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PubMed2005

Enterococcus faecium SF68 enhances the immune response to Giardia intestinalis in mice.

We studied the ability of the probiotic organism Enterococcus faecium SF68 to antagonize Giardia intestinalis infection in mice. Oral feeding of E. faecium strain SF68 starting 7 d before inoculation with Giardia trophozoites significantly increased the production of specific anti-Giardia intestinal IgA and blood IgG. This humoral response was mirrored at the cellular level by an increased percentage of CD4(+) T cells in the Peyer's patches and in the spleens of SF68-fed mice. The improvement of specific immune responses in probiotic-fed mice was associated with a diminution in the number of active trophozoites in the small intestine as well as decreased shedding of fecal Giardia antigens (GSA65 protein). The ability of SF68 to stimulate the immune system at both mucosal and systemic levels highlights mechanisms by which this probiotic might antagonize pathogens in vivo. Taken together, the data demonstrate the strong potential of strain SF68 to prevent protozoa from causing intestinal infections.

Animals↗
PubMed2002

Nucleotide-binding sites in V-type Na+-ATPase from Enterococcus hirae.

Enterococcus hirae V-ATPase, in contrast to most V-type ATPases, is resistant to N-ethylmaleimide (NEM). Alignment of the amino acid sequences of NtpA suggests that the NEM-sensitive Cys of V-type ATPases is replaced by Ala in E. hirae V-ATPase. Consistent with this prediction, the V-ATPase became sensitive upon substitution of the Ala with Cys. The three-dimensional structure of the NtpB subunit of V-ATPase was modeled based on the structure of the corresponding subunit (alpha subunit) of bovine F(1)-ATPase by homology modeling. Overall, the 3D structure of the subunit resembled that of alpha subunit of bovine F(1)-ATPase. The NtpB subunit, which lacks the P-loop consensus sequence for nucleotide binding, was predicted to bind a nucleotide at the modeled nucleotide-binding site. Experimental data supported the prediction that the E. hirae V-ATPase had about six nucleotide-binding sites.

Adenosine Triphosphatases↗
PubMed1999

Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety.

We report here the large-scale purification of vacuolar (V0V1)-type Na+-ATPase from Enterococcus hirae achieved using column anion-exchange and gel filtration chromatographies; 32 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, and K, was obtained from 20 liter culture. This amount is 500-fold larger than that reported in the previous paper [Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1997) J. Biol. Chem. 272, 24885-24890]. The purified enzyme shows a high specific activity of ATP hydrolysis (35.7 micromol Pi released/min/mg protein). ATP-driven 22Na+ uptake by reconstituted V0V1-proteoliposomes exhibited an apparent Kt value for Na+ of 40 microM, which is near the Km value (20 microM) for Na+ of the ATP hydrolytic activity. Denatured gel electrophoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V1 subunit from the V0V1 complex by incubation with ethylenediaminetetraacetic acid; subunit G was not identified. The remaining V0-liposomes containing I and K subunits catalyzed Na+ uptake in response to potassium diffusion potential (Deltapsi, inside negative); the Kt value for Na+ of this reaction was estimated to be about 2 mM. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of the Na+-ATPase activity and Deltapsi-driven Na+ uptake by the V0-liposomes was prevented by the presence of Na+, suggesting that the Na+ binding site overlaps with the DCCD-reactive site.

Dicyclohexylcarbodiimide↗
PubMed1994

Purification and characterization of the catalytic moiety of vacuolar-type Na(+)-ATPase from Enterococcus hirae.

We have previously reported the molecular cloning and sequences of the ntp genes for Enterococcus hirae Na(+)-translocating ATPase [Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044]; the expected structure of this enzyme complex resembles those of the vacuolar H(+)-ATPase complexes in eukaryotes. In this paper we report purification and characterization of the catalytic moiety of Na(+)-ATPase, whose molecular size was about 400 kDa, consisting of polypeptides of 69 kDa (NtpA), 52 kDa (NtpB), and 29 kDa (NtpD) with a probable stoichiometry of 3:3:1. Purified enzyme hydrolyzed GTP as the best substrate (GTP > CTP > UTP > ATP), and the activity was maximal at around pH 6.0. The activity was not stimulated by sodium ions, and was selectively inhibited by nitrate. These properties were different from those of membrane-bound Na(+)-ATPase, suggesting that a significant conformational change of the catalytic moiety may take place upon dissociation from the membrane-embedded moiety and probably also loss of other hydrophilic subunits. Antiserum against purified enzyme inhibited the Na(+)-stimulated ATPase activity of the membranes. Immunoblotting analysis revealed that the change in the amounts of A and B subunits of the membranes paralleled that of the Na(+)-ATPase activity. Furthermore, the A subunit was missing in the membranes of a Na(+)-ATPase mutant, and recovered in those of its revertant. These immunochemical data are consistent with the notion that this enzyme is the hydrophilic catalytic moiety of the V-type Na(+)-ATPase in E. hirae.

Adenosine Triphosphatases↗
PubMed2002

Biochemical and immunological changes in chickens experiencing pulmonary hypertension syndrome caused by Enterococcus faecalis.

Recent studies have proven that Enterococcus faecalis (1.5 x 10(7) live bacteria from a tryptic broth culture given s.c. or intra-abdominally (IA) to 5-wk-old broilers) caused pulmonary hypertension syndrome (PHS) in 97% of the birds within 48 h. Definitive diagnosis of PHS was made at necropsy by observing a cavity on the surface of the right ventricular wall and by increased ratio of left ventricular weight to total ventricular weight. A nonlethal method of diagnosing PHS would enhance the study of PHS and alert production poultrymen to the onset of ascites (waterbelly), which is the cuLminating event of PHS. In the present study, serum hemoglobin, glucose, protein, cholesterol, aspartate amino transferase (AST) and creatine kinase-MB (myocardial in origin) enzymes, differential leukocyte numbers, and specific antibody levels against Ent. faecalis were evaluated as nonlethal diagnostic indicators of PHS. Decreases in serum protein and cholesterol of 3 and 10%, respectively, plus increases in percentages of basophils and monocytes of 18 and 40%, respectively, appear to indicate that PHS has been initiated. An agglutinating antibody, specific against Ent. faecalis, but not against other closely related bacteria, has been developed. Presence of this antibody in a bird means that the bird has previously encountered Ent. faecalis. Thus, this antibody may become a diagnostic for PHS in fast-growing chickens.

Animals↗
PubMed2002

Morphological changes in heart and lungs of broilers experiencing pulmonary hypertension syndrome caused by Enterococcus faecalis.

In a previous report, a method of identification of birds experiencing early symptoms of pulmonary hypertension syndrome (PHS) caused by challenge with Enterococcus faecalis was delineated. This method involved subjective heart scores based on visual observation of a cavity on the external surface of the right ventricular wall (RVW), as well as tonicity and thickness of this wall. Accuracy in identifying birds 48 h postchallenge with E. faecalis was acceptable. However, this method did not attempt to offer other morphological or physiological characteristics for further understanding the etiology of PHS. In the present study, three trials were conducted to establish morphological characteristics of the heart from birds challenged with E. faecalis. In Trials 1 and 2, discrepancies were found in heart length (HL) and thickness of the RVW. In Trial 3, the dry weight of the right ventricle (RV) increased after challenge with E. faecalis, as was the ratio of the mass of the RV to the mass of the total ventricle (TV). Histopathological evidence of hearts and especially lungs of birds challenged with E. faecalis were suggestive of PHS. Results indicated that RV, RV:TV ratio, and histopatholgical evaluation of heart and lungs are complementary to diagnosis of PHS.

Animals↗
PubMed2004

Competitive exclusion of a glycopeptide-resistant Enterococcus faecium in the presence of vancomycin but not equivalent concentrations of tylosin or gentamicin.

The effect of subtherapeutic concentrations of antibiotics (10.0 and 40.0 microg/mL of vancomycin, gentamicin, and tylosin) on the efficacy of a mixed anaerobe culture of chicken microflora (CCF) was studied in a continuous-flow fermentation system. Efficacy of CCF posttreatment was assessed by challenge with glycopeptide-resistant Enterococcus faecium (GRE) at 6.0 log10 cfu/mL. Bacterial enumeration of endogenous CCF isolates, volatile fatty acid (VFA) analysis, and challenge with GRE indicated that CCF efficacy was affected by all antibiotic treatments. Although CCF treated with 10.0 microg/mL of vancomycin eliminated GRE13 at a rate of 0.61 log10 cfu/ mL per day, it was unable to eliminate E. coli, a gram-negative challenge organism. All other antibiotic treatments allowed GRE persistence at approximately 2.0 to 6.5 log10 cfu/mL. All antibiotic-treated cultures had decreased concentrations of acetic and propionic acids. Our data suggest that low concentrations of antimicrobials may adversely affect the microbial ecology of gut microflora with respect to its ability to exclude exogenous bacteria. Moreover, gentamicin had an adverse effect on the inhibitory stringency of CCF even though it showed little anti-anaerobic activity against CCF strict anaerobes in pure culture. Verification of the results in live animals will be necessary to determine if antimicrobial treatment could compromise the effectiveness of normal microflora to serve as a natural host defense against infection.

Animals↗
PubMed2005

Brain abscess due to Enterococcus avium.

We report the first case of brain abscess due to Enterococcus avium in a 19-year-old man with chronic otitis media since childhood. The patient presented to the emergency department in a comatose condition. Contrast-enhanced brain computed tomography scan showed a hypodense area with ring enhancement in the right temporal lobe and mass effect with subfalcine and transtentorial herniation. Emergency temporal burr hole operation was performed and pus drained out, but the patient succumbed to his illness. Culture of the aspirated pus yielded growth of gram-positive cocci identified as E avium, an infrequent pathogen of human infections. The association of E avium with brain abscess further extends the clinical spectrum of this rare pathogen.

Adult↗
PubMed2000

Isolation of Enterococcus faecalis in previously root-filled canals in a Lithuanian population.

The occurrence of Enterococcus faecalis in root canals of previously root filled teeth with apical periodontitis requiring retreatment was studied in Lithuanian patients. Twenty-five asymptomatic teeth were included in the study. Avoiding contamination microbiological samples were taken from the canals before and after preparation and irrigation with sodium hypochlorite and EDTA. Microbes were isolated from 20 of 25 teeth. E. faecalis was isolated from 14 of those 20 culture positive teeth, usually in pure culture or as a major component of the flora. Second samples taken after preparation revealed growth in 7 of the 20 teeth. Five of the seven cases were E. faecalis in pure culture. Isolation of E. faecalis was not related to the use of any particular root filling material in the original root filling. The results indicate that, rather than previous chemical treatment, it is the ecological conditions present in the incompletely filled root canal that are important for the presence of E. faecalis in these teeth.

Bacteriological Techniques↗
PubMed2001

Antimicrobial activity of Ca(OH)2 containing pastes with Enterococcus faecalis in vitro.

Sixty-eight standardized human root specimens were infected with Enterococcus faecalis for 3 wk after removal of the smear layer. After 3 wk of infection, the smear layer was reformed and in half of the specimens, the smear layer was again removed. Aqueous Ca(OH)2 paste and silicone oil based Ca(OH)2 paste were used as the test medications. The specimens were divided into four groups (i.e. (a) nonsmeared aqueous calcium hydroxide group, (b) nonsmeared silicon oil-based calcium hydroxide group, (c) smeared aqueous calcium hydroxide group, and (d) smeared silicon oil-based calcium hydroxide group. Medications were placed in the canals for 7 days. After removal of medications dentin chips were collected and incubated. The quantity of bacteria present was assessed. All calcium hydroxide pastes were effective in the elimination of bacteria in the dentinal tubules, except in the smeared group with silicone oil-based calcium hydroxide.

Calcium Hydroxide↗
PubMed2001

Susceptibilities of Enterococcus faecalis biofilms to some antimicrobial medications.

Enterococcus faecalis has been suggested to be an important etiological agent in endodontic failures. The purpose of this study was to evaluate the effectiveness of chlorhexidine- or antibiotics-based medications in eliminating E. faecalis biofilms. One-day and three-day biofilms of E. faecalis were induced on cellulose nitrate membrane filters. Each biofilm-containing membrane was thoroughly covered with 1 ml of the test medications and incubated for 1 day at 37 degrees C. Treated biofilms were then aseptically transferred to vials containing a neutralizing agent in saline solution and vortexed. Suspensions were 10-fold diluted, seeded onto Mitis salivarius agar plates, and the colony-forming units counted after 48 h of incubation. There were significant differences between the formulations tested. The association of clindamycin with metronidazole significantly reduced the number of cells in 1-day biofilms. However of all medications tested, only 2% chlorhexidine-containing medications were able to thoroughly eliminate most of both 1-day and 3-day E. faecalis biofilms.

Analysis of Variance↗
PubMed2002

Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections.

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).

Actinomyces↗
PubMed2003

Antimicrobial activity of endodontic sealers on Enterococcus faecalis.

Enterococcus faecalis has been shown to be highly resistant once established in the root canal system and may play an important role in endodontic failures. The purpose of this study was to evaluate the antimicrobial activity of four root canal sealers on E. faecalis. Seventeen blood-agar plates were inoculated with E. faecalis using the Lawn technique. Five discs were placed on each plate, four with sealers--Sealapex, Roth 811, Kerr EWT, and AH-Plus--and an ampicillin disc as the control. The plates were incubated at 37 degrees C. The zones of inhibition were measured at 24 and 48 h. Analysis using a one-way ANOVA and Tukey test showed a statistically significance difference (p < 0.05) between all four groups of sealers. Roth 811 showed the largest zone of inhibition (1.1 mm), followed by Sealapex (0.8 mm) and Kerr EWT (0.5 mm), whereas AH-Plus had no antimicrobial activity. There was no difference in the zones of inhibition between the 24- and 48-h time periods.

Analysis of Variance↗
PubMed2003

Effectiveness of stannous fluoride and calcium hydroxide against Enterococcus faecalis.

The objective of this study was to evaluate the antimicrobial activity of stannous fluoride, calcium hydroxide, or the two in combination against Enterococcus faecalis. Fifteen Mueller-Hinton agar plates were used to conduct a disc diffusion susceptibility test. The plates were inoculated with E. faecalis. The groups tested were: (a) stannous fluoride (Stanimax); (b) calcium hydroxide; and (c) a combination of stannous fluoride and calcium hydroxide. Three filter paper discs, each soaked with one of the groups, were placed along with an antibiotic control (ampicillin) on each agar plate. The plates were then incubated at 37 degrees C. The zones of inhibition were measured at 24 and 48 h. One-way ANOVA revealed a statistically significant difference (p < 0.05) between the three groups. Stannous fluoride displayed the widest zone (1.7 mm), followed by the combination of stannous fluoride and calcium hydroxide (1.1 mm), whereas calcium hydroxide by itself displayed a minimal zone of inhibition (0.05 mm).

Analysis of Variance↗
PubMed2003

Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth.

The purpose of this investigation was to compare the antimicrobial effect of MTAD (a mixture of a tetracycline isomer, an acid, and a detergent) with that of NaOCl with and without EDTA. Eighty-five extracted human teeth were contaminated with Enterococcus faecalis for 4 weeks. After biomechanical instrumentation using 1.3% or 5.25% NaOCl as root canal irrigant, the root canal and the external surface of each tooth were exposed to a 5-min application of MTAD, 1.3% NaOCl, 5.25% NaOCl or a 1-min application of EDTA followed by irrigation with 5 ml of 1.3% NaOCl or 5.25% NaOCl. Teeth or dentin shavings were cultured to determine presence or absence of the test bacteria. Fisher's exact test showed that the combination of 1.3% NaOCl as a root canal irrigant and MTAD as a final rinse was significantly more effective against E. faecalis than the other regimens. The chi2 test showed no difference between the other regimens.

Anti-Bacterial Agents↗
PubMed2004

Sonicated extract of Enterococcus faecalis induces irreversible cell cycle arrest in phytohemagglutinin-activated human lymphocytes.

The purpose of this study was to examine whether the sonicated extract of Enterococcus faecalis (SEF) alters the cell cycle transition of lymphocytes and thus regulates the fate of the arrested cells. Human lymphocytes were activated by phytohemagglutinin in the presence or absence of SEF, and cell cycle was assessed by flow cytometry. Seventy-two hours after activation with phytohemagglutinin, cells were activated from G0/G1 to S (6.1%) and G2/M (3.8%) phases of the cell cycle. In contrast, pretreatment with SEF resulted in 90.5% of cells remaining in G0/G1, and cell cycle progression to the S and G2/M phases was consequently inhibited. Caspase assay demonstrated that SEF-treated cells exhibited significantly increased apoptosis (56.7%) compared with phytohemagglutinin alone (28.1%). We propose that if this irreversible cell cycle arrest induced by E. faecalis occurs in vivo, it may result in local immunosuppression and contribute to the pathogenesis of endodontic failure. Our findings that E. faecalis can inhibit lymphocyte responses may be of particular relevance to the pathogenesis of endodontic failure. Although the immunologic mechanism involved in the pathogenesis of persistent periapical lesion is not clearly defined, it is reasonable to predict that the altered immune reaction may be linked to the immunosuppressive potential of E. faecalis or other oral bacteria.

Apoptosis↗
PubMed2004

pH required to kill Enterococcus faecalis in vitro.

Enterococcus faecalis tolerates highly alkaline environments, yet the exact pH required for killing E. faecalis is not known. This study tests growth at 0.5 increments from pH 9.5 to 12. Twelve culture tubes were used in each group. Positive growth was measured using turbidity, a visual scale, and a spectrophotometer. At 24 h, growth was observed in all tubes at pH 9.5 and 10. At 48 h, all pH 10.5 tubes showed growth. At 72 h, six of the pH 11 tubes showed growth. After 7 days, five of the remaining pH 11 tubes were positive. No growth occurred in any of the pH 11.5 or pH 12 tubes. Apparently, pH 10.5 to 11.0 retards growth of E. faecalis, whereas no tubes showed growth at pH 11.5 or greater.

Enterococcus faecalis↗
PubMed2004

Association of Enterococcus faecalis with different forms of periradicular diseases.

Data from culture studies have revealed that Enterococcus faecalis is occasionally isolated from primary endodontic infections but frequently recovered from treatment failures. This molecular study was undertaken to investigate the prevalence of E. faecalis in endodontic infections and to determine whether this species is associated with particular forms of periradicular diseases. Samples were taken from cases of untreated teeth with asymptomatic chronic periradicular lesions, acute apical periodontitis, or acute periradicular abscesses, and from root-filled teeth associated with asymptomatic chronic periradicular lesions. DNA was extracted from the samples, and a 16S rDNA-based nested polymerase chain reaction assay was used to identify E. faecalis. This species occurred in seven of 21 root canals associated with asymptomatic chronic periradicular lesions, in one of 10 root canals associated with acute apical periodontitis, and in one of 19 pus samples aspirated from acute periradicular abscesses. Statistical analysis showed that E. faecalis was significantly more associated with asymptomatic cases than with symptomatic ones. E. faecalis was detected in 20 of 30 cases of persistent endodontic infections associated with root-filled teeth. When comparing the frequencies of this species in 30 cases of persistent infections with 50 cases of primary infections, statistical analysis demonstrated that E. faecalis was strongly associated with persistent infections. The average odds of detecting E. faecalis in cases of persistent infections associated with treatment failure were 9.1. The results of this study indicated that E. faecalis is significantly more associated with asymptomatic cases of primary endodontic infections than with symptomatic ones. Furthermore, E. faecalis was much more likely to be found in cases of failed endodontic therapy than in primary infections.

Adolescent↗