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PubMed2003

Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin.

Enterococcus faecalis is a pathogen that persists in medicated root canals. Here, we tested the hypothesis that the E. faecalis proteases, serine protease and gelatinase, and the collagen-binding protein (Ace) contribute to adhesion to the root canal. Scanning electron microscopy was used to examine dentin binding by four E. faecalis strains: OG1RF, the wild type, and three mutant derivatives of OG1RF, TX5128, TX5243 and TX5256 deficient in serine protease and gelatinase, serine protease, and Ace, respectively. For each strain, 20 root halves were exposed to 3 x 10(9) to 5 x 10(9) cells/ml for 6 h, and 50 fields per root half were examined for adherent bacteria. Statistical analysis revealed that adherence of OG1RF was significantly greater than the mutant strains (P < 0.001), while significant differences were not detected between the protease mutants. The data indicate that serine protease and Ace aid E. faecalis binding to dentin, while the role of gelatinase is uncertain.

Analysis of Variance↗

PubMed2003

Starvation survival, growth and recovery of Enterococcus faecalis in human serum.

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.

Adaptation, Physiological↗

PubMed2003

Linezolid in the treatment of vancomycin-resistant Enterococcus faecium in solid organ transplant recipients: report of a multicenter compassionate-use trial.

Vancomycin-resistant Enterococcus faecium (VRE) is increasing in incidence in solid organ transplant recipients and has a high (up to 83%) associated mortality rate. Until recently, there have been no consistently effective antimicrobial therapies for VRE infection. Linezolid is a new antibiotic that belongs to the class of oxazolidinones approved by the FDA for the treatment of VRE infections, including those with bacteremia. Here, we report the experience with linezolid in an open-label, compassionate-use trial at 53 US centers for the treatment of documented VRE infections in patients with solid organ transplants. Eighty-five patients with solid organ transplants and documented VRE infections were studied. Blood cultures were positive for VRE in 43 patients, while 42 patients had other, non-rectal, sites of infection. Fifty-three patients responded well to treatment, with clinical resolution of the infection (62.4% survival rate). Of these, 47 had documented negative cultures post therapy. The mean duration of therapy for cured patients was 23.5 days. Thirty-two (37.6%) patients died, 28 due to sepsis and organ failure (32.9% failure rate), and 4 due to unrelated causes. Mortality rates for patients with bacteremia were comparable to mortality rates observed with patients who had positive cultures from other sites. Adverse reactions to linezolid included thrombocytopenia (4.7%), decreased leukocyte count (3.5%), and an increase in blood pressure (1.2%), none of which led to discontinuation of therapy. Linezolid appears to be a safe and effective treatment option for VRE, even in the presence of bacteremia, and may lead to decreased mortality in solid organ transplant recipients with VRE infection.

Acetamides↗

PubMed2000

Outbreak of ampicillin-resistant Enterococcus faecium--risk factors for faecal colonisation.

Since January 1995 there has been a nosocomial outbreak at Haukeland University Hospital involving more than 330 patients with clinical infections caused by ampicillin-resistant Enterococcus faecium (ARE) (minimum inhibitory concentration > or =32 mg/l). Rectal carriage of ARE was initially observed on two medical wards only. Here the ARE colonisation rate has remained high. To assess risk factors for ARE colonisation we performed a case-control study including 37 rectal carriers of ARE and 83 non-carriers on these wards. Significant differences were found between cases and controls with respect to the mean number of days on antimicrobial treatment (13.3 for carriers, 5.5 for non-carriers, p<0.001), mean number of different antibiotics prescribed (2.8 for carriers, 2.1 for non-carriers, p= 0.008) and mean number of days in hospital (18.4 vs 10.2, p=0.001). Unadjusted statistical analysis showed that several antibiotics were risk factors for ARE carriage. Logistic regression analysis showed that fluoroquinolone prescription (OR=3.5, p=0.01) and more than 10 days of antibiotic use (OR= 3.3, p=0.01) were significant risk factors. An additional follow-up screening of previous carriers revealed no colonisation 8 to 36 (median 9) months after discharge from hospital (n=17). Prolonged antimicrobial therapy and broad-spectrum antibiotics seem to facilitate nosocomial ARE colonisation.

Ampicillin Resistance↗

PubMed2001

Mechanisms of resistance to imipenem in imipenem-resistant, ampicillin-sensitive Enterococcus faecium.

Enterococcus faecium has six penicillin-binding proteins (PBP), where PBP5 seems to be the main target for beta-lactam antibiotics. The PBP profiles of three imipenem-resistant, ampicillin-sensitive E. faecium strains, isolated from the same patient, were studied using biotinylated ampicillin and chemiluminescence detection. Imipenem resistance in these strains was found to be associated with hyperproduction of PBP5 compared to the ampicillin- and imipenem-susceptible strain ATCC 19434. PBP5 in the imipenem-resistant strains (S1, B2) exhibited a selectively decreased affinity for imipenem. An 854 bp DNA fragment, corresponding to the penicillin-binding domain of pbp5fm, was studied in the resistant strains and the reference strain. Four amino acid substitutions were observed in the resistant strains compared to the susceptible one. The contribution of these substitutions to the increased production of PBP5 in these strains is unclear since the substitution was observed also in a strain without increased production of PBP5. Our results suggest that the moderate imipenem resistance observed in these strains is associated with increased production of PBP5 with relatively decreased affinity for imipenem, and that evolution of imipenem resistance in E. faecium is dinstinct from that of the other beta-lactams such as ampicillin.

Ampicillin↗

PubMed2002

Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis.

Enterococci are members of the healthy human intestinal flora, but are also leading causes of highly antibiotic-resistant, hospital-acquired infection. We examined the genomes of a strain of Enterococcus faecalis that caused an infectious outbreak in a hospital ward in the mid-1980s (ref. 2), and a strain that was identified as the first vancomycin-resistant isolate in the United States, and found that virulence determinants were clustered on a large pathogenicity island, a genetic element previously unknown in this genus. The pathogenicity island, which varies only subtly between strains, is approximately 150 kilobases in size, has a lower G + C content than the rest of the genome, and is flanked by terminal repeats. Here we show that subtle variations within the structure of the pathogenicity island enable strains harbouring the element to modulate virulence, and that these variations occur at high frequency. Moreover, the enterococcal pathogenicity island, in addition to coding for most known auxiliary traits that enhance virulence of the organism, includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates, identifying a class of new targets associated with disease which are not essential for the commensal behaviour of the organism.

Base Composition↗

PubMed2004

Incidence and clinical complications of vancomycin-resistant enterococcus in pediatric stem cell transplant patients.

Vancomycin-resistant enterococcus (VRE) are increasingly important pathogens in stem cell transplant (SCT). In all, 61 pediatric SCT patients had surveillance stool cultures for VRE between July 1999 and November 2002. When VRE was identified, the patients were placed on strict contact isolation. VRE was detected in 15 patients (24.6%). The median age was 3.6 years (range 0.6-18.5 years). Of the 15, 13 (87%) received an allogeneic transplant (six unrelated and seven related). Five of the 15 (33%) colonized patients developed VRE bacteremia. The bacteremia resolved in all five patients after therapy with quinupristin/dalfopristin; three patients required central line removal. Four patients died (38-153 days) post-SCT due to relapse or transplant complication not related to VRE. Of the 11 surviving patients, seven cleared the colonization at a median of 144 days (range 61-198 days) postcolonization. Four patients remain colonized at 68-702 days after the first positive culture. Intestinal colonization with VRE occurred commonly in pediatric SCT patients. Although the morbidity from VRE was not substantial, transplant patients were colonized for prolonged periods. Our results indicate that surveillance for VRE is an effective way to identify colonized patients and may lead to a decrease in transmission to other patients.

Adolescent↗

PubMed2004

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis.

Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix-turn-helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel beta-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.

Bacterial Proteins↗

PubMed1997

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture.

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L-1) was obtained in 9 h with 20 g L-1 glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L-1. Maximum bacteriocin activity (2.9-3.2 MBU L-1) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h-1 and specific bacteriocin production rate increased linearly with dilution rate.

Bacteriocins↗

PubMed2001

Bioactivity of [6R]-5-formyltetrahydrofolate, an unusual isomer, in humans and Enterococcus hirae, and cytochrome c oxidation of 10-formytetrahydrofolate to 10-formyldihydrofolate.

The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H(4)F), is not found in Nature. An oral dose of 13.5 micromol of [6R]-5-HCO-H(4)F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H(4)F) with second-order kinetics and a rate constant of 1.3 x 10(4) M(-1) x s(-1). The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H(2)F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H(4)F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H(4)F from 5-HCO-H(4)F, the unusually large rate constant for the oxidation of 10-HCO-H(4)F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H(4)F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H(4)F to 10-HCO-H(2)F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer.

Cytochrome c Group↗

PubMed2001

Structure-function analysis of purified Enterococcus hirae CopB copper ATPase: effect of Menkes/Wilson disease mutation homologues.

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000-->Arg, changing the conserved Cys-Pro-Cys ('CPC') motif, was mimicked in CopB. The corresponding Cys-396-->Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069-->Gln, was introduced into CopB as His-480-->Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

Adenosine Triphosphatases↗

PubMed1994

Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174.

Vancomycin resistance in enterococci is an increasing clinical problem, and several phenotypes have been identified. We demonstrate here that the resistance mechanism in the constitutively vancomycin-resistant Enterococcus gallinarum BM4174 involves an altered pathway of peptidoglycan synthesis and hydrolysis of the normal precursors in the vancomycin-sensitive pathway. A ligase encoded by the vanC gene catalyses synthesis of D-Ala-D-Ser and substitutes this dipeptide for D-Ala-D-Ala in peptidoglycan precursors. It is presumed that this substitution lowers the affinity of vancomycin for its target site. Destruction of D-Ala-D-Ala (D,D-peptidase activity) and of UDP-MurNAc-L-Ala-D-isoGlu-L-Lys-D-Ala-D-Ala by removal of the terminal D-Ala residue (D,D-carboxypeptidase activity) ensures that the normal vancomycin-sensitive pathway of peptidoglycan synthesis cannot function in the resistant strain.

Amino Acid Sequence↗

PubMed1996

Peptidoglycan structure of Enterococcus faecium expressing vancomycin resistance of the VanB type.

Resistance to glycopeptide antibiotics in enterococci is due to the synthesis of UDP-MurNAc-tetrapeptide-D-lactate (where Mur is muramic acid) replacing the normal UDP-MurNAc-pentapeptide precursor. The peptidoglycan structures of an inducible VanB-type glycopeptide-resistant Enterococcus faecium, D366, and its constitutively resistant derivative, MT9, were determined. Using HPLC, 17 muropeptides were identified and were present regardless of whether resistance was expressed or not. The structures of 15 muropeptides were determined using MS and amino acid analysis. The cross-bridge between D-alanine and L-lysine consisted of one asparagine. No monomer pentapeptide or tetrapeptide-D-lactate could be identified. These results obtained with D366 (non-induced) and MT9 indicate that, in the absence of vancomycin, the cell wall synthetic machinery of E. faecium can process the lactate-containing precursor as efficiently as the normal pentapeptide. In contrast, the presence of subinhibitory inducing concentrations of vancomycin interfered with the synthesis of oligomers.

Amino Acid Sequence↗

PubMed2002

Outbreak of vancomycin-resistant Enterococcus faecium in a haematology unit: risk factor assessment and successful control of the epidemic.

We describe an outbreak of vancomycin-resistant Enterococcus faecium (VRE) on the haematology ward of a Dutch university hospital. After the occurrence of three consecutive cases of bacteraemia with VRE, strains were genotyped and found to be identical. During the next 4 months an intensive surveillance programme identified 21 additional patients to be colonized with VRE, while two more patients developed bacteraemia. A case-control study was carried out to identify risk factors for VRE acquisition. In comparison with VRE-negative control patients (n=49), cases (n=24) had a longer stay on the ward during the year preceding the outbreak (25.8 versus 10.1 d, P=0.02), more cases with acute myeloid leukaemia [11 versus 4, odds ratio (OR) 9.5, 95% confidence interval (CI95) 2.4-32.2] and higher grades of mucositis (P=0.03). Logistic regression analysis identified antibiotic use within 1 month before admission (OR 13.0, CI95 2.1-80.5, P=0.006) and low albumin levels at baseline (OR 1.2, CI95 1.1-1.3, P=0.02) to be independent risk factors. Four patients with VRE-bacteraemia were successfully treated with quinupristin/dalfopristin (Synercid). Control of the outbreak was achieved by step-wise implementation of intensive infection control measures, which included the cohorting of patients, allocation of nurses and reinforcement of hand hygiene.

Adult↗

PubMed1997

Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro.

The effectiveness of 4.0% sodium hypochlorite (NaOCl) used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal was tested in vitro. Root canals contaminated with E. faecalis were treated as follows: (i) irrigation with 2 mL of NaOCl solution and agitation with hand files; (ii) irrigation with 2 mL of NaOCl solution and ultrasonic agitation; (iii) irrigation with NaOCl alternated with hydrogen peroxide. Contaminated canals irrigated with sterile saline solution served as the control. Paper points used to sample bacteria from the root canals were transferred to tubes containing 5 mL of brain heart infusion (BHI) broth. Tubes were incubated and the appearance of broth turbidity was indicative of bacteria remaining in the root canal. There were no statistically significant differences between the experimental groups. However, NaOCl applied by the three methods tested, was significantly more effective than the saline solution (control group) in disinfecting the root canal.

Chi-Square Distribution↗

PubMed1997

Antibacterial activity of calcium hydroxide-containing endodontic sealers on Enterococcus faecalis in vitro.

Antimicrobial activity of endodontic sealers is an important factor in the prognosis of root canal treatment. The aim of this study was to analyse the antimicrobial activity of calcium hydroxide-containing endodontic sealers, Sealapex (SA) and CRCS compared to a ZOE-containing sealer, Roth's cement. The sealers were mixed and placed on the side wall of microtitre plate wells. A 10 microL suspension of Enterococcus faecalis was added to the surface of the sealers 1 h, 24 h, or 7 days after mixture. Bacteria were allowed to come into contact with the sealers for 1 h at 37 degrees C. Fresh media was then added and bacterial growth was measured every 30 min for 16 h. The results showed that in 1-hour-old mixture, CRCS and Roth's cement had a significantly better antimicrobial effect than SA. In 24-hour-old mixtures, ZOE-based sealer showed a more potent antimicrobial activity than calcium hydroxide-containing sealers, whereas SA showed a significantly better antimicrobial effect in the 7-day-old mixture. The antimicrobial activity of each tested sealer changes differently with the time interval between mixing and testing, suggesting different physicochemical properties and potentially diverse clinical applications.

Calcium Hydroxide↗

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

Antilisterial activity of enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 isolated from cheese.

Enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 previously isolated from cheese, exhibited a very narrow spectrum of activity, which is mainly directed against enterococci and Listeria spp. including Listeria monocytogenes. Enterocin 81 activity, which was extremely rapid with maximal effect achieved within 30 min, could not be detected after treatment with various proteolytic enzymes. This activity was bactericidal in nature and induced an important efflux of intracellular material, which was visualized under electron microscopy as filaments coming out of L. monocytogenes cells. However, enterocin 81 did not display bacterial lysis on sensitive cells, as no changes in cell morphology were detected following the bactericidal action. Furthermore, this bacteriocin was shown to be equally active at pH values ranging from 4.0 to 8.0, which, along with the narrow activity spectrum, are two factors of paramount interest with regards to possible use of this bacteriocin in fermented foods.

Bacteria↗