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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 isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented byEnterococcus hirae(56.9%), followed byEnterococcus faecium(25.9%),Enterococcus casseliflavus(10.4%),Enterococcus gallinarum(5.2%) andEnterococcus saccharolyticus(1.7%). All WP isolates were negative forgelE(gelatinase) andsprE(serine protease) as well as thefsrABDCoperon that regulates the two proteases, and only four isolates (7.0%) formed biofilmsin vitro. All SP isolates wereEnterococcus faecalispositive for thefsrABDC, gelE, sprEgenes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed amongE. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticusandEnterococcus mundtii. All NP isolates were negative for thefsroperon and only fourE. hirae(11.1%) formed a biofilm. Of further interest was the loss of the gelatinase 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 thefrsoperon and sequencing of thegelEgene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows thatE. faecaliswith the completefsroperon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especiallyE. hirae,produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype inE. faecalisand its regulation will require additional studies.