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PubMed2002

Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis.

Enterococci are members of the healthy human intestinal flora, but are also leading causes of highly antibiotic-resistant, hospital-acquired infection. We examined the genomes of a strain of Enterococcus faecalis that caused an infectious outbreak in a hospital ward in the mid-1980s (ref. 2), and a strain that was identified as the first vancomycin-resistant isolate in the United States, and found that virulence determinants were clustered on a large pathogenicity island, a genetic element previously unknown in this genus. The pathogenicity island, which varies only subtly between strains, is approximately 150 kilobases in size, has a lower G + C content than the rest of the genome, and is flanked by terminal repeats. Here we show that subtle variations within the structure of the pathogenicity island enable strains harbouring the element to modulate virulence, and that these variations occur at high frequency. Moreover, the enterococcal pathogenicity island, in addition to coding for most known auxiliary traits that enhance virulence of the organism, includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates, identifying a class of new targets associated with disease which are not essential for the commensal behaviour of the organism.

Base Composition↗

PubMed1995

Transcriptional analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Qs RNA and QL RNA), much larger than the prgQ gene, were expressed in the prgQ region. QS RNA was produced constitutively, whereas QL RNA was produced inducibly by pheromone. The lack of any other open reading frame in QL RNA and significant sequence complementarity between the 3' end of QL RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of QL RNA. A model for transcriptional activation of prgB is presented.

Amino Acid Sequence↗

PubMed2001

Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF.

We have previously identified a locus, fsr, a homologue of staphylococcal agr loci, which positively regulates the expression of gelatinase and serine protease (encoded by gelE and sprE, respectively) in Enterococcus faecalis OG1RF. The expression of the three genes in the fsr locus, fsrA, fsrB, and fsrC, appears to be autoregulated, and we have shown that mutants with insertion disruptions in each of these three genes were significantly attenuated in a mouse peritonitis model compared to the parent strain. In the present study, we showed that fsrB and fsrC are highly expressed in the postexponential growth phase and that their expression is cell density dependent. Reverse transcriptase PCR using primers covering the intergenic regions in the fsr/gelE loci confirmed that fsrB and fsrC, as well as gelE and sprE, are cotranscribed. We also showed, using a nonpolar fsrB deletion mutant, that fsrB, the homologue of agrB of staphylococci with unknown function, is required for the regulatory function of fsr. Primer extension and analysis of transcriptional fusions indicated the presence of promoters immediately upstream of fsrA, of fsrB, and of gelE and that the fsrB and gelE promoters are fsr dependent, while the fsrA promoter is an fsr-independent weak constitutive promoter. Two conserved 7-bp direct repeats were found immediately upstream of the fsrB and gelE promoters, similar to the repeats found upstream of P2 and P3 promoters of the agr locus; deletions and mutations in the repeated sequences completely abolished the fsrB and gelE promoter activities, suggesting that the repeats are important for the regulatory function in the fsrB and gelE promoter regions.

Amino Acid Sequence↗

PubMed1997

An in vitro test model for investigation of disinfection of dentinal tubules infected with Enterococcus faecalis.

The aim of the present study was to develop an in vitro test model from human teeth to comparatively examine antibacterial effectiveness of calcium hydroxide, parachlorophenol (PCP) and camphorated parachlorophenol (CPCP) against Enterococcus faecalis in infected root canals. Cylindrical dentin specimens were prepared from freshly extracted human maxillary anterior teeth. The specimens were inoculated with E. faecalis and then medicated with either CPCP, PCP or Ca(OH)2. The disinfecting efficacy of these agents was tested by collecting dentin chips from the inner ("canal") walls of the specimens and counting viable E. faecalis. The dentin chips were diluted and a classical bacterial count technique was used for recovery of E. faecalis strains of 5% sheep blood agar. The effectiveness of CPCP and PCP at one day was superior to the effectiveness of Ca(OH)2. In the three-day group, CPCP was the most effective, followed by Ca(OH)2. The experimental model used in this study may be useful for investigation of the effect of intracanal medicaments on microorganisms lodged in the root dentinal tubules.

Calcium Hydroxide↗

PubMed2006

Antibacterial activity of a new endodontic sealer against Enterococcus faecalis.

AIM: The purpose of this study was to test a new root canal sealer (Epiphany) and 5 other root canal sealers (Diaket, Endomethasone, AH 26, Sealapex, Sultan) for their antimicrobial effect on Enterococcus faecalis. MATERIALS AND METHODS: The antimicrobial effect of 6 root canal sealers was tested by the agar diffusion method. The freshly mixed sealers were placed in prepared wells of agar plates inoculated with E. faecalis. All plates were incubated for 72 hours at 37 degrees C under aerobic conditions, and zones of inhibition were measured at 24, 48 and 72 hours. RESULTS: All of the sealers caused bacterial growth inhibition. Their effectiveness, in descending order of antimicrobial activity, was as follows: Endomethasone, Sultan, Sealapex, Diaket, Epiphany and AH 26. Epiphany sealer had little effect on the tested microorganism. The effectiveness of the root canal sealers decreased marginally with greater duration of incubation. CONCLUSIONS: The Epiphany root canal sealer offered no antibacterial advantage over the other sealers tested.

Analysis of Variance↗

PubMed2000

Survival of a Pseudomonas fluorescens and Enterococcus faecalis aerosol on inert surfaces.

The aim of the work presented here is to study airborne bacteria survival on three inert supports (glass, polyvinyl chloride, and stainless steel). Two Pasteur Institute bacteria were selected: Enterococcus faecalis 10.30.15 and Pseudomonas fluorescens 56.90. We have observed that bacterial aerosol lethality increased proportionally with the relative humidity of the environment, the gram-negative rod appearing more fragile than the cocci. A significant difference in survival rate is measured dependent on the supports tested, the greatest lethality being observed on the PVCs.

Aerosols↗

PubMed1989

Treatment of experimental endocarditis caused by a beta-lactamase-producing strain of Enterococcus faecalis with high-level resistance to gentamicin.

Several antimicrobial regimens were evaluated in the treatment of experimental enterococcal endocarditis due to a beta-lactamase-producing, highly gentamicin-resistant strain of Enterococcus faecalis. Ampicillin alone cleared bacteremia in the majority of rats and reduced titers of bacteria within vegetations (6.84 versus 8.80 log10 CFU/g in controls) but did not sterilize valves. Ampicillin-sulbactam combinations, vancomycin, daptomycin, and imipenem each reduced residual bacterial titers within vegetations to 4.01 log10 CFU/g or less; in 26 to 43% of animals receiving 5 days of therapy, titers of bacteria were reduced to undetectable levels. In a separate experiment, rats received ampicillin-sulbactam, daptomycin, or vancomycin for 10 days and were then observed for 10 days after termination of therapy for evidence of relapse. In surviving rats, valves remained sterile in four of five rats treated with ampicillin-sulbactam, in five of seven treated with daptomycin, but in only one of eight receiving vancomycin.

Animals↗

PubMed2003

Efficacy of ampicillin combined with ceftriaxone and gentamicin in the treatment of experimental endocarditis due to Enterococcus faecalis with no high-level resistance to aminoglycosides.

OBJECTIVE: This study tests the usefulness of ceftriaxone combined with ampicillin as an alternative to ampicillin plus gentamicin for the treatment of experimental endocarditis due to Enterococcus faecalis without high-level resistance to aminoglycosides. It also determines whether adding ceftriaxone to ampicillin and gentamicin increases the effectiveness against experimental enterococcal endocarditis resulting from E. faecalis. METHODS: Animals with catheter-induced endocarditis were infected intravenously with 108 cfu of the EF91 strain of E. faecalis and were treated for 3 days with ampicillin 2 g every 4 h administered as 'human-like' (H-L) pharmacokinetics, plus gentamicin 1 mg/kg every 8 h H-L, or ceftriaxone 2 g every 12 h H-L alone or combined with gentamicin 6 mg/kg every 24 h administered subcutaneously. RESULTS: The results of therapy for experimental endocarditis resulting from EF91 showed that the combination of ampicillin plus ceftriaxone was as effective as ampicillin plus gentamicin. The triple combination did not improve on the overall efficacies of the two-drug combinations. CONCLUSIONS: Because of its lower nephrotoxicity, ampicillin plus ceftriaxone may be a useful alternative therapy for E. faecalis endocarditis in selected patients.

Aminoglycosides↗

PubMed2001

[The effect of a bacterial immunostimulant (human Enterococcus faecalis bacteria) on the occurrence of relapse in patients with].

The following double-blind, placebo-controlled, multicenter study investigated the influence of a bacterial immunostimulant (Symbioflor 1, cells and autolysate of human Enterococcus faecalis) on the occurrence of relapses in patients with chronic recurrent bronchitis (n = 136; placebo n = 66, verum n = 70) in a 6 months treatment period and a follow-up period of 8 months, compared to placebo. Under verum 39 incidents of relapses were recorded, which was about 60% the number observed among the patients treated with placebo (66 incidents). The verum preparation exhibited superior clinical efficacy compared to placebo (p = 0.001) in the Kaplan-Meier test. This better clinical efficiency of the test preparation was particularly observed during the treatment period, with 12 vs. 27 relapses (p = 0.013), but less during the follow-up observation period, with 27 vs. 39 relapses (p = 0.127). In addition, the time span until occurrence of the first relapse was clearly longer under verum (699 days) than under placebo (334 days) and after the end of the observation period 91% of patients under verum experienced only one relapse compared to 62% in the placebo group (p = 0.01). Severity of relapses under verum was also reduced significantly (chi 2; p = 0.001. Only 4 patients under verum required antibiotic therapy compared to 13 patients under placebo. Verum was equally well tolerated as placebo, with no serious side effects in either group. No changes in laboratory tests--haematology and clinical chemistry--were observed. It can be concluded, that previously demonstrated immunomodifying effects of the test preparation have clinical relevance for the treatment of chronic recurrent bronchitis because not only the number but also the severity of acute relapses could be clearly reduced. This is discussed in view of the current literature.

Adjuvants, Immunologic↗

PubMed2001

Adhesion of Enterococcus faecalis 1131 grown under subinhibitory concentrations of ampicillin and vancomycin to a hydrophilic and a hydrophobic substratum.

The effect of two subinhibitory antibiotic concentrations of ampicillin and vancomycin during growth on the adhesion of Enterococcus faecalis 1131 to glass and silicone rubber was studied in a parallel plate flow chamber. Initial deposition rates and numbers of adhering bacteria after 4 h were higher on hydrophilic glass than on hydrophobic silicone rubber, regardless of growth conditions. The presence of 1/4 minimal inhibitory concentration (MIC) of ampicillin during growth reduced enterococcal adhesion to both substrata, but growth in the presence of 1/4 MIC vancomycin did not affect the adhesion of E. faecalis. Moreover, enterococcal adhesion increased after growth in the presence of 1/8 MIC vancomycin. The increased adhesion after growth in the presence of subinhibitory concentrations of vancomycin may have strong implications for patients living with implanted biomaterials, as they may suffer adverse effects from use of this antibiotic, especially since bacteria once adhered are less sensitive to antibiotics.

Ampicillin↗

PubMed2002

Conjugative transfer of the erm(A) gene from erythromycin-resistant Streptococcus pyogenes to macrolide-susceptible S. pyogenes, Enterococcus faecalis and Listeria innocua.

In mating experiments, the erythromycin resistance methylase gene erm(A) was successfully transferred from erm(A)-positive clinical isolates of Streptococcus pyogenes to macrolide-susceptible recipients of S. pyogenes, Enterococcus faecalis and Listeria innocua. Compared with the SmaI macrorestriction pattern of the S. pyogenes recipient, the patterns of S. pyogenes transconjugants shared the lack of a fragment and the appearance of a new, larger fragment. This is the first experimental evidence that the erm(A) gene can be transferred from erythromycin-resistant S. pyogenes to macrolide-susceptible S. pyogenes as well as to other Gram-positive recipients.

Anti-Bacterial Agents↗

PubMed1998

Structural studies of histidine-containing phosphocarrier protein from Enterococcus faecalis.

Based on the complete sequential assignment of the 1H-NMR spectrum by multidimensional NMR techniques the secondary structure and the local geometry of the active site of histidine-containing phosphocarrier protein (HPr) from Enterococcus faecalis were elucidated. We present a comparative analysis of the active site in the seven known structures of HPr from different organisms determined by NMR or X-ray crystallography. In catalysis, HPr is phosphorylated at the ring N61 of His15. No general agreement exists in literature regarding the structure of the active-centre loop. In the crystal structure of HPr from E. faecalis, a torsion strain of the backbone at position 16 was observed, which was assumed to be important to the catalytic mechanism. Coupling constants were determined in order to calculate phi angles to establish whether there are strained torsion angles in HPr from E. faecalis in the solution state. The evaluation of data obtained indicate a stable and well-defined structure of HPr from E. faecalis, with an overall fold similar to that found in HPr from other bacteria. We find that in the active-site region there are relatively large variations in local geometry between the evaluated structures. In HPr from E. faecalis, a particularly detailed view of the phosphate-binding His15 and residues in close spatial proximity was obtained by determination of coupling constants obtained from the double-quantum-filtered COSY spectrum. Our data indicate that in aqueous solution, in the dominant conformational state there is no torsion strain of the backbone at position 16, as observed in the crystal state. The maximum population of a strained conformation in solution can be estimated to be smaller than 23%. The analysis of the data suggests that the active-centre loop is able to adopt different conformations in solution. A similar observation was made for HPr from E. faecalis phosphorylated at its regulatory site (Ser46). 31P-NMR shows that phosphorylated HPr exists in two conformational substates with nearly equal populations.

Bacterial Proteins↗

PubMed2003

Simulation of human gentamicin pharmacokinetics in an experimental Enterococcus faecalis endocarditis model.

Significant differences between animal and human pharmacokinetics may be responsible for the conflicting results of experimental studies. This study determined the impact of human pharmacokinetic simulation (HPS) on gentamicin activity in an Enterococcus faecalis endocarditis model. The decrease in bacterial counts was greater with HPS than with a dose-equivalent regimen without HPS.

Animals↗

PubMed2005

The effectiveness of three instrumentation techniques on the elimination of Enterococcus faecalis from a root canal: an in vitro study.

The in vitro reduction of a bacterial population in a root canal by mechanical instrumentation using three techniques was evaluated. Root canals inoculated with a Enterococcus faecalis (E. faecalis) suspension were instrumented using hand Hedstroem files, Giromatic files, and Hero 642 rotary instruments. Irrigation was performed using sterile saline solution. Root canals were sampled before and after instrumentation. After serial dilutions, samples were plated onto Mitis-Salivarius agar and the colony forming units grown were counted. All instruments tested were able to significantly reduce the number of bacterial cells in the root canal, however, the results of this study indicated that Hedstroem files, Giromatic, and Hero 642 techniques were not significantly different in their ability to reduce intracanal bacteria.

Bicuspid↗

PubMed2006

Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination.

A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

Alleles↗

PubMed2006

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil.

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopeptide resistance by conjugative transfer of VanA elements.

Bacterial Proteins↗

PubMed2004

Crystallization and preliminary crystallographic analysis of an Enterococcus faecalis repressor protein, CylR2, involved in regulating cytolysin production through quorum-sensing.

CylR2 is one of two regulatory proteins associated with the quorum-sensing-dependent synthesis of cytolysin in the common pathogen Enterococcus faecalis. The protein was expressed with a C-terminal six-histidine tag and purified to homogeneity with a cobalt-affinity column followed by size-exclusion chromatography. Both native and SeMet proteins were produced and crystallized. Complete X-ray diffraction data sets were collected from a native crystal, which diffracted to 2.3 angstroms resolution, and a SeMet crystal, which diffracted to 2.1 angstroms. The crystals were tetragonal, belonging to space group P4(1) or P4(3), with unit-cell parameters a = b = 66.2, c = 40.9 angstroms, alpha = beta = gamma = 90 degrees. Based on the calculated Matthews coefficient of 2.6 angstroms3 Da(-1) as well as analysis of anomalous difference Patterson maps, the asymmetric unit most likely contains two molecules of CylR2.

Cloning, Molecular↗

PubMed2005

Pleiotrophic effects of 2 Enterococcus faecalis sagA-like genes, salA and salB, which encode proteins that are antigenic during human infection, on biofilm formation and binding to collagen type i and fibronectin.

BACKGROUND: We have shown previously that Enterococcus faecium SagA has broad-spectrum binding to extracellular matrix (ECM) proteins. In the present study, 2 sagA-like genes, salA and salB, were identified in Enterococcus faecalis. METHODS: We compared the salA and salB mutants; their parental strain, OG1RF; and the salB-complemented strain for binding to ECM proteins and biofilm formation. RESULTS: The salB mutant (TX5123) grew more slowly but showed greater binding (approximately 10%-20% of cells bound) to fibronectin (FN) and collagen type I (CI) than did OG1RF (approximately 1% of cells bound) (P<.001). Although TX5123 showed decreased biofilm formation in tryptic soy broth plus 0.25% glucose (TSBG) (P<.001 vs. OG1RF), a marked increase in biofilm formation was shown by TX5123 but not by OG1RF when they were grown in TSBG plus horse serum (HS) or TSBG plus FN, and the increase was coincident with increased attachment and hydrophobicity of TX5123. Complementation of the salB mutant restored the wild-type phenotypes. CONCLUSIONS: Whether SalB expression is ever sufficiently low in vivo to enhance adherence to ECM proteins or the serum-elicited increase in biofilm formation seen with the salB mutant in vitro is not currently known, but it is a potential way in which this organism could increase its adherence and biofilm formation during infection.

Bacterial Adhesion↗