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PubMed2000

Cloning, sequencing and characterization of the ccpA gene from Enterococcus faecalis.

Enzymes involved in the metabolism of complex carbon and energy sources are unnecessary under conditions of abundant, readily metabolisable nutrients such as glucose or fructose. The repression of these enzymes by glucose has been termed carbon catabolite repression. Mechanisms involved in the carbon catabolite repression in gram-positive bacteria are known to differ from those of gram-negative bacteria such as Escherichia coli. It appears to be mediated by transcriptional repression, requiring trans-acting CcpA, a member of the LacI-GalR family of bacterial regulatory proteins and a cis-acting consensus sequence, designated cre. Here, we report the cloning and characterisation of the chromosomal ccpA gene from Enterococcus faecalis JH2-2. This gene is predicted to encode a 333 amino acids protein with nearly 75% identity to CcpA of Lactobacillus casei.

Amino Acid Sequence↗

PubMed1999

Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR.

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.

ATP-Binding Cassette Transporters↗

PubMed2003

Crystal structure of Enterococcus faecalis SlyA-like transcriptional factor.

The crystal structure of a SlyA transcriptional regulator at 1.6 A resolution is presented, and structural relationships between members of the MarR/SlyA family are discussed. The SlyA family, which includes SlyA, Rap, Hor, and RovA proteins, is widely distributed in bacterial and archaeal genomes. Current evidence suggests that SlyA-like factors act as repressors, activators, and modulators of gene transcription. These proteins have been shown to up-regulate the expression of molecular chaperones, acid-resistance proteins, and cytolysin, and down-regulate several biosynthetic enzymes. The structure of SlyA from Enterococcus faecalis, determined as a part of an ongoing structural genomics initiative (www.mcsg.anl.gov), revealed the same winged helix DNA-binding motif that was recently found in the MarR repressor from Escherichia coli and the MexR repressor from Pseudomonas aeruginosa, a sequence homologue of MarR. Phylogenetic analysis of the MarR/SlyA family suggests that Sly is placed between the SlyA and MarR subfamilies and shows significant sequence similarity to members of both subfamilies.

Amino Acid Sequence↗

PubMed1996

Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

Anti-Bacterial Agents↗

PubMed2002

Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis.

Although symmetrical polyarticular amyloidosis has been described extensively in brown layers, spontaneous unilateral amyloid arthropathy has not been described previously in chickens. Birds from nine flocks of broiler parent stock (PS) had unilateral lameness associated with severe swelling of the left hock joint and the caudal aspect of the metatarsus. Gross pathology was restricted to the left hock joint and the left digital flexor tendons in almost all cases, suggesting an association with administration of Marek's disease vaccine. Amyloid deposits were found in 83% (25/30) of affected joints by histological examination of Congo red stained sections. Systemic amyloidosis, involving mainly the liver and spleen, was found in 59% (10/17) of birds. Enterococcus faecalis was isolated from joints in 77% (23/30) of cases and Staphylococcus aureus was isolated from the joint in one case (1/30). Thirty-five E. faecalis isolates from joints, tendons and blood samples from birds in five affected PS flocks were compared using pulsed-field gel electrophoresis (PFGE) to separate genomic fragments after digestion with SmaI. All but one isolate had identical or closely related restriction endonuclease digestion (RED) patterns that were very similar to a known arthropathic and amyloidogenic E. faecalis isolate. A further 30 E. faecalis isolates from seven grandparent stock (GPS) flocks and two isolates from two unaffected PS flocks of the same genetic background were analysed by PFGE. Among these isolates, 11 originating from four GPS flocks had RED patterns identical to or closely related to the reference amyloid-inducing strain. Moreover, one E. faecalis isolate from amyloidotic joints of brown layers housed in California, USA was included in the analysis and appeared to be identical to the reference strain. This study showed that the E. faecalis isolates involved in these outbreaks of unilateral amyloid arthropathy in broiler breeders belonged to the same clone as that responsible for outbreaks in brown layers.

Amyloidosis↗

PubMed1992

The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate.

Vancomycin resistance plasmids in enterococci carry the genes vanH and vanA, which encode enzymes catalyzing, respectively, the reduction of 2-keto acids to 2-D-hydroxy acids and the addition of D-hydroxy acids to D-alanine. It has therefore been postulated that resistant cells produce peptidoglycan precursors that terminate in the depsipeptide D-alanine-2-D-hydroxy acid rather than the dipeptide D-alanine-D-alanine, thus preventing vancomycin binding (M. Arthur, C. Molinas, T. D. H. Bugg, G. D. Wright, C. T. Walsh, and P. Courvalin, Antimicrob. Agents Chemother. 36:867-869, 1992). In the present work, a cytoplasmic peptidoglycan precursor was isolated from vancomycin-resistant Enterococcus faecalis and analyzed by mass spectrometry, which suggested the structure UDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate.

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↗

PubMed2001

Three-dimensional structure of the histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis in solution.

The histidine-containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate-dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate-response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional (1)H-NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four beta strands, A, B, C, D, encompassing residues 2-7, 32-37, 40-42 and 60-66, form an antiparallel beta sheet lying opposite the two antiparallel alpha helices, a and c (residues 16-26 and 70-83). A short alpha helix, b, from residues 47-53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active-center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94-97], in the solution structure, the active-site His15 rests on top of helix a, and the phosphorylation site N(delta 1) of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X-ray structures shows that the active-center structure derived by X-ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution.

Bacterial Proteins↗

PubMed1998

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in beta-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of beta-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Bacterial Proteins↗

PubMedDate not supplied

Comparison of bactericidal activities of intermittent and continuous infusion dosing of vancomycin against methicillin-resistant Staphylococcus aureus and Enterococcus faecalis.

STUDY OBJECTIVE: To describe the pharmacokinetic profiles of vancomycin administered by continuous infusion and intermittent dosing and compare the duration of activity of the regimens. DESIGN: Randomized, open-label, crossover study. SETTING: Clinical research center at an academic medical center. SUBJECTS: Twelve healthy, nonpregnant volunteers age 27.6 +/- 2.3 years. INTERVENTION: Subjects received the following intravenous vancomycin regimens: 1 g every 12 hours; 2 g continuous infusion over 24 hours; and 1 g continuous infusion over 24 hours. Dosages were administered with and without gentamicin 2 mg/kg. MEASUREMENTS AND MAIN RESULTS: Serum samples were collected, drug concentrations determined, and bactericidal activity measured against two isolates each of methicillin-resistant Staphylococcus aureus and Enterococcus faecalis. Subjects had poor tolerability for continuous infusions. Trough concentration for the intermittent regimen was 5.5 +/- 1.9 mg/ml, and steady-state concentrations were 8.8 +/- 1.6 and 16.9 +/- 1.9 mg/ml for 1 and 2 g continuous infusions, respectively. In general, all regimens provided bactericidal activity throughout the study interval. Against one isolate of E. faecalis, 2 g continuous infusion plus gentamicin provided cidal activity for a significantly greater percentage of the dosing interval (p<0.001). CONCLUSION: Continuous infusion does not greatly improve the activity of vancomycin and should not be routinely administered. However, it may prove useful against isolates with reduced susceptibility to the agent.

Adult↗

PubMed2000

Morphologic changes correlating to different sensitivities of Escherichia coli and enterococcus faecalis to Nd:YAG laser irradiation through dentin.

BACKGROUND AND OBJECTIVE: Previous studies demonstrated the disinfecting potential of Nd:YAG laser irradiation on the root canal system from an overall quantitative viewpoint. The aim of this study was to evaluate the specific effect of irradiation through dentin on gram-negative and gram-positive bacteria with regard to their cell structure. STUDY DESIGN/MATERIALS AND METHODS: Sterile dentin samples of standardized size were divided into two sets of four groups with eight samples each. The first set was inoculated with Escherichia coli as the gram-negative test strain, the second set was inoculated with Enterococcus faecalis, which served as the gram-positive test organism. The samples were then irradiated on the bacteria-free side in contact mode under constant scanning movement at an angle of 10 degrees by use of the fiber optic of the Nd:YAG laser. Upon laser treatment they were critical point dried and subjected to SEM investigation. Another two sets of samples were prepared and irradiated in the same manner and evaluated by standard microbiological procedures to verify whether the observed morphologic alterations correlated to cell death. RESULTS: SEM investigations revealed damage pattens that increased with the amount of energy applied. Whereas the gram-negative test organism showed immediate structural injury, the gram-positive test organism required repeated application of irradiation. The microbiological examination showed reduction of both bacterial strains, yet to different extents. CONCLUSION: Our study demonstrates the different morphologic impact of Nd:YAG laser irradiation through dentin on representatives of the two main groups of bacteria. It shows that the construction of the cell wall is crucial for their individual sensitivity to laser treatment.

Colony Count, Microbial↗

PubMed1999

A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.

Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors.

Conjugation, Genetic↗

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↗

PubMed1996

Treatment of experimental endocarditis due to Enterococcus faecalis using once-daily dosing regimen of gentamicin plus simulated profiles of ampicillin in human serum.

We compared the efficacy of ampicillin, both alone and in combination with gentamicin given once a day (q.d.) or three times a day (t.i.d.), in the treatment of experimental enterococcal endocarditis. Ampicillin was administered by using humanlike pharmacokinetics that simulated the profiles of this drug in human serum. An open one-compartment mathematical model developed in this study was used to estimate the decreasing doses administered with a computer-controlled infusion pump that simulated in rabbits the human serum pharmacokinetics after intravenous administration of 2 g of ampicillin every 4 h. Animals with catheter-induced endocarditis were infected intravenously with 10(8) CFU of Enterococcus faecalis J4 (MICs and MBCs of ampicillin and gentamicin, 2 and 128 and 16 and 64 micrograms/ml, respectively) and were treated for 3 days with ampicillin alone or in combination with gentamicin at 2 mg/kg of body weight subcutaneously t.i.d. or at 6 mg/kg subcutaneously q.d. The serum ampicillin levels and pharmacokinetic parameters of the humanlike pharmacokinetics of ampicillin in rabbits were similar to those found in humans treated with 2 g of ampicillin intravenously. The results of therapy for experimental endocarditis caused by E. faecalis J4 showed that the residual bacterial concentration in aortic valve vegetation was significantly lower in the animals treated with combinations of ampicillin plus gentamicin given q.d. or t.i.d. than in those treated with ampicillin alone (P < 0.01). The dosing interval of gentamicin did not significantly affect (q.d. versus t.i.d.; P = 0.673) the therapeutic efficacy of the combination of ampicillin plus gentamicin.

Ampicillin↗

PubMed1997

Comparison of the bactericidal activities of piperacillin-tazobactam, ticarcillin-clavulanate, and ampicillin-sulbactam against clinical isolates of Bacteroides fragilis, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa.

Owing to the broad spectrum of activity afforded by beta-lactam-beta-lactamase inhibitor preparations, these agents are frequently selected as empiric therapy for the treatment of mixed infections such as intra-abdominal and diabetic foot infections, either alone or in combination with an aminoglycoside. Twelve healthy volunteers were enrolled in a randomized, open-label, four-way crossover trial comparing the bactericidal activities of piperacillin-tazobactam, ticarcillin-clavulanate, and ampicillin-sulbactam against microorganisms commonly isolated from mixed infections. Subjects received the following regimes: (i) 3.375 g of piperacillin-tazobactam intravenously (i.v.) every 6 h (q6h) (ii) 4.5 g of piperacillin-tazobactam i.v. q8h, (iii) 3.1 g of ticarcillin-clavulanate i.v. q6h, and (iv) 3.0 g of ampicillin-sulbactam i.v. q6h. Serum bactericidal titers were determined and used to calculate the duration of measurable bactericidal activity over the dosing interval of each of the regimens against two clinical isolates of Bacillus fragilis, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa. The percentage of the dosing interval over which drug concentrations in serum remained above the MIC for each organism was determined and compared with the observed duration of bactericidal activity was noted (r = 0.78; P < 0.001). All of the regimens demonstrated good activity against B. fragilis and E. coli. Against E. faecalis and P. aeruginosa, however, all of the regimens provided bactericidal activity for less than 50% of the respective dosing intervals. These data suggest that use of shorter dosing intervals or continuous-infusion regimens should be considered in combination with an aminoglycoside to improve the bactericidal profiles of these agents for E. faecalis and P. aeruginosa.

Adult↗

PubMed2003

The Enterococcus faecalis cytolysin: a novel toxin active against eukaryotic and prokaryotic cells.

The enterococcal cytolysin, a two-peptide lytic system, is a divergent relative of a large family of toxins and bacteriocins secreted by pathogenic and non-pathogenic Gram-positive bacteria. This family includes the lantibiotics and streptolysin S. The enterococcal cytolysin is of interest because its activities enhance enterococcal virulence in infection models and, in epidemiological studies, it has been associated with patient mortality. The cytolysin is lethal for a broad range of prokaryotic and eukaryotic cells, and this activity requires two non-identical, post-translationally modified peptides. The smaller of the two peptides also plays a role in a quorum-sensing autoinduction of the cytolysin operon. As a trait that is present in particularly virulent strains of Enterococcus faecalis, including strains that are resistant to multiple antibiotics, it serves as a model for testing the value of developing new virulence-targeting therapeutics. Further, because of the interest in small membrane active peptides as therapeutics themselves, studies of the molecular structure/activity relationships for the cytolysin peptides are providing insights into the physical basis for prokaryotic versus eukaryotic cell targeting.

Animals↗

PubMed2003

Biotransformation of pinoresinol diglucoside to mammalian lignans by human intestinal microflora, and isolation of Enterococcus faecalis strain PDG-1 responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol.

By anaerobic incubation of pinoresinol diglucoside (1) from the bark of Eucommia ulmoides with a fecal suspension of humans, eleven metabolites were formed, and their structures were identified as (+)-pinoresinol (2), (+)-lariciresinol (3), 3'-demethyl-(+)-lariciresinol (4), (-)-secoisolariciresinol (5), (-)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (6), 2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butane-1, 4-diol (7), 3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butane-1, 4-diol (8), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butane-1, 4-diol (9), (-)-enterodiol (10), (-)-(2R, 3R)-3-(3", 4"-dihydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (11), (-)-(2R, 3R)-2-(3', 4'-dihydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (12), (-)-(2R, 3R)-3-(3"-hydroxybenzyl)-2-(4'-hydroxy-3'-methoxybenzyl)butyrolactone (13), 2-(3', 4'-dihydroxybenzyl)-3-(3"-hydroxybenzyl)butyrolactone (14), 2-(3'-hydroxybenzyl)-3-(3", 4"-dihydroxybenzyl)butyrolactone (15) and (-)-(2R, 3R)-enterolactone (16) by various spectroscopic means, including two dimensional (2D)-NMR, mass spectrometry and circular dichroism. A possible metabolic pathway was proposed on the basis of their structures and time course experiments monitored by thin-layer chromatography. Furthermore, a bacterial strain responsible for the transformation of (+)-pinoresinol to (+)-lariciresinol was isolated from a human fecal suspension and identified as Enterococcus faecalis strain PDG-1.

Anticarcinogenic Agents↗

PubMed2005

Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures.

Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.

Animals↗