Enterococcus faecalis provides protection during scavenging in carrion crow (Corvus corone)

The gut microbiota significantly influences host physiology and provides essential ecosystem services.While diet can affect the composition of the gut microbiota,the gut microbiota can also help the host adapt to specific dietary habits.The carrion crow(Corvus corone),an urban facultative scavenger bird,hosts an abundance of pathogens due to its scavenging behavior.Despite this,carrion crows infrequently exhibit illness,a phenomenon related to their unique physiological adaptability.At present,however,the role of the gut microbiota remains incompletely understood.In this study,we performed a comparative analysis using 16S rRNA amplicon sequencing technology to assess colonic content in carrion crows and 16 other bird species with different diets in Beijing,China.Our findings revealed that the dominant gut microbiota in carrion crows was primarily composed of Proteobacteria(75.51%)and Firmicutes(22.37%).Significant differences were observed in the relative abundance of Enterococcus faecalis among groups,highlighting its potential as a biomarker of facultative scavenging behavior in carrion crows.Subsequently,E.faecalis isolated from carrion crows was transplanted into model mice to explore the protective effects of this bacterial community against Salmonella enterica infection.Results showed that E.faecalis down-regulated the expression of pro-inflammatory cytokines tumor necrosis factor alpha(TNF-α),interferon gamma(IFN-γ),and interleukin 6(IL-6),prevented S.enterica colonization,and regulated the composition of gut microbiota in mice,thereby modulating the host’s immune regulatory capacity.Therefore,E.faecalis exerts immunoregulatory and anti-pathogenic functions in carrion crows engaged in scavenging behavior,offering a representative case of how the gut microbiota contributes to the protection of hosts with specialized diets.

Antibiotic Resistance Profile of Enterococcus faecalis and Enterococcus faecium Isolates from Urine and Pleural Fluid in Two Hospitals of Cameroon

Enterococcus faecalis and Enterococcus faecium rank among the leading causes of nosocomial bacteremia and urinary tract infections. They often persist on hospital surfaces due to their ability to withstand adverse environmental conditions (low or high temperatures, high pH, and high salinity). The global Enterococcus faecalis-Enterococcus faecium ratio is currently shifting towards Enterococcus faecium. Enterococci present variable levels of resistance to certain families of antibiotics. This is the case for aminoglycosides, beta-lactams and cephalosporins. In 2017, WHO ranked Enterococci among priority pathogens for research and development of new antibiotics. The objective of our study was to determine the antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolates from urine and pleural fluid in two hospitals in Cameroon. This cross-sectional and analytic study was carried out between June to August 2023 on hospitalized and day patients in which a cytobacteriological test of urine and pleural fluid was done. The samples were inoculated on CLED Agar for urine and on Chocolate + polyvitex and blood agar (prepared from Columbia agar) for pleural fluid samples and incubated at 37℃ for 18 to 24 hours. Identification of isolates was carried out using the API 20 STREP micro gallery (Biomerieux, France) and tested for antimicrobial susceptibility. The data on socio-demographical and potential risk factors were recorded using self-administered questionnaires and data from laboratory analyses of the specimen were collected in a data capture sheet. Potential risk factors associated with the presence of Enterococci, were evaluated using the logistic regression in univariate and multivariate analysis. P value < 0.05 was considered as significant. A total of 511 patients were recruited who were predominantly females. Enterococcus spp were isolated in 27.79% of our samples with Enterococcus faecalis mostly encountered. Enterococcus spp showed a high level of resistance to penicilline (99.3% to Ampicilline), macrolides (66.2% to Erythromycin) and cyclines (85.2% to Doxycycline). Hospitalisation, access to health facilities, contact with urine specimen and hand hygiene practices were risk factors related to infection with Enterococcus spp while hospitalisation, health facility and hand hygiene were related to glycopeptide resistant Enterococcus. Strict compliance with hygiene rules and appropriate antibiotic consumption could help in the fight against these infections.

固定化Alcaligenes faecalis WT14对累积亚硝酸盐的还原

针对好氧反硝化菌在好氧条件有着良好的硝酸盐去除能力,但亚硝酸盐的积累阻碍了TN的去除的问题,采用聚乙烯醇混合海藻酸钠、活性炭粉末和氯化钙的方式对好氧反硝化菌WT14进行固定化,并应用固定化菌去除实际水产养殖废水中的硝酸盐。结果表明,克服了亚硝酸盐积累问题,显著提高了TN去除率。与游离菌相比,固定化菌有着更高的TN去除率(分别为30.2%和55.5%),优化脱氮条件为,20℃、pH 9.0和60 r/min。固定化菌循环使用8个周期均有着稳定的TN去除率(92.6%)。用以处理实际水产养殖废水时,硝酸盐去除率为100%,且不含亚硝酸盐的积累。说明好氧反硝化联合固定化技术在实际应用中具有良好潜力。

Effects of lysedEnterococcus faecalis FK-23 on experimental allergic rhinitis in a murine model

In the current study, we sought to investigate whether lysed Enterococcus faecalis FK-23 （LFK）, a heat-killed probiotic preparation, attenuated eosinophil influx into the upper airway and had immunomodulatory activity in a murine allergic rhinitis model. Eighteen BALB/c mice were divided into three groups; the ovalbumin （OVA）-sen- sitized/challenged group, which received saline orally for 6 weeks （OVA group）, the OVA-sensitized/challenged group, which received LFK orally for 6 weeks （LFK-fed group）, and the non-sensitized group, which received saline for 6 weeks （saline control group）. Nasal rubbing and sneezing were monitored during the study. After the final challenge, interleukin （IL）-4, interferon （IFN）-y, and OVA-specific IgE levels in the sera and splenocyte culture supernatants were determined, eosinophilic infiltrate into the upper airway was quantified, and splenic CD4~CD25＋ regulatory T cells （Tregs） were examined by flow cytometry. We found that nasal rubbing was sig- nificantly reduced in LFK-fed mice compared to the OVA group on d 27 and 35, and sneezing was significantly inhibited by LFK administration for 35 d. LFK-fed mice had significantly less eosinophil influx into the nasal mucosa than the OVA group. There were no significant differences between the LFK-fed group and OVA group in the serum and splenocyte culture supernatant levels of IL-4, IFN-y, and OVA-specific IgE. Interestingly, the LFK-fed mice had a significantly greater percentage of splenic CD4＋CD25＋ Tregs than OVA group. Our results indicate that oral administration of LFK may alleviate nasal symptoms, reduce nasal eosinophilia, and increase the percentage of CD4＋CD25＋ Tregs in experimental allergic rhinitis.

Residual activity of cetrimide and chlorhexidine on Enterococcus faecalis-infected root canals

Effective final irrigation regimen is an important step in order to achieve better disinfection and ensure residual antimicrobial effects after root canal preparation. The aim of this study was to compare the residual antimicrobial activity of 0.2% cetrimide, and 0.2% and 2% chlorhexidine in root canals infected with Enterococcus faecalis. Biofilms of E. faecalis were grown on uniradicular roots for 4 weeks. After root canal preparation, root canals were irrigated with 17% ethylenediaminetetraacetic acid （EDTA） to remove the smear layer. The roots were randomly divided into three experimental groups （n=26） according to the final irrigating solution： Group I, 5 mL 0.2% cetrimide; Group II, 5 mL 0.2% chlorhexidine; and Group III, 5 mL 2% chlorhexidine. Samples were collected for 50 days to denote the presence of bacterial growth. The proportion of ungrown specimens over 50 days was evaluated using the nonparametric Kaplan-Meier survival analysis. Differences among groups were tested using the log-rank test and the level of statistical significance was set at P〈0.05. The highest survival value was found with 2% chlorhexidine, showing statistically significant differences from the other two groups. At 50 days, E. faecalisgrowth was detected in 69.23% specimens in Groups I and II, and in 34.61% specimens of Group III. There were no significant differences between 0.2% cetrimide and 0.2% chlorhexidine. Final irrigation with 2% chlorhexidine showed greater residual activity than 0.2% chlorhexidine and 0.2% cetrimide in root canals infected with E. faecalis.

Infant intestinal Enterococcus faecalis down-regulates inflammatory responses in human intestinal cell lines

AIM: To investigate the ability of Lactic acid bacteria (LAB) to modulate inflammatory reaction in human intestinal cell lines (Caco-2, HT-29 and HCT116). Different strains of LAB isolated from new born infants and fermented milk, together with the strains obtained from culture collections were tested.METHODS: LABs were treated with human intestinal cell lines. ELISA was used to detect IL-8 and TGF-β protein secretion. Cytokines and Toll like receptors (TLRs) gene expression were assessed using RT-PCR. Conditional medium, sonicated bacteria and UV killed bacteria were used to find the effecter molecules on the bacteria. Carbohydrate oxidation and protein digestion were applied to figure out the molecules' residues. Adhesion assays were further carried out.RESULTS: It was found that Enterococcus faecalis is the main immune modulator among the LABs by downregulation of IL-8 secretion and upregulation of TGF-β. Strikingly, the effect was only observed in four strains of E. faecalis out of the 27 isolated and tested. This implies strain dependent immunomodulation in the host. In addition, E. faecalis may regulate inflammatory responses through TLR3, TLR4, TLR9 and TRAF6. Carbohydrates on the bacterial cell surface are involved in both its adhesion to intestinal cells and regulation of inflammatory responses in the host. CONCLUSION: These data provide a case for the modulation of intestinal mucosal immunity in which specific strains of E. faecalis have uniquely evolved to maintain colonic homeostasis and regulate inflammatory responses.

Antimicrobial effect of alexidine and chlorhexidine against Enterococcus faecalis infection

A previous study demonstrated that alexidine has greater affinity for the major virulence factors of bacteria than chlorhexidine.The aim of this study was to compare the antimicrobial activity of 1%alexidine with that of 2%chlorhexidine using Enterococcus faecalis-infected dentin blocks.Sixty bovine dentin blocks were prepared and randomly divided into six groups of 10 each.E.faecalis was inoculated on 60 dentin blocks using the Luppens apparatus for 24 h and then the dentin blocks were soaked in 2%chlorhexidine or 1%alexidine solutions for 5 and 10 min,respectively.Sterile saline was used as a control.The antimicrobial efficacy was assessed by counting the number of bacteria adhering to the dentin surface and observing the degradation of bacterial shape or membrane rupture under a scanning electron microscope.Significantly fewer bacteria were observed in the 2%chlorhexidine-or 1%alexidine-soaked groups than in the control group(P<0.05).However,there was no significant difference in the number of bacteria adhering to the dentinal surface between the two experimental groups or between the two soaking time groups(P>0.05).Ruptured or antiseptic-attached bacteria were more frequently observed in the 10-min-soaked chlorhexidine and alexidine groups than in the 5-min-soaked chlorhexidine and alexidine groups.In conclusion,10-min soaking with 1%alexidine or 2%chlorhexidine can be effective against E.faecalis infection.

Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis C16

Alcaligenes faecalis C16 was found to have the ability to heterotrophically nitrify and aerobically denitrify. In order to further understand its nitrogen removal ability and mechanism, the growth and ammonium removal response were investigated at different C/N ratios and ammonium concentrations in the medium with citrate and acetate as carbon source separately. Furthermore, experiments of nitrogen sources, production of nitrogen gas and enzyme assay were conducted. Results show that the bacterium converts NH+4-N and produces NH2 OH during the growing phase and nitrite accumulation is its distinct metabolic feature. A. faecalis C16 is able to tolerate not only high ammonium concentration but also high C/N ratio, and the ammonium tolerance is associated with carbon source and C/N ratio. The nitrogen balance under different conditions shows that approximately28%–45% of the initial ammonium is assimilated into the cells, 44%–60% is denitrified and several percent is converted to nitrification products. A. faecalis C16 cannot utilize hydroxylamine, nitrite or nitrate as the sole nitrogen source for growth. However, nitrate can be used when ammonium is simultaneously present in the medium. A possible pathway for nitrogen removal by C16 is suggested. The preliminary enzyme assay provides more evidence for this nitrogen removal pathway.

Efficacy of Atmospheric Pressure Plasma as an Antibacterial Agent Against Enterococcus Faecalis in Vitro

Enterococcus faecalis （E. faecalis） is a microorganism that can survive extreme challenges in obturated root canals. The aim of this study was to evaluate the efficacy of a non-thermal atmospheric pressure plasma plume against E. faecalis in vitro. A non-thermal atmospheric pressure plasma jet device which could generate a cold plasma plume carrying a peak current of 300 mA was used. The antibacterial efficacy of this device against E. faecalis and its biofihn un- der different conditions was detected. The antibacterial efficacy of the plasma against E. faecalis and Staphylococcus aureus （S. aureus） was also evaluated. After plasma treatment, the average diameter of inhibition zone on S. aureus and E. faecalis was 2.62±0.26 cm and 1.06±0.30 cm, respectively （P 〈 0.05）. The diameter was increased with prolongation of the treatment dura- tion. The diameters of inhibition zone of the sealed Petri dishes were larger than those of the uncovered Petri dishes. There was significant difference in colony-forming units between plasma group and control group on E. faecalis biofilm （P 〈 0.01）. The transmission electron microscopy revealed that the ultrastructural changes eytoderm of E. faecalis were observed after treatment for 2min. It is concluded that the non-thermal atmospheric pressure plasma could serve as an effective adjunct to standard endodontie microbial treatment.

Prevalence of Enterococcus faecalis in saliva and filled root canals of teeth associated with apical periodontitis

To investigate the prevalence of Enterococcus faecalis in saliva and filled root canals of patients requiring endodontic retreatment for apical periodontitis.Patients with apical periodontitis who were referred for endodontic retreatment were examined.The type and quality of the restoration,symptoms,quality of obturation were recorded.During retreatment,an oral rinse sample and root canal sample were cultured using brain-heart infusion agar and bile esculinazide agar to select for E.faecalis.The 16S rRNA technique was used to identify E.faecalis.A total of 32 women and 22 men（mean age：38 years;s.d.：11 years） and 58 teeth were studied.The prevalence of E.faecalis was 19% in the saliva and 38% in the root canals.The odds that root canals harbored E.faecalis were increased if the saliva habored this bacterium（odds ratio59.7;95% confidence interval51.8-51.6;P,0.05）.Teeth with unsatisfactory root obturation had more cultivable bacterial species in root canals than teeth with satisfactory root obturation（P,0.05）.E.faecalis is more common in root canals of teeth with apical periodontitis than in saliva.The prevalence of E.faecalis in root canals is associated with the presence of E.faecalis in saliva.

Effect of Bacillus subtilis,Enterococcus faecium,and Enterococcus faecalis supernatants on serotonin transporter expression in cells and tissues

BACKGROUND Bacillus subtilis(B.subtilis),Enterococcus faecium(E.faecium),and Enterococcus faecalis(E.faecalis)are probiotics that are widely used in the clinical treatment of irritable bowel syndrome(IBS).Whether the supernatants of these three probiotics can improve gastrointestinal sensation and movement by regulating the serotonin transporter(SERT)expression needs to be clarified.AIM To investigate whether B.subtilis,E.faecium,and E.faecalis supernatants can upregulate SERT expression in vitro and in vivo.METHODS Caco-2 and HT-29 cells were stimulated with probiotic culture supernatants for 12 and 24 h,respectively.A male Sprague-Dawley rat model of post-infectious irritable bowel syndrome(PI-IBS)was established and the rats were treated with phosphate-buffered saline(group A)and three probiotics culture supernatants(groups B,C,and D)for 4 wk.The levels of SERT were detected by quantitative PCR and western blotting.RESULTS The levels of SERT at post-treatment 12 and 24 h were significantly elevated in Caco-2 cells treated with B.subtilis supernatant compared with those in the control group(aP<0.05).Those levels were markedly upregulated in Caco-2 cells stimulated with E.faecium and E.faecalis supernatants at 24 h(aP<0.05).In addition,SERT expression in groups B,C,and D was significantly higher than that in group A in the 2nd wk(aP<0.05).Increased SERT expression was only found in group D in the 3rd wk(aP<0.05).However,there was no significant difference in SERT expression between the groups in the last week(P>0.05).CONCLUSION The supernatants of B.subtilis,E.faecium,and E.faecalis can upregulate SERT expression in intestinal epithelial cells and the intestinal tissues in the rat model of PI-IBS.

乳酸菌Enterococcuse faecalis TN-9低温蛋白酶的提纯及性质

对产自乳酸菌Enterococcuse faecalis TN-9的蛋白酶,进行了硫酸铵沉淀,DEAE-Sephadex A-25以及DEAE Cellulofine A-500离子交换层析的3步纯化和特性研究。纯化酶Native PAGE显示1条蛋白带。SDS-PAGE和凝胶层析分子量分别为30 ku及69 ku。纯化酶最适作用温度为30℃,最适作用pH为7.5～8.0,在pH 6.0～9.5和45℃以下条件下稳定,在0℃下显示了6.1%的相对活性,60℃以上热处理完全失去酶活。该酶被EDTA-2Na,Hg^(2+)、Cu^(2+)、Ni^(2+)、Ag^(2+)、Co^(2+)及Pepstatin A不完全抑制。Zn^(2+)对蛋白酶具有明显的激活作用。纯化酶作用于偶氮酪蛋白的K_(rs)和V_(max)分别为0.098%和72 mg/(h·mg)。该酶为N末端VGSEVTLKNS的明胶酶(Gelatinase)的一种,性质属于低温蛋白酶。

Antimicrobial activity of some essential oils against oral multidrugresistant Enterococcus faecalis in both planktonic and biofilm state

Objective:To evaluate some essential oils in treatment of intractable oral infections,principally caused by hiofilm of multidrug-resistant Enterococcus faecalit(E.faecalis),such as persistent endodontic infections in which their treatment exhibits a real challenge for dentists.Methods:Ten chemically analyzed essential oils by gas chromatography-mass spectrometry were evaluated for antimicrobial activity against sensitive and resistant clinical strains of E.faecalit in both planktonic and hiofilm state using two methods,disk diffusion and broth microdilution.Results:Studied essential oils showed a good antimicrobial activity and high ability in E.faecalit biofilm eradication,whether for sensitive or multidrug-resistant strains,especially those of Origanum glandulosum and Thymbra capitata with interesting minimum inhibitory concentration,biofilm inhibitory concentration,and biofilm eradication concent ration values which doesn't exceed 0.063%,0.75%,and 1.5%,respectively.Conclusions:Findings of this study indicate that essential oils extracted from aromatic plants can be used in treatment of intractable oral infections,especially caused by biofilm of multidrugresistant E.faecalis.

Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm

The purpose of this study was to assess the efficacy of alexidine（ALX）,alone and combined with N-acetylcysteine（NAC）,in eradicating two Enterococcus faecalis strain biofilms.The biofilms of E.faecalis ATCC 29212 and the clinical isolate E.faecalis D1 were grown in the MBEC-high-throughput device for 24 h and were exposed to five twofold dilutions of ALX（2%–0.007 8%）alone and combined with100 mg？mL21NAC,for 1 and 5 min.Eradication was defined as 100%kill of biofilm bacteria.The Student’s t-test was used to compare the efficacy of the associations of the two irrigants.After 1-min contact time,ALX eradicated the biofilms at all concentrations except for 0.007 8%and 0.015 6%–0.007 8%with E.faecalis ATCC 29212 and E.faecalis D1,respectively.Similar results for eradication and concentration were obtained when it was combined with 100 mg？mL21NAC.After 5 min of contact time,ALX alone and combined with NAC eradicated all enterococci biofilms.ALX showed antimicrobial properties against the two E.faecalis strain biofilms tested at very low concentrations,and its combined use with NAC was not seen to enhance its activity.

Effect of Inoculation with Alcaligenes faecalis on the Bacterial Community in Paddy Soils

The rifampin resistant Alcaligenes faecalis strain A1501R survived at a high, initially stable and later slowly declining population size (10 8 to 10 6 CFU per g dry soil) for 60 days in microcosm experiments. Introduction of strain A1501R did not cause great changes in the community profiles generated via microbial community level 16S rDNA based PCR followed by denaturing gradient gel electrophoresis (DGGE) and the Biolog substrate utilization system. The introduction of A1501R had no impact on the dominant bacterial populations in soil. Significant differences in the utilization of some substrates were observed between the control and inoculated soils using a combination of DGGE and Biolog analysis. Results of DGGE and BIOLOG analysis show that introduction of A1501R can affect the lactic acid utilizing bacterial community in case of selection by lactic acid. A specific probe based on the 16S rDNA variable region V6 was constructed via PCR amplification of 16S rDNA using the 971(f) and 1057(r) primers for the detection of A. faecalis strains introduced into soils. Dot blot hybridization of strains or soil DNA and aligment of sequences of the V6 region of 16S rDNA from different bacterial strains show that the V6 probe is very specific for A. faecalis A1501R.

Isolation and Identification of Canine Enterococcus faecalis

With the development of the urban pet industry,suppurative diseases of companion animals are increasing day by day.To explore its associated pathogens,this study was conducted on etilolgical identification from a case of a canine uterus with pus.Bacteriological detection methods were used to isolate and purify the collected disease materials,such as smear staining,microscopic examination and biochemical identification.Then,16S rRNA molecular sequence analysis was used to identify the isolate.It was found that the morphological features of the isolated strain and the biochemical results were consistent with Enterococcus faecalis.The 16S rRNA of the isolate was also as high as 99% homologous with the reference stains.Drug susceptibility test showed that the isolate was resistant to tetracycline,penicillin G,minocycline,erythromycin,ciprofloxacin,norfloxacin,vancomycin,levofloxacin,clindamycin,polymyxin B,chloramphenicol,compound sulfamethoxazole,and clarithromycin,and only susceptible to oxacillin and nitrofurantoin.These results provided reference for the treatment of this type of multidrug-resistant enterococcal infection.

In-vivo Mutagenicity of Protein-modified Enterococcus faecalis

The mutagenicity of the protein-modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test.The test substance was designed with three dose groups (1,2 and 5 g/kg·bw) and intragastrically administrated,with cyclophosphamide as a positive control and normal saline as a normal control.The micronucleus rate and sperm abnormality rate were measured.The results showed that the micronucleus rates and sperm abnormality rates in the different dose groups were not significantly different from those in normal control group ( P >0.05),while the positive control group was significantly higher than the normal control group ( P <0.01).The conclusion is that the protein-modified E.faecalis was tested to be negative in both the mouse bone marrow micronucleus test and mouse sperm abnormality test,suggesting that it has no mutagenicity in vivo.

Ames Test of Protein-modified Enterococcus faecalis

The potential mutagenicity of protein-modified Enterococcus faecalis was evaluated by the Ames test. The test subject was designed with 0. 05,0. 5,5. 0,and 50. 0 mg/dish doses,and a control group was set. With Salmonella typhimurium mutant strains TA97,TA98,TA100 and TA102 as test strains,the Ames test was carried out with( +) or without(-) S9,and the number of revertant colonies was counted. The results showed that for the protein-modified E. faecalis in the case of + S9 and-S9,the average numbers of revertant colonies of the four test strains were less than two times of that of the negative control group,and no dose-response relationship was observed. The protein-modified E. faecalis was tested to be negative in the Ames test,indicating that the test substance was not mutagenic in vitro.

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53

In this work, the optimization of hydrogen production by photo-fermentation bacteria immobilized on agar gel granule was systematic investigated in batch culture. Experiment focus on the effect of some important affecting factors on photo-hydrogen production. Results indicated that immobilized Rhodopseudomonas faecalis RLD-53 exhibited the highest hydrogen yield of 3.15 mol H2/mol acetate under follow optimal condition: agar granule diameter of 2.5 mm, inoculum age of 24 h, agar concentration of 2%, biomass of 4 mg/ml in agar and light intensity of 9000 lux. More importantly, immobilized photo-fermentation bacteria not only can enhance hydrogen production but can increase acids-tolerance capacity, even at pH 5.0 hydrogen also was produced, and thus hopefully immobilized photo-fermentation bacteria can be applied in the combination of dark and photo-fermentation for hydrogen production with high yield.

Virulence properties of a peptide hemolysin produced by Enterococcus faecalis

The characterization and prevalence of virulence factors associated with enterococcal invasiveness and severity of disease are important areas to be investigated. Recently, we described the production of a heat-stable hemolysin by clinical isolates of Enterococcus faecalis cultived in BHI-GA (BHI with glucose and L-arginine). Now, we purified the hemolysin from the culture supernatant by ultra-filtration (PM-10 membrane) and ethanol extraction followed by chromatography in a mBondapak C18 and Superdex Peptide columns. The hemolytic activity was not affected by the proteolytic enzymes. Cholesterol, phospholipids, EDTA and also bivalent ions did not inhibit the hemolytic activity. Among the various carbohydrates, only dextran 4 protected the erythrocytes against lyse. Scanning electron microscopy showed that lyse of erythrocytes occured at once after the exposure to the hemolysin. The mito-chondrial activity and the cell membrane integrity were significantly affected by the hemolysis, within 20 min of exposure and caused apoptosis after 12 h incubation, 51.92% in HeLa and 68% in HEp-2 cells, analyzed by flow cytometry. These results suggest that the heat-stable pore forming hemolysin might be a putative virulence factor in enterococci infections.