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PubMed2002

vanE gene cluster of vancomycin-resistant Enterococcus faecalis BM4405.

Acquired VanE-type resistance to low levels of vancomycin (MIC = 16 microg/ml) in Enterococcus faecalis BM4405 is due to the inducible synthesis of peptidoglyean precursors terminating in D-alanine-D-serine (Fines,M., B. Prichon, P. Reynolds, D. Sahm, and P. Courvalin, Antimicrob. Agents Chemother. 43:2161-2164, 1999). A chromosomal location was assigned to the vanE operon by pulsed-field gel electrophoresis and hybridization, and its sequence was determined. Three genes, encoding the VanE ligase, the VanXYE DD-peptidase, and the VanTE serine racemase, that displayed 43 to 53% identity with the corresponding genes in the vanC operon were found. In addition, two genes coding for a two-component regulatory system, VanRE-VanSE, exhibiting 60 and 44% identity with VanR,-VanS, were present downstream from vanTE. However, because of a stop codon at position 78, VanSE was probably not functional. The five genes, with the same orientation, were shown to be cotranscribed by Northern analysis and reverse transcription-PCR. The vanE, vanXYE, and vanTE genes conferred inducible low-level resistance to vancomycin after cloning in E. faecalis JH2-2, probably following cross talk with a two-component regulatory system of the host.

Anti-Bacterial Agents↗

PubMed1992

A hemolysin-encoding plasmid contributes to bacterial virulence in experimental Enterococcus faecalis endophthalmitis.

Beta-hemolysin production is a variable trait of the Lancefield group D streptococcus, Enterococcus faecalis. The E. faecalis hemolysin is encoded by large transmissible plasmids. The variable nature of this putative virulence factor provided an ideal system for testing its contribution in experimental endophthalmitis. In this study, isogenic E. faecalis strains were compared to determine whether the presence of the hemolysin-encoding plasmid affected the severity of disease in a rabbit endophthalmitis model. Experimental infections (n = 6) with 10(1)-10(4) E. faecalis organisms harboring the hemolysin-encoding plasmid resulted in a 98% loss of retinal function (by electroretinography [ERG]) and white reflex by postoperative day 3. By contrast, infections of similar numbers of plasmid-free E. faecalis organisms (n = 5) resulted in retention of some retinal function (23% per ERG) with a red reflex demonstrated on postoperative day 3. Results of light microscopy, slit-lamp examination, ERG, and indirect ophthalmoscopy indicated that infections with hemolysin-encoding plasmid-containing E. faecalis resulted in a more aggressive endophthalmitis compared with the endophthalmitis caused by plasmid-free E. faecalis. This is the first endophthalmitis model to the authors' knowledge that specifically evaluates bacterial virulence using isogenic strains.

Animals↗

PubMed2006

Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis.

Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

Aminoacyltransferases↗

PubMed2000

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections.

Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

Amino Acid Sequence↗

PubMed2004

Enterococcus faecalis resistant to linezolid: case series and review of the literature.

Reports of linezolid resistance among Enterococcus faecalis have been relatively rare. We describe three patients with linezolid-resistant strains of E. faecalis, discovered after the patients had received long courses (> 30 days) of linezolid therapy. All strains contained the G2576U mutation in 23S rRNA. A literature review revealed one other case in the United States and three cases in Europe; all involved patients who had received long courses of therapy. It appears that recent or extended linezolid therapy may be a risk factor for development of resistant E. faecalis. In patients who have recently been treated with linezolid and have an identified new systemic enterococcal isolate, linezolid sensitivity should be verified as soon as possible to guide therapy. This strategy also should be considered for patients with a breakthrough enterococcal isolate during linezolid therapy.

Acetamides↗

PubMed1997

High-level plasmid-mediated gentamicin resistance and pheromone response of plasmids present in clinical isolates of Enterococcus faecalis.

Eleven pheromone-responding plasmids encoding erythromycin or gentamicin resistance were isolated from multiresistant clinical Enterococcus faecalis isolates. The plasmids were classified into six types with respect to their pheromone responses. The three erythromycin resistance plasmids responded to different pheromones. Of the eight gentamicin resistance plasmids, four plasmids responded to same pheromone. Southern hybridization studies showed that the genes involved in regulation of the pheromone response were conserved in the drug resistance plasmids.

Anti-Bacterial Agents↗

PubMed1998

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins.

The hydrogen peroxide (H2O2) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2.4 mmol l-1 H2O2 pretreatment conferred protection against a lethal concentration (45 mmol l-1) of this agent. The relatively high concentrations of H2O2 used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H2O2 cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H2O2, in addition to the impressive enhancement of synthesis of five H2O2 stress proteins. Combined results suggest that these proteins might play an important role in the H2O2 tolerance response.

Bacterial Proteins↗

PubMed2005

[Molecular characterization of a glycopeptide-resistant Enterococcus faecalis outbreak in an intensive care unit].

INTRODUCTION: Between January and August 2003 glycopeptide-resistant Enterococcus faecalis was isolated from eight patients admitted to the intensive care unit (ICU). METHODS: Antibiotic susceptibility testing was performed by disk diffusion and the Etest, clonal relatedness of the isolates was studied by pulsed-field gel electrophoresis (PFGE), and the presence of vanA was investigated by PCR. RESULTS AND CONCLUSIONS: All the isolates were vanA-positive and had an identical PFGE pattern, showing that an outbreak had occurred in our ICU.

Bacterial Proteins↗

PubMed2003

Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales.

The present study describes the first molecular characterization of environmental and clinical isolates of vancomycin-resistant enterococci (VRE) in Wales. Over a 3-month period (May-July 2000), 134 isolates of VRE (89 Enterococcus faecium and 45 Enterococcus faecalis) were isolated from the patient environment of the University Hospital of Wales (UHW) in Cardiff, Wales, UK. In addition, over the same time-period, 24 clinical isolates of VRE (20 isolates of E. faecium and four isolates of E. faecalis) were obtained from 14 patients. All study isolates were subjected to PFGE typing and their van genotypes were determined by using multiplex PCR. The vanA PCR product (231 bp) was evident in 146 (92 %) of 158 VRE isolates; the remaining 12 isolates (8 %) were positive for the vanB gene. All isolates of E. faecalis were found to be vanA-positive. In total, 16 PFGE banding profiles (pulsotypes) were observed for environmental isolates of E. faecium, whilst eight pulsotypes were found for isolates of E. faecalis. Some of these pulsotypes were isolated from multiple sites, whereas others were more restricted in their distribution. Eleven pulsotypes were evident for clinical isolates and eight of these (representing 11 isolates) were also encountered in environmental isolates. Eleven clinical isolates of E. faecium (55 %) shared an identical pulsotype that was not detected in environmental isolates. These results demonstrate a heterogeneous environmental population of VRE and an association of certain strains with clinical isolates. Predominance of a single pulsotype (not detected in the environment) amongst clinical isolates suggests non-environmental transmission between patients.

Bacterial Proteins↗

PubMed2004

Genetic characterization of high-level gentamicin-resistant strains of Enterococcus faecalis in Iran.

The prevalence of resistance to high levels of gentamicin among 182 isolates of Enterococcus faecalis from 2 Iranian hospitals was 42%. Gentamicin resistance was associated with conjugative plasmids (>70 kb) in most strains. Fingerprinting using EcoRI and HindIII showed genetic variation among these plasmids and gave evidence of nosocomial outbreaks and persistence of infection in different wards of the study hospitals, as well as transfer of plasmids between genetically diverse isolates. Using EcoRI, hospital-based specific plasmid fingerprints were detected for the isolates that had previously proved to be unrelated by multilocus enzyme electrophoresis, suggesting the persistence of related plasmids at each hospital, though minor changes in these related plasmids could be detected with HindIII.

Anti-Bacterial Agents↗

PubMed2004

Effects of glucose on fsr-mediated biofilm formation in Enterococcus faecalis.

Biofilm production is frequently dependent on such environmental factors as cell density and glucose concentration. The Enterococcus faecalis quorum-sensing locus (fsr) increases enterococcal virulence in multiple animal models. To date, fsr has been shown to regulate the transcription of 2 downstream protease genes. We demonstrate that the effect of fsr mutations on biofilm formation, as well as the fsr-mediated catabolite control of biofilm, is mediated via these proteases. The present study provides additional insight into the mechanisms used by E. faecalis to establish nosocomial infection.

Bacterial Proteins↗

PubMed2004

Expression, purification, crystallization and preliminary crystallographic studies of the Enterococcus faecalis cytolysin repressor CylR2.

The expression of an exotoxin called cytolysin contributes to the virulence of Enterococcus faecalis, one of the organisms responsible for antibiotic resistant infections acquired in hospitals. The DNA-binding protein CylR2 is a transcriptional repressor of cytolysin. At a specific cell density, cytolysin triggers signaling events, which result in the dissociation of CylR2 from its DNA-binding site. CylR2 was overexpressed in Escherichia coli and purified and crystals diffracting to 1.9 A were obtained in two different crystal forms. One crystal form belongs to space group P4(1), with unit-cell parameters a = 63.7, b = 63.7, c = 41.2 A, alpha = beta = gamma = 90 degrees, and the other belongs to space group P1, with unit-cell parameters a = 36.9, b = 45.0, c = 47.7 A, alpha = 67, beta = 90, gamma = 66 degrees.

Bacterial Proteins↗

PubMed1990

Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems.

Some effects of visible light on Escherichia coli and Enterococcus faecalis in natural freshwater and seawater were studied by plate counts, colony area measurements, and direct counts. A large number of somnicells (non-culturable cells) were noted in illuminated systems as compared with non-illuminated ones. Colony areas were significantly smaller in illuminated systems. Indirect activity measurements were used to test the effects of visible light on the ability of E. coli and Ent. faecalis to metabolize substrates ([14C]glucose) in natural waters. In illuminated systems, a decrease of glucose uptake was observed. When percentages of assimilation and respiration with respect to the total glucose uptake were analysed a decrease of assimilation percentages and an increase of respiration percentages were observed. In addition, differences in glucose uptake, assimilation and respiration by enteric bacteria were detected for E. coli at the beginning of the experiments between fresh- and seawater and these were interpreted as a toxic effect exerted by seawater on E. coli cells. Differences between species, natural waters and parameters studied (excepting glucose assimilation) were detected in the illuminated systems. We concluded, however, that enteric bacteria under visible light illumination show a general survival strategy characterized by reaching progressively a somnicell stage which can be defined in terms of their (1) inability to form colonies on standard bacteriological media, (2) inability to incorporate substrates, and (3) inactivation of biosynthetic processes.

Colony Count, Microbial↗

PubMed1998

Diversity of structures carrying the high-level gentamicin resistance gene (aac6-aph2) in Enterococcus faecalis strains isolated in France.

Of 24 high-level gentamicin-resistant clinical isolates of Enterococcus faecalis, 20 carried gentamicin resistance (Gmr) plasmids. The plasmids ranged from 65.0 to 80.0 kb in size. Three of these plasmids were nonconjugative, and 17 transferred by conjugation to an E. faecalis recipient at low frequency (10(-5) to 10(-6) transconjugants per donor). The remaining four strains had a nonconjugative chromosomal Gmr determinant. On the basis of restriction enzyme and DNA-DNA hybridization profiles, Tn4001-like alpha elements were located on the chromosome and three types of Tn4001-truncated structures, I, II, and III, were found to be carried by the Gmr plasmids. Structure I lacked IS256 in the right-hand flanking extremity of Tn4001. Structure II was the same as structure I except that it also had a partial deletion of IS256 in the left-hand flanking extremity of Tn4001. Structure III lacked both the right- and left-hand flanking extremities of Tn4001. One of the wild-type strains carried the Gmr determinant both on the chromosome, as a Tn4001-like alpha element, and on a conjugative plasmid, as a Tn4001-truncated type I structure.

Chromosome Mapping↗

PubMed2025

Cellodextrin Metabolism and Phosphotransferase System-Catalyzed Uptake in Enterococcus faecalis.

Two PTS transporters involved in the uptake of cellobiose and short cellooligosaccharides were identified in Enterococcus faecalis. Genes coding for the different EII proteins are found in a locus composed of three operonic structures expressing two distinct EIIC (CelC1 and CelC2), two identical EIIB (CelB1 and CelB2) and a unique EIIA (CelA1). The EIIA plays a central role in β-glucoside uptake because it is required not only for β-homodiholosides but also for the diheteroside N-acetylglucosamine-L-asparagine. Depending on their size, cellooligosaccharides are preferably transported either by CelC1 (di-saccharides) or by CelC2 (4 glycosidic residues and more), with tri-saccharides being taken up by both EIIC transporters. Moreover, CelA1B2C2 require CelGHI to be functional, three small proteins, the function of which remains unknown. CelA1B1C1 is the main but not exclusive transporter of cellobiose and chitobiose. It is involved in the transport of other β-glucodisaccharides, such as laminaribiose and sophorose. This PTS can be complemented by other transporters highlighting the existence of a network for β-glucoside uptake. This locus is under the control of CelR, a LevR-like transcription activator.

Enterococcus faecalis↗

PubMed2001

Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance.

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

Bacterial Adhesion↗

PubMed2005

Enterococcus faecalis sex pheromone plasmid pAM373: analyses of TraA and evidence for its interaction with RpoB.

The Enterococcus faecalis plasmid pAM373 (36.7kb) encodes a mating response to the sex pheromone cAM373 secreted by recipient (plasmid-free) bacteria. Like certain other conjugative enterococcal plasmids, a key regulator of the pheromone response is a negatively acting protein, TraA, which is believed to interact with internalized pheromone to influence expression from a key transcriptional promoter P(0). An earlier report showed that in the case of pAM373 most, but not all, transposon-insertion mutations in traA differed from those in the case of pAD1 and pCF10 in that they did not give rise to the normally characteristic constitutive clumping. We show here that this phenomenon relates to a host effect involving an RpoB-related mutation associated with rifampin resistance. When harboring traA mutants, rifampin-sensitive hosts exhibited constitutive clumping, whereas rifampin-resistant hosts did not-despite the fact that the latter host exhibited a normal pheromone-inducible clumping response when harboring a wild-type plasmid. The data imply that TraA normally remains associated with the transcription complex after induction. In addition the promoter of traA, designated P(a), was shown to be located about 600bp upstream of the translational start site, as clones containing traA required this site to complement traA mutants in trans. Transcription from P(a) also gave rise to a short (130 nt) transcript, mD, expressed at a high level in uninduced cells. An earlier observation suggesting that TraA negatively affected transcriptional readthrough into the 3' end of traA from the t(ac) intrinsic bidirectional terminator between traA and the opposing, adjacent traC was supported by TraA complementation studies. Evidence is also presented suggesting that this regulation at t(ac) also involves an additional, possibly cis-acting, element.

Bacterial Proteins↗

PubMed1996

Detection of genes regulating beta-lactamase production in Enterococcus faecalis and Staphylococcus aureus.

Four beta-lactamase-producing clinical isolates (WH245, WH257, CH570, and DEL) of Enterococcus faecalis were examined for the presence of the staphylococcal beta-lactamase regulatory genes (blaR1 and blaI) by PCR using six primer pairs. All isolates produced small amounts of beta-lactamase constitutively. In WH245, CH570, and DEL, the corresponding regions of the regulatory genes have lost sequences of various lengths. However, the regulatory genes in WH257 appeared to be the same as those in staphylococcal plasmid pI258. The beta-lactamase genes could be transferred to enterococcal and staphylococcal recipients from WH257 and DEL by conjugation or transformation with selection for gentamicin resistance. After transformation, the expression of beta-lactamase from DEL was still constitutive, whereas the gene from WH257 showed inducible expression in Staphylococcus aureus. The gene coding for inducible beta-lactamase production from pI258 showed constitutive expression in E. faecalis. These findings suggest that constitutive beta-lactamase production in E. faecalis is due not only to the absence of functional regulatory genes but to some other factor(s) as well.

Conjugation, Genetic↗