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PubMed1991

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↗
PubMed1991

Serum dependent expression of Enterococcus faecalis adhesins involved in the colonization of heart cells.

Our previous studies have shown that the adhesive ability of Enterococcus faecalis is dependent on the strain and is further modified by growth in serum. The data reported here demonstrate that E. faecalis adherence is mediated by carbohydrate residues present on the bacterial cell surface. Some of these (D-mannose and D-glucose) are expressed by strains isolated from both urinary tract infections (UTI) and endocarditis (EN) when the cells are grown in brain-heart infusion broth (BHIB), and mediate adherence to either urinary tract epithelial cells or the Girardi Heart (GH) cell line. Other residues are present only on EN strains (D-galactose and L-fucose) and mainly mediate adherence to GH cells. These ligands can also be expressed by UTI isolates after growth in serum. D-galactose-bearing adhesins also seem to be involved in internalization of serum grown UTI strains and BHIB or serum grown EN isolates into GH cells.

Bacterial Adhesion↗
PubMed1993

Characterization of the heat shock response in Enterococcus faecalis.

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacterium Enterococcus faecalis. The heat resistance (60 degrees C or 62.5 degrees C, 30 min) of log phase cells of E. faecalis grown at 37 degrees C was enhanced by exposing cells to a prior heat shock at 45 degrees C or 50 degrees C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72kDa) and GroEL (63kDa) heat shock proteins of Escherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La) E. coli proteins and to the Bacillus subtilis sigma 43 factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.

Antigens, Bacterial↗
PubMed1989

Diagnosing endocarditis with the cloned 112 kDa antigen of Enterococcus faecalis.

A new indirect ELISA is presented for the diagnosis of enterococcal endocarditis. It is based on the cloning of Enterococcus faecalis DNA into lambda gt11. The library was screened by antisera from three cases of E. faecalis endocarditis. Three positive clones were found, all of which cross-reacted with the 112 kDa antigen of E. faecalis. One of these was taken and lysogenised into E. coli Y1089 to produce a fusion protein of 125 kDa. This IPTG dependent protein was purified by affinity chromatography and used in an indirect ELISA. The test differentiated between five cases of enterococcal endocarditis (IgG ELISA optical density greater than 0.8) and patients with endocarditis due to staphylococci (IgG ELISA optical density less than 0.243) and other streptococci (IgG ELISA optical density less than 0.656). Patients with a simple E. faecalis septicaemia had a maximum IgG ELISA optical density of 0.636. The only major cross-reaction occurred in patients with Streptococcus bovis endocarditis.

Antibodies, Bacterial↗
PubMed1998

Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis.

The ability of Enterococcus faecalis to metabolically adapt to an oligotrophic environment has been analyzed. E. faecalis is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62 degreesC], acid [pH 3.3], UV254 nm light [180 J/m2], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray, Res. Microbiol. 148:27-35, 1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of E. faecalis.

Autoradiography↗
PubMed2006

Efficiency of NaOCl and laser-assisted photosensitization on the reduction of Enterococcus faecalis in vitro.

OBJECTIVE: To investigate the action of a red laser associated with a photosensitizer on the reduction of Enterococcus faecalis in dental root canal in vitro. STUDY DESIGN: Thirty prepared teeth with single canals were contaminated. The chemical group was irrigated with 0.5% NaOCl and left flooded for 30 minutes. In the laser group, a paste-based photosensitizer was maintained in the root canals for 5 minutes, and then irradiated with a laser at 685 nm using an optical fiber for an E of 1.8 J during 3 minutes. After treatment, the canal content was collected, serially diluted, and cultured to determine the number of colony-forming units. RESULTS: Photosensitizer alone or laser alone did not have any bactericidal effect. Chemical solution reduced viable bacteria in 93.25%. Laser photosensitization resulted in a reduction of 99.2%, a significantly higher bacterial reduction than NaOCl. CONCLUSION: Laser photosensitization was effective for reducing E. faecalis in root canals and could be an adjunct to endodontic treatment.

Azulenes↗
PubMed1995

Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes.

Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown.

Amino Acid Sequence↗
PubMed1998

Regulation of the Enterococcus faecalis pAD1-related sex pheromone response: analyses of traD expression and its role in controlling conjugation functions.

The Enterococcus faecalis haemolysin plasmid pAD1 (60 kb) confers a conjugative mating response to an octapeptide sex pheromone (cAD1) secreted by plasmid-free strains. The response involves two plasmid-borne regulatory determinants: traE1, whose product positively regulates all or most conjugation-related structural genes; and traA, whose product negatively regulates traE1 by controlling transcriptional readthrough of an upstream termination site (TTS1/TTS2). TraA binds to the promoter region of iad, which encodes a pheromone-inhibitor peptide, iAD1; and TTS1/TTS2 tightly terminates transcription arriving from this promoter during the uninduced state. A determinant, traD, appearing to encode a small peptide (23 amino acids), is located just downstream of iad and is in the opposite orientation. Transcripts of traD were identified and found to be present at a relatively high level in cells not expressing conjugation functions; the amount of RNA was greatly reduced, however, upon induction of the pheromone response. The decrease in traD RNA was not a consequence of the induced activity of TraE1, as it also occurred in a traE1 insertion mutant. A mutation in traD that would eliminate translation but that did not affect transcription had no apparent effect on the cell phenotype, indicating that RNA was likely to be the functional product. This was consistent with our finding that synthesis of traD RNA containing the translational defect was able to complement, in trans, a temperature-sensitive traD mutation. Thus, transcription of the traD determinant is significantly involved in downregulation of the pAD1 pheromone response.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of highly gentamicin-resistant Enterococcus faecalis isolates lacking high-level streptomycin resistance.

Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.

DNA Restriction Enzymes↗
PubMed2004

Effects of sonicated Enterococcus faecalis extracts on interleukin-2 and interleukin-4 production by human T cells.

In the present authors' previous study, sonicated Enterococcus faecalis extracts were shown to suppress the cell cycle progression of human lymphocytes. To study the effect of this microorganism on the function of lymphocytes, the authors investigated the levels of interleukin-2 (IL-2) and interleukin-4 (IL-4) production by T lymphocytes before and after the addition of sonicated E. faecalis extracts. In this study, levels of IL-2 and IL-4 produced from T cells were evaluated by using the quantitative sandwich enzyme immunoassay technique. In response to phytohemagglutinin (PHA) stimulation, T cells produced increased levels of IL-2 and IL-4. However, the expressions of both cytokines were significantly inhibited when PHA-activated T cells were preexposed to 12.5 microg/ml and 25 microg/ml of sonicated E. faecalis extracts (p < 0.05). This effect was concentration-dependent, because the levels of IL-2 and IL-4 expressions were not affected by the addition of a low concentration (5 microg/ml) of sonicated extract. These findings suggest that Th1 and Th2 immunosuppression mediated by E. faecalis could be a part of the pathogenic mechanism of the endodontic failure associated with this microorganism.

Cells, Cultured↗
PubMed2005

In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis.

OBJECTIVE: The aim of this study was to assess in vitro the antimicrobial activity of 2% chlorhexidine gel against Enterococcus faecalis , comparing it to other endodontic irrigants (2% chlorhexidine liquid and 5.25% sodium hypochlorite). STUDY DESIGN: Eighty roots of human mandibular premolars were prepared by serial instrumentation technique, autoclaved, and contaminated for 7 days with E faecalis monocultures. The roots were then divided into 5 groups according to the irrigant substance used during the standardized biomechanical preparation. In order to evaluate the antimicrobial action of the irrigant substances, 3 microbial samples were taken: initial (before the biomechanical preparation); post-treatment (immediately after the biomechanical preparation), and final (7 days after the biomechanical preparation). The microbiological samples were plated to count the colony-forming units (CFU). RESULTS: The 2% chlorhexidine gel and 2% chlorhexidine liquid significantly reduced the E faecalis CFU in the post-treatment and final microbiological samples. The 5.25% sodium hypochlorite also reduced the E faecalis CFU immediately after the root canal instrumentation, but it was not able to keep the root canal free of detectable E faecalis in the final sample. CONCLUSIONS: The 2% chlorhexidine gluconate (gel and liquid) antimicrobial ability was more effective than 5.25% sodium hypochlorite in keeping low CFU of E faecalis for 7 days after the biomechanical preparation.

Anti-Infective Agents, Local↗
PubMed2006

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages.

The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to H(2)O(2) stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM H(2)O(2). They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by H(2)O(2) was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.

Animals↗
PubMed2006

Prevalence of Enterococcus faecalis at multiple oral sites in endodontic patients using culture and PCR.

There are conflicting data on the occurrence of Enterococcus faecalis in the oral cavity of endodontic patients. This study investigated the prevalence of E. faecalis in multi-site oral samples (n = 136) from 41 endodontic patients using culture and polymerase chain reaction (PCR). Additionally, culturable strains were investigated for virulence traits. Overall, E. faecalis was detected in at least one tongue, oral rinse, or gingival sulcus sample in 68% of patients and in the root canals only in 5% of patients. In 21 patients from whom samples were obtained from all four sites, E. faecalis was detected in more tongue than gingival sulcus, oral rinse, and root canal samples (43, 14, 10, and 10%, respectively; p = 0.0148, chi(2)), and in proportionally greater numbers of patients with gingivitis/periodontitis compared to healthy periodontium (73% versus 20%; p = 0.03, Fisher's exact test). PCR was more sensitive than culture in detecting E. faecalis in oral samples (32% and 4%, respectively; p < 0.0001, McNemar's test). Multiple virulence traits were identified in culturable strains.

Adult↗
PubMed2004

A putative sugar-binding transcriptional regulator in a novel gene locus in Enterococcus faecalis contributes to production of biofilm and prolonged bacteremia in mice.

A biofilm-negative transposon mutant was created from an Enterococcus faecalis strain that produces a lot of biofilm. The transposon had been inserted in the second gene of a locus consisting of 4 open-reading frames, designated bop (biofilm on plastic surfaces). A nonpolar deletion of this gene and of parts of the 2 flanking genes was created; production of biofilm by this deletion mutant was significantly enhanced, compared with that by the wild-type strain. Expression of a downstream gene was significantly lower in the transposon mutant than in the wild-type strain and the biofilm-enhanced deletion mutant. Transformation of this gene into the transposon mutant partially restored production of biofilm. Mice challenged by intravenous injection with the biofilm-negative mutant strain showed significantly reduced numbers of colony-forming units in the blood, compared with mice challenged with the biofilm-enhanced deletion mutant and the wild-type. These results indicate that bop is involved in production of biofilm and probably regulates expression of biofilm in the E. faecalis strain tested.

Animals↗
PubMed1999

Encapsulated Enterococcus faecalis: role of encapsulation in persistence in mouse peritoneum in absence of mouse lethality.

Two nonhemolytic, mucoid, encapsulated strains of Enterococcus faecalis lacked lethality for 23 white mice when inoculated (10(9) cells/mL) intraperitoneally. Bacteremia was short lived (2 to 3 days), but peritoneal cultures remained positive for 7 days postinoculation. Although encapsulation did not result in animal lethality, encapsulation may have delayed peritoneal clearance by interference with phagocytosis.

Animals↗
PubMed2003

Efficacy of chlorhexidine- and calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro.

OBJECTIVE: We sought to assess the efficacy of chlorhexidine (CHX) and calcium hydroxide, Ca(OH)(2), against Enterococcus faecalis in vitro. STUDY DESIGN: The effect of CHX (0.2% and 2% in gel or solution) and Ca(OH)(2) (alone or with 0.2% CHX gel) was evaluated by using the agar diffusion test and an in vitro human root inoculation method, to measure zone of inhibition or bacterial growth with optical density analysis, respectively. For optical density analysis, samples from infected root canals were collected after 7 days of medication and were cultured for 24 hours in brain-heart infusion to detect viable bacteria. RESULTS: In the agar diffusion test, CHX was effective against E faecalis in a concentration-dependent fashion, but Ca(OH)(2) alone had no effect. In the root canal inoculation test, CHX was significantly more effective against E faecalis than Ca(OH)(2) was (P < .05), but there were no significant differences between the modes of medication or concentrations of CHX. CONCLUSIONS: CHX is effective against E faecalis in vitro. Further in vivo studies are needed to confirm the value of CHX in clinical treatment.

Anti-Infective Agents, Local↗
PubMed2003

Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units.

We studied 45 isolates of Enterococcus faecalis with high-level gentamicin resistance (HLGR), all but one concomitantly resistant to ciprofloxacin, and 25 ciprofloxacin-resistant isolates without HLGR for genetic relatedness using pulsed-field gel electrophoresis (PFGE). E. faecalis were isolated from patients admitted to intensive care units at eight hospitals in southern Sweden from December 1996 through December 1998. Genomic analysis by PFGE resulted in three clusters of genetically related isolates (designated clusters I, II and III) and 23 unique clones. Cluster I was found predominantly in the eastern and central parts of southern Sweden and clusters II and III in south-western Sweden. Among the 45 isolates with HLGR, 69% belonged to cluster I, 20% to cluster II, and 11% had unique PFGE patterns, which suggests that the majority of isolates with HLGR are closely related. Among the 25 ciprofloxacin-resistant isolates without HLGR, 68% had unique PFGE patterns, 12% belonged to cluster I and 20% to cluster III, which suggests the ciprofloxacin-resistant isolates are not related. All isolates with HLGR contained the aac(6')Ie-aph(2")Ia gene, which was carried on a Tn5281-like transposon in all isolates except one. We conclude that HLGR in E. faecalis was mainly due to dissemination of genetically related clones during the time studied, and that HLGR in these isolates was due to the presence of the aac(6')Ie-aph(2")Ia gene.

Cross Infection↗