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PubMed1991

Bacteremia caused by hemolytic, high-level gentamicin-resistant Enterococcus faecalis.

Between 1 January 1984 and 31 December 1987, 206 enterococcal blood isolates at the University of Wisconsin Hospital and Clinics were analyzed for high-level aminoglycoside resistance (hereafter high-level aminoglycoside resistance is simply referred to as "resistance") and hemolysin production. Of 190 Enterococcus faecalis isolates, 68 (35.8%) were resistant to gentamicin. Of these 68 strains, 67 (98.5%) contained a gene coding for the bifunctional aminoglycoside-modifying 6'-aminoglycoside acetyltransferase-2"-aminoglycoside phosphotransferase [AAC(6')-APH(2")] enzyme. Of 190 isolates, 85 (44.7%) were hemolytic and contained a gene coding for component A of the enterococcal hemolysin. Sixty-two of 68 (91.2%) gentamicin-resistant isolates but only 23 of 122 (18.8%) gentamicin-susceptible isolates were hemolytic (P less than 0.001). Twelve of the hemolytic, gentamicin-resistant E. faecalis blood isolates, but only 2 of 9 nonhemolytic or gentamicin-susceptible isolates, had identical chromosomal DNA restriction endonuclease digestion patterns, suggesting a common derivation for these strains. A historical cohort study from 1 July 1985 to 31 March 1987 identified by regression analysis postsurgical intensive care unit status (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.1 to 22.8) and prior treatment with an expanded- or broad-spectrum cephalosporin (OR, 3.0; 95% CI, 0.9 to 10.1) as risk factors for gentamicin-resistant E. faecalis bacteremia. Patients with hemolytic, gentamicin-resistant E. faecalis bacteremia had a fivefold-increased risk for death within 3 weeks of their bacteremia compared with patients with nonhemolytic, gentamicin-susceptible strains (95% CI, 1.0 to 25.4).

Bacteremia↗

PubMed2005

The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis.

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.

Bacterial Proteins↗

PubMed2004

In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on Enterococcus faecalis.

Several studies have shown a higher success rate of root canal therapy when the canal is free from bacteria at the time of obturation. Treatment strategies that are designed to eliminate this microflora should include agents that can effectively disinfect the root canal. Enterococcus faecalis is often associated with persistent endodontic infections. While in vivo studies have indicated calcium hydroxide to be the most effective all-purpose intracanal medicament, iodine potassium iodide (IKI) and chlorhexidine (CHX) may be able to kill calcium hydroxide-resistant bacteria. Supplementing the antibacterial activity of calcium hydroxide with IPI or CHX preparations was studied in bovine dentine blocks. While calcium hydroxide was unable to kill E. faecalis in the dentine, calcium hydroxide combined with IKI or CHX effectively disinfected the dentine. The addition of CHX or IKI did not affect the alkalinity of the calcium hydroxide suspensions. It may be assumed that combinations also have the potential to be used as long-term medication. Cytotoxicity tests using the neutral red method indicated that the combinations were no more toxic than their pure components.

Animals↗

PubMed2004

Aerosolization of Mycoplasma synoviae compared with Mycoplasma gallisepticum and Enterococcus faecalis.

In order to study the airborne transmission of an arthropathic strain of Mycoplasma synoviae, preliminary aerosol experiments were performed. They were conducted in duplicate in an empty isolator (1.3 m3) to assess the yield and viability of M. synoviae with time compared with Mycoplasma gallisepticum and Enterococcus faecalis. After aerosol generation air samples were taken with two different devices using gelatine or cellulose nitrate filters. There was no difference between the devices, but cellulose nitrate filters yielded very low bacterial counts. The aerosolized dose per isolator for M. synoviae was 3.4 x 10(10) colony-forming units (cfu), for M. gallisepticum was 2.6 x 10(10) cfu and for E. faecalis was 3 x 10(10) cfu. Immediately after aerosolization, concentrations of about 10(6) to 10(7) cfu/m3, 10(7) to 10(8) cfu/m3 and 10(8) to 10(9) cfu/m3 air of M. synoviae, M. gallisepticum and E. faecalis were found, respectively. At 25 min M. synoviae concentrations dropped below the detection level (<4 x 10(4) cfu), while 10(5) to 10(6) and 10(8) to 10(9) cfu were found for M. gallisepticum and E. faecalis, respectively. The average M. synoviae concentration during the experiment was estimated at 10(2) to 10(3) cfu/l. The M. gallisepticum and E. faecalis aerosol generated an average of approximately 10(3) to 10(4) cfu/l air and 10(5) to 10(6) cfu/l air, respectively. Thus mycoplasma and E. faecalis aerosols were successfully generated despite considerable initial loss as measured by culture. The loss was greater in the mycoplasma aerosols, especially those of M. synoviae.

Aerosols↗

PubMed1998

Enterococcus faecalis glutathione reductase: purification, characterization and expression under normal and hyperbaric O2 conditions.

Glutathione reductase is found ubiquitously in eukaryotes and Gram-negative bacteria, and plays a significant role in bacterial defense against oxidative stress. Glutathione reductase from the Gram-positive bacterium Enterococcus faecalis was purified to homogeneity using anion exchange, hydrophobic interaction, and affinity chromatography. The homogeneous 49-kDa enzyme contained 1 mol bound FAD per subunit. The determined N-terminal amino acid sequence of the E. faecalis enzyme displays significant identity with glutathione reductases from other Gram-negative and Gram-positive bacteria, as well as yeast and human erythrocyte reductases. The kinetic mechanism is ping-pong, and the determined kinetic parameters exhibited by the E. faecalis glutathione reductase are similar to those found for glutathione reductases from yeast, Escherichia coli, and human erythrocyte. A two-fold increased expression of glutathione reductase activity and a three-fold induction of glutathione peroxidase activity were observed under hyperbaric O2 growth conditions without a corresponding change in the total glutathione and soluble thiol content. The difference in the expression of the enzyme, and its cognate substrate's intracellular concentration, under these conditions suggest that the gene encoding glutathione reductase is responsive to oxygen concentration, but that the genes encoding the glutathione synthesizing enzymes are not linked to an oxygen-sensitive promoter.

Amino Acid Sequence↗

PubMed2006

Impact of Enterococcus faecalis on the bactericidal activities of arbekacin, daptomycin, linezolid, and tigecycline against methicillin-resistant Staphylococcus aureus in a mixed-pathogen pharmacodynamic model.

We inoculated an in vitro pharmacodynamic model simultaneously with clinical isolates of methicillin-resistant Staphylococcus aureus and an enterocin-producing enterococcus (vancomycin-resistant Enterococcus faecalis, ampicillin susceptible) at 7 log10 CFU/ml to examine enterocin effects and antimicrobial activity on staphylococci. The investigated antimicrobial regimens were 100 mg arbekacin every 12 h (q12h), 6 mg daptomycin per kg of body weight/day, 600 mg linezolid q12h, and 100 mg tigecycline q24h alone and in combination (daptomycin, linezolid, and tigecycline) with arbekacin. Simulations were performed in triplicate; bacterial quantification occurred over 48 h, and development of resistance was evaluated throughout. When we evaluated the impact of antimicrobial activity against S. aureus alone, daptomycin demonstrated bactericidal activity (>or=3 log10 CFU/ml kill), whereas arbekacin, linezolid, and tigecycline displayed bacteriostatic activities (<3 log10 CFU/ml kill). In the mixed-pathogen model, early and distinctive stunting of S. aureus growth was noted (1.5 log CFU/ml difference) in the presence of enterocin-producing E. faecalis compared to growth controls run individually (P=0.02). Most noteworthy was that in the presence of enterocin-producing E. faecalis, bactericidal activity was observed with arbekacin and tigecycline and with the addition of arbekacin to linezolid. Antagonism was noted for the combination of tigecycline and arbekacin against S. aureus in the presence of enterocin-producing E. faecalis. Our research demonstrates that the inhibitory effect of E. faecalis contributed significantly to its overall antimicrobial impact on S. aureus. This contribution was enhanced or improved compared to the activity of each antimicrobial alone. Further research is warranted to determine the impact of polymicrobial infections on antimicrobial activity.

Acetamides↗

PubMed1995

The closely related ermB-ermAM genes from Clostridium perfringens, Enterococcus faecalis (pAM beta 1), and Streptococcus agalactiae (pIP501) are flanked by variants of a directly repeated sequence.

The Clostridium perfringens macrolide-lincosamide-streptogramin B resistance gene, ermBP, was sequenced and shown to be identical to the ermB-ermAM gene from the promiscuous Enterococcus faecalis plasmid pAM beta 1 and to have at least 98% nucleotide sequence identity to other ermB-ermAM genes. Flanking the ermBP structural gene were almost identical directly repeated 1,341-bp sequences (DR1 and DR2). These repeats potentially encoded a 298 (or 284)-amino-acid protein that had sequence similarity to chromosomal and plasmid partitioning proteins. The pAM beta 1 and Streptococcus agalactiae (pIP501) erm determinants appeared to have DR2 but had similar internal 973- or 956-bp deletions in DR1, respectively. Some of the other ermB-ermAM class determinants had small portions of DR1, but none had complete copies. It is postulated that the C. perfringens ermBP determinant was derived from an enterococcal or streptococcal determinant that had complete copies of both DR1 and DR2.

Amino Acid Sequence↗

PubMed2002

Enterococcus faecalis exacerbates burn injury-induced host responses in rats.

Pathophysiology of burn injury with complications of gram-positive infections is not well characterized. We have developed an in vivo rat model to study the effects of burn injury along with intra-abdominal inoculation of Enterococcus faecalis. We hypothesized that although burn injury or E. faecalis inoculation by itself may not induce significant pathophysiological responses, the combination of the two can lead to adverse pathophysiological consequences. Sprague-Dawley rats were divided into 4 groups: group 1(C), controls; group 2(B), burn injury on 30% total body surface area; group 3(EF), intra-abdominal implantation of bacterial pellet impregnated with E. faecalis; group 4(B+EF), burn injury plus bacterial pellet implantation. The mortality was 25% and 60% on day 1 and 2 in Group 4(B+EF), respectively; no significant mortality was observed in other groups. In group 4(B+EF), metabolic acidosis, respiratory alkalosis, and a hyperdynamic state developed on day 1, and metabolic and respiratory acidosis and a hypodynamic state on day 2. There were no significant alterations in metabolic or hemodynamic measurements in other groups. Intestinal microvascular permeability to albumin on day 1 and 2 was increased in group 4(B+EF). In group 2(B), microvascular permeability was not increased significantly. Although the permeability was increased on day 1 in group 3(EF), it declined on day 2. The metabolic and hemodynamic alterations were correlated with increased intestinal microvascular permeability to albumin. E. faecalis appeared to be involved in initiating a vicious cycle of burn injury-mediated disruption of intestinal integrity along with metabolic and hemodynamic derangements.

Albumins↗

PubMed2002

The Enterococcus faecalis fsrB gene, a key component of the fsr quorum-sensing system, is associated with virulence in the rabbit endophthalmitis model.

We used a rabbit endophthalmitis model to explore the role of fsrB, a gene required for the function of the fsr quorum-sensing system of Enterococcus faecalis, in pathogenicity. A nonpolar deletion mutant of fsrB had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of fsrB in E. faecalis pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.

Animals↗

PubMed2004

Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication.

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.

Amino Acid Sequence↗

PubMed1999

Effects of the addition of Enterococcus faecalis in Cebreiro cheese manufacture.

Ten batches of Cebreiro, a fresh or short-ripened acid-curd cheese, produced in the Galician mountains (NW Spain) were prepared from pasteurized milk inoculated with microorganisms isolated from raw-milk cheese. Two control batches were made with a Lactococcus lactis subsp. lactis starter; 8 batches were made with the lactococcal starter plus one of eight Enterococcus faecalis cultures: 4 E. faecalis var. liquefaciens (EFLB) and 4 E. faecalis var. faecalis (EFFB). Whey dry matter in the EFLB was notably higher than in the control batches and this was related to lower cheese yields. After over 15 days storage the highest counts of both aerobic bacteria and lactic acid bacteria were observed for the EFLB. The lower content in protein on dry matter was found in the EFLB. The beta-casein broke down to a greater extent in the EFLB than in the EFFB, the lowest values being obtained for the control batches. The higher level of hydrolysis of alphax1-casein and maximum peptide alpha(s1) - I/alpha(s1)-casein ratio were obtained for EFFB at day 15 of storage. In all the batches made with enterococci soluble nitrogen was higher than in the control batches, with the highest values in the EFLB. In all the batches made with enterococci, volatile free fatty acid, long-chain free fatty acids and diacetyl and acetoin contents at days 10 and 15 of storage were higher than in the control batches, the highest values being obtained for EFLB. Acetic acid in all batches accounted for the main proportion of the volatile free fatty acids. Butyric and caproic acids were not detected in the volatile free fatty acids fractions of the control batches, but both acids were detected in most of the batches made with enterococci. The more intense acid taste was found in the EFFB and control batches, the most bitter taste being found in the EFLB. Buttery, rancid and spicy flavors were more evident in the EFLB. The rancid and spicy flavors were positively correlated with the contents of volatile free fatty acids and long-chain free fatty acids. The cheeses of EFLB proved to be more crumbly than the EFFB, whereas the stickiness and deformability were higher in the EFFB. The batches with similar organoleptic characteristics to those of traditional cheese were the batch IV made with the less proteolytic strain of E. faecalis var. liquefaciens, and the batch VI made with a moderate lipolytic activity strain of E. faecalis var. faecalis.

Animals↗

PubMed2006

Horizontal transfer of tet(M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats.

Two wild-type strains of Lactobacillus plantarum previously isolated from fermented dry sausages were analysed for their ability to transfer antibiotic resistance plasmids in the gastrointestinal tract. For this purpose, we used gnotobiotic rats as an in vivo model. Rats were initially inoculated with the recipient Enterococcus faecalis JH2-2 at a concentration of 10(10) CFU mL(-1). After a week, either of the two donors L. plantarum DG 522 (harbouring a tet(M)-containing plasmid of c. 40 kb) or L. plantarum DG 507 [harbouring a tet(M)-containing plasmid of c. 10 kb and an erm(B)-containing plasmid of c. 8.5 kb] was introduced at concentrations in the range of 10(8)-10(10) CFU mL(-1). Two days after donor introduction, the first transconjugants (TCs) were detected in faecal samples. The detected numbers of tet(M)-TCs were comparable for the two donors. In both cases, this number increased to c. 5 x 10(2) CFU g(-1) faeces towards the end of the experiment. For erm(B)-TCs, the number was significantly higher and increased to c. 10(3) CFU g(-1) faeces. To our knowledge, this is the first study showing in vivo transfer of wild-type antibiotic resistance plasmids from L. plantarum to E. faecalis.

Animals↗

PubMed1999

Interferon-gamma effects on activities of gentamicin and vancomycin against Enterococcus faecalis resistant to the drugs: an in vitro study with human neutrophils.

The emergence of multidrug-resistant enterococci presents a major therapeutic challenge since there is currently no clearly effective antimicrobial therapy for these infections. The combinatorial effects of interferon-gamma (IFN-gamma) with gentamicin and/or vancomycin against a clinical isolate of drug-resistant Enterococcus faecalis, were evaluated in an in vitro system with human neutrophils. Following inoculation of cultures of human neutrophils with the organism, treatments were initiated immediately after the infection and the number of viable bacteria was determined at 12, 18 and 24 h. Antibiotics were applied at concentrations close to their clinically achievable serum trough and peak levels. Treatment with IFN-gamma alone induced a maximal growth inhibition of up to 40% at a concentration of 100 U/ml. Addition of the cytokine to either therapeutic trough or peak concentrations of gentamicin and vancomycin, or a combination of both antimicrobials, was associated with a significant (P < 0.01) enhancement of anti-enterococcal activity compared with the effects of the agents alone. Investigation of a potential underlying mechanism of anti-enterococcal action of IFN-gamma reveals that it is, most probably, largely due to an activated secretion of the microbicidal reactive oxygen intermediates by neutrophils. The results of this study show that there is a possibility that IFN-gamma could be a useful adjunct in the treatment of multidrug-resistant E. faecalis.

Combined Modality Therapy↗

PubMedDate not supplied

Enterococcus faecalis: in vitro activity of antimicrobial drugs, singly and combined, with and without defibrinated human blood, against high-level-gentamicin-resistant isolates.

Teicoplanin (4 micrograms/ml) combined with 4 micrograms/ml of ampicillin, mezlocillin, and piperacillin, respectively, proved more efficacious than vancomycin (4 micrograms/ml) combined with the three beta-lactam antibiotics against three representative clinical isolates of Enterococcus faecalis that showed high-level-gentamicin resistance (HLGR). In the presence of 65% (v/v) of fresh defibrinated human blood, teicoplanin (4 micrograms/ml) alone afforded significant bactericidal activity, but failed to completely sterilize assay tube contents, whereas teicoplanin combined with any of the three beta-lactam antibiotics consistently and completely killed HLGR E. faecalis inocula.

Ampicillin↗

PubMed1992

Sequence analysis of the beta-lactamase repressor from Staphylococcus aureus and hybridization studies with two beta-lactamase-producing isolates of Enterococcus faecalis.

The putative beta-lactamase (Bla) repressor gene, blaI, from the staphylococcal plasmid pI524 was isolated, and the DNA sequence was determined. The sequence of blaI was found to be identical to the blaI sequence from pI9789 (blaI blaZ seg-1), a plasmid related to pI524. A blaI probe from pI524 was hybridized with plasmid and genomic DNA from Bla+ Enterococcus faecalis isolates HH22 and PA. The Bla structural gene of HH22 has been previously shown to be of staphylococcal origin, but DNA homologous to the staphylococcal Bla repressor was not found, indicating that the constitutive production of beta-lactamase in these E. faecalis isolates may be the result of a missing repressor protein.

Base Sequence↗

PubMed2000

Enterococcus faecalis adhesin, ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I.

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46 degrees C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46 degrees C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single approximately 105-kDa band of mutanolysin extracts from OG1RF grown at 46 degrees C, while no band was detected in extracts from OG1RF grown at 37 degrees C, nor from the OG1RF ace mutant grown at 37 or 46 degrees C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 microg/ml, and also inhibited the 46 degrees C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

Animals↗

PubMed2005

Isolation of Enterococcus faecalis from a case of nonpuerperal breast abscess.

Nonpuerperal breast abscess (NPBA) has different etiology as compared to the mastitis occurring in post partum women. The condition presents either as acute suppurative infection or chronic type. Organisms usually implicated are Staphylococcus aureus, coagulase negative staphylococci, and anaerobes. Mostly the infection is polymicrobial in nature. Herein, we report the isolation of Enterococcus faecalis from a case of acute suppurative NPBA.

Abscess↗

PubMed2000

Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing.

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 has been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcus faecalis to serve as a donor for the conjugative transfer of transposon Tn916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of > or =6 x 10(-8) per recipient CFU. To determine the transposon insertion site, an oligonucleotide primer corresponding to the 3' end of Tn916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown to insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepticum genome. The ability to determine transposon insertion sites in mycoplasmas by genomic sequencing has not been previously described and allows rapid sequence analysis of transposon-generated mutants.

Animals↗