Identification of a capsular polysaccharide from Enterococcus faecium U0317 using a targeted approach to discover immunogenic carbohydrates for vaccine development.

Enterococcus faecium, a gram-positive opportunistic pathogen, has become a major concern for nosocomial infections due to its resistance to several antibiotics, including vancomycin. Finding novel alternatives for treatment prevention, such as vaccines, is therefore crucial. In this study, we used various techniques to discover a novel capsular polysaccharide. Firstly, we identified an encapsulated E. faecium strain by evaluating the opsonophagocytic activity of fifteen strains with antibodies targeting the well-known lipoteichoic acid antigen. This activity was attributed to an unknown polysaccharide. We then prepared a crude cell wall glycopolymer and fractionated it, guided by immunodot-blot analysis. The most immunoreactive fractions were used for opsonophagocytic inhibition assays. The fraction containing the inhibitory polysaccharide underwent structural characterization using NMR and chemical analyses. The elucidated structure presents a branched repeating unit, with the linear part being: →)-ß-d-Gal-(1 â†’ 4)-ß-d-Glc-(1 â†’ 4)-ß-d-Gal-(1 â†’ 4)-ß-d-GlcNAc-(1→, further decorated with a terminal α-d-Glc and a d-phosphoglycerol moiety, attached to O-2 and O-3 of the 4-linked Gal unit, respectively. This polysaccharide was conjugated to BSA and the synthetic glycoprotein used to immunize mice. The resulting sera exhibited good opsonic activity, suggesting its potential as a vaccine antigen. In conclusion, our effector-function-based approach successfully identified an immunogenic capsular polysaccharide with promising applications in immunotherapy.

Untargeted metabolomic analysis reveals the mechanism of Enterococcus faecium agent induced CaCO3 scale inhibition.

In this study, a lactic acid bacterium, Enterococcus faecium, was found to prevent CaCO3 precipitation through its metabolism. On analysis of all stages of E. faecium growth, static jar tests demonstrated that stationary phase E. faecium broth possessed the highest inhibition efficiency of 97.3% at a 0.4% inoculation dosage, followed by the decline and log phases with efficiencies of 90.03% and 76.07%, respectively. Biomineralization experiments indicated that E. faecium fermented the substrate to produce organic acid, which resulted in modulation of the pH and alkalinity of the environment and thus inhibited CaCO3 precipitation. Surface characterization techniques indicated that the CaCO3 crystals precipitated by the E. faecium broth tended to be significantly distorted and formed other organogenic calcite crystals. The scale inhibition mechanisms were revealed by untargeted metabolomic analysis on log and stationary phase E. faecium broth. In total, 264 metabolites were detected, 28 of which were differential metabolites (VIP ≥ 1 and p < 0.05). Of these, 15 metabolites were upregulated in stationary phase broth, and 13 metabolites were downregulated in log phase broth. Metabolic pathway analysis suggested that improved glycolysis and the TCA cycle were the main reasons for enhancement of the antiscaling performance of E. faecium broth. These findings have significant implications for microbial metabolism-induced CaCO3 scale inhibition.

Potential of Enterococcus faecium LM5.2 for lipopeptide biosurfactant production and its effect on the growth of maize (Zea mays L.).

This study characterized the lipopeptide biosurfactants produced by lactic acid bacteria isolated from milk and milk products, in addition to their effect on maize plant growth. The oil displacement test was performed as a primary screening method to select the BS producing bacteria. The strain LM5.2 had the maximum emulsification index of 45.1 ± 3, reduced the surface tension to 32.98 ± 0.23% among all the isolates, and efficiently produced 945.26 ± 4.62 mg/l of biosurfactants within 48 h in MRS broth under the optimum conditions. The isolate LM5.2 was identified using physiochemical tests and 16S rRNA gene sequencing as E. faecium. The biosurfactant was purified by TLC and identified as lipopeptide-like iturins and surfactins based on Rf values. Mass spectroscopy, NMR, and FTIR analysis also confirmed the biosurfactant's identity as the derivatives of iturin and surfactin. In vitro biosurfactant application significantly enhanced seedling growth at an optimal concentration of 450 µg/ml. Plant treatment (pot experiment) with E. faecium LM5.2 significantly promoted Zea mays growth compared to the control. To the best of our knowledge, this is the first report of lipopeptide biosurfactant production by E. faecium. Moreover, the study also showed that the biosurfactant and biosurfactant-producing E. faecium LM5.2 could be an eco-friendly plant growth-promoting agent.

Optimization of cultivation strategy and medium for bacteriocin activity of Enterococcus faecium HDX-2.

A lactic acid bacteria (LAB) isolated from pickled Chinese cucumber was screened for bacteriocin production. The strain was identified to be Enterococcus faecium HDX-2. Based on the Plackett-Burman (PB) experiment, glucose, Ca2+, and initial pH were found to be the most significant parameters of bacteriocin production. Afterward, effects of the three main parameters on bacteriocin activity were further investigated by central composite design (CCD) and the optimum composition was glucose 22 g/L, Ca2+ 0.6 mM, and initial pH 7.2. Optimum results showed that bacteriocin activity was increased to 1337.60 ± 36.71 AU/mL, 2.23-fold higher than in MRS medium without parameters optimization. The bacteriocin also showed significant antimicrobial activity against Listeria monocytogenes in meat and cheese model system.

Effect of enterocins against methicillin-resistant animal-derived staphylococci.

The occurence and spread of animal-derived methicillin resistant staphylococci (MRS) worldwide is a current problem, especially due to their increasing incidence in food animals and their products, with possible contamination of food consumers and handlers. Staphylococci isolated from animals (n = 123) were identified with MALDI-TOF mass spectrometry and screened for methicillin/oxacillin/cefoxitin resistance (MR) using the disk diffusion method. Twenty-three phenotypically MRS strains were analysed using PBP2' Latex Agglutination Test Kit to confirm the phenotypic MR and PCR was performed for mecA gene detection; mecA gene positive strains were furtherly confirmed by means of sequencing. The susceptibility of MRS to 11 partially-purified enterocins (Ent) produced by E. faecium, E. durans and E. mundtii strains of animal, feed/food and environmental origin was checked using agar spot tests. Out of 23 MRS, PBP testing confirmed MR in 17 strains. Three Staphylococcus epidermidis and one S. vitulinus were mecA positive. The majority of MRS, including two mecA gene-positive strains S. epidermidis R44/1 and P3/Tr2a, were susceptible to the tested enterocins, mainly to Ent7420, EntA(P)/EK13, Ent412, Ent55 and Ent9296 (in the range 100 - 12,800 AU/mL). The most susceptible strains appeared to be the mecA gene-positive S. epidermidis SE R44/1 and SE P3/Tr2a strains, inhibited by eight enterocins out of 11 tested (100-200 AU/mL). Only four strains (including mecA gene positive S. epidermidis SE P3/Tr1 and S. vitulinus SV K12PL/1) were resistant to the tested antimicrobial substances. These results indicate that the enterocins used offer a promising option for prevention and treatment of bacterial infection caused by MRS in animals.

Physical and antibacterial properties of bacterial cellulose films supplemented with cell-free supernatant enterocin-producing Enterococcus faecium TJUQ1.

In this study, a composite film was prepared with bacterial cellulose (BC) of Gluconacetobacter xylinus and cell-free supernatant (CFS) of Enterococcus faecium TJUQ1, which was named BC-E. The optimum conditions for the preparation of the composite film with a minimal antibacterial activity were the soak of BC in 80 AU/mL CFS for 6 h. By scanning electron microscope observation, the surface network structure of BC-E was denser than that of BC. The tensile strength of BC and BC-E was 4.65 ± 0.88 MPa and 16.30 ± 0.92 MPa, the elongation at break of BC and BC-E was 3.33 ± 0.89% and 31.60 ± 1.15%, respectively, indicating the mechanical properties of BC-E were significantly higher than that of BC (P < 0.05). The swelling ratio of BC-E (456.67 ± 7.20%) was lower than that of BC (1377.78 ± 9.07%), demonstrating BC-E films presented better water resistance. BC-E films were soaked with 320 AU/mL CFS, and then used to pack the ground meat with 6.55 log10 CFU/g of Listeria monocytogenes. After 8 days of storage, the number of bacteria decreased by 3.16 log10 CFU/g. Similarly, total mesophilic bacterial levels in the ground meat decreased by 2.41 log10 CFU/g compared to control groups.

Anticancer and antimicrobial potential of enterocin 12a from Enterococcus faecium.

BACKGROUND: Increase in the number of infections caused by Gram-negative bacteria in neutropenic cancer patients has prompted the search for novel therapeutic agents having dual anticancer and antimicrobial properties. Bacteriocins are cationic proteins of prokaryotic origin that have emerged as one of the most promising alternative antimicrobial agents with applications as food preservatives and therapeutic agents. Apart from their antimicrobial activities, bacteriocins are also being explored for their anticancer potential. RESULTS: In this study, a broad-spectrum, cell membrane-permeabilizing enterocin with a molecular weight of 65 kDa was purified and characterized from the culture supernatant of vaginal Enterococcus faecium 12a. Enterocin 12a inhibited multidrug-resistant strains of various Gram-negative pathogens such as Salmonella enterica, Shigella flexneri, Vibrio cholerae, Escherichia coli and Gram-positive, Listeria monocytogenes, but had no activities against different strains of gut lactobacilli. The mass spectrometric analysis showed that the enterocin 12a shared partial homology with 4Fe-4S domain-containing redox protein of E. faecalis R712. Further, enterocin 12a selectively inhibited the proliferation of various human cancer cell lines in a dose-dependent manner but not that of normal human peripheral blood mononuclear cells. Enterocin 12a-treated cancer cells showed apoptosis-like morphological changes. CONCLUSION: Enterocin 12a is a novel bacteriocin that has anticancer properties against human cell lines and negligible activity towards non-malignant cells. Therefore, it should be further evaluated for its anticancer potential in animal models.

Thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 on dried basil leaves.

The enhanced heat resistance of Salmonella developed at low water activity makes it a serious challenge to eliminate them during thermal processing. The objectives of this research are to (i) investigate the effect of water activity on thermal inactivation of Salmonella cocktail (Agona, Tennessee, Mbandaka, Montevideo, and Reading) in dried basil leaves, and (ii) evaluate Enterococcus faecium NRRL B-2354 as an appropriate surrogate for Salmonella in dried basil leaves. Dried basil leaves, inoculated with a Salmonella cocktail and E. faecium separately, were equilibrated to different water activities (aw: 0.40, 0.55, and 0.70) in a humidity-controlled chamber. The basil samples were packed (1.6 ± 0.1 g) in aluminum pouches and thermally treated at 70, 75, and 80 °C using a dry heating method for 0-180 min to obtain the thermal death curve. The microbial survival data was fit using two primary models (Log-linear and Weibull model). Results from AICc showed that the log-linear model fits well for thermal inactivation of both microorganisms. As the aw decreases from 0.70 to 0.40 at 75 °C, the D-value increases from 3.30 to 9.14 min for Salmonella and 6.53 to 14.07 min for E. faecium. Based on the AICc values, the modified Bigelow model fits the D-values better than the response surface model for both the microorganisms. The kill ratio of surrogate to pathogen ranged from 1.4 to 2.8, indicating that it is a conservative surrogate for Salmonella for performing validation of the thermal pasteurization process. The identification of suitable surrogate and development of modified Bigelow model will help the spice industry in developing the thermal processes for improving the safety of basil leaves.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

In ovo inoculation of an Enterococcus faecium-based product to enhance broiler hatchability, live performance, and intestinal morphology.

Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium-based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 µL of Marek's vaccine (MV), 2) MV and 1.4 × 105 cfu GH/50 µL, 3) MV and 1.4 × 106 cfu GH/50 µL, 4) MV and 1.4 × 107 cfu GH/50 µL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 105 and 107 GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 107 GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 107 GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 105 and 107 GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study.

Structural analysis of a novel substrate-free form of the aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6')-Ii enzymes target the amino group linked to the 6' C atom in an aminoglycoside. Several structures of the AAC(6')-Ii from Enterococcus faecium [Ef-AAC(6')-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6')-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6')-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6')-Ii.

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses.

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

Enterococcus faecium L-15 Extract Enhances the Self-Renewal and Proliferation of Mouse Skin-Derived Precursor Cells.

Lactic acid bacteria (LAB) in the gastrointestinal tract have beneficial health effects. LAB activate the proliferation of intestinal stem cells and speed the recovery of damaged intestinal cells, but little is known about effect of LAB on other adult stem cells. In this study, a cell-free extract of Enterococcus faecium L-15 (L15) was exposed to mouse skin-derived precursor cells (SKPs), and the changes in characteristics associated with proliferation and self-renewal capacity were investigated. L15 increased the size of the spheres and the proliferation rate of SKPs. Cell cycle analysis revealed that cells in the S-phase increased after treatment with L15. In the L15-treated group, the total number of spheres significantly increased. The expression level of pluripotency marker genes also increased, while the mesenchymal lineage-related differentiation marker genes significantly decreased in the L15-treated group. The PI3K/Akt signaling pathway was activated by L15 in SKPs. These results indicate that L15 enhances proliferation and self-renewal of SKPs and may be used as a supplement for stem cell maintenance or application of stem cell therapy. This is the first report to investigate the functional effects of E. faecium on the proliferation and self-renewal capacity of SKPs.

Comparative study of host-associated and commercial probiotic effects on serum and mucosal immune parameters, intestinal microbiota, digestive enzymes activity and growth performance of roach (Rutilus rutilus caspicus) fingerlings.

The study aimed to isolate host-associated probiotic (HAP) lactic acid bacteria from intestine of adult Caspian roach and compare the efficacy of HAP with a commercially available probiotic strain (Pediococcus acidilactici) on the growth and feed utilisation, digestive enzymes and systemic and mucosal immune system of roach fingerling. The HAP strain isolated from roach intestine was Enterococcus faecium strain CGMCC1.2136. The experiment was a simple completely randomized design and lasted for eight weeks. Two hundred and seventy fish with an average weight of 12 g randomly distributed into nine tanks. The trial consisted of three treatments with three respective replications. During the experimental period, fish received basal diet without any bacterial supplementation (as the control group), basal diet enriched with 108 CFU g-1 HAP or 107 CFU g-1 CP. At the end of the experiment, serum immune parameters of those fish fed HAP including alkaline phosphatase activity, total protein content, total immunoglobulin level, lysozyme activity and complement activity (ACH50) were significantly higher that other experimental groups (P < 0.05). Similarly, dietary supplementation of HAP resulted in better mucosal immune parameters in comparison to control group and commercial probiotic administration (P < 0.05). Intestinal heterotrophic bacteria and autochthonous LAB counts of those fish fed HAP were significantly higher than other experimental groups at the end of the experiment as well as 15 days seizing probiotic administrations (P < 0.05). Fish fed with HAP containing diet presented significantly higher amylase, lipase and protease activity in comparison to the CP fed fish and the control group (P < 0.05). Growth indices of those fish fed HAP were significantly higher than other treatments (P < 0.05). The highest carcass protein and ash content along with the lowest body moisture content belonged to those fish received HAP (P < 0.05). In conclusion, the use host-HAP resulted in better immune competence and growth performance and it seems aquaculture sector should probably focus on the development of probiotics isolated from the cultured species instead of using terrestrial probiotics with greatly different requirements and environmental conditions.

Enterococcus faecium supplementation in sows during gestation and lactation improves the performance of sucking piglets.

The aim of this study was to evaluate the effects of Enterococcus faecium DSM 7,134 supplementation on the performance of sows and their litters. A total of 15 primiparous sows (Landrace × Yorkshire) were randomly divided into three treatments with five replicates. Dietary treatments were: CON, basal diet; E1, CON + 0.025% E. faecium; E2, CON + 0.05% E. faecium. No significant differences were observed on body weight and feed intake of lactating sows with E. faecium supplementation, but linearly increased the sow apparent total tract digestibility (ATTD) of dry matter (DM), nitrogen (N) and gross energy (GE; p < .05), and decreased piglets pre-weaning mortality (p < .05). Piglets from E. faecium-supplemented sows linearly increased weaning weight, average daily gain (ADG) and gain:feed ratio (p < .05), as well as linearly decreased diarrhoea score (p < .05) in the first weaning week. Piglets from E. faecium-supplemented sows linearly increased faecal Lactobacillus and Enterococci counts (p < .05), while linearly decreased faecal Escherichia coli counts (p < .05) after weaning. In conclusion, dietary supplementation of E. faecium improved the ATTD of DM, N and GE in lactating sows, as well as improved body weight, ADG and shifted faecal microbiota in their litters.

[Evaluation of anti-candida activity of metabolites of enterococcal clinical isolates.]

When studying the effect of the metabolic products of clinical isolates of enterococci on the viability of Candida albicans, it was found that metabolites of all tested strains of Enterococcus faecium, E. faecalis had a fungistatic effect. At the same time a reliable fungicidal effect is a strain-specific feature. It is better to use the method of delayed antagonism on double-layer agar to assess the antifungal effect of enterococcal metabolism products.

Detection and characterization of bacterial polysaccharides in drug-resistant enterococci.

Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.

The impact of dietary supplementation of different feed additives on performances of broiler breeders characterized by different egg-laying rate.

This study was conducted to determine the impact of different feed additives on reproductive performance, egg quality, intestinal morphology, and blood metabolic profile of broiler breeder with different egg-laying rate. A total of 512 AA broiler breeders (48 wk old) were used in a 2 × 4 factorial design which encompassed 2 egg-laying rate levels [average (AR) and low (LR)] and 4 different dietary groups [control (no additive), 6 × 108 CFU/kg Enterococcus faecium (EF), 200 mg/kg apple pectic oligosaccharide (APO), and 1,000 mg/kg tributyrin (TRI)]. As expected, the LR breeders presented higher egg weight, eggshell thickness (P < 0.05), and feed conversion ratio as well as lower egg-laying and qualified egg rate than the AR breeders (P < 0.01). Dietary supplementation with the 3 additives improved egg weight (P ≤ 0.01). Dietary APO addition improved albumen height and Haugh units (P < 0.05) in both AR and LR breeders. Compared with APO and TRI, dietary EF addition increased eggshell thickness (P ≤ 0.01). An effect of the egg-laying rate and dietary additives on eggshell thickness (P < 0.01) was noted, with the addition of EF enhancing the eggshell thickness, which is more pronounced in the AR group. The duodenum of AR breeders presented a lower crypt depth and a higher villus/crypt ratio (P < 0.05); moreover, an effect of the laying rate and dietary additives on crypt depth was noted (P < 0.05), with the addition of APO to the diet resulting in a lower crypt depth. Compared with the APO and TRI, dietary EF addition increased follicle-stimulating hormone (FSH) level in serum (P < 0.05). Overall, the results gathered in this study indicate that LR breeders have lower production performance, eggshell thickness and decreased gastrointestinal tract functionality in compared with the AR breeders. Dietary supplementation with APO might improve albumen quality and decrease duodenal morphology, while EF improved eggshell quality and FSH secretion, and the improvement was more pronounced in the breeders with an average egg-laying rate.

Enterocin BacFL31 from a Safety Enterococcus faecium FL31: Natural Preservative Agent Used Alone and in Combination with Aqueous Peel Onion (Allium cepa) Extract in Ground Beef Meat Storage.

Safety aspects and probiotic properties of Enterococcus faecium FL31 strain producing an enterocin, named BacFL31 were previously demonstrated. Taking into account its originality, the enterocin BacFL31 was added alone at 200 AU/g or in combination with the aqueous peel onion (Allium cepa) extract (APOE) at 1.56 ± 0.3 mg/mL to ground beef meat. Its biopreservative effect was evaluated by microbiological, physicochemical and sensory analyses during 14 days at 4°C. The APOE was characterized for its phytochemical content: total phenolic (TPC), flavonoids (TFC) and tannins contents (TAC), its antioxidant capacity using the in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) and its antilisterial activity. APOE had a high TPC, TFC and TAC respectively with 140 ± 2.05 (mg GAE/g), 35 ± 0.5 (mg QE/g) and 20.6 ± 1.4 (mg CE/g). Equally, APOE showed a potential radical scavenging activity compared to the butylated hydroxytoluene (BHT), with an anti-radical power (ARP) of 46 ± 1.5. During 14 days of storage at 4°C, the combination between APOE and BacFL31 limited the microbial deterioration (P < 0.05), led to a decrease in thiobarbituric acid reactive substances (TBARS) values and slowed down the metmyoglobin (MetMb) and carbonyl group accumulation and delayed the disappearance of sulfphydryl proteins (P < 0.05). The combination was also efficient (P < 0.05) against microflora proliferation, decreased primary and secondary lipid oxidation (P < 0.05), reduced protein oxidation and enhanced significantly (P < 0.05) the sensory attributes. Thus, the enterocin BacFL31 use from a safe Enterococcus faecium combined with APOE as a potential natural preservative to biocontrol ground beef was promising as it was effective at low concentration. The data lay bases for new tests to be carried out in other food matrices.

Virulence profiles of vancomycin-resistant enterococci isolated from surface and ground water utilized by humans in the North West Province, South Africa: a public health perspective.

Vancomycin-resistant enterococci (VRE) have been responsible for numerous outbreaks of serious infections in humans worldwide. Enterococcus faecium and Enterococcus faecalis are the principal species that are frequently associated with vancomycin resistance determinants, thus usually implicated in hospital- and community-acquired infections in humans. The study aim was to determine the antibiotic resistance and virulence profiles of VREs isolated from surface and groundwater samples that are used by humans in the North West Province, South Africa. A total of 170 water samples were collected and analyzed. Eighty-one potential isolates were screened for characteristics of Enterococcus species using preliminary biochemical tests, PCR assays and sequence analysis. The antimicrobial resistance profiles of the isolates against nine antibiotics were determined and a dendrogram was generated to access the relatedness of the isolates. The isolates were screened for the presence of antibiotic resistance and virulence genes by multiplex PCR analysis. A total of 56 isolates were confirmed as Enterococcus species and the proportion of E. faecium (46.9%) was higher than E. faecalis (29%) and E. saccharolyticus (1.2%). Sequence data of E. faecium, E. faecalis, and E. saccharolyticus isolates revealed 97 to 98% similarities to clinical strains deposited in NCBI Genbank. Large proportions (44; 78.6%) of the isolates were resistant to vancomycin while 16 and 3.6% of the isolates possessed the vanA and vanB genes respectively. The MAR phenotype Vancomycin-Nalidixic Acid-Streptomycin-Chloramphenicol-Ampicillin-Oxytetracycline-Gentamycin-Nitrofurantoin-Sulphamethoxazole indicated that some isolates were resistant to all of the nine antibiotics tested. Cluster analysis of antibiotic resistance data revealed two major clusters. Sixteen (36.4%), 14 (27.3%), 3 (6.8%), and 2 (4.5%) of the VRE isolates possessed the gel, asa1, hyl, and esp virulence genes respectively while the cylA gene was not detected in the study. Multiple antibiotic-resistant enterococci were also resistant to vancomycin and possessed virulence determinants indicating that they can pose severe public health complications on individuals who consume contaminated water.