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PubMed1991

Characterization of the gentamicin resistance transposon Tn5281 from Enterococcus faecalis and comparison to staphylococcal transposons Tn4001 and Tn4031.

In Enterococcus faecalis, the genetic determinant encoding gentamicin resistance (Gmr) on the conjugative plasmid pBEM10 previously has been shown to be on a mobile element. In the current study, this element, termed Tn5281, was shown to relocate in the absence of homologous recombination in E. faecalis UV202. On the basis of restriction endonuclease analysis and DNA-DNA hybridization studies, Tn5281 was shown to be similar, if not identical, to the Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in U.S. isolates of Staphylococcus epidermidis, since all three of these transposons have symmetrically located HindIII (2.5 kb apart), ClaI (slightly more than 2.5 kb apart), and HaeIII (3.9 kb apart) sites. Restriction endonuclease digestion patterns of Tn5281 generated with HincII, ScaI, and AluI were also consistent with Tn4001 and Tn4031. By using a probe specific for the external portion of the terminal inverted repeat of Tn4031, it was determined that each terminus of Tn5281 contained a 0.35-kb HaeIII fragment and a 0.7-kb HindIII-HaeIII fragment. The sizes of these fragments are identical to those found in the staphylococcal transposons, which is a further indication that inverted repeats like IS256 are present in Tn5281. A 1-kb HaeIII fragment in pBEM10 also hybridized with this probe, which indicates that Tn5281 in pBEM10 contains a double copy of the inverted repeat at one end.

Conjugation, Genetic↗
PubMed2002

The Enterococcus faecalis gene encoding the novel general stress protein Gsp62.

The Enterococcus faecalis general stress protein Gsp62 was purified using two-dimensional gel electrophoresis and its 25 N-terminal amino acid sequence determined. Analysis of the corresponding gene revealed that the gsp62 product is a 172 aa protein. Transcriptional analysis of gsp62 gave evidence for a monocistronic mRNA, the synthesis of which was induced at the onset of stationary phase and in response to heat shock, acid pH, detergents (i.e. SDS or bile salts), ethanol, tert-butyl hydroperoxide, sodium chloride and, to a lesser extent, hydrogen peroxide. 5' rapid amplification of cDNA ends by PCR experiments showed that gsp62 transcription initiates 30 nt upstream of the ATG start codon. Although gsp62 expression was induced in response to various stresses, its disruption had no significant effect on the cell survival after each individual stress. Two-dimensional protein gels from wild-type and mutant cells revealed no pleiotropic effect of the mutation on protein synthesis. Transcriptional fusions with the lacL lacM beta-galactosidase genes showed that an inverted repeat located upstream of the promoter is required for transcriptional induction by environmental stresses but not by entrance into stationary phase. Two distinct mechanisms responding to different signals are thus involved in gsp62 induction.

Amino Acid Sequence↗
PubMed2005

In vitro assessment of the host response against Enterococcus faecalis used in probiotic preparations.

Along with other lactic acid bacteria, enterococci are used in food products and as health promoting agents. The safety of these products must be ensured, because they contain potentially pathogenic microorganisms. Here we present an in vitro opsonophagocytic assay that closely mimics the protective human immune response to Enterococcus faecalis and Enterococcus faecium. A collection of closely related E. faecalis isolates used as probiotics showed different susceptibilities to opsonic killing, suggesting that some of these isolates possess a capsule while other do not. This information may be helpful in assessing the safety of a given bacterial isolate used and could detect likely enterococcal candidates for probiotic preparations.

Bacterial Capsules↗
PubMed2002

Pre-inoculation of urinary catheters with Escherichia coli 83972 inhibits catheter colonization by Enterococcus faecalis.

PURPOSE: The capacity of a preexisting coating of Escherichia coli 83972 to reduce catheter colonization by Enterococcus faecalis 210 was investigated. Enterococcus was chosen for these trials since it is a common urinary pathogen in patients with an indwelling urinary catheter. MATERIALS AND METHODS: Each experiment tested 3 growth conditions. Group 1 or E. coli plus Enterococcus catheters were exposed to E. coli 83972 for 24 hours and then to Enterococcus for 30 minutes. Group 2 or E. coli alone catheters were incubated in E. coli for 24 hours and then in sterile broth for 30 minutes. Group 3 or Enterococcus alone catheters did not undergo the initial incubation with E. coli before the 30-minute incubation with Enterococcus: All catheters were then incubated in sterile human urine for 24 hours. Catheters were washed with saline and cut into 5, 1 cm. segments. Each segment was sonicated and the sonication fluid was diluted and plated. The results of each of the 5 segments were averaged and the set of experiments was repeated 7 times. RESULTS: A preexisting coating of E. coli 83972 reduced catheter colonization by E. faecalis 210 more than 10-fold. Enterococcus alone catheters had a median of 9.7 x 10(5) enterococci per cm., whereas E. coli plus Enterococcus catheters had a median of 0.38 x 10(5) enterococci per cm. (p = 0.016). CONCLUSIONS: Pre-inoculating urinary catheters with E. coli 83972 significantly impedes catheter colonization by Enterococcus: These promising in vitro results prompt the clinical investigation of this particular application of bacterial interference.

Antibiosis↗
PubMed2004

A case of Enterococcus faecalis endophthalmitis with corneal ulcer.

Although there have been a few reported cases of Enterococcal endophthalmitis, this is an unusual case of endophthalmitis complicated with corneal ulcer caused by Enterococcus faecalis. A 67-year-old male patient with diabetes mellitus underwent secondary intraocular lens implantation. Post-operative recovery was uneventful until a wound rupture was noted 3 weeks after the operation. On day 12 after the repair of the wound, endophthalmitis accompanied by wound necrosis and a full-thickness corneal ulcer was detected. His vision was light perception, and Enterococcus faecalis was identified by culture in samples of conjunctival sac, anterior chamber and vitreous humor. After 3 rounds of intravitreal antibiotics injection, the vitreous opacity disappeared on ultrasonographic finding but corneal opacity and corneal neovascularization still remained.

Aged↗
PubMed2004

Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.

Conjugation, Genetic↗
PubMed1996

Gentamicin resistance gene transfer from Enterococcus faecalis and E. faecium to Staphylococcus aureus, S. intermedius and S. hyicus.

Transfer of gentamicin resistance from Enterococcus faecalis and E. faecium to Staphylococcus aureus, S. hyicus and S. intermedius was accomplished by filter mating. Transfer from E. faecalis was plasmid mediated and a probe to the gentamicin resistance gene aac(6')-aph(2") hybridized to HindIII fragments of various sizes (7.5, 6.2 and 4.7 kbp) of these plasmids. Transfer from E. faecium was apparently to the chromosome in 9 of 10 instances with HindIII fragments of 3.3 or 3.0 kbp hybridizing to the probe. These results are consistent with transposon mediated resistance. Plasmid transfers to S. intermedius were at much lower frequencies than to S. aureus or to S. hyicus but presumed transposon transfer direct to the chromosome was equal in all three species.

Burns↗
PubMed1992

Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis.

The contribution of the pAD1-encoded cytolysin to Enterococcus faecalis virulence in an experimental endophthalmitis model was studied by using isogenic strains differing only in the location of transposon Tn917. The course of experimental endophthalmitis in New Zealand White rabbits was evaluated by postoperative reduction in retinal neuroresponsiveness, thin-section histopathology, and transmission electron microscopy. Infections caused by cytolytic E. faecalis resulted in 99% loss of retinal function at postoperative day 3, while isogenic, noncytolytic strains produced reductions of only 74.2%. Light microscopy revealed near-total destruction of retinal architecture at 24 h postinfection with cytolytic E. faecalis, while noncytolytic strains produced few or no destructive changes. Transmission electron microscopy revealed tissue destruction in retinal layers as early as 6 h postinfection with cytolytic E. faecalis. In vivo and in vitro growth rates of cytolytic and noncytolytic E. faecalis showed similar kinetics. These data demonstrate the contribution of the pAD1-encoded cytolysin to both the course and the severity of experimental E. faecalis endophthalmitis.

Animals↗
PubMed2000

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071.

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.

Amino Acid Sequence↗
PubMed1990

[The virulence of Enterococcus faecalis in experimental urinary tract infection in mice].

The virulence of Enterococcus faecalis in the urinary tract in mice was studied. Pyelonephritis was distinctly caused by E. faecalis which have a high affinity to mouse kidneys. This infection was continuous, but did not turn into sepsis. The relationship between the production of hemolysin or protease and the virulence in the urinary tract was investigated without finding any distinct association. It is concluded that E. faecalis is a pathogenic organism in the urinary tract and presumably induces a severe infection in the compromised host.

Animals↗
PubMed2005

Biofilm formation by Enterococcus faecalis on intraocular lens material.

PURPOSE: To compare biofilm formation by Enterococcus faecalis on different intraocular lens (IOL) materials. METHODS: E. faecalis biofilms were cultivated on disks of IOL material (silicone, PMMA [polymethylmethacrylate], or acrylic). Biofilms were stained with crystal violet (CV), which served as an index of biofilm formation. The bacterial population was enumerated after biofilm homogenization. Biofilms were also examined by confocal microscopy. RESULTS: At 24 hr, there was no significant difference in biofilm formation, or the population within biofilms, among the three materials tested. However, after 48 and 72 hr, it was observed that silicone supported the least amount of biofilm formation (p < 0.05); biomass on PMMA and acrylic IOLs continued to increase, with acrylic demonstrating the greatest amount of stainable biofilm (p < 0.0005). The population on PMMA was significantly greater than the other IOL materials (p < 0.005). Confocal microscopy confirmed the lack of biomass on silicone. CONCLUSIONS: Among three IOL materials, E. faecalis biofilms formed more readily on PMMA and acrylic compared to silicone.

Acrylic Resins↗
PubMed1993

Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faecalis sex-pheromone plasmids.

The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sex-pheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N- and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed.

Antibodies↗
PubMed1996

[Characteristic clinical features of pyelonephritis caused by Enterococcus faecalis].

A clinical study was performed in 13 cases of refractory Enterococcus faecalis pyelonephritis that were detected in the Department of Urology, Hiratsuka Municipal Hospital, from April 1975 to March 1995. The characteristic features were that pyelonephritis was commonly seen in females, and clinical symptoms (low-grade intermittent fever, low back pain, general malaise, etc.) were continuously refractory. Bacteriuria or polymicrobial infections were often found, and bacterial count was often as high as 10(2)-10(4)/ml. Nevertheless we considered that E. faecalis caused of refractory pyelonephritis must not have week adherence and pathogenesis to the kidney, owing to the clinical symptoms and the basic subjects. Furthermore this infection was difficult to cure completely with antimicrobials having activity against E. faecalis, and long-term treatment was needed. Therefore, we recommend that treatment for refractory E. faecalis pyelonephritis be carefully selected according to clinical symptoms and the bacterial density of this strain.

Adolescent↗
PubMedDate not supplied

Antimicrobial resistance from enterococci in a pediatric hospital. Plasmids in Enterococcus faecalis isolates with high-level gentamicin and streptomycin resistance.

BACKGROUND: Enterococcus spp. is an important nosocomial and community-acquired pathogen. Recent studies have documented the increasing importance of this pathogen in children, particularly in the hospital setting. Our objective in this study was to report the frequency of antimicrobial resistance in enterococci and to determine the characteristics of high-level gentamicin resistance (HLGR) plasmids in Enterococcus faecalis clinical isolates. METHODS: Two hundred eighty-nine enterococcal isolates were collected during an 18-month period from a tertiary-care pediatric hospital in Mexico City. Isolates were screened for antibiotic resistance, including HLGR. High-level, gentamicin-resistant E. faecalis strains were selected for pulsed-field electrophoresis (PFGE) typing and plasmid analysis. Transferability of resistance markers was carried out using filter matings. RESULTS: Seventy-six percent of isolates were E. faecalis, 10% were E. avium, 5.2% E. faecium, 5.2% E. raffinossus, 1.38% E. malodoratus, 0.6% E. hirae, and 0.6% E. casseliflavus. Antimicrobial resistance was ampicillin and penicillin 29%, imipenem 17%, and vancomycin 3%, HLGR 5%. The following 15 high-level, gentamicin-resistant isolates were identified: six E. faecalis; four E. avium; three E. faecium, and two E. casseliflavus. Five of the six E. faecalis isolates were different by PFGE and transferred gentamicin and streptomycin resistance on filter membranes. Transfer frequencies ranged from 8.2 x 10(-4) to 6.92 x 10(-5) transconjugants/recipient cell. The plasmid content of donors and transconjugants were homogeneous (one plasmid of 47 kb). CONCLUSIONS: In this pediatric hospital, antimicrobial resistance in Enterococcus spp. is common. Frequency of high-level, gentamicin-resistant strains is low. Mechanism of HLGR appears to be due to a single plasmid dissemination.

Drug Resistance, Microbial↗
PubMed2006

Coaggregation interactions between oral and endodontic Enterococcus faecalis and bacterial species isolated from persistent apical periodontitis.

Interactions between Enterococcus faecalis and other species found in root canal infections might be important for the development and persistence of periapical disease. The aim of this study was to investigate the coaggregation interactions between E. faecalis clinical isolates and species previously shown to survive and induce apical periodontitis in monkeys: Peptostreptococcus anaerobius, Prevotella oralis, Fusobacterium nucleatum, and Streptococcus anginosus. Intergeneric coaggregation assays were conducted in duplicate with observations scored immediately at 0 h, 1 h and 24 h after mixing of combinations of strains. All E. faecalis strains (n = 53) coaggregated with F. nucleatum; E. faecalis did not coaggregate with P. anaerobius or S. anginosus. One strain, E. faecalis E1, coaggregated with P. oralis, with aggregates visible at 1 h. Coaggregation interactions between E. faecalis and F. nucleatum observed in this study suggest a potential role for this combination in endodontic infections.

Animals↗
PubMed1997

The joint of Tn916 circular intermediates is a homoduplex in Enterococcus faecalis.

Tn916 is a 18-kb conjugative transposon originally identified in Enterococcus faecalis. The first step for Tn916 movement is its excision from the donor replicon with the formation of a nonreplicating covalently closed circular intermediate. Studies on formation of circular intermediates in Escherichia coli have shown that the joint between the Tn916 termini is a 6-bp heteroduplex formed by the two regions flanking the transposon before its excision (coupling sequences). In this work we studied the joint of Tn916 termini in circular intermediates formed in both E. coli and E. faecalis. Our strategy was to use direct sequencing of amplification products obtained from the joint region of single target molecules. In E. coli, 50% of circular intermediates contained a heteroduplex joint, while the remaining 50% displayed a homoduplex joint formed by one of the two coupling sequences. In E. faecalis, we could not demonstrate the presence of any heteroduplex joint. In this case 77.7% of the analyzed joints were formed by the left coupling sequence.

DNA Replication↗
PubMed2003

Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage.

Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies), and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora.

Animals↗