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Comparative analysis of 18 sex pheromone plasmids from Enterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family.

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3'-terminus of the surface exclusion gene, sep1, of sex pheromone plasmid pPD1 in Enterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.

Amino Acid Sequence↗

Measurement of active constitutive L-pyrrolidonyl-peptidase from the genera Streptococcus and Enterococcus.

In the family Streptococceae the ability to measure L-pyrrolidonyl-peptidase is limited to Lancefield group D Enterococcus and group A Streptococcus pyogenes. A number of methods exist to assay this enzyme. All measure pyrrolidonyl-peptidase by the ability of the bacterium to cleave L-pyrrolidonyl-beta-naphthylamide to form free beta-naphthylamine and L-pyrrolidone-carboxylic acid. Free beta-naphthylamine is then reacted with N,N-dimethylamino-cinnamaldehyde to form a red color complex. These methods are generally expensive and require a 2-4 h incubation period before results are available. A method, which employs the substrate dried on paper discs and can be easily made, is described herein. It is simple to perform, inexpensive, rapid, and has a long storage life. The results of this constitutive method are equivalent to those obtained using a commercial system.

Aminopeptidases↗

Does aggregation substance of Enterococcus faecalis contribute to development of endocarditis?

Aggregation substance (AS) of Enterococcus faecalis which is encoded by so-called sex pheromone plasmids enables the bacteria to bind to in vitro-cultured pig kidney tubular cells. It is reported that the presence of AS is not of pivotal importance for the ability of E. faecalis to cause infective endocarditis (EN). The lines of evidence for this are twofold: 1) sex pheromone plasmids and, therefore, the gene for AS were not present more often in epidemiologically unrelated strains of E. faecalis isolated from human cases of EN than in isolates from well-water (26 vs. 18%); 2) the presence of the adhesin did not correlate with the establishment of EN in an animal (rat) model. The data are discussed with respect to the specificity of interaction of AS with eukaryotic cells and the results of other studies.

Adhesins, Bacterial↗

The effect of salinomycin and lasalocid on laboratory cultures of Enterococcus faecium and Staphylococcus gallinarum strains.

The growth of Enterococcus faecium strains CCM 4231 and EF 26, and Staphylococcus gallinarum SG 31 was inhibited by salinomycin and lasalocid at concentrations of 25 and 50 mg/L. Staphylococcus gallinarum was more sensitive to the additives used than were enterococci. Maximum inhibition (90%) was measured after the growth with the SG 31 strain in the presence of both ionophores. Growth of organisms was more inhibited by salinomycin at 25 mg/L (67.5%) than at 50 mg/L (63%). The inhibitory effect in enterococcal strains reached after the addition of salinomycin and lasalocid (on average) 63 and 58%, respectively. The CCM 4231 strain was more inhibited by salinomycin as well as by lasalocid than was the EF 26 strain.

Enterococcus faecium↗

Occurrence of Enterococcus spp. in waters.

We studied 630 bacterial strains isolated from surface waters and determined as enterococci on the basis of their growth on Slanetz-Bartley agar in typical colonies. The strains were tested and characterized by several key conventional tests for basic differentiation of enterococci and by commercial test kits. We identified 135 strains of E. foecium (21%), 115 E. faecalis (18%), 30 E. mundtii (5%), 27 E. hirae (4%), 22 E. casseliflavus (3%), 21 E. gallinarum (3%), 17 E. durans-E. hirae complex (3%), 5 E. durans (1%), and 1 strain of E. avium. 150 strains were classified only as Enterococcus sp. (25%) and 107 strains (17%) isolated from Slanetz-Bartley agar were not enterococci. We found that the non-enterococcal group consisted of other Gram-positive cocci and Gram-positive and Gram-negative rods. Based on the identification we tried to find a relation between taxonomic position of isolated strains and their colony morphology on Slanetz-Bartley agar. Our of the total of 523 identified enterococci, 345 strains (66%) formed purple colonies, 136 red colonies (26%), 37 pink colonies (7%) and 5 cream colored colonies (1%). There was no correlation among the color, size or colony morphology and the taxonomic characterization of enterococcal strains.

Bacteriological Techniques↗

Antimutagenicity of milk fermented by Enterococcus faecium.

The diethyl ether extracts isolated from unfermented milk and milk fermented by Enterococcus faecium exhibited dose-dependent inhibition of mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitrovin (NIT), 5-nitro-2-furylacrylic acid (NFA) and UV-irradiation on the Ames bacterial test (Salmonella typhimurium strains TA97 and TA100) and the unicellular flagellate Euglena gracilis. Overall, the fermented milk extract was the most active against UV-irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria. The highest antibleaching effects were observed against MNNG. The differences between antimutagenic effects from fermented and unfermented milk extracts were determined to be statistically significant at the 0.95 CI level.

Acrylates↗

Hypocholesterolemic and immunostimulatory effects of orally applied Enterococcus faecium M-74 in man.

Lyophilized Enterococcus faecium M-74 was administered to 12 adult subjects in a daily oral dose of 5 x 10(9) bacteria for six weeks. The bacterium temporarily colonized the host intestine and its excretion with stool persisted for five weeks after the last does. The mean levels of serum cholesterol and LDL showed a a biphasic effect--an elevation followed by a sharp decrease (on day 64 of investigation). The decrease corresponded in time with a significant increase in the ability to reduce iodonitrotetrazolium and superoxide production by peripheral neutrophils incubated with zymosan or phorbol myristate acetate, and also with an elevated production of IgG by peripheral blood mononuclear cells. Hence, intake of E. faecium may have a hypocholesterolemic and immunostimulatory effect. It was also demonstrated that E. faecium significantly reduced the average activity of beta-D-glucuronidase in stools.

Adjuvants, Immunologic↗

Effects of nitrogen sources on bacteriocin production by Enterococcus faecium A 2000.

The production of a novel broad-spectrum antimicrobial peptide enterococcin A 2000, active against Gram-positive and Gram-negative microorganisms including Listeria subsp. and Escherichia coli, by Enterococcus faecium strain A 2000 isolated from the surface of traditional Bulgarian yellow cheese "kash-kaval" is considerably influenced by complex nitrogen sources in the production medium. Medium components, especially peptone and yeast extract, and their concentration contributed to the increase in bacteriocin production during the stationary phase (16-46 h) of cultivation even in the absence of one of the components present in the basal cultivation MRS medium.

Bacteriocins↗

Isolation and partial characterization of an antibacterial substance produced by Enterococcus faecium.

A strain of Enterococcus faecium isolated from Bulgarian yellow cheese "kashkaval" produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (< 2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti-Listeria motif YGNGV.

Amino Acid Sequence↗

Treatment of experimental adjuvant arthritis with the combination of methotrexate and lyophilized Enterococcus faecium enriched with organic selenium.

The efficacy of combination therapy with methotrexate (MTX) and probiotic bacteria Enterococcus faecium enriched with organic selenium (EFSe) in rats with adjuvant arthritis was determined. Rats with adjuvant arthritis were given MTX (0.3 mg/kg 2-times weekly, orally); lyophilized E. faecium enriched with Se (15 mg/kg, 5 d per week, orally); and a combination of MTX plus EFSe for a period of 50 d from the immunization. Levels of serum albumin, serum nitrite/nitrate concentrations, changes in hind paw swelling, arthrogram score, bone erosions, whole body bone mineral density (BMD) and bone mineral content (BMC) were assayed in the rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX and with the combination MTX + EFSe significantly inhibited markers of both inflammation and arthritis. Significant differences in favor of combination therapy with MTX + EFSe as compared to MTX alone were seen in serum albumin concentration, hind paw swelling and arthrogram score. Reductions in radiographic scores were also more pronounced in the combination therapy group. Combination therapy, but not MTX alone, inhibited the reduction of BMD and BMC; treatment with lyophilized EFSe alone had no significant effect on adjuvant arthritis in rats. The potent therapeutic effect of low dosage MTX therapy in combination with lyophilized EFSe on adjuvant arthritis in rats was shown.

Animals↗

Genetic stability of the antagonistic character of Enterococcus faecalis ssp. liquefaciens and the detection of a new inhibitory bacteriocin-like substance.

The inhibitory capacity of strain S-48 of Enterococcus faecalis ssp. liquefaciens was studied. The strain produces a broad-spectrum peptide antibiotic (AS-48) that has been characterized elsewhere. The isolation of mutants from S-48 after mutagenic treatment revealed another inhibitory substance which remained masked in the wild strain. The protein nature and restricted spectrum of this substance points to its being a bacteriocin.

Acridine Orange↗

Induction of inhibitory agent produced by Enterococcus faecalis.

The effect of treatment with inducing agents, such as mitomycin C, hydrogen peroxide and UV irradiation on the production of two inhibitors by different mutants from Enterococcus faecalis S-48 was studied. With hydrogen peroxide and UV light no increase in either the absolute or the relative amount of antagonistic substances was observed. With mitomycin C, a significant increase in the individual cell capacity for inhibitor production was detected.

Anti-Bacterial Agents↗

Extracellular proteinase from Enterococcus faecalis subsp. liquefaciens. I. Growth and extracellular proteinase production under different culture conditions.

Growth and extracellular proteinase production by Enterococcus faecalis subsp. liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37 degrees C. Proteinase production was not affected by temperature in the range studied (7-45 degrees C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.

Bacteriological Techniques↗

Extracellular proteinase from Enterococcus faecalis subsp. liquefaciens. II. Partial purification and some technological important properties.

An extracellular proteinase from Enterococcus faecalis subsp. liquefaciens has been purified 780-fold by a method including gel filtration on Sephadex G-50 and affinity chromatography with gramicidin J as ligand. Approximately 15% of the original enzyme activity was recovered. A purification of 14,800-fold, with 11.4% yield, may be reached using chromatofocusing as final step in the purification procedure. The molar mass of the enzyme has been estimated to be approximately 30 kDa by Sephadex gel filtration and approximately 26 kDa by SDS-PAGE. The isoelectric point has been found to be 4.6. Maximum enzyme activity of the proteinase has been observed at pH 7.5 and 45 degrees C. The enzyme hydrolyzed bovine serum albumin, alpha-lactoalbumin, beta-lactoglobulin, casein and pork myofibrillar and sarcoplasmic proteins. The extracellular proteinase was very stable; the enzyme maintained its activity in cell-free extracts over a very wide range of temperatures (-25 to 37 degrees C) for at least 2 months. At 12 degrees C, it was stable in the pH range of 5.5 to 8.0.

Cheese↗

Synergic activity of selenium and probiotic bacterium Enterococcus faecium M-74 against selected mutagens in Salmonella assay.

Concentrated extracts of MRS (De Man-Rogosa-Sharpe) media in which probiotic bacterium Enterococcus faecium strain M-74 was grown exerted different antimutagenic activity against ofloxacin-, N-methyl, N'-nitro-N-nitrosoguanidine- and sodium 5-nitro-2-furylacrylate-induced mutagenicity in Salmonella typhimurium assay depending on the presence (+Se) or absence of disodium selenite pentahydrate (-Se). The antimutagenicity of MRS(+Se) extract was higher than that of MRS(-Se) extract. Selenium enhanced also the antimutagenic effect of both live and killed cells of E. faecium M-74, respectively. The live bacteria decreased the mutagenicity of selected substances more than killed cells. Synergic activity of selenium with the bacterium was also manifested.

Antimutagenic Agents↗

Inductive effects of environmental concentration of atrazine on Escherichia coli and Enterococcus faecalis.

Atrazine solutions (0.1, 1, 10 and 100 microg/L) inoculated with Escherichia coli and Enterococcus faecalis under natural conditions significantly increased (p < or = 0.05) the population levels of both test bacteria; it indicates the ability of bacterial cells to degrade atrazine and to use the original compound or its degradation products as nutrient(s). In some cases, alterations in the morphology of the colonies were also observed on selective solid media. Biochemical differentiation was also found and, on the other hand, a loss of culturability was recorded; this suggests that bacteria have entered in a viable but nonculturable state. A re-appearance of the colonies occurred after inoculation on tryptone-soy agar with atrazine.

Atrazine↗

Occurrence of aminoglycoside-modifying-enzyme genes aac(6')-aph(2"), aph(3'), ant(4') and ant(6) in clinical isolates of Enterococcus faecalis resistant to high-level of gentamicin and amikacin.

The genes coding for 4 aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3'), ANT(4') and ANT(6) were determined in 44 Slovak clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin (HLGR, collection 1) and 48 E. faecalis isolates with resistance to amikacin (AR, collection 2). The occurrence of spotted genes was (collection 1 vs. collection 2): aac(6)-aph(2") 81.8 vs. 8.3 %, ant(4') 52.3 vs. 81.3 %, aph(3') 50 vs. 56.3 % and ant(6) 6.8 vs. 4.2 %, the most frequent combinations of genes in the HLGR collection were aac(6')-aph(2") + ant(4') and aac(6')-aph(2") + aph(3). In contrast, the aph(3') + ant(4') gene profile was predominant in AR isolates. None of the isolates contained all four AGME genes simultaneously.

Acetyltransferases↗

Oral application of Enterococcus faecium strain EE3 in healthy dogs.

The ability of canine strain Enterococcus faecium EE3 to survive in healthy dogs and its effect on microbiological and biochemical parameters was determined. The strain was individually applied to 11 dogs per os at a dose of 10(9) CFU/mL (differed from 2 to 3 mL) for 1 week and persisted in feces for 3 months after cessation of its administration (reaching average concentration of 6.83 +/- 0.95 log CFU/g). Seven d after administration, a decrease in staphylococci and a significant decrease in Pseudomonas-like bacteria was observed. On the other hand, concentration of lactic acid bacteria increased but the growth of E. coli was not influenced. In the blood samples of dogs after 0-1 d (before application) and the blood samples 1 week after application, total lipids decreased in 8 dogs; the total protein also decreased. The levels of cholesterol were brought to the physiological level, i.e. in blood samples with low cholesterol values it increased to the physiological level and in those with high levels it decreased; cholesterol was not influenced in 3 dogs.

Animals↗