific to vanA for 2‐24hrs at 50oC.A texas red probe targeted to E. faecalis 16s rRNA was used as a control.ResultsThe gene frame method allowed for the reproducible assay of weak to moderate biofilmproducing enterococci.Control FISH probes to 16s rRNA gave a strong intracellular signal in cells grown bothplanktonically and in biofilm.The vanA FISH probe demonstrated intercellular specificity, although in planktonic cells thestaining pattern was diffuse and less intense than the 16s staining. In biofilm the vanA proberesulted in a significant increase in extracellular signal associated with bacterial extracellularpolymeric substances. This was observed regardless of nutrient content used for biofilmdevelopment and it was not present in controls.ConclusionsThis study utilised gene frames® for the novel analysis of biofilm directly on microscopeslides. FISH probes designed to specifically bind to the plasmid‐associated vanA gene,demonstrate staining patterns not observed in planktonic cells.