Putrescine production by Lactococcus lactis subsp. cremoris CECT 8666 is reduced by NaCl via a decrease in bacterial growth and the repression of the genes involved in putrescine production

The reduction of NaCl in food is a public health priority; high NaCl intakes have been associated with serious health problems. However, it is reported that reducing the NaCl content of cheeses may lead to an increase in the content of biogenic amines (BAs). The present work examines the effect of NaCl on the accumulation of putrescine (one of the BAs often detected at high concentration in cheese) in experimental Cabrales-like cheeses containing Lactococcus lactis subsp. cremoris CECT 8666, a dairy strain that catabolises agmatine to putrescine via the agmatine deiminase (AGDI) pathway. The genes responsible for this pathway are grouped in the AGDI cluster. This comprises a regulatory gene (aguR) (transcribed independently), followed by the catabolic genes that together form an operon (aguBDAC). Reducing the NaCl concentration of the cheese led to increased putrescine accumulation. In contrast, increasing the NaCl concentration of both pH-uncontrolled and pH-controlled (pH 6) cultures of L. lactis subsp. cremoris CECT 8666 significantly inhibited its growth and the production of putrescine. Such production appeared to be inhibited via a reduction in the transcription of the aguBDAC operon; no effect on the transcription of aguR was recorded. The present results suggest that low-sodium cheeses are at risk of accumulating higher concentrations of putrescine.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and by the GRUPIN14-137 project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2013–2017 and the European Regional Development Funds.Peer reviewe

Biofilm-forming capacity in biogenic amine-producing bacteria isolated from dairy products

Biofilms on the surface of food industry equipment are reservoirs of potentially food-contaminating bacteria-both spoilage and pathogenic. However, the capacity of biogenic amine (BA)-producers to form biofilms has remained largely unexamined. BAs are low molecular weight, biologically active compounds that in food can reach concentrations high enough to be a toxicological hazard. Fermented foods, especially some types of cheese, accumulate the highest BA concentrations of all. The present work examines the biofilm-forming capacity of 56 BA-producing strains belonging to three genera and 10 species (12 Enterococcus faecalis, 6 Enterococcus faecium, 6 Enterococcus durans, 1 Enterococcus hirae, 12 Lactococcus lactis, 7 Lactobacillus vaginalis, 2 Lactobacillus curvatus, 2 Lactobacillus brevis, 1 Lactobacillus reuteri, and 7 Lactobacillus parabuchneri), all isolated from dairy products. Strains of all the tested species - except for L. vaginalis-were able to produce biofilms on polystyrene and adhered to stainless steel. However, the biomass produced in biofilms was strain-dependent. These results suggest that biofilms may provide a route via which fermented foods can become contaminated by BA-producing microorganisms.This work was funded by the Spanish Ministry of the Economy and Competitiveness (AGL2013-45431-R) and the Plan for Science, Technology and Innovation 2013–2017 financed by the European Regional Development Fund and the Principality of Asturias (GRUPIN14-137). MD is a beneficiary of an FPI fellowship from the Spanish Ministry of the Economy and Competitiveness.Peer Reviewe

Putrescine biosynthesis in Lactococcus lactis is transcriptionally activated at acidic pH and counteracts acidification of the cytosol

Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH 5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter P. However, the external pH had no significant effect on the activity of the aguR promoter P, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH 5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and by the GRUPIN14-137 Project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2014–2017 and the European Regional Development Funds. The authors thank Adrian Burton for language and editing assistance.Peer Reviewe

Is256 abolishes gelatinase activity and biofilm formation in a mutant of the nosocomial pathogen enterococcus faecalis v583

Enterococcus faecalis is one of the most controversial species of lactic acid bacteria. Some strains are used as probiotics, while others are associated with severe and life-threatening nosocomial infections. Their pathogenicity depends on the acqui-sition of multidrug resistance and virulence factors. Gelatinase, which is required in the first steps of biofilm formation, is an important virulence determinant involved in E. faecalis pathogenesis, including endocarditis and peritonitis. The gene that codes for gelatinase (gelE) is controlled by the Fsr quorum-sensing system, whose encoding genes (fsrA, fsrB, fsrC, and fsrD) are located immediately upstream of gelE. The integration of a DNA fragment into the fsr locus of a derived mutant of E. faecalis V583 suppressed the gelatinase activity and prevented biofilm formation. Sequence analysis indicated the presence of IS256 integrated into the fsrC gene at nucleotide position 321. Interestingly, IS256 is also associated with biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus. This is the first description of an insertion sequence that prevents biofilm formation in E. faecalis. Copyright de NRC Research PressPeer Reviewe

A PCR-DGGE method for the identification of histamine-producing bacteria in cheese

Histamine is the biogenic amine (BA) most frequently involved in food poisoning. Cheese is among the foods in which it is most commonly found, and in some of the highest concentrations. Its accumulation in cheese is mainly due to the presence of lactic acid bacteria (LAB) that produce histidine decarboxylase, an enzyme coded by the gene hdcA. This gene has been sequenced in several histamine-producing LAB. This paper reports a new culture-independent method based on PCR-DGGE for detecting and identifying, at the species level, the histaminogenic bacteria present in cheese. Primers were designed based on the hdcA gene sequences available for Gram positive bacteria, and PCR and DGGE optimized in order to differentiate between amplicons corresponding to different histamine-producing species. The proposed method provides a rapid and simple means of detecting and identifying histamine-producing Gram positive bacteria in foods with complex microbial communities, such as cheese.This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and the Plan for Science, Technology and Innovation 2013–2017 of the Principality of Asturias, which is co-funded by the European Regional Development Fund (GRUPIN14-137). M.D. was a beneficiary of an FPI fellowship from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and by the GRUPIN14-137 project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2014-2017 and the European Regional Development Funds.Peer reviewe

Q69 (an E. faecalis-infecting bacteriophage) as a biocontrol agent for reducing tyramine in dairy products

Biogenic amines (BAs) are low molecular weight nitrogenous compounds with biological activity, formed from amino acids by decarboxylation. BAs are naturally present in all living organisms playing essential roles. However, their accumulation in food through the metabolic activity of certain microorganisms constitutes a toxicological hazard. Among foods, cheeses accumulate some of the highest concentrations of BAs since they provide an ideal environment for their accumulation. Most of the methods proposed for reducing BAs in cheese, such as milk pasteurization, have not only failed to completely solve the problem, they also affect non-BA producing lactic acid bacteria, i.e., the bacteria that participate in the development of the organoleptic characteristics of cheese. Novel technologies specifically targeted against BA producers are therefore needed to control BA accumulation. Bacteriophages have been proposed as agents for specifically controlling the presence of foodborne pathogens in food. Due to its specificity, they could be used as a biotechnological tool targeted to reduce the population of BA-producing bacteria. The present work reports the isolation, from cheese, and the characterization of bacteriophage Q69, which infects specifically Enterococcus faecalis, the species mainly responsible of the accumulation of the BA tyramine in foods. Furthermore, its capacity to reduce the accumulation of tyramine in different conditions -including a model cheese- was proven. The obtained results open up the possibility of use bacteriophages to prevent BA accumulation in fermented foods.This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R), and by the GRUPIN14-137 project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2013–2017 and European Regional Development Funds.Peer Reviewe

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R) and by the GRUPIN14-137 project, which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2014-2017 and the European Regional Development Funds.Peer reviewe

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis

[Background] Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes.[Results] This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P aguB . The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten.[Conclusion] The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R), the Principality of Asturias Plan for Science, Technology and Innovation 2013-2017, and FEDER funds (GRUPIN14-137). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). P.A.S. was the beneficiary of a fellowship from the FICYT, Principality of Asturias, Spain (BP09093). D.M.L. and B.d.R. were beneficiaries of JAE DOC contracts (CSIC).Peer Reviewe

Mastitis modifies the biogenic amines profile in human milk, with significant changes in the presence of histamine, putrescine and spermine

Biogenic amines (BAs) are low molecular weight nitrogenous organic compounds with different biological activities. Putrescine, spermidine and spermine are essential for the development of the gut and immune system of newborns, and are all found in human milk. Little is known, however, about the role of histamine, tyramine or cadaverine in breast milk. Nor is it known whether mastitis alters the BA composition of milk. The BA profile of human milk, and the influence of mastitis on BA concentrations, were therefore investigated. Putrescine, spermidine and spermine were the main BAs detected. In mastitis-affected milk, the concentrations of putrescine, spermine and histamine were higher.This work was performed with the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2013-45431-R and AGL2013-41980P) and the GRUPIN14-137 project (which is co-financed by the Plan for Science, Technology and Innovation of the Principality of Asturias 2014-2017 and the European Regional Development Funds). M.P. was the recipient of an FPU (Programa de Formacion del Profesorado Universitario) fellowship from the Spanish Ministry of Education, Culture and Sport.Peer Reviewe