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PubMed2005

Quantification of initial adhesion of Enterococcus faecalis to medical grade polymers using a DNA-based fluorescence assay.

This paper reports on the use of a DNA-based fluorescence assay to study and quantify the initial interactions of the uropathogen Enterococcus faecalis with different polymers commonly used for the fabrication of medical devices and implants, including polyurethane (PU), silicone (SI), high-density polyethylene (HDPE), polyamide (PA), poly(methyl methacrylate) (PMMA) and polytetrafluoroethylene (PTFE). To follow the kinetics of E. faecalis adhesion, polymer samples were incubated in bacterial solution for various times and the relative concentration of adhered bacteria was obtained using two methods: commonly used CFU plate counting and a DNA quantification assay. Results obtained from DNA-based fluorescence assays showed that E. faecalis adhesion on PU is 3-times higher than that on PTFE following exposure to bacteria for 180 min. Neither surface wettability nor surface roughness of the studied polymers was found to correlate with E. faecalis adhesion, suggesting the involvement of much more complex adhesion mechanisms of bacteria onto surfaces. SEM micrographs of adhered bacteria illustrated that adhesion was different depending on the type of polymeric substrate: adhesion on PU samples was characterized by the aggregation of bacterial cells in dense clusters, as well as by the presence of fimbriae between cells and the substrate, which could explain the high adhesion to PU compared to the other polymers. This work demonstrated that the bacterial adhesion to polymers occurs at an early stage of the contact and suggests that the initial adhesion stage should be controlled, in order to prevent subsequent biofilm formation and, thus, reduce the risk of implant-associated infections.

Bacterial Adhesion↗
PubMed1992

In vivo activity of the combination of daptomycin and fosfomycin compared with daptomycin alone against a strain of Enterococcus faecalis with high-level gentamicin resistance in the rat endocarditis model.

The in vivo activity of the combination of daptomycin and fosfomycin against a beta-lactamase-producing, highly gentamicin-resistant strain of Enterococcus faecalis in a relapse model of rat endocarditis was studied. Minimum inhibitory concentrations (MICs) (micrograms per milliliter) for these agents against this strain were 4 (daptomycin) and 16 (fosfomycin). Time-kill studies demonstrated synergistic bactericidal activity when daptomycin (0.5 micrograms/ml) and fosfomycin (32 micrograms/ml) were combined. There was no significant difference between the number of valves sterilized by daptomycin alone [six (35%) of 17 valves sterilized] and daptomycin+fosfomycin [ten (59%) of 17 valves sterilized] p = 0.3. These results suggest that the in vitro bactericidal synergism demonstrable between these two agents against strains of enterococci will not necessarily translate into greater therapeutic efficacy in clinical infections.

Animals↗
PubMed2001

Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules.

Comparative analysis of the conjugative transposons Tn5397 from Clostridium difficile and Tn916 from Enterococcus faecalis, and the CW459tet(M) element from Clostridium perfringens, has revealed that these tetracycline-resistance elements are closely related. All three elements contain the tet(M) resistance gene and have sequence similarity throughout their central region. However, they have very different integration/excision modules. Instead of the int and xis genes that are found in Tn916, Tn5397 has a large resolvase gene, tndX. The C. perfringens element encodes the putative Int459 protein, which is a member of the integrase family of site-specific recombinases but is not closely related to Int from Tn916. Based on these studies it is concluded that the clostridial elements have a modular genetic organization and were derived independently from distinct mobile genetic elements.

Base Sequence↗
PubMed1996

Analyses of traA, int-Tn, and xis-Tn mutations in the conjugative transposon Tn916 in Enterococcus faecalis.

The conjugative transposon Tn916 moves intercellularly via an excision/insertion mechanism that involves products of int-Tn and xis-Tn. Tn5-insertion mutations in these genes were found to be complemented in an Enterococcus faecalis host by specific coresident transposons harboring the corresponding wild-type allele. A determinant designated traA, partially overlapping and divergently transcribed from xis-Tn, is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express a trans-acting product.

Amino Acid Sequence↗
PubMed1991

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.

Chromosomes, Bacterial↗
PubMed2000

Cloning, characterization and expression of an Enterococcus faecalis gene responsive to heavy metals.

Gene encoding stress response proteins are induced by a variety of environmental stimuli including the presence of heavy metals. To address the utility of this response for pollutant detection, one cadmium-induced gene in Enterococcus faecalis was isolated, sequenced and studied at the transcriptional level. csrA contains an open reading frame encoding a protein of 168 amino acids with homology to the enzyme peptide methionine sulfoxide reductase. The csrA mRNA was barely present in unstressed E. faecalis cells grown in M17-glucose medium, but accumulated at higher levels in cadmium-treated cells. Mercury also had an effect on csrA expression, whereas lead, copper and manganese induced csrA expression only at the highest doses tested. Our results suggest that biosensors based on E. faecalis may have potential applications for environmental monitoring and should be constructed.

Bacterial Proteins↗
PubMed2007

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized.

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.

Bacteriological Techniques↗
PubMed2006

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.

Amino Acid Sequence↗
PubMed2002

Enterococcus faecalis surface proteins determine its adhesion mechanism to bile drain materials.

An important step in infections associated with biliary drains is adhesion of micro-organisms to the surface. In this study the role of three surface proteins of Enterococcus faecalis (enterococcal surface protein, aggregation substances 1 and 373) in the adhesion to silicone rubber, fluoro-ethylene-propylene and polyethylene was examined. Four isogenic E. faecalis strains with and without aggregation substances and one strain expressing enterococcal surface protein were used. The kinetics of enterococcal adhesion to the materials was measured in situ in a parallel plate flow chamber. Initial deposition rates were similar for all strains, whereas the presence of surface proteins increased the total number of adhering bacteria. Nearest neighbour analysis demonstrated that enterococci expressing the whole sex-pheromone plasmid encoding aggregation substances 1 or 373 adhered in higher numbers through mechanisms of positive cooperativity, which means that adhesion of bacteria enhances the probability of adhesion of other bacteria near these bacteria. Enterococci with the enterococcal surface protein did not adhere through this mechanism. These findings indicate that the surface proteins of E. faecalis play a key role in the adhesion to bile drains and bile drain associated infections.

Bacterial Adhesion↗
PubMed1989

Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones. An ultrastructural comparison using immuno labelling, transmission and high resolution scanning electron microscopic techniques.

The distribution of sex pheromone induced aggregation substance was studied on the cell surface of various Enterococcus faecalis strains. In the accompanying paper we have shown that the aggregation substance appears as a layer of hairlike structures. Using direct and indirect immunogold technique, transmission electron microscopy and high resolution scanning electron microscopy we investigated the appearance and distribution of the aggregation substance. The "hairs" increase in number with increasing exposure to sex pheromones (maximum density: 1300/microns2). We show that these structures are unequally distributed over the cell surface, even if the cells were induced by sex pheromones for a long period of time. Statistical analysis of the unequal distribution indicates that aggregation substance is incorporated into pre-existing "old" cell-walls and that this incorporation shows a saturation ca. 40 min after addition of sex pheromones.

Biological Factors↗
PubMed1987

Homology of a transferable tetracycline resistance determinant of Clostridium difficile with Streptococcus (Enterococcus) faecalis transposon Tn916.

In several tetracycline-resistant (Tetr) Clostridium difficile strains, homology with the Tn916 part of plasmid pAM120 DNA was observed. This 15-kilobase transposon, carrying a Tetr determinant, was originally found in Streptococcus (Enterococcus) faecalis. Hybridization experiments revealed that at least six of seven HincII fragments of Tn916, representing greater than 95% of its length, showed homology with DNA of Tetr C. difficile strains. Therefore, a close relationship of the C. difficile Tetr-determining element with the entire Tn916 transposon can be assumed, although differences were observed concerning the number of HindIII cleavage sites within the transposon. In addition to strong hybridization of Tetr determinants of C. difficile with Tn916, weak signals were detected when DNA of Tets C. difficile was hybridized with Tn916. These weak signals could be attributed to a single internal HincII fragment of Tn916.

Clostridium↗
PubMed1992

Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.

Acetates↗
PubMed2000

Modeling of Enterococcus faecalis D-alanine:D-alanine ligase: structure-based study of the active site in the wild-type enzyme and in glycopeptide-dependent mutants.

A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was produced using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template. The model passed critical validation criteria with an accuracy similar to that of the template crystallographic structure and showed that ADP and methylphosphinophosphate were positioned in a large empty pocket at the interface between the central and the C-terminal domains, as in E. coli. It evidenced the residues important for substrate binding and catalytic activity in the active site and demonstrated a large body of conserved interactions between the active sites of the E. faecalis and the E. coli D-Ala:D-Ala ligase, the major differences residing in the balance between the hydrophobic and aromatic environment of the adenine. The model also successfully explained the inactivity of four spontaneous mutants (D295 --> V, which impairs interactions with Mg2+ and R293, which are both essential for binding and catalytic activity; S319 --> I, which perturbs recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in the vicinity of the deletion remains unaltered but phosphate transfer from ATP is perturbed because of lack of K253; T316 --> I, which causes the loss of a hydrogen bond affecting the positioning of S319 and therefore the binding of D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this approach, combining molecular modeling and molecular biology, may help in the design of specific ligands which could inhibit the enzyme and serve as novel antibiotics.

Adenosine Diphosphate↗
PubMed2021

Removal of peptidoglycan and inhibition of active cellular processes leads to daptomycin tolerance in Enterococcus faecalis.

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.

Anti-Bacterial Agents↗
PubMed1992

New high-content disks for determination of high-level aminoglycoside resistance in clinical isolates of Enterococcus faecalis.

Disks impregnated with 500 and 1000 micrograms of streptomycin, 1000 micrograms of kanamycin and 250 and 500 micrograms of gentamicin were used for detection of high-level resistance to aminoglycosides in 120 clinical isolates of Enterococcus faecalis. Fifty-seven strains were highly resistant to streptomycin, 80 to kanamycin and 41 to gentamicin. Using disks containing 500 micrograms of streptomycin, 1000 micrograms of kanamycin and 500 micrograms of gentamicin strains resistant to high levels of these drugs (97.9%, 100% and 100%, respectively) were accurately detected. Better discrimination between high-level and low-level resistance was achieved with a 500 micrograms streptomycin or gentamicin disk. Zone-size breakpoints are proposed for detection of high-level resistance by disk diffusion.

Drug Resistance, Microbial↗
PubMed1990

Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion.

Pheromone-induced conjugal transfer of the hemolysin-bacteriocin plasmid pAD1 of Enterococcus faecalis is regulated by a cluster of determinants designated traA, traB, and regions C and E. The E region is believed to include a positive regulator that controls many structural genes related to conjugation. The pheromone-inducible Tn917-lac fusion NR5, located in the E region, is regulated by the products of traA, traB, and the C region. To more closely examine the effects of these genes on the induction of E region products, inserts in each of these genes were combined with the NR5 fusion in a novel approach involving triparental matings with a pAD1 miniplasmid and recombinational mutagenesis. Results indicate that (i) the traA gene product is a key repressor of the pheromone response; (ii) the traB gene product, in cooperation with a gene within or regulated by the E region, controls pheromone shutdown; (iii) a primary function of the C region gene product is in pheromone sensing, with secondary functions in pheromone shutdown and negative regulation; and (iv) the host in which the plasmid resides has a dramatic effect on the regulation of the NR5 fusion in traB and C region mutants. Numerous parallels were observed between the regulation of the NR5 fusion and the regulation of the aggregation and transfer response. These parallels aided in further defining the functions of particular regulatory determinants as well as further establishing the link between the regulation of the E region and the regulation of the aggregation and transfer response.

Cloning, Molecular↗
PubMed1999

Detection of a streptomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of Enterococcus faecalis that was streptomycin resistant but did not appear to contain the 6'-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 microg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coli C600(pHP45Omega) known to contain the aadA gene. The aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-alpha competent cells. D-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadA genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundstr om et al. for E. coli plasmid R6-5 (L. Sundström, P. Râdström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191-201, 1988), by Fling et al. for the aadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095-7106, 1985), and by Hollingshead and Vapnek for E. coli R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17-30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe an aadE gene, since the isolates were reported to be susceptible to spectinomycin.

Amino Acid Sequence↗