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PubMed2004

Relations between the consumption of antimicrobial growth promoters and the occurrence of resistance among Enterococcus faecium isolated from broilers.

The present study investigates, at farm level, the effect of the time-span between sampling and the last time a particular antimicrobial growth promoter (AGP) was included in the feed on the probability of selecting an AGP-resistant Enterococcus faecium isolate from a broiler flock. The probability that a randomly selected E. faecium isolate was resistant to avilamycin, erythromycin or virginiamycin was 0.91, 0.92 and 0.84, respectively if the isolate originated from a broiler flock fed either avilamycin- or virginiamycin-supplemented feed. As the time-span between sampling and the last AGP consumption increased, the probability of isolating an E. faecium isolate resistant to a particular AGP decreased (probability <0.2 within 3-5 years after last exposure to AGPs). The decrease in probability over time showed little farm-to-farm variation. The number of times a particular AGP was given to previous flocks reared in the same house had no effect on the probability of isolating a resistant isolate.

Animal Feed↗
PubMed1997

Molecular epidemiology of glycopeptide-resistant Enterococcus faecium on a renal unit.

The clinical and molecular epidemiology of glycopeptide-resistant Enterococcus faecium was investigated during an outbreak on a renal unit. Forty-nine patients were colonized or infected during a 15-month period. Sites of colonization included faeces, urine, intravenous (IV) catheter tips and wound swabs. Ten patients had infections, which included five bacteraemias and three episodes of peritonitis. Pulsed-field gel electrophoresis of 43 patient isolates of glycopeptide-resistant E. faecium identified seven strains during the first 7 months of the outbreak. Three of these strains affected five or more patients. One strain accounted for 17/43 isolates. Isolates that were possibly related to another renal unit strain were cultured from patients at two other Manchester hospitals. These isolates were epidemiologically-related, and may represent a single Manchester epidemic strain. Of five patients who had multiple isolates of glycopeptide-resistant E. faecium, three had isolates representing a single strain and two were colonized or infected by more than one strain.

Adult↗
PubMed2000

Eradication of vancomycin resistant Enterococcus faecium from a paediatric oncology unit and prevalence of colonization in hospitalized and community-based children.

We previously reported an outbreak of vancomycin resistant enterococci (VRE) in a paediatric oncology unit in December 1995 which was associated with widespread environmental contamination of the unit with VRE. We undertook this study to evaluate the effectiveness of the infection control policy instituted subsequent to the outbreak and to investigate the underlying prevalence of VRE colonization in hospitalized, outpatient and community-based children. We sought to establish the molecular similarity of VRE isolates from the study. Stool specimens were obtained from outpatients at risk of VRE, hospital inpatients and from healthy community-based children. VRE colonization was eradicated from the inpatient unit within 11 months, but in outpatients, 16 months after the outbreak, 4 of 137 (2.9 %) attending oncology outpatients, 5 of 65 (7.7%) with cystic fibrosis and 1 of 12 (8.3 %) with liver disease were found to be colonized with VRE. The isolates were all Enterococcus faecium, Van A phenotype except one E. casseliflavus of the Van C phenotype. All were unique in SmaI DNA macrorestriction patterns with the exception of two isolates, which were similar to the original outbreak strain and three further isolates of a single strain but which differed from the outbreak strain. Of 315 hospital inpatients, 2.5 % were colonized with VRE of the Van C resistance phenotype but VRE was not detected in 116 healthy, community-based children. We conclude that effective strategies can successfully control spread of VRE but despite a low prevalence of VRE colonization in hospital patients and in community-based children, outbreaks can occur when infection control practices are not optimal. Continued vigilance to detect VRE and limit spread within hospitals is therefore necessary.

Adolescent↗
PubMed1995

Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147.

Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.

Amino Acid Sequence↗
PubMed1994

Identification of the DNA-binding site for the phosphorylated VanR protein required for vancomycin resistance in Enterococcus faecium.

The vancomycin resistance operon of Enterococcus faecium encodes a two-component regulatory system comprising VanS and VanR. In vitro experiments showed that about 5% of a labile phosphorylated VanR (P-VanR) was accumulated from ATP and a maltose-binding protein-VanS fusion protein (MBP-VanS). Alternatively, about an 8% abundance of P-VanR was produced with acetyl phosphate. In such incubations, gel shift experiments revealed that P-VanR selectively bound to a 254-bp DNA fragment that contains the vanH promoter for the vanH, vanA, and vanX structural genes. When VanS was added with a mole ratio for VanS:VanR of higher than 1:1, VanS competed with DNA for P-VanR and abolished the gel shift. P-VanR bound 500-fold more tightly to the vanH promoter region, with an estimated EC50 of 40 nM, than the unphosphorylated VanR. A second DNA fragment of 197 bp containing the proposed vanR promoter for the vanR and vanS regulatory genes also exhibited gel shift, but with much lower affinities. A mutant VanR(D53A) was shown to be incompetent for phosphorylation by phosphorylated MBP-VanS or by acetyl phosphate; however, it still bound DNA specifically, albeit with low affinity. DNase I footprinting by P-VanR revealed that a ca. 80-bp region was protected on the vanH promoter and a ca. 40-bp region was protected on the vanR promoter. The unphosphorylated VanR footprinted the same 80 bp on the vanH promoter, but only 20 bp on the vanR promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed2003

Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity.

The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.

Acetyltransferases↗
PubMed1999

Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism.

Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.

Amino Acid Sequence↗
PubMed1999

Influence of a probiotic adjunct culture of Enterococcus faecium on the quality of cheddar cheese.

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.

Cheese↗
PubMed2004

Probing surface adhesion forces of Enterococcus faecalis to medical-grade polymers using atomic force microscopy.

The aim of this study was to compare the initial adhesion forces of the uropathogen Enterococcus faecalis with the medical-grade polymers polyurethane (PU), polyamide (PA), and poly(tetrafluoroethylene) (PTFE). To quantify the cell-substrate adhesion forces, a method was developed using atomic force microscopy (AFM) in liquid that allows for the detachment of individual live cells from a polymeric surface through the application of increasing force using unmodified cantilever tips. Results show that the lateral force required to detach E. faecalis cells from a substrate differed depending on the nature of the polymeric surface: a force of 19 +/- 4 nN was required to detach cells from PU, 6 +/- 4 nN from PA, and 0.7 +/- 0.3 nN from PTFE. Among the unfluorinated polymers (PU and PA), surface wettability was inversely proportional to the strength of adhesion. AFM images also demonstrated qualitative differences in bacterial adhesion; PU was covered by clusters of cells with few cell singlets present, whereas PA was predominantly covered by individual cells. Moreover, extracellular material could be observed on some clusters of PU-adhered cells as well as in the adjacent region surrounding cells adhered on PA. E. faecalis adhesion to the fluorinated polymer (PTFE) showed different characteristics; only a few individual cells were found, and bacteria were easily damaged, and thus detached, by the tip. This work demonstrates the utility of AFM for measurement of cell-substrate lateral adhesion forces and the contribution these forces make toward understanding the initial stages of bacterial adhesion. Further, it suggests that initial adhesion can be controlled, through appropriate biomaterial design, to prevent subsequent formation of aggregates and biofilms.

Bacterial Adhesion↗
PubMed1999

Structure and function of vacuolar Na+-translocating ATPase in Enterococcus hirae.

A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa proteolipid), were highly similar to those of A, B, and proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.

Adenosine Triphosphatases↗
PubMed1999

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part.

Previously, we showed that quantitatively minor several glycolipids totaling only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97-112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779-86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.

Carbohydrate Sequence↗
PubMed2003

The Enterococcus hirae paradigm of copper homeostasis: copper chaperone turnover, interactions, and transactions.

The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. The cop operon is induced by copper, which allows uncompromised growth in up to 5 mM ambient copper. Copper uptake appears to be accomplished by the CopA ATPase, a member of the heavy metal CPx-type ATPases and closely related to the human Menkes and Wilson ATPases. The related CopB ATPase extrudes copper when it reaches toxic levels. Intracellular copper routing is accomplished by the CopZ copper chaperone. Using surface plasmon resonance analysis, it was demonstrated that CopZ interacts with the CopA ATPase where it probably becomes copper loaded. CopZ in turn can donate copper to the copper responsive repressor CopY, thereby releasing it from DNA. In high copper, CopZ is proteolyzed. Cell extracts were found to contain a copper activated proteolytic activity that degrades CopZ in vitro. This post-translational control of CopZ expression presumably serves to avoid the accumulation of detrimental Cu-CopZ levels.

Adenosine Triphosphatases↗
PubMed2003

Real-time PCR method to detect Enterococcus faecalis in water.

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.

DNA, Bacterial↗
PubMed2003

Reduction of Salmonella in gnotobiotic Japanese quails caused by the enterocin A-producing EK13 strain of Enterococcus faecium.

The effect of the enterocin A-producing EK strain of Enterococcus faecium on Salmonella dusseldorf SA31 was tested in gnotobiotic Japanese quails. Sixteen 3-day-old gnotobiotic Japanese quails, hatched from disinfected eggs placed in sterile boxes, were divided into two groups of 8 birds: a control group, which was inoculated orally with the SA31 strain (1 x 10(7) CFU/ml), and the experimental group, which was inoculated orally with 200 microl of E. faecium (1 x 10(9) CFU/ml), 16 h before infection with the S. dusseldorf. The latter group then received the same average dose of E. faecium daily in drinking water. Faecal samples were taken 8, 24, 48 and 168 h after the inoculation of S. dusseldorf and examined for S. dusseldorf and E. faecium (EK13). The quails were then killed and the number of the EK13 strain of E. faecium and of S. dusseldorf in the caecum and ileum were estimated. A reducing effect of the EK13 strain against the SA31 strain in faeces was detected in the samples taken at 24 and 48 h from the group with the EK13 strain. Significant reductions were also found in the numbers of S. dusseldorf SA31 strain in the caecum but not in the ileum.

Animals↗
PubMed1997

Contribution of antibody to neutrophil-mediated killing of Enterococcus faecalis.

Late-onset septicemia due to Enterococcus faecalis is common among very low-birth weight neonates. These infants have low concentrations of placentally derived IgG and developmentally low levels of complement. The aim of the present study was to determine the contribution of antibody to in vitro neutrophil-mediated phagocytosis of E. faecalis. Antibody alone, as contained in an adult serum pool heated to inactivate complement, promoted only a modest reduction in the initial bacterial inoculum (50 +/- 12%) for 6 of 10 E. faecalis bacterial strains tested and allowed growth of the other four strains. In the presence of complement, NHS promoted > or = 90% reduction in the initial bacterial inoculum of two representative strains at serum concentrations as low as 0.5%. Hypogammaglobulinemic serum supported similar activity only at concentrations above 5%. Purification of IgG and IgM fractions from NHS revealed that IgM had the higher specific activity to promote phagocytic activity. Absorption to remove specific antibody significantly reduced bactericidal activity by normal human serum, complement-deficient sera, and hypogammaglobulinemic serum. Reconstitution of hypogammaglobulinemic serum with antibody as contained in 1% heated normal human serum or in immune globulin for intravenous use (1200 mg/dl) restored phagocytic activity. Thus, E. faecalis-specific antibody enhances PMN-mediated killing of this organism. Adjunctive therapy with intravenous immunoglobulin could augment the host response to enterococcal infections in infancy.

Adult↗
PubMed2004

Biotechnological production of L(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis.

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).

Adaptation, Physiological↗
PubMed2003

Spontaneous vegetative endocarditis due to Enterococcus faecalis in a rottweiler puppy.

A 5-month-old female Rottweiler was referred because of a 5-week diarrhea and a sudden onset of a cardiac murmur auscultated by its veterinarian. Definitive diagnosis of bacterial endocarditis was based on ultrasonographic visualization of vegetative cardiac lesions and positive cultures of Enterococcus faecalis in blood and urine. Complicating findings were suppurative nephritis and renal infarction. Despite intensive supportive care, the endocarditis and clinical condition deteriorated and the dog had to be euthanized.

Animals↗