The Capability of Tyramine Production and Correlation between Phenotypic and Genetic Characteristics of Enterococcus faecium and Enterococcus faecalis Strains.

Eleonora Bargossi,Chiara Montanari,Fausto Gardini,Veronica Gatto,Sandra Torriani,Giulia Tabanelli

doi:10.3389/fmicb.2015.01371

Abstract

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.

Highlights

IntroductionTyramine is a biogenic amine (BA) which can have severe acute effects if ingested in excessive amounts with food consisting in peripheral vasoconstriction, increased cardiac output, accelerated respiration, elevated blood glucose and release of norepinephrine, symptoms known as “cheese reaction” (Shalaby, 1994; McCabe-Sellers et al, 2006; Marcobal et al, 2012).Lactic acid bacteria (LAB) are among the most efficient producers of tyrosine decarboxylase (TDC), the enzyme responsible for tyramine formation (Marcobal et al, 2012)
The tyramine production by cells re-suspended in buffered systems highlighted the heterogeneity of tyrosine decarboxylase (TDC) activity within enterococci
The study of the genetic characteristics of the E. faecium strains allowed to correlate specific mutations in the tyrDC gene sequence to their different tyraminogenic activity, and suggested the involvement of another gene annotated as putative tyrosine decarboxylase in the complete genome of E. faecium

Summary

Introduction

Tyramine is a biogenic amine (BA) which can have severe acute effects if ingested in excessive amounts with food consisting in peripheral vasoconstriction, increased cardiac output, accelerated respiration, elevated blood glucose and release of norepinephrine, symptoms known as “cheese reaction” (Shalaby, 1994; McCabe-Sellers et al, 2006; Marcobal et al, 2012).Lactic acid bacteria (LAB) are among the most efficient producers of tyrosine decarboxylase (TDC), the enzyme responsible for tyramine formation (Marcobal et al, 2012). Among LAB, species belonging to the genus Enterococcus are recognized as the most efficient tyramine producers (Leuschner et al, 1999; Suzzi and Gardini, 2003; Ladero et al, 2012; Marcobal et al, 2012). Enterococci occur in many different habitats and they are often contaminant in food of animal origin (Franz et al, 2011) Due to their salt and pH tolerance and to their ability to grow over a wide temperature range, these LAB are competitive in harsh environmental conditions, and can be a relevant component of the ripening microbiota of cheeses and sausages (Franz et al, 1999, 2011; Giraffa, 2003). The ability to produce tyramine is considered a species characteristic of Enterococcus faecalis and it is extremely widespread among strains of E. faecium and E. durans (Ladero et al, 2012)

Objectives

Methods

Results

Conclusion