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PubMed1992

Selective intracellular lactate invisibility in Enterococcus faecalis.

In this article we will demonstrate that differences in Hahn T2 relaxation of the 1H NMR signal from cytosolic and extracellular lactate can be exploited to monitor lactate concentration gradients in bacterial cells and provide information on lactate transport mechanisms. As a by-product of this study we have determined that there are at least three pools of lactate in bacterial cells with differing visibility in the NMR experiment. This has serious implications for the spectral editing techniques that are so vital for in vivo spectroscopy.

Biological Transport↗

PubMed2004

Contribution of gelatinase, serine protease, and fsr to the pathogenesis of Enterococcus faecalis endophthalmitis.

Gelatinase and serine protease were found to contribute in concert to pathogenesis in a rabbit model of endophthalmitis. However, a mutant defective in the fsr regulator was observed to be more attenuated than a mutant rendered defective in the expression of gelatinase and serine protease as the result of a polar transposon insertion into the former. This increased attenuation suggests that there are possible additional pleiotropic effects of the defect in fsr on expression of traits contributing to the pathogenesis of enterococcal infection.

Animals↗

PubMed2005

Decreased virulence of a gls24 mutant of Enterococcus faecalis OG1RF in an experimental endocarditis model.

In the current study, the gls24 disruption mutant TX10100, previously shown to be more sensitive to bile salts and attenuated in a mouse peritonitis model, showed an approximately fivefold higher 50% infective dose than wild-type OG1RF in a rat endocarditis model. When administered as a mixture, TX10100, unlike a downstream glsB mutant, was significantly outnumbered by OG1RF in vegetations, organs, and blood, despite being inoculated in greater numbers. These results indicate that gls24 is important in the pathogenesis of enterococcal endocarditis.

Animals↗

PubMed1996

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos.

The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.

Conjugation, Genetic↗

PubMed2000

Identification of general stress genes in Enterococcus faecalis.

The susceptibility and the acquisition of tolerance of E. faecalis ATCC 19433 to heat, ethanol, bile salts, NaCl, H2O2 and pH shifts were determined. During exposure to these environmental stresses, protein synthesis analysed by 2-D electrophoresis revealed 167 stress proteins. Six stress protein were found to be induced by at least six of the eight treatments and considered to be general stress proteins (Gsp). Western blotting identified two of these Gsp as DnaK and GroEL. Analysis of the four other Gsp revealed that at least two of them were not previously described.

Bacterial Proteins↗

PubMed2006

Expression of virulence-related genes by Enterococcus faecalis in response to different environments.

Enterococci are ubiquitous organisms used to both improve the flavor and texture of fermented foods, and provide protective mechanisms as either a probiotic or antimicrobial additive. However, two species, E. faecalis and E. faecium, are also associated with 10% of nosocomial infections of the bloodstream, wounds, urinary tract and heart. While the genes involved in the pathogenicity of these organisms are slowly identified along with the mechanisms behind their regulation, the environmental signals involved in the conversion to pathogenicity remain unclear. The distribution of virulence genes was determined in 13 E. faecalis isolates from medical, food and animal sources. Regardless of their source of isolation, all isolates harbored between eight and thirteen virulence genes. Relative differences in expression of the virulence associated genes clpP, clpX, gls24, agg, efaA, gelE, and cylBL(L) were examined in E. faecalis TMW 2.63 and TMW 2.622 exposed to different environments (LB, BHI, respective supernatants, pig fecal extract, LB+6.5% NaCl, LB+pH5, LB+6.5% NaCl+pH5, and sausage medium) using RT-PCR and Lightcycler technology. Significant differences in expression were influenced by growth phase, environment, and isolate, which suggests that these three factors be taken into consideration during the selection of enterococci for use in foods or as probiotics rather than their source of isolation or set of virulence genes.

Animals↗

PubMed1989

Purification and amino acid composition of peptide antibiotic AS-48 produced by Streptococcus (Enterococcus) faecalis subsp. liquefaciens S-48.

Peptide antibiotic AS-48 was purified to homogeneity by ion-exchange chromatography, gel filtration chromatography, and reversed-phase liquid chromatography. The purified fraction was active against gram-positive and gram-negative bacteria. AS-48 is a basic protein with an isoelectric point of ca. 10.5 and a molecular mass of 7.4 kilodaltons. Its inhibitory activity was markedly affected by sodium dodecyl sulfate and cardiolipin but not by neuraminidase, pectinase, beta-glucosidase, or beta-glucuronidase. Differential scanning calorimetry data suggested that AS-48 molecules lack a compact structure.

Amino Acids↗

PubMed1993

Construction of a slime negative transposon mutant in Staphylococcus epidermidis using the Enterococcus faecalis transposon Tn917.

The slime-producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn 917. Using this transposon mutagenesis system we obtained the Tn917-inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the transposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime-negative phenotype compared with spontaneous slime-negative mutants of S. epidermidis strain CNS23. In slime-ELISA no slime-associated antigen could be detected in extracts of the transposon mutant. Compared to slime-positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.

DNA Transposable Elements↗

PubMed1994

Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583.

Acquired resistance to glycopeptides in enterococci is associated with the production of D-Alanine:D-Alanine ligase-related proteins. The VanA protein associated with high-level vancomycin and teicoplanin resistance (VanA phenotype) synthesizes a new peptidoglycan precursor, D-alanine-D-lactate, that has reduced glycopeptide affinity. Production of a similar protein, VanB, is induced in strains that display variable levels of vancomycin resistance but remain susceptible to teicoplanin (VanB phenotype). This paper describes the over-production, purification and characterization of VanB. Comparison of kinetic parameters of the two Van enzymes suggests that differences in catalytic efficiency could account, at least in part, for the various levels of vancomycin resistance.

Amino Acid Sequence↗

PubMed2003

Evaluation of the antibacterial potential of tetracycline or erythromycin mixed with calcium hydroxide as intracanal dressing against Enterococcus faecalis in vivo.

OBJECTIVE: We evaluated the intracanal antibacterial potential of tetracycline or erythromycin mixed with calcium hydroxide (CH) against enterococci in vivo. STUDY DESIGN: Fifty-five teeth in which enterococci were present were dressed for 1 month with either tetracycline (28 teeth) or erythromycin (27 teeth) mixed with CH. RESULTS: The tetracycline mixture was effective against enterococci in 22 teeth (79%). In 7 teeth, other microorganisms were recovered, resulting in a total antimicrobial effect of 54%. The corresponding results for erythromycin were 96% and 56%. CONCLUSIONS: The antimicrobial treatment of CH in combination with either erythromycin or tetracycline had a significant effect on enterococci, but the overall antimicrobial effect was relatively weak. Erythromycin mixed with CH seems to be a valuable option in the battle against monoinfections of enterococci.

Adult↗

PubMed1991

Epidemiology of an endemic strain of beta-lactamase-producing Enterococcus faecalis.

Three previously reported beta-lactamase-producing (Bla+) enterococci with distinct but related antibiotic resistance phenotypes, plasmid profiles, and plasmid restriction endonuclease digestion patterns were isolated at the West Haven Veterans Administration Medical Center in Connecticut (WH245, WH257, WH571) in July 1986 and March 1987. In this study, we analyzed the whole-cell DNA of these isolates by using pulsed-field gel electrophoresis of large chromosomal fragments generated by SmaI digestion. The three West Haven isolates showed very similar chromosomal restriction endonuclease digestion patterns; these patterns were distinct from those of Bla+ and Bla- enterococci from other geographic areas and from those of other West Haven enterococci, suggesting an endemic Bla+ strain at this institution. Clinical information regarding these isolates suggests that exposure to the genitourinary clinic, obstructive urinary tract disease, and frequent antibiotic therapy may have been common risk factors for acquisition of this endemic strain.

Aged↗

PubMed2006

Enterococcus faecalis adhesin, Ace, mediates attachment to particulate dentin.

An enzyme linked immunosorbent assay was developed to assess E. faecalis adhesion to particulate dentin. E. faecalis, OG1RF, which expresses the collagen binding protein (Ace+), and a derivative of OG1RF, TX5256, deficient in the collagen binding protein (Ace-) were grown at 46 degrees C, necessary for in vitro expression of Ace, and at 37 degrees C. E. faecalis binding to dentin was measured at 0, 15, 30, 60, 120, and 360 minutes. Compared to TX5256 and OG1RF grown at 37 degrees C, OG1RF grown at 46 degrees C adhered significantly better at all time points except 15 minutes (p < 0.001) exhibiting maximum binding at 120 minutes (17.4% of a positive control). Type I collagen at 100 microg/ml inhibited dentin binding by OG1RF grown at 46 degrees C in both competition (p < 0.005) and displacement assays (p < 0.046). Immunoaffinity purified anti-Ace IgG at 200 microg of protein inhibited adhesion of OG1RF grown at 46 degrees C to dentin.

Adhesins, Bacterial↗

PubMed1991

[Bacteremia caused by vancomycin-resistant Enterococcus faecalis. Report of 2 cases].

The rising concern about nosocomial bacteremia due to vancomycin-resistant E. faecalis in Spanish hospitals. Retrospective review of the medical records of two patients with nosocomial bacteremia due to E. faecalis resistant to vancomycin. Both patients (a 78 years-old male and a 65 years-old female) were admitted in two separate hospital units. None of them had been previously treated with vancomycin, and both patients had severe underlying diseases. The two strains isolated showed high-level vancomycin and teicoplanin resistance, although they were sensitive to daptomycin. No other strain with high level glycopeptide antibiotic resistance was isolated in the hospital during the following 23 months. This occasional resistance to glycopeptides among enterococci has been shown also in other Spanish hospitals. The increasing incidence of penicillin and aminoglycoside resistance among enterococci in our environment could have a practical impact, either for the clinical laboratory and/or for therapeutic decisions in patients with infections due to this microorganism.

Aged↗

PubMed1991

Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.

Base Sequence↗

PubMed1996

Functional analysis of the Enterococcus faecalis plasmid pAD1-encoded stability determinant par.

The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.

Base Sequence↗

PubMed2004

Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater.

The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.

Animals↗

PubMed1997

Induction of VanA vancomycin resistance genes in Enterococcus faecalis: use of a promoter fusion to evaluate glycopeptide and nonglycopeptide induction signals.

To characterize induction of VanA resistance a plasmid was constructed in which the gene for firefly luciferase lucA was placed under the control of the promoter for the VanA resistance genes, the vanH promoter. This system afforded convenient quantitative measurement of induction of the VanA genes. Glycopeptide antibiotics and antibiotics representing 19 different mechanisms of action were evaluated for their ability to induce. Antibiotics that acted as inducers were all inhibitors of late steps of peptidoglycan synthesis. These included moenomycin, bacitracin, tunicamycin, ramoplanin and glycopeptides, but not penicillin or other beta-lactam antibiotics. Glycopeptide antibiotics were the most potent inducers. Both glycopeptides with little or no antimicrobial activity and semisynthetic glycopeptides active against VanA resistant enterococci were inducers. Overall, results suggest that an induction response may involve both an internal signal, such as precursor accumulation, and the glycopeptide molecule itself as a signal. The system may be useful as a screen for new antimicrobial agents.

Anti-Bacterial Agents↗

PubMed1989

Construction of Enterococcus faecalis pAD1 miniplasmids: identification of a minimal pheromone response regulatory region and evaluation of a novel pheromone-dependent growth inhibition.

Several pAD1 miniplasmids were constructed that consisted of all or a portion of the pAD1 EcoRI B fragment with pheromone-inducible "E region" lacZ transcriptional fusions. Miniplasmids containing the entire EcoRI B fragment (the "E miniplasmids") were found to regulate LacZ expression normally, indicating that sufficient information is present on this fragment to regulate a pheromone response. These plasmids also encoded normal replication functions. The E miniplasmids were further reduced by deleting a KpnI fragment. These "K miniplasmids" were able to perform most of the functions central to a pheromone response but failed to induce the fused transcripts to levels observed in the parental plasmids. This defect was found to be due to a pheromone-dependent growth inhibition of cells containing the K miniplasmids. Evidence indicated that this inhibition was due to transcriptional readthrough beyond the lacZ gene and into the putative replication region of the plasmid. Possible mechanisms of this inhibition as well as its potential usefulness in further examining the characteristics of the pheromone response are discussed.

Chromosome Mapping↗