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.     

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.     

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.     

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.     

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 characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations

Presumptive lactic acid bacterial cocci were found in six sourdoughs (out of 20) from the Abruzzo region (central Italy) and subjected to phenotypic and genotypic characterization. A total of 21 isolates, recognized as seven strains by randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing, were identified by a polyphasic approach, consisting of 16S rRNA gene sequencing, multiplex PCR assays and physiological features, as Enterococcus faecium and Pediococcus pentosaceus. Four strains belonging to those species and previously isolated from wheat kernels were inoculated in sterile flour to verify their capacity to grow in sourdough environment. Doughs with several dual bacterial combinations, including Lactobacillus sanfranciscensis, were propagated for 11 days and pH measurements and bacterial counts were carried out.     

Effects of Enterococcus faecium (SF68) on immune function in mice

Probiotics exert health benefits on human and animals when administered in adequate amounts. The objective of this study was to assess the effects of Enterococcus faecium (SF68) on intestinal colonisation and immune function of BALB/c mice. Six-week-old female BALB/c mice were orally administered with E. faecium (SF68). Results showed that the total anaerobe and lactobacilli in the faeces increased (P < 0.05), while the number of faecal enterobacteria decreased (P < 0.05) in E. faecium-fed mice. Furthermore, supplementation of E. faecium (SF68) increased the percentage of double positive (DP) cells in peripheral blood, the concentration of plasma IgG, and the levels of interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-γ (IFN-γ) in splenocytes of the mice (P < 0.05). This study demonstrated that E. faecium SF68 affects the intestinal microbial flora and modulates the immune responses, which indicates a viable probiotic characteristic of E. faecium SF68 in modification of immune function.     

Technological and safety properties display biodiversity among enterococci isolated from two Egyptian cheeses, "Ras" and "Domiati"

The technological and safety properties of 35 indigenous strains of Enterococcus faecium isolated from two Egyptian cheeses were characterised in order to determine their ability for rational manufacture of diversified and typical dairy products in the Middle East. A great diversity was observed within the 35 strains on the basis of their technological properties. A statistical analysis made it possible to distribute the 35 strains of E. faecium into different groups. Three groups were identified in terms of their acidification activity, measured by the Cinac system: a group of strains that quickly acidified milk, a second that moderately acidified milk, and a last cluster that revealed weak acidification activity. On the basis of texturing properties that were evaluated using a texturometer and a viscometer, a cluster of strains produced viscous and firm gels, a second cluster included fairly viscous, firm and cohesive gels, and a last group generated slightly viscous, but firm and very cohesive gels. By considering the aroma profiles that were determined by gas chromatography coupled with mass spectrometry, four clusters were identified. One cluster displayed a high dimethyl disulfide level, a second group of strains was highly aromatic, a third cluster led to typical “lactic” products, and the last cluster made it possible to obtain low aromatic products. None of the 35 strains proved to be β-haemolytic on the basis of the characterisation of their safety properties. The resistance to 20 antibiotics was assessed by the disc diffusion method. The 35 isolates were sensitive to 12 antibiotics, and among them, one isolate was resistant to only two antibiotics (nalixidic acid and streptomycin). The resistance to eight antibiotics was strain-dependant. Finally, this study demonstrates that some indigenous strains of E. faecium displayed interesting technological properties for cheese manufacture, together with good safety characteristics. They could be useful for the manufacture of typical products in Egypt.     

Contributions to selected phenotypic characteristics of large species- and lineage-specific genomic regions in Listeria monocytogenes

We hypothesized that genomic regions specific to Listeria monocytogenes or selected L. monocytogenes strains may contribute to virulence and phenotypic differences among the strains. A whole genome alignment of two completed L. monocytogenes genomes and the one completed Listeria innocua genome initially identified 28 genomic regions of difference (RD) > 4 kb that were found in one or both L. monocytogenes genomes, but absent from the non-pathogenic L. innocua. In silico analyses using an additional 18 draft L. monocytogenes genomes showed that (i) 15 RDs were found in all or most L. monocytogenes genomes; (ii) three RDs were found in all or most lineage I genomes, but absent from lineage II genomes; and (iii) four RDs were found in all lineage II genomes, but no lineage I genomes. Null mutants in two L. monocytogenes-specific RDs (RD16 and RD30; found in most L. monocytogenes) and the lineage II-specific RD25 showed no evidence for impaired invasion or intracellular growth in selected tissue culture cells. Although, in pH 5.5 minimal media, the ΔRD30 null mutant showed reduced ability to compete with its parent strain, indicating that RD30 may have a role in L. monocytogenes growth under limited nutrient conditions at acidic pH.     

Effects of organic acids on thermal inactivation of acid and cold stressed Enterococcus faecium

In this study the adaptative response to heat (70 °C) of Enterococcus faecium using fresh and refrigerated (at 4 °C for up to 1 month) stationary phase cells grown in Brain Heart Infusion (BHI) buffered at pH 7.4 (non-acid-adapted cells) and acidified BHI at pH values of 6.4 and 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells) was evaluated. In all cases, the survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetic. The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, whereas the subsequent cold storage of cells reduced E. faecium thermal tolerance. Fresh acid-adapted cells showed t_2.5-values (time needed to obtain an inactivation level of 2.5 log₁₀ cycles) ranging from 2.57 to 9.51 min, while non-acid-adapted cells showed t_2.5-values of 1.92 min. The extent of increased heat tolerance varied with the acid examined, resulting in the following order: citric ≥ acetic > malic ≥ lactic > hydrochloric ≥ ascorbic. In contrast, cold storage progressively decreased E. faecium thermal resistance. The t_2.5 values found at the end of the period studied were about 2–3-fold lower than those corresponding to non-refrigerated cells, although this decrease was more marked (about 5-fold) when cells were grown in buffered BHI and BHI acidified at pH 5.4 with hydrochloric acid. These findings highlight the need for a better understanding of microbial response to various preservation stresses in order to increase the efficiency of thermal processes and to indicate the convenience of counterbalancing the benefits of the hurdle concept.     

Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10 °C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.     

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.     

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

The effects of temperature,chlorine and acids on the survival of Listeria and Salmonella strains associated with uncooked shrimp carapace and cooked shrimp flesh

The purpose of this study was to assess the influence of the association of Listeria and Salmonella with shrimp surfaces on the effects of temperature, chlorine and acids on their survival. Planktonic, attached and colonized cells of Listeria monocytogenes Scott A, L. monocytogenes V7, Salmonella Senftenberg 1734b and S. Typhimurium ATCC 14028 were challenged with high (50°, 60° and 70 °C) and low (4 °C) temperature, 100 ppm sodium hypochlorite solution, and acetic, hydrochloric and lactic acids (pH 4.0). Attached and colonized Listeria and Salmonella showed significantly greater (p < 0.05) resistance to heat (∼1.3–2.6 fold increase in D-values), hypochlorite (∼6.6 ≥ 40.0 fold) and acids (∼4.0–9.0 fold) than their planktonic counterparts. There were no significant differences (p > 0.05) in the survival of planktonic, attached or colonized cells of Listeria and Salmonella stored under refrigerated conditions. The association of Listeria and Salmonella with shrimp surfaces enhances their resistance to heat, chlorine and acids. Both attachment to, and subsequent colonization of, shrimp surfaces by pathogens may reduce the efficacy of methods used in their control. Strategies to reduce attachment of these pathogens to shrimp are required to assure safety of this product.     

Assessment of the anti-listerial activity of microfloras from the surface of smear-ripened cheeses

The anti-listerial activity of microfloras from the surface of various smear-ripened cheeses was evaluated using four methods that were then compared. Method A measured the anti-listerial potential of supernatants from short-time liquid cultures, whereas in Method B, a model cheese was co-inoculated with the microflora and Listeria innocua test strains. Method C was based on successive propagations of the microfloras on this model cheese, and Method D on successive propagations of the microfloras together with Listeria test strains. Anti-listerial activity considerably depended on the microflora used. Significant correlations were obtained between Methods A and B and Methods C and D. With Methods C and D, the highest anti-listerial activity was obtained with the microflora from a Livarot-type cheese (FC12). To investigate the cause of the anti-listerial activity of FC12, Fourier Transform InfraRed (FTIR) analyses of microbial populations were performed on the original microflora as well as on the microflora after propagations on the model cheese. The composition of FC12 had changed considerably upon propagation, and in the propagated microflora, the population of yeasts was dominated by Yarrowia lipolytica strains, whereas the population of bacteria was dominated by Vagococcus species.     

Chemical composition,antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone

We investigate in this work the chemical composition by GC–EIMS, the antibacterial and the cytotoxic activities of Tunisian Nigella sativa essential oil and its bioactive compound, thymoquinone, were tested against various clinical cariogenic bacteria (n = 30). Eighty-four compounds were identified in the essential oil. The major one was p-cymene (49.48%) whereas thymoquinone represented only 0.79%. The essential oil (2.43 mg/disc) containing only 3.35 μg of thymoquinone showed pronounced dose dependant antibacterial activity against Streptococcus mitis, Streptococcus mutans, Streptococcus constellatus and Gemella haemolysans (15.5 ± 0.707 mm). However, pure thymoquinone compound (150 μg/disk) was active against all the studied strains especially S. mutans and S. mitis (24.5 ± 0.71 and 22 ± 1.41 mm inhibition zones, respectively).     

Effects of nisin, EDTA and salts of organic acids on Listeria monocytogenes, Salmonella and native microflora on fresh vacuum packaged shrimps stored at 4 °C

Nisin (500 IU ml⁻¹), EDTA (0.02 M), potassium sorbate (PS) (3%, w/v), sodium benzoate (SB) (3%, w/v) or sodium diacetate (SD) (3%, w/v); alone or in combination were used to dip uninoculated shrimps and shrimps inoculated with Listeria monocytogenes or Salmonella (∼4.0–5.0 log CFU g⁻¹). Shrimps were then drip-dried, vacuum packaged and stored at 4 °C for 7 days. Untreated shrimps were used as a control. Numbers of L. monocytogenes, Salmonella and native background microflora were determined on uninoculated and inoculated shrimps on days 0, 3 and 7. Nisin–EDTA–PS and nisin–EDTA–SD significantly reduced (p < 0.05) L. monocytogenes numbers by 1.07–1.27 and 1.32–1.36 log CFU g⁻¹, respectively, on day 0 and 3. However, all treatments failed to significantly reduce (p > 0.05) Salmonella counts on shrimps throughout storage. On day 7, numbers of aerobic bacteria, psychrotrophic bacteria and Pseudomonas on combined nisin–EDTA–salt of organic acids treated shrimps were significantly lower (p < 0.05) by 4.40–4.60, 3.50–4.01, and 3.84–3.99 log CFU g⁻¹ respectively, as compared to the control. Dipping in organic acids solutions followed by vacuum packaging and chilled storage can help reduce L. monocytogenes and native microflora, but not Salmonella, on fresh shrimps.     

Cold atmospheric pressure plasma treatment of ready-to-eat meat: inactivation of Listeria innocua and changes in product quality

The application of cold atmospheric pressure plasma for decontamination of a sliced ready-to-eat (RTE) meat product (bresaola) inoculated with Listeria innocua was investigated. Inoculated samples were treated at 15.5, 31, and 62 W for 2–60 s inside sealed linear-low-density-polyethylene bags containing 30% oxygen and 70% argon. Treatments resulted in a reduction of L. innocua ranging from 0.8 ± 0.4 to 1.6 ± 0.5 log cfu/g with no significant effects of time and intensity while multiple treatments at 15.5 and 62 W of 20 s with a 10 min interval increased reduction of L. innocua with increasing number of treatments. Concentrations of thiobarbituric acid reactive substances (TBARS) increased with power, treatments and storage time and were significantly higher than those of control samples after 1 and 14 days of storage at 5 °C. However, the levels were low (from 0.1 to 0.4 mg/kg) and beneath the sensory threshold level. Surface colour changes included loss of redness of ∼40% and 70% after 1 and 14 days of storage, respectively, regardless of plasma treatment. The results indicate that plasma may be applicable in surface decontamination of pre-packed RTE food products. However, oxidation may constitute an issue in some products.     

Chemical basis of anti-listerial effects of rosemary herb during stomaching with fresh-cut vegetables

Fresh rosemary herb has demonstrated much stronger anti-listerial effects when stomached with contaminated fresh-cut vegetables than those obtained by application of rosemary essential oils. Various types of rosemary extracts (hydrodistillates, CO₂ extracts, hexane/acetone extracts) were analysed by GC–MS and tested for anti-listerial effects in vitro. Principal component analysis showed that verbenone levels were most correlated with anti-listerial effects, followed by levels of camphor. Stomaching rosemary herb greatly increased headspace levels of verbenone and camphor (by up to 20 times) compared to intact or chopped fresh rosemary. It is speculated that by crushing the herb under water, stomaching released enhanced levels of these components as lipophilic nanosomes which rapidly migrated to lipophilic surfaces, including the membranes of Listeria cells.