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PubMed2006

Linezolid in prophylaxis against experimental aortic valve endocarditis due to Streptococcus oralis or Enterococcus faecalis.

There are no experimental studies regarding the prophylactic efficacy of linezolid against infective endocarditis. Nonbacterial thrombotic endocarditis of the aortic valve was induced in rabbits by the insertion of a polyethylene catheter. Twenty-four hours later, animals were randomly assigned to a control group, and groups receiving either ampicillin (two doses of 40 mg/kg of body weight each, given intravenously, 2 h apart) or linezolid (a single per os dose of 75 mg/kg). The first dose of ampicillin and the single dose of linezolid were administered 0.5 and 1 h, respectively, prior to the intravenous inoculation of approximately 10(7) CFU of Streptococcus oralis or Enterococcus faecalis. Linezolid peak levels in rabbit serum were similar to the peak serum levels in humans following a 600-mg oral dose of linezolid. Linezolid prevented endocarditis in 87% of S. oralis-challenged rabbits (P < 0.001 versus controls; P = 0.026 versus ampicillin). In rabbits challenged with E. faecalis, linezolid prevented endocarditis in 73% (P = 0.003 versus controls; P = 0.049 versus ampicillin). Ampicillin prevented endocarditis due to S. oralis or due to E. faecalis in 47% (P = 0.005 versus controls) and in 30% (P = not significant versus controls) of the challenged animals, respectively. In conclusion, linezolid was effective as prophylaxis against endocarditis caused by a strain of S. oralis and to a lesser degree against that caused by a strain of E. faecalis. Its prophylactic efficacy was superior to that of ampicillin.

Acetamides↗

PubMedDate not supplied

Potential of Enterococcus faecalis as a human fecal indicator for microbial source tracking.

Regulatory agencies are interested in a fecal indicator bacterium with a host range limited to humans because human fecal contamination represents the greatest hazard to humans, yet is a relatively easy nonpoint source to remedy. Watersheds with human fecal contamination could be given first priority for cleanup. A fecal indicator bacterium with a host range limited to humans and a few other warm-blooded animal species would also simplify microbial source tracking because only a few animal species would be required for any host origin database. The literature suggests that the fecal indicator bacterium Enterococcus faecalis has a limited host range. On this basis, we selected this bacterium for study. Of 583 fecal streptococcal isolates obtained on Enterococcosel agar from Canada goose, cattle, deer, dog, human, chicken, and swine, 392 were considered presumptive enterococci and were subsequently speciated with the API 20 Strep system. Of these isolates, 22 were Ent. durans (5.6%), 61 were Ent. faecalis (15.6%), 98 were Ent. faecium (25.0%), 86 were Ent. gallinarum (21.9%), and 125 were unidentified (31.9%). The host range of the Ent. faecalis isolates was limited to dogs, humans, and chickens. Media were developed to isolate and identify Ent. faecalis quickly from fecal samples and this scheme eliminated Ent. faecalis isolates from dogs. When the remaining Ent. faecalis isolates were ribotyped, it was possible to differentiate clearly among the isolates from human and chicken. It may be that combining the potentially limited host range of Ent. faecalis with ribotyping is useful for prioritizing watersheds with fecal contamination.

Animals↗

PubMed2003

Expression of glycopeptide-resistance gene in response to vancomycin and teicoplanin in the cardiac vegetations of rabbits infected with VanB-type Enterococcus faecalis.

VanB-type resistance in enterococci corresponds to resistance to vancomycin but not to resistance to the related glycopeptide teicoplanin, because the vanB gene cluster is activated by the VanR(B)-VanS(B) 2-component regulatory system in response to vancomycin but not to teicoplanin. Mutations in the vanS(B) gene allow for constitutive or teicoplanin-inducible expression of the resistance genes. To analyze in vivo expression of the van genes in rabbits with experimental endocarditis, a VanB-type Enterococcus faecalis with a transcriptional fusion between the P(YB) promoter of resistance genes and the gfpmut1 gene for the green-fluorescent protein in the chromosome was constructed. Rounded heaps containing fluorescent bacteria were detected in vegetation slides from rabbits treated with vancomycin but not in those from control rabbits, revealing induction of a tightly regulated vanB gene cluster. Teicoplanin-resistant mutants were detected as fluorescent bacteria in rabbits treated with teicoplanin. Thus, the reporter system monitored expression of a glycopeptide-resistance gene in vivo at a single-cell level.

Animals↗

PubMed2007

Bactericidal efficacy of Er,Cr:YSGG laser irradiation against Enterococcus faecalis compared with NaOCl irrigation: an ex vivo pilot study.

AIM: To compare the efficacy of a standard NaOCl irrigation procedure with that of Er,Cr:YSGG laser irradiation in contaminated root canals having small and large apical foramina. METHODOLOGY: Forty root canals of extracted central incisor teeth with straight roots were chosen so that their apical foramina just permitted the tip of a size 20-K file to pass through. The canals were then enlarged with files to size 60 and randomly divided into four groups of 10 teeth each. The apical foramina of one group were widened further so that the tip of a size 45-K file could just pass through. After sterilization, all roots were inoculated with Enterococcus faecalis for 48 h at 37 degrees C. The first group was used as a control, the second group was irrigated with 3% NaOCl solution for 15 min, and the last two groups having different sizes of apical foramina were irradiated with the Er,Cr:YSGG laser at output power from 0.5 W, with 20% air and water levels. The disinfecting efficacy of the groups was tested by collecting dentine chips from the inner canal walls of the specimens and counting viable E. faecalis on Mueller-Hinton agar plates. RESULTS: The differences in the mean number of viable colonies between the control and laser groups were statistically significant (P < 0.05). The control specimens had the highest number of microorganisms (153 x 10(3) +/- 39 x 10(3)). Complete sterilization was achieved in the 3% NaOCl group. The mean colony forming units (CFU) values obtained after Er,Cr:YSGG laser irradiation were 6.6 x 10(3) CFU and 6.5 x 10(3) CFU in root canals having large and small apical foramina respectively. CONCLUSION: In teeth with straight roots the Er,Cr:YSGG laser reduced the viable microbial population in root canals with small and large apical foramina but did not eradicate all bacteria. Three percent NaOCl inhibited the growth of E. faecalis and effectively sterilized all root canals.

Chromium↗

PubMed1989

Postantibiotic effect of penicillin plus gentamicin versus Enterococcus faecalis in vitro and in vivo.

A persistent suppression of bacterial growth following a brief exposure to an antibiotic (postantibiotic effect [( PAE]) has been described for a variety of antibiotics and microorganisms. If a PAE is present in vivo, antibiotic levels in tissue at the site of infection may decrease below the MIC without bacterial regrowth in the latter portion of the dosing interval. In the present studies, a PAE was sought in vitro and in vivo for penicillin G plus gentamicin versus Enterococcus faecalis. The studies demonstrated that increasing concentrations of gentamicin caused an increased rate of bactericidal action and an increasingly prolonged PAE in vitro. The combination of penicillin and gentamicin, in addition to more rapid killing, exhibited a more prolonged PAE than did penicillin alone. However, unlike these in vitro findings, no PAE could be demonstrated in vivo in rats with experimental left-sided enterococcal endocarditis treated with penicillin plus gentamicin. This suggests that antibiotic vegetation levels should be maintained above the MIC throughout the dosing interval to prevent loss of efficacy as a result of bacterial regrowth.

Animals↗

PubMed2001

Expression of the major heat shock proteins DnaK and GroEL in Streptococcus pyogenes: a comparison to Enterococcus faecalis and Staphylococcus aureus.

One of the outstanding problems in the field of heat shock response has been to elucidate the mechanism underlying the induction of heat shock proteins (HSPs). In this work, we initiate an analysis of the expression of heat shock groEL and dnaK genes and their promoters in S. pyogenes. The synthesis of total cellular proteins was studied upon transfer of a log-phase culture from 37 degrees C to 42 degrees C by performing 5-min pulse-labeling experiments with (35)S-Met. The heat shock responses in the pathogenic Gram-positive cocci, Enterococcus faecalis and Staphylococcus aureus, were also analyzed.

Bacterial Proteins↗

PubMed2006

Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis.

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg(2+)-citrate, Ca(2+)-citrate or Fe(3+)-citrate. The Fe(3+)-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca(2+)-citrate complex with an affinity constant of 3.5 microm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca(2+)-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe(3+)-citrate transporter, no transport activity with Fe(3+) (or Fe(2+)) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca(2+), Sr(2+), Mn(2+), Cd(2+) and Pb(2+) and not with Mg(2+), Zn(2+), Ni(2+) and Co(2+). The specificity appears to correlate with the size of the metal ion in the complex.

Bacterial Proteins↗

PubMed2005

Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity.

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of beta-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.

Amino Acid Sequence↗

PubMed2000

Evaluation of chlorhexidine and povidone iodine activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis using a surface test.

Most published studies of the activity of biocides against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) have been based on suspension tests. This study was undertaken to provide information on the effect of chlorhexidine and povidone iodine on bacteria dried on to surfaces, a situation in which biocide activity is known to be reduced. The inactivation of MRSA (10 strains), methicillin-sensitive Staphylococcus aureus (MSSA, 10 strains), VRE (nine strains) and vancomycin-sensitive Enterococcus faecalis (VSE, 10 strains) by 0.5% aqueous chlorhexidine gluconate or 10% povidone iodine was evaluated by applying the European surface test method. Povidone iodine was equally active against resistant and sensitive strains of both species with microbicidal effects (ME), i.e. the log(10)concentration of micro-organisms compared with controls treated with distilled water, after 1.5 min of 3.14 and 3.49 for VRE and VSE respectively, and 3.47 and 3.78 for MRSA and MSSA. Chlorhexidine was equally active against VRE and VSE (ME 3.37 vs. 3. 56 after 7 min, respectively), but was significantly less active against MRSA as opposed to MSSA (ME 3.07 vs. 3.83 after 10 min, P= 0. 017).

Anti-Infective Agents, Local↗

PubMed2006

Cell preparation of Enterococcus faecalis strain EC-12 prevents vancomycin-resistant enterococci colonization in the cecum of newly hatched chicks.

The use of antimicrobials in broilers is considered to be a cause of the appearance of vancomycin-resistant enterococci (VRE). Once VRE penetration occurs, whatever its origin, it is difficult to expel the enterococci from the intestine because of their multiple resistance, whether natural or acquired. In this study, we evaluated the prevention of VRE colonization by the dietary supplementation of a cell-wall preparation of Enterococcus faecalis strain EC-12 (EC-12) in newly hatched broilers that were challenged by experimental infection with VRE. The chicks were fed a basal diet supplemented with 0.05% (wt/wt) EC-12 powder for 15 d. The control group and that administered Lactobacillus sp. were fed the basal diet. The VRE challenge was administered orally when the chicks were 2 d old (d 0). Dietary EC-12 reduced VRE colonization in the intestine from d 3 to 14. Total IgA in the cecal digesta and total IgG in the serum were higher on d 14 in the EC-12 treatment group. However, VRE-specific and EC-12-specific antibodies were not affected in serum. Hence, it appeared that dietary EC-12 stimulated the gut immune system and reinforced the immune reaction against the VRE challenge to accelerate its defecation from the chick intestine.

Animals↗

PubMed1998

Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth.

Entry into the viable but nonculturable (VNC) state is a survival mechanism that bacteria can adopt when they find themselves in an adverse environment. When in this state, bacteria are still viable but are unable to form colonies on growth medium. The possibility of Gram-positive species entering the VNC state when environmental conditions are adverse and remaining viable and capable of resuming active growth is demonstrated for the first time in this study by using exponential-phase cultures of Enterococcus faecalis inoculated in filtered, sterilized water from Lake Garada (Italy). Over the 60-day study, the number of total cells stained with a fluorescent or counted with a Coulter Counter remained constant, while the number of cells capable of forming colonies on Tryptic Soy Agar (TSA) declined rapidly from 10(6) CFU/ml on day 0 to 10(3) CFU/ml on day 4. On day 14 no colonies could be observed when 50 ml of inoculated lake water were plated. E. faecalis cells conserved their viability while in the VNC state, as can be demonstrated by active uptake of amino acids, which are also incorporated into proteins, and by continuous detection of E. faecalis specific DNA by PCR throughout the experiment. The possibility of revival of the E. faecalis cells in the VNC state when returned to conditions supporting its cell growth has also been demonstrated. The data obtained in this study lend further support to recent criticisms of the traditional methods used to evaluate water quality based on plate counts, assessing fecal contamination indicators such as Escherichia coli and fecal streptococci.

Colony Count, Microbial↗

PubMed1996

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Amino Acid Sequence↗

PubMed2005

Persistence of Enterococcus faecalis in aquatic environments via surface interactions with copepods.

Several human pathogens and fecal-pollution indicators may persist as viable organisms in natural environments, owing to their ability to activate different types of survival strategies. These strategies include adhesion on both abiotic and biotic surfaces and the entrance to the so-called viable but nonculturable (VBNC) state. In an 18-month survey for the detection of enterococci in both lake water and seawater, C. Signoretto et al. (Appl. Environ. Microbiol. 70:6892-6896, 2004) have shown that Enterococcus faecalis was detected mostly bound to plankton and in the VBNC state. In the present study, we show that in vitro adhesion of E. faecalis to copepods accelerated the entry of cells into the VBNC state relative to that of planktonic bacteria. VBNC E. faecalis cells maintained their adhesive properties to copepods and chitin (the main component of the copepod carapace), though to a reduced extent in comparison with growing cells. Sugar competition experiments showed interference with adhesion to both copepods and chitin by GlcNAc and only to copepods by D-mannose. Four enterococcal cell wall proteins present in both growing and VBNC cells and lipoteichoic acid were shown to be capable of binding chitin. The results indicate that copepods may represent an additional environmental reservoir of enterococci, thus suggesting the advisability of redesigning the protocols currently used for microbial detection during the evaluation of the microbiological quality of environmental samples.

Bacterial Adhesion↗

PubMed2004

The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase.

Bacterial growth as a biofilm on solid surfaces is strongly associated with the development of human infections. Biofilms on native heart valves (infective endocarditis) is a life-threatening disease as a consequence of bacterial resistance to antimicrobials in such a state. Enterococci have emerged as a cause of endocarditis and nosocomial infections despite being normal commensals of the gastrointestinal and female genital tracts. We examined the role of two-component signal transduction systems in biofilm formation by the Enterococcus faecalis V583 clinical isolate and identified the fsr regulatory locus as the sole two-component system affecting this unique mode of bacterial growth. Insertion mutations in the fsr operon affected biofilm formation on two distinct abiotic surfaces. Inactivation of the fsr-controlled gene gelE encoding the zinc-metalloprotease gelatinase was found to prevent biofilm formation, suggesting that this enzyme may present a unique target for therapeutic intervention in enterococcal endocarditis.

Adaptation, Physiological↗

PubMed2004

Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts.

OBJECTIVES: Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors. METHODS: We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells. RESULTS: Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed. CONCLUSION: The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.

Adult↗

PubMed2000

mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm.

The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of Enterococcus faecalis pbp5 mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.

Acclimatization↗

PubMed1997

Alterations in the chemical composition of peptidoglycan of Escherichia coli DH5 alpha induced by the expression of Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of membrane proteins involved in penicillin resistance in Enterococci and other micro-organisms. This PBP is thought to be capable of taking over the activity of all other PBPs during peptidoglycan synthesis. Unfortunately, nothing is known about the enzymatic activity catalyzed by this PBP, but a transpeptidase/transglycosylase action can be postulated to allow complete peptidoglycan synthesis. Recently, we cloned and expressed in Escherichia coli the PBP5 (a low-affinity PBP) of Enterococcus faecalis (Signoretto, C., Boaretti, M., and Canepari, P.: FEMS Microbiol. Lett. 123, 99-106, 1994). Here we describe some of the effects of this PBP when expressed in E. coli, in terms of increased growth rate and autolysis, and particularly its effects on the fine chemical composition of the E. coli peptidoglycan. A distinct increase in the di- and tripeptide monomers and a parallel decrease in the tetrapeptide monomer are described. The results presented here are explained in terms of a partial action of the postulated transpeptidase/ transglycosylase enzymatic complex which leads to the cleavage of one, two or three amino-acids from the pentapeptide monomer, but is incapable of performing the cross-linking between two side-chains due to lack of the natural substrate which is different from that of E. coli.

Amino Acid Sequence↗

PubMed2002

Formation of vegetations during infective endocarditis excludes binding of bacterial-specific host antibodies to Enterococcus faecalis.

Infectious endocarditis is a microbial infection of the endothelial lining of the heart that typically occurs on damaged or prosthetic heart valves. The characteristic lesion seen with infective endocarditis, termed "the vegetation," is composed of bacteria surrounded by a platelet/fibrin layer attached to the underlying endothelium. The vegetation has long been believed to exclude or hinder host defenses from clearing bacteria, although formal demonstration of mechanisms by which this occurs are lacking. This study investigated the ability of the vegetation to exclude host antibodies specific for the bacterial surface protein aggregation substance in vivo during experimental endocarditis caused by Enterococcus faecalis. The results demonstrate that, once the vegetation encloses bacteria, they are no longer accessible to high-titer bacterial-specific host antibodies, establishing a mechanism by which the vegetation functions to protect the bacteria from the humoral immune response.

Animals↗