Rapid Detection and Quantification of Tyrosine Decarboxylase Gene (tdc) and Its Expression in Gram-Positive Bacteria Associated with Fermented Foods Using PCR-Based Methods

Abstract

In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

Characterization of Tetracycline-Resistant Streptococcus thermophilus Isolates from Italian Soft Cheeses

“…RNA extraction and reverse transcription-PCR procedures were performed as previously described (20). At the tetracycline concentration tested, tet(S) was always expressed, while expression of the genes tet(M) and tet(L) was found only in a few strains (Table 3).…”

Identification of a Tyrosine Decarboxylase Gene ( tdcA ) in Streptococcus thermophilus 1TT45 and Analysis of Its Expression and Tyramine Production in Milk

“…Tyramine formation is the result of bacterial decarboxylation of tyrosine that is carried out by several microorganisms, including strains of lactic acid bacteria (LAB) belonging to different genera and species (7,23). Among LAB, Streptococcus thermophilus is of major importance for the dairy industry, where it is commonly used in starter cultures for the manufacture of yogurt, fermented milks, and many varieties of cheese.…”

“…3, as follows: 1 volume of 26.8 mM EDTA, pH 12.0 (18), was added to 4 ml of the milk culture, and the mixture was centrifuged and washed twice with sterile water. The extraction was carried out as described by Torriani et al (23). RNA integrity, concentration, and purity were checked by electrophoresis on 2% (wt/vol) agarose gel and by measurement of the OD 260 /OD 280 ratio.…”

Origin of the Putrescine-Producing Ability of the Coagulase-Negative Bacterium Staphylococcus epidermidis 2015B

“…Recently, histidine decarboxylase genes have been characterized in S. capitis (16) and S. epidermidis (K. Yokoi and K. Kodaira, unpublished data; GenBank accession no. AB378754), while a fragment of a putative tyrosine decarboxylase gene was described in an S. epidermidis strain (46). Genome sequencing has demonstrated that putrescine production by ornithine decarboxylation is a trait that is abundantly found in the Gram-negative Gammaproteobacteria but rarely in Gram-positive bacteria.…”