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.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Description of the bacteriocins produced by two strains of Enterococcus faecium isolated from Italian goat milk

In this study two strains of Enterococcus faecium, M241 and M249, isolated from goat milk, were studied for their capability to produce antibacterial compounds. It was determined that the bacteriocins produced by both strains were active towards Listeria monocytogenes and Clostridium butyricum, and they did not have any activity with respect to other species of lactic acid bacteria. Enterocins A and B were targeted by polymerase chain reaction (PCR) and sequenced, after cloning, in both strains. The bacteriocins contained in the cell free supernatants were stable when subjected to treatments at high and low temperatures or with lipase, catalase and alpha-amylase. Whereas, the activity was lost when proteinases were used. Lastly, a co-culture experiment with L. monocytogenes in skimmed milk was performed. In the presence of the E. faecium strains, the pathogen showed a delay in the growth of about 6h and it reached a maximum counts of about 10(6) colony forming unit, two orders of magnitude low with respect to the control. This result suggests the possibility to use the strains studied as starter cultures to enhance food safety of dairy products.     

Isolation and characterization of vanA genotype vancomycin-resistant Enterococcus cecorum from retail poultry in Japan

The isolation rate of high-level vancomycin-resistant enterococci (VRE) from poultry samples in Japan has increased in recent years. As this raises concerns for the potential spread of genes encoding vancomycin resistance, poultry is routinely screened for VRE. Here, we report the isolation and characterization of a vanA genotype vancomycin-resistant Enterococcus cecorum strain (E. cecorum IPHa84) from retail domestic poultry in September 2009. The species identification was performed by biochemical testing and sequencing of the 16S rRNA and manganese-dependent superoxide dismutase genes. The vancomycin and teicoplanin susceptibility tests showed that E. cecorum IPHa84 was resistant to vancomycin and susceptible to teicoplanin, demonstrating that this isolate was VanB phenotype-vanA genotype VRE. Moreover, a vanA gene cluster was found in a chromosomally encoded Tn1546-related element, which exhibited the characteristic structure of the prototype Tn1546 element, but contained eight point mutations. The vanS sequence of E. cecorum IPHa84 contained three point mutations and was 100% identical to those of VRE isolated from different broiler droppings in Japan prior to the banning of avoparcin, indicating that the Tn1546-related element may be stable in poultry production environments, even in the absence of selective pressure. The isolation of a novel enterococcal species harboring the vanA gene reconfirms that poultry can serve as a reservoir of VanA-type VRE or vancomycin resistance genes, and suggests that the transmission of these risk factors from poultry to humans through the food chain remains a potential threat in Japan.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.     

Isolation and selection of yeasts from wine grape ecosystem secreting cold-active pectinolytic activity

The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12 °C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751 U/mL) at pH 3.5 and 12 °C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production.     

Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for the detection, quantification and identification of bacteria with the ability to produce tyramine. This method was found to be specific and to show a wide dynamic range, thus allowing the quantification of these tdc+ bacterial groups among the complex microbiota of cheese. tdc qPCR was used to follow the development of tdc+ microbiota during the manufacture of a blue-veined cheese (Cabrales) made from raw milk. In this type of cheese, tdc+ enterococci seem to be responsible for the high concentrations of tyramine detected. The method was also used to identify and quantify tdc+ enterococci and lactobacilli in 18 commercially available cheeses. Different types and numbers of these microorganisms were found. Their relationships with the concentration of tyramine and technological factors are discussed.     

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.     

Tyrosine- and histidine-decarboxylase positive lactic acid bacteria and enterococci in dry fermented sausages

Lactic acid bacteria (LAB) and enterococci were isolated immediately after stuffing (day 0), at the end of ripening (28th day) and at the end of storage (112th day) from dry fermented sausages produced by two different producers (K; R) in two diameters (4.5 and 7 cm) using either of two spice mixtures (P; H) and either of two starter cultures (Pediococcus pentosaceus, C; Lactobacillus curvatus + Staphylococcus carnosus, F), resulting in a total of 16 different combinations. Tyrosine-decarboxylase DNA sequence (tyrdc) was identified on average in 88% and 44% of enterococci and LAB isolates, respectively at the end of ripening, the corresponding figures regarding histidine-decarboxylase gene sequence (hisdc) was 71% and 16%, respectively. Lactobacillus plantarum, L. brevis and L. casei/paracasei, and Enterococcus faecium and Enterococcus faecalis were identified as tyramine/histamine producers in the sausages.     

屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究

为了开发谷氨酸脱羧酶(glutamate decarboxylase,GAD),以Enterococcus faecium为gadB基因供体、纤维素结合域(cellulose-binding domain,CBD)为亲和标签,利用内含肽DnaB自剪切作用分离GAD,对融合酶CBD-DnaB-GAD的构建、表达、GAD纯...     

Occurrence of biogenic amines in wine: The role of grapes

The evolution of biogenic amines from must to wine has been studied in seven different grape cultivars before and after malolactic fermentation. Alcoholic and malolactic fermentations have been carried out using selected yeasts and bacteria that, in a previous study, were unable to produce biogenic amines. The study has been performed under aseptic conditions to exclude possible interferences due to uncontrolled contaminating microorganisms present in grapes and/or in the environment. The goal of this work was to investigate the influence of grape on biogenic amines content of the wine. The results obtained showed that grape variety is related to the presence of some biogenic amines in wines and that, climatic conditions also affect the accumulation of these compounds in grapes.     

Experimental challenge of bovine mammary glands with Enterococcus faecium during early and late lactation

Mammary glands of early and late lactation cows were challenged with Enterococcus faecium of bovine origin to determine in vivo pathogenicity and milk somatic cell count (SCC) responses. A total of 20 early lactation and 18 late lactation mammary glands were challenged. Two isolates highly adaptive and 2 isolates poorly adaptive for in vitro growth in mammary secretion were used as challenge strains of bacteria. Challenged quarters of early lactation cows were more susceptible to intramammary infection caused by E. faecium than those of late lactation cows. Intramammary challenge with isolates poorly adaptive for in vitro growth in mammary secretions resulted in 94.7% of quarters infected compared with 36.8% of the quarters infused with the isolates highly adaptive for in vitro growth in mammary secretions. Milk from quarters infused with the isolates poorly adaptive for in vitro growth had higher SCC and bacterial counts compared with quarters challenged with the isolates highly adaptive for in vitro growth. A stage of lactation effect within treatment groups was measured when milk SCC were compared between early and late lactation cows. Milk SCC in uninfused (negative control) quarters were lower in early lactation cows compared with late lactation cows. Conversely, in quarters infused with isolates poorly adaptive for in vitro growth, SCC were higher in early lactation cows compared with late lactation cows on d 2, 3, 4, 15, 16, and 17 postchallenge. In quarters infused with isolates highly adaptive for in vitro growth, SCC response did not differ between early and late lactation cows. In vitro growth of E. faecium in mammary secretion was inversely related to in vivo pathogenicity in the mammary glands of early and late lactation cows.     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

Transport of glycerol by Pediococcus pentosaceus isolated from wine

Pediococcus pentosaceus N(5)p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis-Menten relationship with an observed apparent K(m) of 33 microM and a V(max) of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl(2) and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide-m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.     

葡萄酒中生物胺的产生与工艺控制

生物胺存在于多种发酵食品中，人体吸收过量的生物胺后会引起不良的生理反应。在葡萄酒苹果酸—乳酸发酵(MLF)过程中，有些乳酸菌能够对氨基波脱投产生生物胺。利用PCR与DNA探针技术能够快速检测葡萄酒中的组胺产生菌。工艺上采用接种法进行MLF，并在MLF完成后对乳酸菌进行有效清除、可以显著降低葡萄酒中生物胺的含量。     

In vitro synthesis of biogenic amines by Brochothrix thermosphacta isolates from meat and meat products and the influence of other microorganisms

Twenty Brochothrix thermosphacta strains tested for biogenic amines (BAs) production, formed histamine (6.6-16.2 mg/kg) and tyramine (18.7-35.4 mg/kg) but neither putrescine nor cadaverine. Six of the twenty strains were also investigated in respect of their influence on the synthesis of BAs by Escherichia coli, Pseudomonas sp., Proteus mirabilis and Lactobacillus sakei. In pure culture Escherichia coli produced all of the studied amines (histamine, tyramine, putrescine and cadaverine) with a total concentration of 167.7 mg/kg, P. mirabilis produced a total of 56.7 mg/kg histamine, tyramine and cadaverine, while Lactobacillus sakei and Pseudomonas sp. produced histamine and tyramine, totaling 37.9 and 35.2 mg/kg respectively. All B. thermosphacta promoted cadaverine production by Escherichia coli which increased by 12-68%, and some of them contributed to the appearance of this amine among the metabolites of Pseudomonas. The presence of B. thermosphacta decreased the potential ability of P. mirabilis to produce BAs.     

Impact of Australian Dekkera bruxellensis strains grown under oxygen-limited conditions on model wine composition and aroma

Spoilage of red wine by the yeast species Dekkera bruxellensis is a common problem for the global wine industry. When conditions are conducive for growth of these yeasts in wine, they efficiently convert non-volatile hydroxycinnamic acids into aroma-active ethylphenols, thereby reducing the quality of the wine. It has been demonstrated previously that dissolved oxygen is a key factor which stimulates D. bruxellensis growth in wine. We demonstrate that whereas the presence of oxygen accelerates the growth of this species, oxygen-limited conditions favour 4-ethylphenol production. Consequently, we evaluated wine spoilage potential of three D. bruxellensis strains (AWRI1499, AWRI1608 and AWRI1613) under oxygen-limited conditions. Each strain was cultured in a chemically-defined wine medium and the fermentation products were analysed using HPLC and HS-SPME–GC/MS. The strains displayed different growth characteristics but were equally capable of producing ethylphenols. On the other hand, significant differences were observed for 18 of the remaining 33 metabolites analysed and duo-trio sensory analysis indicated significant aroma differences between wines inoculated with AWRI1499 and AWRI1613. When these wines were spiked with low concentrations of 4-ethylphenol and 4-ethylguaiacol, no sensorial differences could be perceived. Together these data suggest that the three predominant D. bruxellensis strains previously isolated during a large survey of Australian wineries do not differ substantively in their capacity to grow in, and spoil, a model wine medium.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.