The gene cluster associated with strong biofilm-formation capacity by histamine-producing Lentilactobacillus parabuchneri encodes a sortase-mediated pilus and is located on a plasmid.

Histamine is a biogenic amine synthesized through the enzymatic decarboxylation of the amino acid histidine. It can accumulate at high concentrations in foods through the metabolism of certain bacteria, sometimes leading to adverse reactions in consumers. In cheese, histamine can accumulate at toxic levels; Lentilactobacillus parabuchneri has been identified the major cause of this problem. Previous studies have shown some L. parabuchneri strains to form biofilms on different surfaces, posing a contamination risk during cheese production, particularly for cheeses that are processed post-ripening (e.g., grating or slicing). The food contamination they cause can result in economic losses and even foodborne illness if histamine accumulates in the final product. The aim of the present work was to identify the genes of L. parabuchneri involved in biofilm formation, and to determine their function. The genomes of six strains with different biofilm-production capacities (strong, moderate and weak) were sequenced and analysed. A cluster of four genes, similar to those involved in sortase-mediated pilus formation, was identified in the strong biofilm-producers, suggesting it to have a role in surface adhesion. Cloning and heterologous expression in Lactococcus cremoris NZ9000 confirmed its functionality and involvement in adhesion and, therefore, in biofilm formation. PacBio sequencing showed this cluster to be located on a 33.4 kb plasmid, which might increase its chances of horizontal transmission. These findings provide insight into the genetic factors associated with biofilm formation in histamine-producing L. parabuchneri, and into the risks associated with this bacterium in cheese production.

Isolation and Characterization of Enterococcus faecalis-Infecting Bacteriophages From Different Cheese Types.

Enterococci are a diverse group of Gram-positive, lactic acid bacteria (LAB). They are found in many environments, including fermented foods, in which they could constitute a health threat since they produce biogenic amines, which consumption can lead to intoxication. Moreover, enterococci has also emerged as an important hospital-acquired pathogens via its acquisition of antimicrobial resistance. Bacteriophages possess features that make them optimal biotechnological weapons for controlling bacterial growth in disease and food spoilage contexts. However, no silver bullet bacteriophage exists that can eliminate all the undesirable bacteria in a complex environment. Rather, a combination of phages with different host ranges would be required which implies the need for large collections of diverse phages. This work reports the isolation of several Enterococcus faecalis-infecting bacteriophages from different types of cheese, along with the range of E. faecalis strains of diverse origin (from dairy to clinical environments) they are able to infect. The isolated phages showed a large diversity regarding the number and origin of strains they infect. Some of these phages were subjected to morphological and genomic characterization which confirmed their diversity and showed they belong to different families and genera. The present findings increase the potential arsenal for the bacteriophage-based biocontrol of harmful E. faecalis populations.

Enterococcus faecalis Bacteriophage 156 Is an Effective Biotechnological Tool for Reducing the Presence of Tyramine and Putrescine in an Experimental Cheese Model.

Biogenic amines (BA) - nitrogenous compounds of low molecular weight - are the result of metabolism of certain amino acids. They are biologically present in all living organisms and play essential physiological roles. However, their accumulation in foodstuffs due to the metabolic activity of certain microorganisms represents a toxicological risk. Containing such microorganisms, and with an abundance of precursor substrate amino acids, fermented foods in general, and cheeses in particular, provide an ideal matrix for the accumulation of these toxic compounds. Unfortunately, the main microorganisms responsible for BA accumulation are members of the lactic acid bacteria (LAB) group, which are also essential for the development of the organoleptic characteristics of the final product. The methods used to reduce the BA content of cheese, such as milk pasteurization, commonly fail to do so, and affect desirable non-BA-producing LAB as well. Bacteriophages have been proposed as biotechnological tools for diminishing the presence of undesirable microorganisms in dairy products. Given their specificity, they could be used to target the population of BA-producing bacteria. In this work, we aimed to explore the use of Enterococcus faecalis infecting phages as a tool to reduce the content of BA in dairy products. For this, we proceeded to the isolation and characterization of E. faecalis bacteriophage 156, a member of the family Myoviridae. Its genome was sequenced and compared with that of E. faecalis family Myoviridae phages available in public databases. Its capacity to decrease the accumulation of the BA tyramine and putrescine in an experimental laboratory-scale cheese model was proven.

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.

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.

Nucleotide sequence alignment of hdcA from Gram-positive bacteria.

The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 〈10.2174/157340110791233256〉 [1], Linares et al. 2016 〈http://dx.doi.org/10.1016/j.foodchem.2015.11.013〉〉 [2]). The reaction is catalyzed by a pyruvoyl-dependent histidine decarboxylase (Linares et al. 2011 〈10.1080/10408398.2011.582813〉 [3]), which is encoded by the gene hdcA. In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see 〈http://dx.doi.org/ 10.1016/j.foodcont.2015.11.035〉〉 [4].

Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model.

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.

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.