The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using ¹H, ¹³C, and ³¹P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→     

Comparison of the effects of Enterococcus faecium and aromatic oils from sage and oregano on growth performance and diarrhoeal diseases of weaned pigs

The influence of a probiotic additive and aromatic oils from medicinal and spicy plants on the basic production parameters and on the incidence of intestinal disorders of weaned pigs (weaning at the age of 30–32 days) was compared in two trials in a commercial pig fattening farm (Michalovce, Slovakia). In the first trial, an experimental (n _ex = 38) and a control group (n _co = 32) were used. A commercial probiotic additive containing Enterococcus faecium M-74 (6 × 10⁸ CFU/g) and IgY antibodies (920 mg/g dried activated egg matter) against pig Rotavirus and different strains of Escherichia coli (K88, K99, 987P, F18) were added to feed mixture of the experimental group from 30^th to 40^th day (12 × 10⁸ CFU/g) and subsequently from 40^th to 50^th day (6 × 10⁸ CFU/g) of age. The second trial was performed with three groups of pigs (n _ex1 = 28, n _ex2 = 24, n _co = 28). Aromatic oils isolated from sage (Salvia officinalis L., Labiateae) or oregano (Origanum vulgare L., Lamiaceae) were added to feed mixtures of the 1^st (90 mL/100 kg) and 2^nd experimental group (35 mL/100 kg), respectively, from 32^th to 53^th day of age. A statisticaly non-significant decrease of the incidence of diarrhoeal diseases was observed in the experimental groups of both trials as follows: in the 1^st trial by 43.41%, whereas in the 2^nd trial by 20.03% (1^st exp. group) and 6.72% (2^nd exp. group) in comparison with the control groups. The daily weight gains increased in the 1^st trial by 42.95 g/day, while in the 2^nd trial by 69.52 g/day (1^st exp. group) and 105.23 g/day (2^nd exp. group; p < 0.05) compared with controls. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Selenium enhances the antimutagenic activity of probiotic bacterium Enterococcus faecium M-74

The antibacterial activity of the probiotic bacterium Enterococcus faecium M-74 was assessed on De Man–Rogosa–Sharpe (MRS), Todd–Hewitt (T–H), M17 (M-17) and brain heart infusion (BHI) media with sodium selenite pentahydrate (+Se) and without sodium selenite pentahydrate (–Se) under aerobic or anaerobic conditions against nine bacterial pathogens. The highest antibacterial activity was found to be in the MRS medium under anaerobic conditions. There were no differences in the antibacterial activity between MRS(+Se) and MRS(–Se) media. The antimutagenic activity of MRS(+Se) and MRS(–Se) extracts after culture with E. faecium M-74 as well as of live and killed cells of E. faecium M-74 grown in the presence or absence of Se against the genotoxicity of ofloxacin (OFL) and acridine orange (AO) was determined in the Euglena gracilis assay. The MRS(+Se) extracts showed a significantly higher activity in reducing the genotoxicity of OFL and AO than MRS(–Se) extracts. The live cells of the probiotic strain M-74 exhibited higher antimutagenic activity than the killed bacterial cells, but differed depending on the mutagen used. However, the live bacterial cells grown in the presence of Se showed significantly higher antimutagenic activity. These results suggest a potential benefit for the future development of new Se-enriched probiotics exhibiting higher antimutagenic properties.     

Bacteriocin-producing strain of Enterococcus faecium EK 13 with probiotic character and its application in the digestive tract of rabbits

Enterococcus faecium EK 13 is a bacteriocin-enterocin A producing strain with probiotic properties. In this study its colonization, stability and effect on microflora in rabbits was studied as well as its influence on zootechnical parameters. Fifty rabbits of both sexes (HYPLUS, 30-day old; after weaning) were divided into control (CG) and experimental (EG) groups. They were fed a standard diet. Moreover, 25 rabbits in EG were fed daily (for 4 weeks) 15 g (separate doses ∼1.6 g) of lyophilized EK13 strain (rifampicin resistant variant — rif^R; 10⁹ cfu/g) dissolved in drinking water. After cessation of EK13 (rif^R) strain application, the rabbits in both groups were fed a standard diet for the next 2 weeks. Sampling was performed in double on day 0 (at the beginning of experiment), weekly during EK13 (rif^R) strain application as well as on week 1 and 2 after cessation of EK13 (rif^R) strain application. The counts of EK13 (rif^R) strain reached 7.1 ± 2.6 log₁₀ cfu/g after 4 weeks and even on week 2 after its cessation the counts 5.6 ± 2.3 log₁₀ cfu/g were determined. The total counts of enterococci in the rabbits were already increased in EG comparing with CG (p < 0.05); even 2 weeks after EK13 (rif^R) strain cessation, their counts in EG were 7.2 ± 2.6 log₁₀ cfu/g (p < 0.001). Enterococci in CG reached at the same time the value 3.7 ± 2.6 log₁₀ cfu/g. The counts of E. coli were significantly reduced in EG during 4 weeks (p < 0.05, p < 0.001). Even 2 weeks after EK13 (rif^R) strain cessation significant difference in E. coli counts between CG and EG was detected (p < 0.001). Enterobacteria in EG were significantly reduced (p < 0.001). Average daily gain in EG was 41.0 ± 3.83 in comparison to CG (40.6 ± 3.72); it means almost the same; although rabbits in EG showed higher feed intake per kg of gain than rabbits in CG. Preliminary results demonstrated that EK13 is a perspective probiotic candidate for rabbits. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Enterococcus faecium EK13--an enterocin a-producing strain with probiotic character and its effect in piglets

The experiment was conducted to determine the effects of the inoculation of the probiotic and enterocin A-producing strain Enterococcus faecium EK13 on selected parameters of metabolic profile, gut microflora, growth, and health in newborn piglets of Slovak White Improved. Piglets for study were divided into two groups: one group (EK13 group, n=8) received strain EK13 per os once daily for 7 days (2ml per piglet, 10(9)CFU/mL of saline buffer). The control group of piglets (n=7) was given placebo-saline buffer. The experiment lasted 14 days. After 7 days, strain EK13 reached 9.8 log(10) CFU/g in faeces of E. faecium EK13 treated piglets while counts of Escherichia coli were significantly lower (P<0.01) than in piglets of the control group. The concentrations of total serum protein, calcium, haemoglobin, haematocrit, red blood cell count and index of phagocytic activity of leukocytes were significantly higher after application of strain EK13. On the other hand, cholesterol was significantly lower in the EK13 group of animals. On day 14, piglets were killed and samples of intestinal contents were taken. Total counts of bacteria in the intestinal contents (jejunum, ileum, caecum, colon) were not significantly influenced. The pH value was significantly lower (P<0.05) only in duodenum of piglets receiving E. faecium EK13. There was a significant higher concentration of lactic acid (P<0.01) and propionic acid in the colon (P<0.001) of the EK13 group. Application of E. faecium EK13 did not influence the daily body weight gain significantly.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, Iran

Aims:  Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE).
Methods and Results:  During 1 year study (September 2005–2006), a total of 106 HLGR-EF isolates were collected from clinical ( n  = 48) and STP ( n  = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di  = 0·97) among the clinical and 21 PhP types ( Di  = 0·91) among the STP isolates. Representative isolates of each PhP type ( n  = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 ( Di  = 0·96) and 16 ( Di  = 0·93) types among the strains isolated from clinical and STP samples, respectively.
Conclusions:  We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates.
Significance and Impact of the Study:  The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances.     

Identification of new sex pheromone plasmids in Enterococcus faecalis.

Summary We describe the identification of the following new sex pheromone plasmids inEnterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

A rapid DNA extraction method for sugarcane and its relatives

A simple DNA extraction method based on CTAB precipitation was used to obtain DNA from members of the genusSaccharum and related species. DNA yields and purities were similar for allSaccharum species sampled. The method described here resulted in high quality total DNA suitable for polymerase chain reaction (PCR)-based techniques as well as restriction endonuclease digestion, Southern hybridization, and DNA cycle-sequencing.     

A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species

Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality, restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems.     

Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium

Background

Supplementation of broiler chicken diets with probiotics may improve carcass characteristics and meat quality. However, the underlying molecular mechanism remains unclear. In the present study, 2D-DIGE-based proteomics was employed to investigate the proteome changes associated with improved carcass traits and meat quality of Arbor Acres broilers (Gallus gallus) fed the probiotic Enterococcus faecium.

Results

The probiotic significantly increased meat colour, water holding capacity and pH of pectoral muscle but decreased abdominal fat content. These meat quality changes were related to the altered abundance of 22 proteins in the pectoral muscle following E. faecium feeding. Of these, 17 proteins have central roles in regulating meat quality due to their biological interaction network. Altered cytoskeletal and chaperon protein expression also contribute to improved water holding capacity and colour of meat, which suggests that upregulation of chaperon proteins maintains cell integrity and prevents moisture loss by enhancing folding and recovery of the membrane and cytoskeletal proteins. The down-regulation of β-enolase and pyruvate kinase muscle isozymes suggests roles in increasing the pH of meat by decreasing the production of lactic acid. The validity of the proteomics results was further confirmed by qPCR.

Conclusions

This study reveals that improved meat quality of broilers fed probiotics is triggered by proteome alterations (especially the glycolytic proteins), and provides a new insight into the mechanism by which probiotics improve poultry production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1167) contains supplementary material, which is available to authorized users.     

A simple, rapid and effective method for total RNA extraction from Lentinula edodes

A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms.     

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.