Histamine-producing Lactobacillus parabuchneri strains isolated from grated cheese can form biofilms on stainless steel.

The consumption of food containing large amounts of histamine can lead to histamine poisoning. Cheese is one of the most frequently involved foods. Histamine, one of the biogenic amines (BAs) exhibiting the highest safety risk, accumulates in food contaminated by microorganisms with histidine decarboxylase activity. The origin of these microorganisms may be very diverse with contamination likely occurring during post-ripening processing, but the microorganisms involved during this manufacturing step have never been identified. The present work reports the isolation of 21 histamine-producing Lactobacillus parabuchneri strains from a histamine-containing grated cheese. PCR revealed that every isolate carried the histidine decarboxylase gene (hdcA). Eight lineages were identified based on the results of genome PFGE restriction analysis plus endonuclease restriction profile analysis of the carried plasmids. Members of all lineages were able to form biofilms on polystyrene and stainless steel surfaces. L. parabuchneri is therefore an undesirable species in the dairy industry; the biofilms it can produce on food processing equipment represent a reservoir of histamine-producing bacteria and thus a source of contamination of post-ripening-processed cheeses.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins.

An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.

The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria.

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 10(7)), Lactobacillus plantarum (5.7 × 10(5)), Lactobacillus casei (2.3 × 10(5)), Leuconostoc citreum (2.7 × 10(5)), and Enterococcus faecalis (2.4 × 10(5)). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

Molecular basis of antimicrobial drug resistance in Staphylococcus aureus isolates recovered from young healthy carriers in Spain.

The frequency and genetic bases of antimicrobial drug resistance was determined for 111 Staphylococcus aureus recovered from young healthy carriers in a Spanish region. Resistances to ampicillin (84.7%), kanamycin (27%), erythromycin (25.2%), clindamycin (22.5%), tetracycline (11.7%), amikacin and tobramycin (6.3% each), gentamicin (5.4%), chloramphenicol (2.7%), ciprofloxacin (0.9%; MIC 4 µg/ml), moxifloxacin (0.9%) and mupirocin (0.9%; MIC 60 µg/ml) were found, and all were susceptible to methicillin (MSSA). Nearly 50% of the isolates were resistant to one antibiotic, 30% to two, 15.3% to three and 1.8% to four, while only 6.3% remained fully susceptible. A total of 31 profiles were found. For each phenotypic resistance, at least one gene accounting for it was identified. The detected genes were blaZ; erm(A)-erm(B)-erm(C)-msr(A)-msr(B)-lnu(A), aphA-aadE-sat4-aacA + aphD-aadD, tet(K), cat, and qacA/B, for resistance to ampicillin, macrolides and/or lincosamides, aminoglycosides, tetracycline, chloramphenicol, and quaternary ammonium compounds, respectively. In all isolates carrying cat genes, in all except one of the isolates positive for tet(K), and in most isolates with blaZ, erm(C), msr(A), or msr(B), the gene(s) mapped on resistance plasmids, which were detected in 69.2% of the resistant isolates (65% of the total). The S. aureus from young healthy carriers analysed in the present study do not constitute a reservoir of MRSA, but they represent a repository of multiple determinants conferring resistance to "old" antimicrobials. Some of these have still clinical applications and, considering the increasing resistance to recently introduced antimicrobials, none of them can be disregarded.

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis.

BACKGROUND: Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium's regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR. RESULTS: This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells. CONCLUSION: pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.

Is the production of the biogenic amines tyramine and putrescine a species-level trait in enterococci?

Biogenic amines (BA) are toxic nitrogenous compounds that can be accumulated in foods via the microbial decarboxylation of certain amino acids. Lactic acid bacteria (LAB) strains belonging to different species and genera have been described as BA producers and are mainly responsible for their synthesis in fermented foods. It is generally accepted that the capacity to produced BAs is strain-dependent. However, the large number of enterococci identified as BA producers suggests that the aminogenic trait may be a species-level characteristic. Enterococcus faecalis, Enterococcus faecium and Enterococcus durans strains of different origin were analysed to determine their capacity to produce tyramine and putrescine. The presence of the genes responsible for this and the identity of their flanking regions were checked by PCR. The results suggest that tyramine biosynthesis is a species-level characteristic in E. faecalis, E. faecium and E. durans. Putrescine synthesis was found to be a species-level trait of E. faecalis, with production occurring via the agmatine deamination pathway. Some E. faecium strains of human origin also produced putrescine; this trait was probably acquired via horizontal gene transfer.

Comparative phenotypic and molecular genetic profiling of wild Lactococcus lactis subsp. lactis strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes, isolated from starter-free cheeses made of raw milk.

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies in this study appear to be good starter candidates.

Integrative expression system for delivery of antibody fragments by lactobacilli.

A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.

Tacrolimus modulates liver and pancreas nitric oxide synthetase and heme-oxygenase isoforms and cytokine production after endotoxemia.

Cytoprotective effects of tacrolimus are due to its unspecific anti-inflammatory and anti-oxidant properties. Neither the exact mechanisms nor if there is any organ-specificity or dose-dependent response have not been yet elucidated. Our aim was to evaluate the effect of tacrolimus on oxidative stress and mediator production in liver and pancreatic tissue secondary to endotoxemia. Wistar rats were pretreated with intraperitoneal injection of tacrolimus (0.07, 0.15, and 0.3mg/kg) 24h before Escherichia coli LPS was administrated. Animals were sacrificed 24h after LPS administration and iNOS, eNOS, and nNOS and type 1 and 2 heme-oxygenase (HO) expression were measured. TNF-α and IL-1 tissue expression and plasmatic NO, CO, TNF-α, and IL-1 were also determined. LPS exposure increased iNOS expression in both organs, eNOS did not show variations and liver nNOS expression was significantly lower. Tacrolimus diminished both pancreas and liver iNOS and nNOS expression. Both liver and pancreatic eNOS expression augmented when tacrolimus was administrated. High doses of tacrolimus were correlated with ameliorated liver HO-1 plus HO-2 and pancreas HO-1 expression after LPS stimulation. Tacrolimus treatment diminished TNF-α but not IL-1 expression increase after LPS challenge in hepatic tissue. Pancreatic TNF-α and IL-1 values diminished partially when high doses were employed. Plasmatic NO, CO, TNF-α, and IL-1 concentrations increase after LPS challenge was diminished when highest doses of tacrolimus were given. In conclusion, tacrolimus exerts a protective effect on commonly observed harmful phenomena after LPS stimulation by modulating liver and pancreas oxidative enzyme expression and cytokine production.

Biogenic amines in dairy products.

Biogenic amines (BA) are organic, basic, nitrogenous compounds with biological activity, mainly formed by the decarboxylation of amino acids. BA are present in a wide range of foods, including dairy products, and can accumulate in high concentrations. In some cheeses more than 1000 mg of BA have been detected per kilogram of cheese. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding the BA content in dairy products, it is generally assumed that they should not be allowed to accumulate. Greater knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods. This article focuses on the factors that affect BA production, in particular environmental conditions, the microorganisms that produce them, the genetic organization and regulation of the biosynthetic pathways involved, and the available methods for detecting the presence of BA or BA-producing microorganisms in dairy products.

Biogenic amines content in Spanish and French natural ciders: application of qPCR for quantitative detection of biogenic amine-producers.

Biogenic amines (BA) are low molecular weight nitrogenous bases commonly found in fermented foods and beverages and their consumption can induce undesirable reactions. In this work, the BA content in natural cider from Spain and France was determined. Samples from commercially available cider or obtained during the elaboration process were analyzed. A different profile and BA concentration was observed depending on cider origin. qPCR tools developed for the quantitative detection of BA producers from cheese and wine were tested in the cider samples. A good connection between the BA content and the presence of BA-producing microorganisms was observed. Based on these tools, BA-producing bacteria were isolated from the analyzed cider samples, including new potential histamine- and putrescine-producing Lactobacillus paracollinoides strains.

Extraction of RNA from fermented milk products for in situ gene expression analysis.

This work describes a simple method for extracting high-quality microbial RNA from fermented milk. Pretreatment consists merely of adding a concentrated solution of sodium citrate to the milk product. The proposed method provides consistently better yields of high-quality RNA than the conventional method independent of the texture of the fermented milk. Furthermore, it can be used with small amounts of starting material, requiring only the use of Eppendorf-size centrifuges-a great advantage for analytical laboratories. The RNA obtained is suitable for the detection of live microorganisms and for transcriptional studies based on quantitative reverse transcription polymerase chain reaction (RT-qPCR).

The biogenic amine tryptamine, unlike ß-phenylethylamine, shows in vitro cytotoxicity at concentrations that have been found in foods.

ß-phenylethylamine and tryptamine are biogenic amines (BA) often found in foods. In general, BA are assumed to be toxic and their accumulation in food is not recommended. However, present knowledge regarding the toxicity of ß-phenylethylamine and tryptamine is limited; more information is needed if qualitative and quantitative risk assessments of foods are to be successfully conducted. This study describes a real-time analysis of ß-phenylethylamine and tryptamine toxicity on a human intestinal epithelial cell line. Both BA caused cell necrosis and apoptosis, although the former was the main mode of action of ß-phenylethylamine, and the latter the main mode of action of tryptamine. Only tryptamine was cytotoxic at concentrations found in BA-rich foods. The results presented in this work may contribute to establish legal limits for ß-phenylethylamine and tryptamine in food.

Construction and characterization of a double mutant of Enterococcus faecalis that does not produce biogenic amines.

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes.

The biogenic amines putrescine and cadaverine show in vitro cytotoxicity at concentrations that can be found in foods.

Putrescine and cadaverine are among the most common biogenic amines (BA) in foods, but it is advisable that their accumulation be avoided. Present knowledge about their toxicity is, however, limited; further research is needed if qualitative and quantitative risk assessments for foods are to be conducted. The present work describes a real-time analysis of the cytotoxicity of putrescine and cadaverine on intestinal cell cultures. Both BA were cytotoxic at concentrations found in BA-rich foods, although the cytotoxicity threshold for cadaverine was twice that of putrescine. Their mode of cytotoxic action was similar, with both BA causing cell necrosis; they did not induce apoptosis. The present results may help in establishing legal limits for both putrescine and cadaverine in food.

Spermine and spermidine are cytotoxic towards intestinal cell cultures, but are they a health hazard at concentrations found in foods?

Spermine and spermidine are polyamines (PA) naturally present in all organisms, in which they have important physiological functions. However, an excess of PA has been associated with health risks. PA accumulates at quite high concentrations in some foods, but a quantitative assessment of the risk they pose has been lacking. In the present work, the cytotoxicity of spermine and spermidine was evaluated using an in vitro human intestinal cell model, and employing real-time cell analysis. Both spermine and spermidine showed a dose-dependent cytotoxic effect towards the cultured cells, with necrosis the mode of action of spermidine and perhaps also that of spermine. Spermine was more cytotoxic than spermidine, but for both PA the concentrations found to be toxic were above the maximum at which they have been found in food. The present results do not, therefore, support the idea that spermine or spermidine in food is harmful to healthy people.

The Relationship among Tyrosine Decarboxylase and Agmatine Deiminase Pathways in Enterococcus faecalis.

Enterococci are considered mainly responsible for the undesirable accumulation of the biogenic amines tyramine and putrescine in cheeses. The biosynthesis of tyramine and putrescine has been described as a species trait in Enterococcus faecalis. Tyramine is formed by the decarboxylation of the amino acid tyrosine, by the tyrosine decarboxylase (TDC) route encoded in the tdc cluster. Putrescine is formed from agmatine by the agmatine deiminase (AGDI) pathway encoded in the agdi cluster. These biosynthesis routes have been independently studied, tyrosine and agmatine transcriptionally regulate the tdc and agdi clusters. The objective of the present work is to study the possible co-regulation among TDC and AGDI pathways in E. faecalis. In the presence of agmatine, a positive correlation between putrescine biosynthesis and the tyrosine concentration was found. Transcriptome studies showed that tyrosine induces the transcription of putrescine biosynthesis genes and up-regulates pathways involved in cell growth. The tyrosine modulation over AGDI route was not observed in the mutant Δtdc strain. Fluorescence analyses using gfp as reporter protein revealed PaguB (the promoter of agdi catabolic genes) was induced by tyrosine in the wild-type but not in the mutant strain, confirming that tdc cluster was involved in the tyrosine induction of putrescine biosynthesis. This study also suggests that AguR (the transcriptional regulator of agdi) was implicated in interaction among the two clusters.

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.

Data on recovery of 21 amino acids, 9 biogenic amines and ammonium ions after spiking four different beers with five concentrations of these analytes.

A novel chromatographic method for the simultaneous analysis of nine biogenic amines, 21 amino acids and ammonium ions in beer has been recently described in "A UHPLC method for the simultaneous analysis of biogenic amines, amino acids and ammonium ions in beer" (Redruello et al., 2017) [1]. The present article provides recovery data of the 31 analytes after spiking four different beers with five concentrations of each analyte (15, 30, 60, 120 and 240 µM).