The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

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The aim of this study was to investigate the diversity of tyramine production capability of four Enterococcus strains in buffered systems in relation to their genetic characteristics and environmental conditions. Cells of the strains Enterococcus faecalis EF37 and ATCC 29212, and E. faecium FC12 and FC643 were re-suspended in phosphate/citrate buffers with different pH, NaCl concentration and incubation temperature. At intervals, cell viability and tyramine production were assessed by plate counting and HPLC analysis, respectively. The activity of a purified tyrosine decarboxylase (TDC) was determined under the same conditions, as a reference. Reduced loss in cell viability was observed in all the tested conditions, except for pH 4 after 24 h. The TDC activity was greatly heterogeneous within the enterococci: EF37 and FC12 produced the higher tyramine concentrations, ATCC 29212 showed a reduced decarboxylase activity, while EF643 did not accumulate detectable amounts of tyramine in all the conditions assayed. Among the considerate variables, temperature was the most influencing factor on tyramine accumulation for enterococcal cells. To further correlate the phenotypic and genetic characteristics of the enterococci, the TDC operon region carrying the genes tyrosine decarboxylase (tyrDC), tyrosine/tyramine permease (tyrP), and Na+/H+ antiporter (nhaC-2) was amplified and sequenced. The genetic organization and nucleotide sequence of this operon region were highly conserved in the enterococcal strains of the same species. The heterogeneity in tyramine production found between the two E. faecalis strains could be ascribed to different regulation mechanisms not yet elucidated. On the contrary, a codon stop was identified in the translated tyrDC sequence of E. faecium FC643, supporting its inability to accumulate tyramine in the tested conditions. In addition, the presence of an additional putative tyrosine decarboxylase with different substrate specificity and genetic organization was noticed for the first time. Concluding, the high TDC activity heterogeneity within enterococci determined different accumulation of tyramine, depending on different genetic determinants, regulation mechanisms, and environmental factors. The present research contributes to elucidate the genetic characteristics of enterococcal strains and correlate specific mutations to their different strain-dependent tyraminogenic activity.

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