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PubMed2000

Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state.

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.

Bacterial Proteins↗
PubMed2001

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bacterial Adhesion↗
PubMed2001

Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States.

Vancomycin-resistant Enterococcus spp. (VRE) were isolated from sewage and chicken feces but not from other animal fecal sources (dog, cow, and pig) or from surface waters tested. VRE from hospital wastewater were resistant to > or =20 microg of vancomycin/ml and possessed the vanA gene. VRE from residential wastewater and chicken feces were resistant to 3 to 5 microg of vancomycin/ml and possessed the vanC gene.

Animals↗
PubMed2001

Citrate metabolism by Enterococcus faecalis FAIR-E 229.

Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.

Cheese↗
PubMed2001

pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis.

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless beta-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. beta-Galactosidase was expressed in E. coli and E. faecalis at levels of 10(3) and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

Affinity Labels↗
PubMed2001

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136.

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.

Adenosine Triphosphate↗
PubMed2002

Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs.

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.

Animals↗
PubMed2002

Serum as a factor influencing adhesion of Enterococcus faecalis to glass and silicone.

The purpose of this work was to analyze the effect of serum on the physicochemical surface properties and adhesion to glass and silicone of Enterococcus faecalis ATCC 29212 at 37 degrees C. As is presented using thermodynamics analysis, serum minimizes the interaction of cells with water, which correlates well with the increase in hydrophobicity and in bacterial adhesion to glass and silicone.

Bacterial Adhesion↗
PubMed2002

Two forms of NAD-dependent D-mandelate dehydrogenase in Enterococcus faecalis IAM 10071.

Two forms of NAD-dependent D-mandelate dehydrogenase (D-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different K(m) and V(max) values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

Alcohol Oxidoreductases↗
PubMed2002

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity.

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.

Animals↗
PubMed2002

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309.

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.

Amino Acid Sequence↗
PubMed2002

Vancomycin-resistant Enterococcus faecium (VREF) from Norwegian poultry cluster with VREF from poultry from the United Kingdom and The Netherlands in an amplified fragment length polymorphism genogroup.

The genetic relationship between 197 vancomycin-resistant Enterococcus faecium (VREF) isolates and 21 vancomycin-susceptible E. faecium isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of E. faecium and clustered in a previously defined poultry AFLP genogroup.

Animals↗
PubMed2002

Description of a 23.9-kilobase chromosomal deletion containing a region encoding fsr genes which mainly determines the gelatinase-negative phenotype of clinical isolates of Enterococcus faecalis in urine.

Expression of virulence-related extracellular proteases, gelatinase, and serine protease of Enterococcus faecalis is regulated by a quorum-sensing system encoded by the fsr gene cluster. In this study, a 23.9-kb chromosomal deletion containing the fsr gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of E. faecalis from urine.

Bacterial Proteins↗
PubMed2002

Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production.

Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 microg ml(-1) for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.

Amino Acid Sequence↗
PubMed2002

Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393.

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

Amino Acid Sequence↗
PubMed2002

Adhesion to bile drain materials and physicochemical surface properties of Enterococcus faecalis strains grown in the presence of bile.

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.

Adhesins, Bacterial↗
PubMed2003

Predominance of vanA genotype among vancomycin-resistant Enterococcus isolates from poultry and swine in Costa Rica.

The use of avoparcin as a growth promoter is considered to have selected for vancomycin-resistant enterococci (VRE). In Costa Rica, the use of avoparcin for poultry and swine was intensive until the product was withdrawn from the market in 2000. We evaluated the presence of VRE in poultry, swine, and cattle fecal samples obtained during 1998 and 1999. A total of 185 VRE isolates were recovered from 116 out of 893 samples. Enterococcus faecium was the most frequently isolated species (50.8%), being predominant among poultry (71.6%) and swine (37.7%) isolates, but it was not recovered from the bovine samples. The second-most-frequently-isolated species from poultry and swine, respectively, were E. durans (23.2%) and E. faecalis (21.7%). E. casseliflavus was the only species obtained from bovine samples, but it was not found among the avian isolates. An evident predominance of the vanA determinant among vancomycin-resistant enterococcal species from poultry and swine, but not from cattle, was observed and was similar to the situation in European countries before avoparcin was forbidden. The diversity of the vanA determinant in the isolates was assessed by detection of the IS1251 insertion in the vanSH intergenic region and of the IS1476 insertion in the vanXY intergenic region. However, in none of the 154 vanA+ isolates recovered in this study were those insertions detected.

Animals↗
PubMed2003

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid.

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.

Amino Acid Sequence↗