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PubMed2026

Emergence of a Novel, Phenotypically Difficult-to-Detect Vancomycin-Resistant Enterococcus faecium Clone (ST117/CT7799).

A significant increase of vancomycin-resistant Enterococcus faecium (VREfm) infections was observed in South-Eastern Austria since 2024. The prolonged outbreak is caused by a novel vanB-VREfm clone (ST117/CT7799, "VREfmstyr"). This study characterizes the atypical difficult-to-detect resistance phenotype and assesses the genomic relatedness of the isolates. Patient and outbreak characteristics were investigated including whole genome sequencing of the isolates. Sensitivity of broth microdilution (BMD), gradient tests (GT), disk diffusion (DD), and automated susceptibility testing (VITEK2) was compared. The performance of commercial screening media was evaluated. From sporadic detections in early 2024 case numbers began to rise during the year. In 30/31 (97%) of all cases, intra-hospital transmission was considered likely and an association with invasive procedures was identified in most cases. Core genome multilocus sequence typing revealed only six allelic differences between VREfmstyr isolates collected in a 12-month period, all belonging to the E. faecium ST117/CT7799 lineage. BMD detected vancomycin resistance (MIC > 4 mg/L) in no more than 16/31 (52%) of isolates after 24 h incubation, while GT and DD misclassified all isolates. Only prolonged incubation improved the performance of these assays. VITEK2 analysis, however, correctly classified all 31 isolates. Of four commercially available VRE-screening agars, only one was capable of detecting VREfmstyr after 24 h incubation. The emergence and clonal dissemination of VREfm ST117/CT7799 reveals a serious diagnostic gap as commonly used diagnostic algorithms fail to reliably detect this resistance phenotype. Our findings should help to further evaluate the true geographical distribution and clinical significance of this novel VREfm clone.

Enterococcus faecium↗
PubMed2006

Single nucleotide polymorphism analysis of the enterocin P structural gene of Enterococcus faecium strains isolated from nonfermented animal foods.

The bacteriocins produced by two lactic acid bacteria isolated from nonfermented fresh meat and fish, respectively, and exhibiting a remarkable antilisterial activity, were characterized. Bacteriocinogenic strains were identified as Enterococcus faecium and the maximum bacteriocin production by both strains was detected in the stationary phase of growth. The activity against Listeria monocytogenes was maintained in pH range of 3-7 and was stable in both strains after heating at 100 or 121 degrees C. The genes coding for enterocin P were detected, isolated, and sequenced in both E. faecium strains. They exhibited DNA/DNA homology in the 87.1-97.2% range with respect to the other four enterocin P genes reported so far. Three single nucleotide polymorphism events, silent at the amino acid level, were detected at nucleotide positions 45 (G/A), 75 (A/G), and 90 (T/C) in E. faecium LHICA 28-4 and may explain the differences reported for those loci in other enterocin P-producing E. faecium strains. This work provides the first description of enterocin P-producing E. faecium strains in nonfermented foodstuffs and, in the case of E. faecium LHICA 51, the first report of an enterocin P-producing strain isolated from fish so far.

Amino Acid Sequence↗
PubMed1997

Improvement of diffraction quality upon rehydration of dehydrated icosahedral Enterococcus faecalis pyruvate dehydrogenase core crystals.

Members of the family of 2-oxoacid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of alpha-keto acids and are among the most remarkable enzymatic machineries in the living cell. These multienzyme complexes combine a highly symmetric (cubic or icosahedral) core with a dynamic and flexible arrangement of numerous subunits and domains surrounding the core. The center of the complex is formed by either 24 or 60 copies of dihydrolipoamide acetyltransferase (E2)-a multidomain enzyme. The hollow icosahedral cores are composed of 60 identical subunits of the catalytic domain of E2 with a molecular weight of about 1.8 million Da. Bipyramidal crystals suitable for X-ray diffraction of the icosahedral core of the pyruvate dehydrogenase multienzyme complex from Enterococcus faecalis were grown up to 0.7 mm in each dimension. The crystals belong to space group R32 with a = b = 244.3 A (hexagonal setting), and have a solvent content of 73%. The asymmetric unit contains one-third of the molecule, i.e., 20 of the 60 subunits. Initial X-ray crystallographic data to 7 A resolution were collected at cryotemperatures at synchrotron facilities. Interestingly, the diffraction was improved significantly upon rehydrating dehydrated crystals and extended to 4.2 A.

Crystallography, X-Ray↗
PubMed2005

Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis.

The composition of 19 garlic natural health products (NHPs) and fresh garlic extracts were determined, as was their antibacterial activity. The 19 NHPs and 5 fresh garlic extract standards were analysed for their principal active constituents. They were also extracted for 5, 10 or 15 min in water to fresh garlic equivalents of 200 mg/mL. The extract's minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against three indicator microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis) were determined by the broth microdilution method. While 47% of the aqueous garlic NHP extracts exhibited activity against N. gonorrhoeae, only 16% of the aqueous extracts inhibited S. aureus or E. faecalis at all three timepoints. Generally, products with high antimicrobial activity contained higher levels of garlic constituents with comparable activity to fresh garlic extracts, while products with marginal antibacterial activity often contained lower concentrations of constituents than their product labels indicated. Different extraction times affected antibacterial activity only against N. gonorrhoeae and tended to be correlated with levels of allicin. Thus, many extracts showed discrepancies in both composition, allicin:alliin ratio and antimicrobial activity, raising concerns as to standards of preparation and quality control for these products.

Anti-Bacterial Agents↗
PubMed2003

Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry.

Metallochaperones are soluble proteins involved in metal transport and regulation in vivo. Copper metallochaperones belong to a structural family of metal binding domains displaying a ferredoxin-like fold (betaalphabetabetaalphabeta) and a consensus metal-binding motif MXCXXC. The metal-binding selectivities for this class of proteins are poorly documented so far. The present study focuses on the measurement of the selectivity of the copper metallochaperone CopZ from Enterococcus hirae for different metal ions using an experimental approach based on electrospray ionization mass spectrometry (ESI-MS). All the metal cations tested, i.e. Cu(I), Cu(II), Hg(II), Cd(II) and Co(II), form specific metal complexes with CopZ. The study of a chemically modified CopZ as well as variants of CopZ in the active site demonstrated that the complexes observed by ESI-MS, i.e. in the gas phase, corresponded to the complexes previously observed by other analytical methods in solution. Competition experiments allowed the classification of the metal ions by increasing affinities for CopZ as follows: Co << Cd < Hg < Cu. A dissociation constant in the range of 20 microM was determined for cobalt. The affinity of CopZ for the other metals tested was found to be higher, with dissociation constants smaller than micromolar.

Bacterial Proteins↗
PubMed2000

A second dihydroorotate dehydrogenase (Type A) of the human pathogen Enterococcus faecalis: expression, purification, and steady-state kinetic mechanism.

We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern.

Amino Acid Sequence↗
PubMed1993

A gene encoding the 16-kDa proteolipid subunit of Enterococcus hirae Na(+)-ATPase complex.

By further sequencing the cloned genome DNA of Enterococcus hirae that retains the genes encoding two major subunits of Na(+)-transport ATPase (Takase, K., Yamato, I., and Kakinuma, Y. (1993) J. Biol. Chem. 268, 11610-11616), we found a gene, ntpK, encoding a very hydrophobic protein of 156 amino acids (Mr = 16,036). The amino acid sequence deduced from the ntpK gene matched with the partial sequence of a 16-kDa protein purified as the proteolipid component of Na(+)-ATPase. The amino acid sequence and the hydropathy profile of the NtpK product were very similar with those of the 16-kDa proteolipids of vacuolar (V-) H(+)-ATPases in eukaryotes. The amino-terminal half of this sequence was highly homologous to the carboxyl-terminal half, suggesting that it was evolved from a common ancestral gene through duplication. The proteolipid of E. hirae Na(+)-ATPase belongs to that of the eukaryotic V-ATPase.

Adenosine Triphosphatases↗
PubMed1994

The Enterococcus faecalis extracellular metalloendopeptidase (EC 3.4.24.30; coccolysin) inactivates human endothelin at bonds involving hydrophobic amino acid residues.

The extracellular metalloendopeptidase (EC 3.4.24.30; coccolysin) from Enterococcus faecalis (Strain OG1-10) inactivates human endothelin-1 by hydrolyzing the peptide primarily at the Ser5-Leu6 and the His16-Leu17 bonds and the human big endothelin at several bonds involving hydrophobic amino acid residues. The big endothelin fragment 22-38 was also hydrolyzed at a high rate. The degradation of endothelin by coccolysin resembles the peptidolytic processing of endothelin by thermolysin. Because E. faecalis is associated with a large number of infectious diseases, it is possible that the manifestation of inflammatory conditions in the presence of this organism is related to the coccolysin-catalyzed inactivation of endothelin.

Amino Acid Sequence↗
PubMed1994

Induction of the putative copper ATPases, CopA and CopB, of Enterococcus hirae by Ag+ and Cu2+, and Ag+ extrusion by CopB.

The two P-type ATPases CopA and CopB are effecting regulation of cellular copper activity in Enterococcus hirae. With antibodies against these ATPases, we showed on Western blots the simultaneous induction of CopA and CopB by copper or silver ions. Copper contents of wild type and mutant cells lacking either CopA, CopB or both enzymes were measured by atomic absorption. Strains disrupted in copB showed clearly enhanced copper contents. Mutants lacking CopB also lost the ability of energy dependent efflux of silver ions. Our results demonstrate that CopA and CopB are under the same genetic control and support the proposal that CopB is a copper and silver exporting ATPase.

Adenosine Triphosphatases↗
PubMed1999

Effects of promoter mutations on the in vivo regulation of the cop operon of Enterococcus hirae by copper(I) and copper(II).

The cop operon of Enterococcus hirae encodes a repressor, CopY, a copper chaperone, CopZ, and two copper ATPases, CopA and CopB. Regulation of the cop operon is bi-phasic, with copper addition as well as copper chelation leading to induction. Using a plasmid-borne system with a reporter gene, induction of wild-type and mutant cop promoters by high and low copper conditions was investigated. Only mutations that impaired the interaction of CopY with both DNA binding sites had a marked effect on regulation, leading to hyperinduction by copper(I) or copper(II). Chelation of copper(II), but not copper(I), also induced the operon, but induction by copper chelation was not significantly affected by the mutations. E. hirae mutants with reduced extracellular copper reductase activity exhibited the same induction kinetics as wild-type cells. These results show that copper addition and copper chelation induce the cop operon by different routes.

Adenosine Triphosphatases↗
PubMed2000

Cytokine-inducing macromolecular glycolipids from Enterococcus hirae: improved method for separation and analysis of its effects on cellular activation.

Previously, we showed that several minor macromolecular glycolipids accounting for less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possess cytokine-inducing activity, whereas the purified LTA does not. In other words, the immunobiological activity of the LTA fraction reported in the 1980s was not attributable to LTA itself, but to other glycolipids coexisting in the fraction. In the present study, we improved the procedure of separation of the active glycolipids and evaluated their effects on cellular activation. The immunobiologically active glycolipids were separated from the crude glycolipid fraction obtained by hot phenol-water extraction of the cells. The total yield of active glycolipids was about fivefold higher than that separated by the previous method. Interleukin-6-inducing activities of the active glycolipids from 1,25-dihydroxy vitamin D(3)-differentiated human monocytic leukemia cells, THP-1, were inhibited by anti-CD14 mAbs in a dose-dependent manner. Macrophages from Toll-like receptor (TLR)-2-deficient or -4-deficient mice completely lacked the ability to produce tumor necrosis factor-alpha on stimulation with active glycolipids. These observations indicated that the cellular activation by the active glycolipids from E. hirae is mediated by CD14 and by both TLR2 and TLR4.

Animals↗
PubMed2001

Purification and functional analysis of the copper ATPase CopA of Enterococcus hirae.

The Enterococcus hirae ATPase CopA is a member of the recently discovered heavy metal ATPases and shares 43% sequence identity with the human Menkes and Wilson copper ATPases. To study CopA biochemically, it was overexpressed in E. coli with an N-terminal histidine tag and purified to homogeneity by nickel affinity chromatography. The purified CopA catalyzed ATP hydrolysis with a V(max) of 0.15 micromol/min/mg and a K(m) for ATP of 0.2 mM and had an optimum pH of 6.25. The activity was 3- to 4-fold stimulated by reconstitution into proteoliposomes. The enzyme formed an acylphosphate intermediate. Its kinetics of formation and the effects of inhibitors and metal ions upon it support a function of CopA in copper transport. Purification and functional reconstitution of CopA provides the basis to study copper transport in vitro.

Adenosine Triphosphatases↗
PubMed2001

Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance.

Intracellular copper routing in Enterococcus hirae can be accomplished by the CopZ metallochaperone. Using surface plasmon resonance analysis, we show here that CopZ interacts with the CopA copper ATPase. The binding affinity of CopZ for CopA was increased in the presence of copper, due to a 15-fold lower dissociation rate constant. Mutating the N-terminal copper binding motif of CopA from CxxC to SxxS abolished this copper-induced effect. Moreover, CopZ failed to show an interaction with an unrelated copper binding protein used as a control. These results show that (i) the CopA copper ATPase specifically interacts with the CopZ chaperone, (ii) this interaction is based on protein-protein interaction, and (iii) surface plasmon resonance is a novel tool for quantitative analysis of metallochaperone-target interactions.

Adenosine Triphosphatases↗
PubMed2000

Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10.

Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides+259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone-responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Qa promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion analyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction.

Amino Acid Sequence↗
PubMed2000

The 1.9 A resolution structure of phospho-serine 46 HPr from Enterococcus faecalis.

The histidine-containing phosphocarrier protein HPr is a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which transfers metabolic carbohydrates across the cell membrane in many bacterial species. In Gram-positive bacteria, phosphorylation of HPr at conserved serine 46 (P-Ser-HPr) plays several regulatory roles within the cell; the major regulatory effect of P-Ser-HPr is its inability to act as a phosphocarrier substrate in the enzyme I reaction of the PTS. In order to investigate the structural nature of HPr regulation by phosphorylation at Ser46, the structure of the P-Ser-HPr from the Gram- positive bacterium Enterococcus faecalis has been determined. X-ray diffraction analysis of P-Ser-HPr crystals provided 10,043 unique reflections, with a 95.1 % completeness of data to 1.9 A resolution. The structure was solved using molecular replacement, with two P-Ser-HPr molecules present in the asymmetric unit. The final R-value and R(Free) are 0.178 and 0.239, respectively. The overall tertiary structure of P-Ser-HPr is that of other HPr structures. However the active site in both P-Ser-HPr molecules was found to be in the "open" conformation. Ala16 of both molecules were observed to be in a state of torsional strain, similar to that seen in the structure of the native HPr from E. faecalis. Regulatory phosphorylation at Ser46 does not induce large structural changes to the HPr molecule. The B-helix was observed to be slightly lengthened as a result of Ser46 phosphorylation. Also, the water mediated Met51-His15 interaction is maintained, again similar to that of the native E. faecalis HPr. The major structural, and thus regulatory, effect of phosphorylation at Ser46 is disruption of the hydrophobic interactions between EI and HPr, in particular the electrostatic repulsion between the phosphoryl group on Ser46 and Glu84 of EI and the prevention of a potential interaction of Met48 with a hydrophobic pocket of EI.

Bacterial Proteins↗
PubMed2000

Aggregation substance promotes colonic mucosal invasion of Enterococcus faecalis in an ex vivo model.

BACKGROUND: Bacterial translocation through the gastrointestinal tract is the crucial step in the pathogenesis of intraabdominal infections. We assessed whether aggregation substance (AS), a bacterial adhesin and virulence factor of Enterococcus faecalis, promotes bacterial translocation and colonic mucosal invasion in an ex vivo experiment. METHODS: Colonic mucosa of male Wistar rats was placed in a modified Ussing system. The mucosal side of the chamber was filled with a suspension of E. faecalis OG1X:pAM721 (AS-positive) or E. faecalis OG1X (AS-negative). The serosal side was filled with sterile Dulbecco's modified Eagle's medium. For assessment of colonic mucosal invasion the mucosal side was incubated for 2.5 h with a suspension of AS-positive or AS-negative enterococci. After being washed, a solution of gentamicin and penicillin G in tissue culture medium was added on both sides in order to kill extracellular bacteria. Subsequently, the mucosa was removed from the system, washed, lysed with Triton X-100, and homogenized. Viable intramural bacteria were quantified by plating serial dilutions of the homogenate on Todd-Hewitt broth agar plates. To quantify bacterial translocation samples which were taken at various time points from the serosal side were plated on Todd-Hewitt broth agar plates and colony forming units (CFU) were determined. RESULTS: Invasion of the AS-positive E. faecalis strain OG1X:pAM721 into the colonic mucosa was significantly higher than invasion rates of the AS-negative strain OG1X (2.88 log(10) CFU/ml vs 1.73 log(10) CFU/ml; P = 0.02). However, none of the tested strains was found to translocate from the mucosal to the serosal side within the incubation time of 4 h. CONCLUSIONS: The aggregation substance promotes invasion of E. faecalis into colonic mucosa.

Animals↗
PubMed2001

Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins.

Aggregation substance (AS) of Enterococcus faecalis (E. faecalis), a sex pheromone plasmid encoded cell surface protein, mediates the formation of bacterial aggregates, thereby promoting plasmid transfer. The influence of pAD1-encoded AS, Asa1, on binding to immobilized extracellular matrix proteins was studied. The presence of AS increased enterococcal adherence to fibronectin more than eight-fold, to thrombospondin more than four-fold, to vitronectin more than three-fold, and to collagen type I more than two-fold (P<0.001). In contrast, binding to laminin and collagen type IV occurred independently of AS. Adherence of the constitutively AS expressing E. faecalis OG1X(pAM721) to immobilized fibronectin was found to be approximately five times higher than that of Staphylococcus aureus Cowan and approximately 30 times higher than that of Streptococcus bovis. Investigation of strains with various deletions within the structural gene of asa1 suggests that attachment to immobilized fibronectin is mainly mediated by amino acids within the variable region or by neighbouring residues. Thus, AS may promote adherence to injured epithelium and endothelium, where extracellular matrix proteins are exposed, thereby facilitating colonization and infection.

Bacterial Proteins↗
PubMed1994

Conjugal transfer of transposon Tn916 from Enterococcus faecalis to Bacillus licheniformis.

Transposon Tn916 was conjugally transferred from Enterococcus faecalis to Bacillus licheniformis bacterial strains ATCC 10716 and ATCC 9945A by filter and broth matings. The Tn916 transfer frequencies to B. licheniformis ranged from 10(-7) to 10(-5) selecting for tetracycline-resistant (Tcr) transconjugants depending on broth or filter mating. Movement of Tn916 was demonstrated when Tcr B. licheniformis transconjugants were mated with Bacillus subtilis strain W23. Tn916 insertion caused several auxotrophic and bacitracin deficient mutants. Southern blot analyses of HindIII chromosomal digests extracted from Tcr transconjugants showed that the transposon inserted at different sites in the B. licheniformis chromosome and the copy number of Tn916 varied. This approach should be useful for genetic studies of B. licheniformis.

Bacillus↗