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PubMed1996

Augmented production of extracellular superoxide by blood isolates of Enterococcus faecalis.

To assess the frequency of extracellular superoxide (O-2) production by enterococci, multiple species were surveyed in a whole organism assay for their ability to reduce ferricytochrome c in a superoxide dismutase-inhibitable fashion. For stool and clinical enterococcal isolates and 12 type strains, only Enterococcus faecalis (87/91 isolates and ATCC 19433), Enterococcus faecium (5/13 isolates), Enterococcus casseliflavus (ATCC 25778), and Enterococcus gallinarum (ATCC 35038) produced extracellular O-2. Among 106 strains comprising 13 species of enteric gram-negative bacilli and gram-positive cocci, only Lactococcus lactis subspecies lactis produced extracellular O-2. Mean (+/-SE) rates of extracellular O-2 production in vitro by E . faecalis for isolates associated with bacteremia and endocarditis and for isolates from stool were 2.4+/-0.2, 1.9+/-0.2, 1.5+/-0.3 nmol of O-2 min(-1) 10(9) cfu(-1), respectively (P=.025, analysis of variance), suggesting an association between invasiveness and extracellular O-2 production.

Bacteremia↗

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↗

PubMed1995

Conjugative transposition of Tn916-related elements from Enterococcus faecalis to Escherichia coli and Pseudomonas fluorescens.

We studied the ability of transposons Tn916, Tn1545, and Tn916-Km, a Tn916 derivative expressing kanamycin resistance, to be conjugatively transferred from Enterococcus faecalis to various gram-negative bacteria. Our results demonstrate that these types of elements can carry out conjugative transposition from the chromosome of E. faecalis to those of Escherichia coli and Pseudomonas fluorescens and that the accomplishment of this event depends on the donor potential of the E. faecalis transposon delivery strain. Since the tet(M) gene does not confer a selectable level of tetracycline resistance to gram-negative bacteria such as E. coli, the presence of another marker(s) readily expressed in these recipients is required for the detection of this type of transfer. Conjugal transfer of Tn916-Km from E. faecalis to E. coli is not restricted by the EcoK restriction system, nor does it depend on the presence of functional homologous recombination system and integration host factor proteins in the recipient bacteria.

Base Sequence↗

PubMed2003

Cytolysin gene expression in Enterococcus faecalis is regulated in response to aerobiosis conditions.

Here we investigate the expression of cylL(L)and cylL(S), the genes that encode the structural subunits of the cytolysin/haemolysin of Enterococcus faecalis, in response to aerobiosis conditions. Haemolysis assays of E. faecalis strains cultured under aerobic and anaerobic conditions revealed three different haemolytic phenotypes, one of which exhibited greater haemolysis under anaerobic conditions than under aerobic conditions, and was shown to be associated with the presence of the cyl genes. Reporter gene studies revealed that cylL(L) L(S) promoter activity was significantly greater (up to 8.6-fold) under anaerobic compared to aerobic conditions throughout batch growth, demonstrating that these genes are regulated in response to the degree of aerobiosis. Band shift assays confirmed the binding of a protein factor to the region between 202 and 37 bp upstream of the cylL(L)start codon, and a higher level of binding was observed with anaerobically derived cell-free extracts than with extracts of aerobically grown cells. This is the first report of an oxygen-regulated virulence factor in E. faecalis (that is distinct from the quorum-sensing regulatory system reported previously), and may be of in vivo relevance for the bacterium in biofilms and other environments characterised by oxygen gradients.

Aerobiosis↗

PubMed1994

Enterococcus faecalis aggravates pyelonephritis caused by Pseudomonas aeruginosa in experimental ascending mixed urinary tract infection in mice.

Mixed urinary tract infection was caused by simultaneous inoculation of 10(4) CFU each of Enterococcus faecalis TN2005 and Pseudomonas aeruginosa P9 into the bladders of CBA/J mice. Both organisms proliferated in the kidneys, and viable cell counts of E. faecalis TN2005 reached a peak level of 4.1 x 10(5) CFU per pair of kidneys within the first 24 h, while P. aeruginosa P9 counts increased more slowly. The number of P. aeruginosa P9 cells peaked at 8.3 x 10(6) CFU per pair of kidneys 5 days after infection. Five days after mixed infection, infiltration of neutrophils into the renal pelvis and renal medulla was observed. Immunohistochemical staining revealed the presence of E. faecalis antigen in the renal medulla. P. aeruginosa antigen was detected mainly in the renal pelvis 5 days after infection and in the renal medulla as well as the renal pelvis 14 days after infection. Mixed infection induced pyelonephritis within 5 days after mixed infection, while it was not observed until 14 days after infection with P. aeruginosa P9 alone. P. aeruginosa P9 inoculated together with E. faecalis TN2005 was more resistant to eradication from the kidneys by beta-lactam antibiotics than P. aeruginosa P9 inoculated alone. These results suggest that E. faecalis TN2005 invades the renal medulla first in mixed urinary tract infection and induces histological changes which lead to aggravation of the pyelonephritis caused by P. aeruginosa P9.

Animals↗

PubMed2001

Consequences of VanE-type resistance on efficacy of glycopeptides in vitro and in experimental endocarditis due to Enterococcus faecalis.

The consequences on glycopeptide activity of low-level resistance to vancomycin due to VanE-type resistance were evaluated in vitro and in experimental endocarditis caused by Enterococcus faecalis BM4405 (MICs of vancomycin and teicoplanin: 16 and 0.5 microg/ml, respectively), its susceptible derivative BM4405-1, and susceptible E. faecalis JH2-2. After 24 h of incubation, vancomycin at 8 microg/ml was not active against E. faecalis BM4405 whereas it was bacteriostatic against strains BM4405-1 and JH2-2. Against all three strains, vancomycin at 30 microg/ml and teicoplanin at 8 or 30 microg/ml were bacteriostatic but bactericidal when combined with gentamicin. In rabbits with aortic endocarditis due to VanE-type resistant strain BM4405, treatment with a standard dose of vancomycin generated subinhibitory plasma concentrations (i.e., peak of 36.3 +/- 2.1 microg/ml and trough of 6.0 +/- 2.2 microg/ml) and led to no significant reduction in mean aortic valve vegetation counts compared to no treatment of control animals. In contrast, a higher dosing regimen of vancomycin (i.e., resulting in a peak of 38.3 +/- 5.2 microg/ml and a trough of 15.0 +/- 8.3 microg/ml), providing plasma concentrations above the MIC for the entire dosing interval, led to significant and similar activities against all three strains, which were enhanced by combination with gentamicin. Treatment with teicoplanin led to results similar to those obtained with vancomycin at a high dose. No subpopulations with increased resistance to glycopeptides were selected in vitro or in vivo. In conclusion, the use of a high dose of vancomycin was necessary for the treatment of experimental enterococcal endocarditis due to VanE-type strains.

Animals↗

PubMed2003

Characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolate from a dog with mastitis: further evidence of a clonal lineage of VREF in New Zealand.

We report here on the characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolated from a dog with mastitis. The isolate was positive for the vanA, ermB, and tet(M) genes, with vanA and ermB carried on the same transferable plasmid. Comparison of this isolate with VREF from poultry and human sources in New Zealand demonstrated identical SmaI macrorestriction patterns and Tn1546-like elements. This is further evidence of a clonal lineage of VREF in New Zealand.

Animals↗

PubMed2006

Enterococcus faecalis strains show culture heterogeneity in cell surface charge.

Adhesion of micro-organisms to biotic and abiotic surfaces is an important virulence factor and involves different types of interactions. Enterococcus faecalis, a human commensal and an important opportunistic pathogen, has the ability to adhere to surfaces. Biliary stents frequently become clogged with bacterial biofilms, with E. faecalis as one of the predominant species. Six E. faecalis strains isolated from clogged biliary stents were investigated for the presence of specific biochemical factors involved in their adhesion: aggregation substances (Aggs) and the enterococcal surface protein (encoded by the esp gene). In addition, physico-chemical factors involved in adhesion (zeta potential and cell surface hydrophobicity) were determined, as well as the influence of ox bile on these properties. Two-thirds of the biliary stent isolates displayed culture heterogeneity in the pH dependence of their zeta potentials. Moreover, 24 out of 46 clinical isolates of E. faecalis, including 11 laboratory strains, also displayed such heterogeneity. The culture heterogeneity was demonstrated to be a stable trait, not caused by quorum sensing, not plasmid mediated, and independent of the presence of esp and Agg. Data presented show that culture heterogeneity in zeta potential enhances adhesion to an abiotic surface. A higher prevalence of culture heterogeneity in zeta potential in pathogenic as compared to non-pathogenic isolates could indicate that this phenomenon might play a role in virulence and putatively in pathogenesis.

Animals↗

PubMed1991

The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis.

The effects of tetracycline on transfer of the conjugative, tetracycline-resistance transposon. Tn925, as well as the ability of the transposon to promote the transfer of chromosomal genes was examined in Enterococcus faecalis and Bacillus subtilis. To test for chromosomal transfer, multiply-marked strains of each organism, each carrying a single chromosomal copy of Tn925, were mated on filters with suitable recipient strains, under conditions where transformation and transduction were precluded. In both cases, transfer of a variety of chromosomal genes, at frequencies comparable to the frequency of Tn925 transfer, was detected readily. The presence of Tn925 in one of the members of the mating pair was absolutely required for chromosomal transfer, but transfer of Tn925 did not accompany every chromosomal transfer event. The results were consistent with a mating event resembling a type of cell fusion, allowing for extensive recombination between the genomes of the mating partners. Growth of Tn925-containing donor cells in the presence of tetracycline increased the transfer frequency of Tn925 by about tenfold in E. faecalis, but not in B. subtilis.

Bacillus subtilis↗

PubMed2000

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

Bacterial Adhesion↗

PubMed2004

Synergistic activity of vancomycin and teicoplanin alone and in combination with streptomycin against Enterococcus faecalis strains with various vancomycin susceptibilities.

The synergy between two glycopeptides, vancomycin (Vm) and teicoplanin (Tec) and streptomycin (Sm) was studied by time-kill method. Five clinical vanB resistant Enterococcus faecalis (ENC) isolates with variable Vm-susceptibility were used. Different concentrations of Vm, Tec and Sm representing therapeutic concentrations were combined. Antibacterial activity was related to the concentrations of Vm and Sm, and Vm susceptibility to ENC. For strains with Vm MIC up to 64 mg/l, synergy was achieved with higher concentrations of Vm and Sm, while all combinations of Tec and Sm were synergistic against all strains except ENC 29. For ENC 29 with Vm MIC of 512 mg/l and Tec MIC of <1 mg/l, none of the combinations was synergistic. The significance of these in vitro results needs further investigation in vivo.

Anti-Bacterial Agents↗

PubMed2001

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery.

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.

ATP-Binding Cassette Transporters↗

PubMed1996

The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.

Bacterial Proteins↗

PubMed1993

Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function.

In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ability of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E. faecalis cell with the ability to specifically remove (apparently by irreversible binding) cCF10 activity from culture medium. The pCF10 DNA encoding this ability was localized to a 3.4-kb segment within a region involved in negative control of expression of conjugal transfer functions. This segment also encoded ability to bind the pheromone inhibitor peptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an adjacent region of pCF10 and could encode a protein with significant similarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducible hemolysin plasmid pAD1, according to F.Y. An and D.B. Clewell (Abstr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene product showed significant relatedness to binding proteins encoded by oligopeptide permease (opp) operons in gram-positive and gram-negative bacteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An, and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.

Amino Acid Sequence↗

PubMed1995

Analysis of Enterococcus faecalis isolates from intercontinental sources by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE) were compared in this study of 65 Enterococcus faecalis isolates recovered over a 20-year period from diverse geographic sources. Clonal relationships recognized by PFGE were also recognized by MLEE; however, MLEE recognized as greater number of isolates as belonging to clonal groups than did PFGE. Both techniques were reproducible and discriminatory, but MLEE more readily recognized relationships among large numbers of isolates. MLEE confirmed the previously reported clonal spread of beta-lactamase-producing E. faecalis to six hospitals in five states. MLEE provided a useful population framework of the E. faecalis isolates in this sample, while PFGE was able to differentiate among isolates within some MLEE clonal groups.

Alleles↗

PubMed2006

In vitro assessment of the effectiveness of chlorhexidine gel and calcium hydroxide paste with chlorhexidine against Enterococcus faecalis and Candida albicans.

OBJECTIVE: The purpose of this study was to compare the effectiveness of various medicaments, including Ca(OH)2/2% chlorhexidine, 2% chlorhexidine gel, and Ca(OH)2 alone, against Enterococcus faecalis and Candid albicans in vitro. STUDY DESIGN: Eighty extracted single-rooted human maxillary teeth were used. After removing the crown, each root was instrumented up to size 50 by using a conventional technique. The root canal was irrigated with ethylenediamine tetra-acetic acid (EDTA) solution to remove smear layer. Then, roots were infected with E faecalis and C albicans. Subsequently, the roots were divided into 4 treatment groups: group 1 was treated with calcium powder hydroxide in distilled water, group 2 was treated with calcium hydroxide powder in 2% chlorhexidine, group 3 was treated with 2% chlorhexidine gel, and group 4 was treated with 0.9% sterile saline serving as negative control. Microbial samples were taken after 7, 15, and 30 days. After incubation, dentine chips were obtained form each root canal and examined microbiologically. The microbiological samples were plated to count colony-forming units in per milligram of dentin. RESULTS: The results showed that the 2% chlorhexidine gel was significantly more effective than calcium hydroxide with 2% chlorhexidine, calcium hydroxide, and control saline solution (P < .05). CONCLUSION: Under the conditions of this study, 2% chlorhexidine gel is effective in the elimination of E faecalis and C albicans from the root canal system. However, to support this in vitro observation, further in vivo studies are needed.

Anti-Infective Agents, Local↗

PubMed2001

Analysis of sigma(54)-dependent genes in Enterococcus faecalis: a mannose PTS permease (EII(Man)) is involved in sensitivity to a bacteriocin, mesentericin Y105.

The sigma(54) RNA polymerase subunit has a prominent role in susceptibility of Listeria monocytogenes and Enterococcus faecalis to mesentericin Y105, a class IIa bacteriocin. Consequently, sigma(54)-dependent genes as well as specific activators also required for expression of these genes were sought. Five putative sigma(54)-associated activators were detected in the genome of E. faecalis V583, and all but one could activate the transcription of permease genes belonging to sugar phosphotransferase systems (PTSs). Interestingly, these activators display a helicase signature not yet reported in this activator family, which could explain the ATP-dependent mechanism of DNA unwinding preceding the start of transcription. To find which activator is linked to susceptibility of E. faecalis to mesentericin Y105, their respective genes were subsequently interrupted. Among them, only mptR gene interruption led to a resistance phenotype. Immediately downstream from mptR, a putative sigma(54)-dependent operon was found to encode a mannose PTS permease, namely EII(t)(Man). Moreover, in liquid culture, glucose and mannose induced the sensitivity of E. faecalis to mesentericin Y105. Since sugars have previously been reported to induce PTS permease expression, it appears that EII(t)(Man) expression, presumably induced in the presence of glucose and mannose, leads to an enhanced sensitivity of E. faecalis to the bacteriocin. Additional information was gained from knockouts within the permease operon. Interruption of the distal mptD gene, which encodes the IID subunit of EII(t)(Man), strikingly led to resistance to mesentericin Y105. Moreover, MptD appears to be a peculiar membrane subunit, bearing an additional domain compared to most known IID subunits. According to these results, EII(t)(Man) is clearly involved in susceptibility to mesentericin Y105 and could even be its receptor at the E. faecalis surface. Finally, it is hypothesized that MptD could be responsible for the targeting specificity, via an interaction between its additional domain and mesentericin Y105.

Amino Acid Sequence↗

PubMed2001

Chemical synthesis and biological activity of the gelatinase biosynthesis-activating pheromone of Enterococcus faecalis and its analogs.

An 11-residue peptide lactone, termed the gelatinase biosynthesis-activating pheromone (GBAP), triggers the production of the pathogenicity-related extracellular proteases, gelatinase and serine protease, in Enterococcus faecalis. In this study, we synthesized GBAP and its analogs and examined their gelatinase biosynthesis-inducing activity. This study on the structure-activity relationship shows that a lactone ring was indispensable for the activity.

Amino Acid Sequence↗