Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environment.

Summary The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd-Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of iso- lates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP iso- lates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Entero- coccus casseliflavus (10.4%), Enterococcus galli- narum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelati- nase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Entero- coccus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelati- nase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4-8°C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural envi- ronment and may represent a source/reservoir of clinically relevant strains. In addition, many environ- mental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies.