Effects of Sodium Chloride on Tyramine Production in a Fermented Food Model and its Inhibition by Tyrosine-degrading Lactobacillus plantarum JA-1199.

Tyramine is a health-adverse biogenic amine, which can accumulate in fermented foods like cheese by decarboxylation of the free amino acid tyrosine by either starter cultures or resident microbes such as lactic acid bacteria including Enterococcus spp., respectively. Our study aimed to show the effect of sodium chloride concentrations on tyramine production as well as to characterise bacterial strains as anti-tyramine biocontrol agents in a 2 mL micro-cheese fermentation model. The effect of sodium chloride on tyramine production was assayed with tyramine producing strains from eight different species or subspecies. Generally, an increase in sodium chloride concentration enhanced tyramine production, e.g. from 0% to 1.5% of sodium chloride resulted in an increase of tyramine of 870% with a Staphylococcus xylosus strain. In the biocontrol screening among lactic acid bacteria, a Lactobacillus plantarum JA-1199 strain was screened that could consume in successful competition with other resident bacteria tyrosine in the micro-cheese model as a source of energy gain. Thereby tyramine accumulation was reduced between 4% to 99%. The results of this study disclose a feasible strategy for decreasing tyramine concentration and increasing the safety level of fermented food. It is an example of development and application of bacterial isolates as starter or protective cultures in food, a biocontrol topic, which Oreste Ghisalba - in his project evaluation function of SNF and later on CTI - was promoting with great emphasis in our ETH Food Biotechnology research group.

Detection of Biogenic Amines and Tyramine-Producing Bacteria in Fermented Sausages from Switzerland.

ABSTRACT: Fermented foods can cause human illness because of the unhealthy effect of biogenic amines (BAs) that accumulate by decarboxylation of free amino acids. Salami-type fermented sausages can contain BAs, but which bacteria and environmental factors contribute to BA production is not clear. Sixty-two sausages purchased from Swiss markets were evaluated for decarboxylating bacterial strains and concentrations of the BAs cadaverine, histamine, putrescine, and tyramine. Based on the size and number of employees of the meat processing plants, sausages were separated into two groups: artisanal and industrial. Concentrations of all four BAs were higher in industrial sausages than in artisanal sausages. Tyramine was the major BA detected in 46 of 62 sausages, at a maximum concentration of 785.22 mg/kg. Enterococci and coagulase-negative staphylococci (mainly the meat starter culture Staphylococcus xylosus) were the main tyramine producers. Putrescine was found in 20 of 62 samples, at a maximum concentration of 707.77 mg/kg. Concentrations of these two BAs were significantly correlated (P = 0.0407). Cadaverine and histamine were detected in nine and eight samples, respectively, and both were found in significantly higher concentrations (P = 0.019 and 0.036, respectively) in industrial sausages. Based on the tyramine concentration, five groups of fermented sausages were identified: group 1, very high concentrations (>700 mg/kg); group 2, high concentrations (400 to 700 mg/kg); group 3, moderate concentrations (200 to 400 mg/kg); group 4, low concentrations (<200 mg/kg); group 5, concentrations below the detection limit (0.05 mg/kg). Product samples with tyramine concentrations >200 mg/kg were considered of lower quality because consumption of such samples could be unhealthy for sensitive consumers.

Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme.

BACKGROUND: The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). RESULTS: Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. CONCLUSION: Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.

pDB2011, a 7.6 kb multidrug resistance plasmid from Listeria innocua replicating in Gram-positive and Gram-negative hosts.

pDB2011, a multidrug resistance plasmid isolated from the foodborne Listeria innocua strain TTS-2011 was sequenced and characterized. Sequence analysis revealed that pDB2011 had a length of 7641 bp and contained seven coding DNA sequences of which two were annotated as replication proteins, one as a recombination/mobilization protein and one as a transposase. Furthermore, pDB2011 harbored the trimethoprim, spectinomycin and macrolide-lincosamide-streptogramin B resistance genes dfrD, spc and erm(A), respectively. However, pDB2011 was only associated with trimethoprim and spectinomycin resistance phenotypes and not with phenotypic resistance to erythromycin. A region of the plasmid encoding the resistance genes spc and erm(A) plus the transposase was highly similar to Staphylococcus aureus transposon Tn554. The dfrD gene was 100% identical to dfrD found in a number of Listeria monocytogenes isolates. Additionally, assessment of the potential host range of pDB2011 revealed that the plasmid was able to replicate in Lactococcus lactis subsp. cremoris MG1363 as well as in Escherichia coli MC1061 and DH5α. This study reports the first multidrug resistance plasmid in L. innocua. A large potential for dissemination of pDB2011 is indicated by its host range of both Gram-positive and Gram-negative bacteria.

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes.

OBJECTIVES: To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. METHODS: The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. RESULTS: Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. CONCLUSIONS: This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with ß-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.