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PubMed2006

The antimicrobial effect of MTAD, sodium hypochlorite, doxycycline, and citric acid on Enterococcus faecalis.

This study compared the antimicrobial effect of MTAD, two of its components, doxycycline and citric acid, and sodium hypochlorite (NaOCl) in two in vitro models on Enterococcus faecalis. In the bovine tooth model, the lumens of 30 bovine dentin discs were infected with E. faecalis for 2 weeks before treating with either one of the experimental irrigants or saline. Bacteria in the shavings were collected with two sizes of burs and enumerated after overnight culturing. Zones of inhibition were recorded in the agar diffusion model for each irrigant. In the tooth model, NaOCl and doxycycline were more effective than control in killing E. faecalis at the shallow bur depth, but at the deeper bur depth only NaOCl was superior. In the agar diffusion model, NaOCl produced less inhibition than MTAD or doxycycline.

Animals↗

PubMed2005

A vancomycin-dependent VanA-type Enterococcus faecalis strain isolated in Japan from chicken imported from China.

AIMS: The characterization of KC122.1, which is a vancomycin-dependent VRE (Vancomycin-resistant enterococci) (Enterococcus faecalis) and the first case in Japan of a VRE isolate obtained from chicken meat imported from China. METHODS AND RESULTS: PCR amplification of vanA, vanS and ddl gene and direct sequencing of the PCR products were performed. KC122.1 was a VanA-type VRE showing high-level vancomycin resistance and low-level teicoplanin resistance, and its vanS gene had three point mutations. The ddl gene of KC122.1 was sequenced and two changes were found at the ninth codon (GCC-GAC) and the stop codon (TAA-CAA). The latter change was also found in the laboratory strain E. faecalis FA2-2. CONCLUSIONS: Three point mutations in vanS resulted in high-level vancomycin resistance and low-level teicoplanin resistance. The change at the ninth codon resulted in the inactivation of the ddl gene and vancomycin-dependent growth. An eight amino acid extension at the C-terminal did not impair the function of the D-Ala : D-Ala ligase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first example of the isolation of VRE from chicken meat imported from China and the first vancomycin-dependent VRE from a nonhuman source.

Animals↗

PubMed2001

Study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis in a flock of brown layer chickens.

Enterococcus faecalis-related amyloid arthropathy was diagnosed in a sample of birds from a flock of brown layer parent chickens aged 57 weeks. E. faecalis was isolated from amyloidotic knee joints and from blood samples from lame birds. From this flock a smaller one was kept for production purposes to study the vertical transmission of arthropathic and amyloidogenic E. faecalis. Three batches of non-soiled and thoroughly disinfected eggs, to prevent egg-shell contamination, were collected for 6 weeks and submitted for incubation every 2 weeks. No joint pathology was found in the offspring chicks (n = 458) monitored for joint disease until 16 or 21 weeks of age. Fresh and candled eggs (infertiles and dead embryos) obtained at day 18 of the incubation period had negative results at bacteriological examination of the yolk sac, except one non-hatched egg (late embryonal death in shell) from which E. faecalis was isolated. Genotyping by pulsed-field gel electrophoresis (PFGE) of all E. faecalis isolates obtained from blood and joints (except one) of the parent birds and of the non-hatched egg after Sma I digestion showed that all isolates were genetically closely related or identical to a known arthropathic and amyloidogenic E. faecalis strain. In this study, vertical transmission of E. faecalis, although it may occur on a small scale as shown by PFGE, did not seem to play a significant role. Conversely, the chronicity of the condition and the development of an immune response may have affected the efficiency of its transmission.

Amyloidosis↗

PubMed2005

Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications.

This study aimed to investigate and compare the efficacy of selected root canal irrigants and a medicament on a clinical isolate of Enterococcus faecalis grown as biofilm or planktonic suspension phenotype. A cell-dense pellet "presentation" prepared from planktonic phenotype was also tested. Each bacterial presentation was exposed to calcium hydroxide (pH 12.3), 0.2% chlorhexidine gluconate, 17% ethylene-diamine-tetra-acetic acid, 10% povidone iodine, or 3.0% sodium hypochlorite (NaOCl) for a range of time periods (1, 2, 4, 8, 15, 30, and 60 min). Phosphate buffered saline was used as a control agent. The difference in gradients of bacterial killing among the biofilm, planktonic suspension or pellet presentation was significant (p < 0.05) and dependent upon the test agent except in the case of NaOCl and calcium hydroxide where no difference could be detected. NaOCl was the most effective agent and achieved 100% kills for all presentations of E. faecalis after a 2 min contact time.

Biofilms↗

PubMed2001

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bacterial Adhesion↗

PubMed2006

In vitro evaluation of the antimicrobial effects of a root canal sealer-antibiotic combination against Enterococcus faecalis.

The purpose of this in vitro study was to evaluate the antimicrobial effects of five antibiotics when added to Kerr Pulp Canal Sealer EWT against Enterococcus faecalis. Five antibiotics: amoxicillin, penicillin, clindamycin, metronidazole, and doxycycline, were added separately to Kerr sealer. Thirty brain heart infusion agar plates were inoculated with E. faecalis and sterile paper discs containing a sealer-antibiotic combination were randomly assigned to the inoculated plates. Kerr sealer on a sterile paper disc and a blank sterile paper disc served as the controls. Fifteen plates were incubated aerobically and the remaining 15 plates were incubated anaerobically at 37 degrees C. The zones of inhibition were measured at 48 hours. No significant differences (p > 0.05) were found between aerobic and anaerobic groups. The groups were combined and data analysis using a one-way ANOVA and Tukey's post hoc test was performed. Results revealed that sealer-antibiotic combinations containing amoxicillin, penicillin, clindamycin, and doxycycline had a significant difference (p < 0.001) in the mean zones of inhibition when compared to Kerr EWT sealer alone. No significant differences (p > 0.05) were found between the metronidazole-sealer combinations and Kerr EWT sealer alone.

Aerobiosis↗

PubMed2002

[The efficacy of intraventricularly administered vancomycin in the case of central nervous system infection caused by enterococcus faecalis].

The case of 40 years old man with subarachnoid hemorrhage with intraventricular bleeding and with consecutive cerebro-meningitis is presented. The bacterial pathogen was Enterococcus faecalis sensitive only to glycopeptide antibiotics. The standard therapy with intravenously administered Vancomycin and Teicoplanin was not effective. Because of the worsening of patient's clinical status and clinical symptoms of sepsis the intraventricular Vancomycin (20 mg/day) was introduced. At the second day of the therapy the gradual patient's recovery was observed. The symptoms typical for meningitis diminished as well as cerebro-spinal fluid (CSF) parameters normalized. There was no bacterial growth in the blood serum and in CSF. As we can observe the intraventricular administration of Vancomycin is efficient method of cerebro-meningitis treatment. In our opinion the blood-brain barrier, even pathologically changed by infection, do not allow antibiotics to penetrate CSF, even in the maximal intravenous doses. In the cases of cerebro-meningitis caused by bacteria sensitive only to glycopeptide antibiotics, the intraventricular administration of the drug might be an alternative way of therapy especially when the doses of intravenous antibiotics need to be reduced.

Adult↗

PubMed2004

Enterococcus faecalis pheromone-responsive protein PrgX: genetic separation of positive autoregulatory functions from those involved in negative regulation of conjugative plasmid transfer.

The pCF10 plasmid in Enterococcus faecalis transfers from donor cells to recipients upon induction via peptide pheromone. Two plasmid-encoded negative regulators produced from the same transcript, PrgX protein and Qa RNA, repress conjugation genes in uninduced donor cells. PrgX positively autoregulates production of both itself and mature Qa RNA, and is believed to repress the prgQ promoter in a pheromone-sensitive fashion. Previous analysis of PrgX was complicated because mutations in prgX affecting regulation of conjugation also disrupted PrgX autoregulation, suggesting the two functions might be inseparable. In this study, we isolated 14 single amino acid substitutions in PrgX that reduced or eliminated repression of prgQ, without affecting autoregulation or DNA binding. PrgX was shown to bind to its cognate pheromone, cCF10, and most of the mutations lowered the affinity of PrgX for cCF10. Dimerization was affected by five of the mutations and the data indicate that it is required, but insufficient for pheromone induction. We propose a new model for the mechanism used by PrgX for regulation of the prgQ promoter, PrgX autoregulation, and Qa RNA processing.

Bacterial Proteins↗

PubMed1990

Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1.

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N-terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13,160) acidic protein of unknown function. The gene for aggregation substance (named asa1) was found to code for a protein of 1296 amino acids (Mr 142,248). The protein has a signal peptide of 43 amino acids (the resulting Mr for mature aggregation substance is 137,429) and contains in its C-terminal region a proline-rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram-positive organisms, but no other similarities to surface proteins from Gram-positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1-specific aggregation substance. The protein contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.

Amino Acid Sequence↗

PubMed1990

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↗

PubMed2004

An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid.

Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44-331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10((156-358)) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.

Bacterial Adhesion↗

PubMed2003

Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro.

AIM: To evaluate the effectiveness of 2% chlorhexidine gluconate gel and calcium hydroxide (Ca(OH)2) as intracanal medicaments against Enterococcus faecalis. METHODOLOGY: One hundred and eighty dentine tubes prepared from intact freshly extracted bovine maxillary central incisors were infected in vitro for 7 days with E. faecalis. The specimens were divided into four groups, according to the intracanal medicament used, as follows: Group 1: 2% chlorhexidine gluconate gel; Group 2: calcium hydroxide in a viscous vehicle (polyethyleneglycol 400); Group 3: 2% chlorhexidine gluconate gel + calcium hydroxide and Group 4: Brain Heart Infusion (BHI) broth (control group). The medicaments were placed into the canal lumen and left there for experimental times of 1, 2, 7, 15 and 30 days. After each period, irrigation with sterile saline to remove the medicament was performed and the canals were dried with sterile paper points. Dentine chips were removed from the canals with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing BHI broth. The tubes were incubated at 37 degrees C and daily observed for microbial growth, visualized by the medium turbidity. RESULTS: Chlorhexidine gel alone completely inhibited the growth of E. faecalis after 1, 2, 7 and 15 days. Calcium hydroxide allowed microbial growth at all experimental times. The combination of chlorhexidine and Ca(OH)2 was effective after 1 and 2 days demonstrating 100% antibacterial action; however, its antibacterial activity reduced between 7 and 15 days. CONCLUSION: Under the conditions of this study, it can be concluded that 2% chlorhexidine gel alone was more effective against E. faecalis than calcium hydroxide (P < 0.05). However, its antibacterial activity depended on how long it remained inside the root canal.

Animals↗

PubMed1998

Fatal suppurative mesenteric thrombophlebitis caused by an Enterococcus faecalis highly resistant to aminoglycosides: case report.

A 56-year old man with non-Hodgkin's lymphoma and biliary tract endoprosthesis, developed chronic bacteremia caused by Enterococcus faecalis with high-level resistance to gentamicin and streptomycin. The sources of bacteremia were a device-associated biliary tract infection, a suppurative thrombophlebitis of the confluence of the superior mesenteric vein with the splenic vein as well as multiple liver and pancreatic abscesses. Despite antibiotic therapy and multiple drainages of abscesses, the patient died due to overwhelming infection.

Anti-Bacterial Agents↗

PubMed1996

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Amino Acid Sequence↗

PubMed1994

[Fundamental and clinical studies on MIC breakpoint of SY5555 for Enterococcus faecalis in complicated urinary tract infections of moderate severity].

A multicenter clinical study was carried out to investigate the clinical efficacy of SY5555 from viewpoint of its MIC breakpoint for Enterococcus faecalis. SY5555 was administered in dosages of 150 mg x 3/day and 300 mg x 3/day, for 7 days, to patients with complicated urinary tract infections of moderate severity (no indwelling catheter). Based primarily on the bacteriological efficacy (eradication) as the criterion for evaluation, the MIC breakpoint of SY5555 was investigated. SY5555 showed potent antimicrobial activity towards E. faecalis, and its MIC80 value for the clinical isolates of this bacterial species was 1.56 micrograms/ml. Analysis was carried out to determine the extent to which this in vitro activity is corroborated by the drug's clinical efficacy in eradicating E. faecalis. 1. Experimental study In a study employing an experimental model of complicated bladder infections of moderate severity, it was judged that the MIC breakpoints of SY5555 administered at 150 mg x 3/day and 300 mg x 3/day were 2 micrograms/ml and 8 micrograms/ml. 2. Clinical study SY5555 was administered at 150 mg x 3/day (7 days) to six patients and at 300 mg x 3/day (7 days) to 32 patients, and the clinical efficacy of these treatments was investigated. 2.1. Investigation of the clinical MIC breakpoint showed it to be 12.5 micrograms/ml for both the 150 mg x 3/day and 300 mg x 3/day regimens. However, since the number of cases in this study was small, analysis was performed of the data from a large number of cases by including the cases registered in a nation-wide Phase II study of SY5555. That analysis showed the clinical MIC breakpoint for the two dosages to be 3.13 micrograms/ml and 6.25 micrograms/ml. Thus, this analysis of large number of cases yielded clinical MIC breakpoint that are in agreement with the abovementioned experimental MIC breakpoints. 2.2. In the evaluation of the efficacy rate using the UTI drug efficacy evaluation criteria, cases rated as "good" or "excellent" numbered five of the six patients in the 150 mg x 3/day treatment group and 25 of the 32 (78.1%) patients in the 300 mg x 3/day treatment group, or 30/33 (78.9%) cases overall. These efficacy rates are about the same as the 82.0% reported for the nation-wide Phase II study. 2.3. Each of the strains of E. faecalis isolated from the patients was found to be eradicated by the SY5555 treatment: four strains in the 150 mg dosage group (the MIC of each was 3.13 micrograms/ml or less) and 18 strains in the 300 mg dosage group (the MIC of each was 6.25 micrograms/ml or less). Moreover, in both dosage groups, E. faecalis was not found to recur after the SY5555 treatment. 3. Conclusions In consideration of the above findings in the investigations of treatment of patients with complicated urinary tract infections of moderate severity, the following conclusions can be drawn. 3.1. In antimicrobial treatment employing SY5555 in a regimen consisting of 300 mg x 3/day for 7 days, the clinical breakpoint is in the range of 6.25-12.5 micrograms/ml. 3.2. In particular, as would be surmised from the fact that the MIC80 value of SY5555 for E. faecalis isolated is 1.56 micrograms/ml, this drug shows quite strong efficacy in the eradication of E. faecalis from cases of complicated urinary tract infection.

Adult↗

PubMed1992

Conjugal transfer of plasmid DNA from Enterococcus faecalis to Escherichia coli in digestive tracts of gnotobiotic mice.

We have studied the transfer of the conjugative shuttle plasmid pAT191, which confers resistance to kanamycin, from Enterococcus faecalis to Escherichia coli in the digestive tracts of six gnotobiotic mice. Colonies of E. coli resistant to kanamycin were isolated from the feces of two mice, respectively, on days 25 and 35 after the beginning of the experiment and never thereafter. The transfer frequency of pAT191, expressed as the number of transconjugants per donor cell isolated from intestines of sacrificed mice, was 3 x 10(-9). These results indicate that conjugation is a mechanism that could account for the resistance gene flux from gram-positive to gram-negative bacteria observed in nature.

Animals↗

PubMed1993

Regulation of glycerol metabolism in Enterococcus faecalis by phosphoenolpyruvate-dependent phosphorylation of glycerol kinase catalyzed by enzyme I and HPr of the phosphotransferase system.

Using a polyclonal antibody against glycerol kinase from Enterococcus faecalis, we could demonstrate that glycerol kinase is inducible by growth on glycerol-containing medium and that during growth on glycerol the enzyme is mainly phosphorylated. Glucose and other sugars metabolized via the Embden-Meyerhof pathway strongly repressed the synthesis of glycerol kinase, while if glycerol was also present during growth, low activity, reflecting partial induction and the presence of mainly unphosphorylated, less active enzyme, was found. With gluconate, which is also a substrate of the phosphotransferase system, repression of glycerol kinase was less severe, but the enzyme was mainly present in the less active, unphosphorylated form. Effects of growth on different carbon sources on glycerol uptake are also reported.

Bacterial Proteins↗

PubMed2006

Effect of smear layer and chlorhexidine treatment on the adhesion of Enterococcus faecalis to bovine dentin.

The aim of this in vitro study was to determine the effects of a smear layer and chlorhexidine (CHX) treatment on the adhesion of Enterococcus faecalis to bovine dentin. Forty dentin blocks from bovine incisors were prepared and randomly divided into four groups of 10 each. The blocks in group 1 were placed in sterile saline for 5 minutes, while those in group 2 were treated with 17% EDTA for 5 minutes. The blocks in group 3 were placed in 2% CHX for 7 days. The blocks in group 4 were treated with 17% EDTA for 5 minutes, and then placed in 2% CHX for 7 days. All the blocks were immersed in a suspension of E. faecalis for 3 hours. The bacteria adhering to the dentin surface were counted by examination using a scanning electron microscope. The most significant amount of bacteria was retained on the samples from group 1 (p < 0.05) and the smallest amount of bacteria adhered to the samples from group 4. These results suggest that a smear layer enhances the adherence of E. faecalis to the dentin, and CHX is effective in reducing the adherence of microorganisms.

Analysis of Variance↗