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PubMed2003

The aggregation domain of aggregation substance, not the RGD motifs, is critical for efficient internalization by HT-29 enterocytes.

Aggregation substance (AS), a surface protein encoded on the pheromone-inducible plasmids of Enterococcus faecalis, has been shown to increase adherence and internalization into a number of different cell types, presumably through integrin binding mediated by the N-terminal RGD motif of AS. Here, defined mutations constructed in Asc10, the AS encoded by the plasmid pCF10, are analyzed for their ability to promote increased internalization levels into HT-29 enterocytes. The results clearly show that the previously identified Asc10 functional domain, not the RGD motifs, is critical for Asc10-directed internalization of E. faecalis into HT-29 enterocytes. Also, expression of Asc10 in the nonaggregating E. faecalis strain INY3000 is unable to mediate HT-29 internalization. However, Asc10-expressing E. faecalis cells are not internalized as bacterial aggregates, suggesting bacterial aggregation is not a prerequisite for HT-29 internalization. These data show that Asc10 directs internalization of E. faecalis into HT-29 enterocytes through a non-RGD-dependent mechanism.

Bacterial Proteins↗
PubMed2002

DNA amplification and hybridization assays in integrated plastic monolithic devices.

PCR amplification, DNA hybridization, and a hybridization wash have been integrated in a disposable monolithic DNA device, containing all of the necessary fluidic channels and reservoirs. These integrated devices were fabricated in polycarbonate plastic material by CO2 laser machining and were assembled using a combination of thermal bonding and adhesive tape bonding. Pluronics polymer phase change valves were implemented in the devices to fulfill the valving requirements. Pluronics polymer material is PCR compatible, and 30% Pluronics polymer valves provide enough holding pressure to ensure a successful PCR amplification. By reducing the temperature locally, to approximately 5 degrees C, Pluronics valves were liquefied and easily opened. A hybridization channel was made functional by oligonucleotide deposition, using Motorola proprietary surface attachment chemistry. Reagent transport on the device was provided by syringe pumps, which were docked onto the device. Peltier thermal electrical devices powered the heating and cooling functionality of the device. Asymmetrical PCR amplification and subsequent hybridization detection of both Escherichia coli K-12 MG1655 and Enterococcus faecalis DNAE genes have been successfully demonstrated in these disposable monolithic devices.

DNA↗
PubMed1992

Continuous intravenous versus intermittent ampicillin therapy of experimental endocarditis caused by aminoglycoside-resistant enterococci.

We studied the efficacy of continuous intravenous infusion of ampicillin compared with that of intermittent administration of ampicillin alone or in combination with gentamicin for the therapy of highly aminoglycoside-resistant enterococcal experimental endocarditis. Rabbits were infected with a gentamicin-susceptible (MIC, 256 micrograms/ml) strain of Enterococcus faecalis or a strain of E. faecalis which was highly resistant to gentamicin in vitro (MIC, greater than 2,000 micrograms/ml). Administration of ampicillin by continuous intravenous infusion did not significantly enhance the killing of enterococci in vivo compared with that by intermittent administration of ampicillin for either the aminoglycoside-susceptible or the aminoglycoside-resistant strain. In combination with gentamicin, there were no significant differences in efficacies obtained with intermittent versus continuous intravenous infusion of ampicillin therapy for experimental endocarditis caused by either strain of E. faecalis.

Ampicillin↗
PubMed2003

[Prevalence of AA(6')-APH(2")Ia gene among high-level aminoglycoside resistant Enterococci and Staphylococci].

According to new reports the AAC (6')-APH (2")Ia gene is no longer the only gene encoding resistance to gentamycin in Gram-positive cocci and therefore the current method for predicting synergism aminoglycosides with bacterial cell wall active agents in this bacteria may need revision. To further our knowledge of aminoglycoside resistance mechanism in Gram-positive cocci in Gdańsk region we tested presence of AAC (6')-APH (2")Ia gene among 22 enterococcal (E. faecalis) and 41 staphylococcal (S. haemolyticus, S. aureus, S. epidermidis) gentamycin-resistant isolates. Presence of AAC (6')-APH (2")Ia gene varied from 50% (n = 6) in gentamycin-resistant S. epidermidis, 80% (n = 10) in gentamycin resistant S. haemolyticus 88% in methicillin-resistant Staphylococcus aureus (MRSA) (n = 25). In Enterococcus faecalis this gene was noticed only in 59% (n = 22) of gentamycin-resistant isolates. These results suggest that spread of resistance gene among different species is limited and AAC (6')-APH (2")Ia mediated gentamycin-resistance mechanism is more common among MRSA and Staphylococcus haemolyticus.

Aminoglycosides↗
PubMed1994

Intravenous streptomycin: a useful route of administration.

Streptomycin is an aminoglycoside antibiotic that is indicated for the treatment of tuberculous and nontuberculous infections. Intramuscular injection is the recommended route of administration. There are few reports on intravenous administration of streptomycin. We describe the use of intravenous streptomycin to treat endocarditis due to a strain of Enterococcus faecalis with high-level resistance to gentamicin. Physicians should consider the intravenous route as an alternate method of administering streptomycin.

Aged↗
PubMed2004

Antibacterial efficacy of calcium hydroxide and chlorhexidine gluconate irrigants at 37 degrees C and 46 degrees C.

This study investigated the ability of two endodontic irrigants to eliminate Enterococcus faecalis from dentinal tubules, and whether their antimicrobial action was enhanced by heat. The lumens of disks prepared from extracted bovine roots were infected with E. faecalis and incubated for 72 h. Specimens were then filled with saline, 10% calcium hydroxide (Ca(OH)2), or 0.12% chlorhexidine gluconate (CHX) at 24 degrees C or 46 degrees C and incubated at 37 degrees C or 46 degrees C. The samples were then pulverized and plated to quantify residual bacteria. No statistical difference (p > 0.05) in bacterial growth was seen between the two saline groups, or between the two medication groups at a given temperature. CHX and Ca(OH)2 at either temperature produced significantly less growth than either saline group, and CHX or Ca(OH)2 at 46 degrees C produced significantly less growth than either group at 37 degrees C. Heat enhanced the antibacterial action of both experimental irrigants against E. faecalis, but heating saline produced no increase in bactericidal effect.

Animals↗
PubMed1993

Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis.

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

Animals↗
PubMed1987

Disk diffusion susceptibility testing and broth microdilution quality control guidelines for BMY-28100, a new orally administered cephalosporin.

The BMY-28100 30-micrograms-disk test was evaluated by using 615 clinical isolates. Regression analyses and error rates were determined, leading to the recommendation of greater than or equal to 18-mm zone diameters (MIC correlate, greater than or equal to 8.0 micrograms/ml) for susceptibility and less than or equal to 14-mm zone diameters (MIC correlate, greater than or equal to 32 micrograms/ml) for resistance. Nearly all false-susceptible disk test results were among the Providencia spp. and the beta-lactamase-positive Haemophilus influenzae strains. Susceptibility disk test results for these species should be interpreted with caution. The following broth microdilution MIC quality control guidelines were determined from results of a multilaboratory trial: Escherichia coli ATCC 25922, 1.0 to 4.0 micrograms/ml; Enterococcus faecalis ATCC 29212, 4.0 to 16 micrograms/ml; Staphylococcus aureus ATCC 29213, 0.25 to 1.0 microgram/ml; and Pseudomonas aeruginosa ATCC 27853, greater than 32 micrograms/ml.

Cephalosporins↗
PubMed2002

Biofilm formation in medicated root canals.

The hypothesis that Enterococcus faecalis resists common intracanal medications by forming biofilms was tested. E. faecalis colonization of 46 extracted, medicated roots was observed with scanning electron microscopy (SEM) and scanning confocal laser microscopy. SEM detected colonization of root canals medicated with calcium hydroxide points and the positive control within 2 days. SEM detected biofilms in canals medicated with calcium hydroxide paste in an average of 77 days. Scanning confocal laser microscopy analysis of two calcium hydroxide paste medicated roots showed viable colonies forming in a root canal infected for 86 days, whereas in a canal infected for 160 days, a mushroom-shape typical of a biofilm was observed. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed no differences between the protein profiles of bacteria in free-floating (planktonic) and inoculum cultures. Analysis of biofilm bacteria was inconclusive. These observations support potential E. faecalis biofilm formation in vivo in medicated root canals.

Bacterial Proteins↗
PubMedDate not supplied

Bactericidal effect of levofloxacin on strains with equal susceptibility in an in vitro urinary bladder model.

The in vitro bactericidal effects of therapeutic concentrations of levofloxacin (LVFX) on clinical isolates of Pseudomonas aeruginosa and Enterococcus faecalis were assessed. Starting from 10(7) CFU/ml, the bacterial count was determined serially during the exposure to LVFX present at concentrations automatically simulated to its clinically achievable urinary levels at the recommended dosage (100 mg 3 times daily). An isolate of P. aeruginosa for which the MIC of LVFX was 16 microgram/ml was completely eradicated after 10 h of exposure to LVFX. On the other hand, 40 h of exposure to LVFX were required for complete eradication of an isolate of E. faecalis with equal susceptibility, although the bacterial count was rapidly reduced to 10(2) CFU/ml in the early phase of the exposure. It was concluded that LVFX exerts a more potent bactericidal effect on P. aeruginosa than on E. faecalis despite a similar susceptibility of these organisms to LVFX.

Anti-Infective Agents, Urinary↗
PubMed1993

Blockage of urethral catheters by bacterial biofilms.

We report the case of a 63-year-old woman whose indwelling urethral catheter became blocked regularly at 4-5 day intervals over a period of 10 weeks. 'Worm-like' structures 25-30 cm in length were found either in the catheter, completely occluding the lumen, or in the drainage tube thereby blocking the valve of the drainage bag. Electron microscopy showed that these structures were composed of bacteria, while culture revealed them to be mixed communities of Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis and Proteus mirabilis. Following treatment with ciprofloxacin, catheter drainage continued freely for a further period of 10 weeks.

Catheters, Indwelling↗
PubMed2006

Isolation and identification of the icosalides--cyclic peptolides with selective antibiotic and cytotoxic activities.

Three cyclic peptolides have been isolated from two different fungal species and their structures determined. Icosalides A1 (1a), A2 (1b), and B (1c) each contain two serine and two leucine amino acid residues and incorporate two fatty acid moieties as part of the central twenty-member ring. 1a contains L-serine and both D- and L-leucine residues, while 1b and 1c contain only L-amino acid residues. Icosalide A1 displays antimicrobial activity against Streptococcus pyogenes, S. pneumoniae (Felton), and Enterococcus faecalis. Icosalides A2 and B are cytotoxic to replicating MDCK cells.

Anti-Bacterial Agents↗
PubMed2006

Endogenous and exogenous glucocorticoids in experimental enterococcal infection.

The potentially protective role of the host adrenal-glucocorticoid response to enterococcal infection was evaluated in an experimental model in which mice were infected intraperitoneally with two distinct Enterococcus faecalis strains (K9 and CP-1). We demonstrated that corticosterone levels in serum and peritoneal-lavage fluid were elevated within 1 hour of infection with either E. faecalis strain. We also demonstrated that adrenalectomized mice generated a more robust localized peritoneal tumor necrosis factor alpha (TNF-alpha) response to both E. faecalis strains than did sham-adrenalectomized mice but that neither E. faecalis strain induced a systemic TNF-alpha response. Further, peritoneal TNF-alpha production in adrenalectomized mice infected with either E. faecalis K9 or CP-1 was suppressed by prior treatment with an exogenous glucocorticoid (dexamethasone). The potential clinical significance of these results was suggested by our findings that adrenalectomy markedly increased susceptibility (a>100-fold decrease in the 50% lethal dose) to lethal infections with E. faecalis CP-1 and that prior dexamethasone treatment partially compensated for adrenalectomy. In marked contrast to these findings, however, adrenalectomy did not substantially increase susceptibility to lethal E. faecalis K9 infection. Further, preinfection with E. faecalis CP-1 1 hour before infection with E. faecalis K9 did not protect mice from lethal E. faecalis K9 infections. Collectively, these studies indicate that the host can generate a glucocorticoid response to E. faecalis infection that suppresses TNF-alpha production. Further, this glucocorticoid response can protect the host from potentially lethal E. faecalis infections, but different strains show heterogeneity with respect to the extent of protection afforded by the adrenal-glucocorticoid response.

Adrenalectomy↗
PubMed2005

Occurrence of pS86/pEF47-related plasmids in gram-positive cocci.

A small, low-copy-number cryptic 5.5-kb plasmid, designated pEF47, was isolated from a rumen bacterium Enterococcus faecalis 47 and partially characterized. Comparisons by restriction and partial sequence analysis of pEF47 demonstrated high homology to pS86 plasmid, recently isolated from a human clinical strain E. faecalis S-86. PCR followed by sequence analysis and Southern hybridization showed that pS86/pEF47-related replicons are regularly encountered in plasmids from gram-positive cocci.

Animals↗
PubMed2001

Antimicrobial activity of several calcium hydroxide preparations in root canal dentin.

The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)2) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degrees C in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)2 USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)2 USP (0.1 g/ml H2O); group 3, Pulpdent TempCanal paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)2 can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)2 may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.

Analysis of Variance↗
PubMed2005

Identification of enterococcal, streptococcal and Weissella species in the faecal flora of individually owned dogs.

AIMS: To improve the limited information on the composition of the faecal Gram-positive coccal flora of healthy dogs by the use of a molecular identification method. METHODS AND RESULTS: Faecal swabs were collected for the selective isolation of Gram-positive coccal strains. Colonies with enterococcal- and streptococcal-like morphology were identified by tRNA intergenic length polymorphism analysis (tDNA-PCR). Fourteen known species belonging to three genera (Enterococcus, Streptococcus and Weissella) and one alleged new enterococcal species were found. CONCLUSIONS: The faecal flora of dogs comprises an unusually broad diversity of culturable Gram-positive coccal species with Enterococcus faecalis being most frequently present followed by not less than six other species of about equal importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Many human- and animal-associated enterococci and streptococci are also present in dog faeces together with the largely uncharacterized Weissella cibaria and a group of strains resembling Enterococcus dispar, but representing a distinct and hitherto unknown species. Phenotypic characteristics of the latter two species were determined and the test results were compared with the species descriptions of W. cibaria and E. dispar respectively.

Animals↗
PubMed1994

Roles of antibodies and complement in phagocytic killing of enterococci.

The contributions of complement and antibodies to polymorphonuclear leukocyte (PMN)-mediated killing of enterococci were investigated with pooled normal human serum (PNHS) or immune human sera (IHS) from patients with serious enterococcal infections. Each IHS containing antienterococcal antibodies demonstrated by enzyme-linked immunosorbent assay and Western blotting (immunoblotting) was examined with the enterococcus strain isolated from the same patient. PNHS promoted PMN-mediated killing of enterococci similar to that for IHS. PMN-mediated killing was consistently abrogated after preopsonization with heat-inactivated PNHS, but some heat-inactivated IHS supported neutrophil bactericidal activity. Inhibition of the classical pathway of complement by chelation of either PNHS or IHS with Mg-EGTA [Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not alter PMN-mediated killing, suggesting that activation of the alternative pathway of complement is sufficient to promote killing of enterococci by PMNs. PMN-mediated killing assays were also performed with normal rabbit serum and immune rabbit serum against enterococci. Preopsonization with heat-inactivated immune rabbit serum resulted in PMN-mediated killing of enterococci, which was ablated after adsorption of the serum with the same isolate used for immunization. The influence of different phenotypic enterococcal traits on neutrophil-mediated killing was also investigated. Similar kinetics of killing were observed for derivatives of Enterococcus faecalis strains regardless of resistance to antimicrobial agents or production of beta-lactamase, hemolysin, gelatinase, or surface proteins involved in the aggregative response to pheromones. In summary, PMN-mediated killing of enterococci appears to depend primarily on complement activation by either the classical or the alternative pathway. Human antienterococcal antibodies generated during infection variably promoted neutrophil bactericidal activity, while antibody raised in a rabbit supported PMN-mediated killing of the organism examined. Finally, the different phenotypic properties of E. faecalis examined did not influence the neutrophil-mediated killing of these organisms.

Adult↗