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.

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.

Pretreatment with probiotics Enterococcus faecium NCIMB 11181 attenuated Salmonella Typhimurium-induced gut injury through modulating intestinal microbiome and immune responses with barrier function in broiler chickens

Background:Preventing Salmonella infection and colonization in young birds is key to improving poultry gut health and reducing Salmonella contamination of poultry products and decreasing salmonellosis for human consumption(poultry meat and eggs).Probiotics can improve poultry health.The present study was conducted to investigate the impact of a probiotics,Enterococcus faecium NCIMB 11181(E.faecium NCIMB 11181)on the intestinal mucosal immune responses,microbiome and barrier function in the presence or absence of Salmonella Typhimurium(S.Typh-imurium,ST)infection.Methods:Two hundred and forty 1-day-old Salmonella-free male broiler chickens(Arbor Acres AA+)were randomly allocated to four groups with 6 replicate cages of 10 birds each.The four experimental groups were follows:(1)nega-tive control(NC),(2)S.Typhimurium,challenged positive control(PC),(3)the E.faecium NCIMB 11181-treated group(EF),(4)the E.faecium NCIMB 11181-treated and S.Typhimurium-challenged group(PEF).Results:Results indicated that,although continuous feeding E.faecium NCIMB 11181 did not obviously alleviate growth depression caused by S.Typhimurium challenge(P>0.05),E.faecium NCIMB 11181 addition significantly blocked Salmonella intestinal colonization and translocation(P<0.05).Moreover,supplemental E.faecium NCIMB 11181 to the infected chickens remarkably attenuated gut morphological structure damage and intestinal cell apoptosis induced by S.Typhimurium infection,as evidenced by increasing gut villous height and reducing intes-tinal TUNEL-positive cell numbers(P<0.05).Also,E.faecium NCIMB 11181 administration notably promoting the production of anti-Salmonella antibodies in intestinal mucosa and serum of the infected birds(P<0.05).Addition-ally,16S rRNA sequencing analysis revealed that E.faecium NCIMB 11181 supplementation ameliorated S.Typhimu-rium infection-induced gut microbial dysbiosis by enriching Lachnospiracease and Alistipes levels,and suppressing Barnesiella abundance.Predicted function analysis indicated that the functional genes of cecal microbiome involved in C5-branched dibasic acid metabolism;valine,leucine and isoleucine biosynthesis;glycerolipid metabolism and lysine biosynthesis were enriched in the infected chickens given E.faecium NCIMB 11181.While alanine,asparate and glutamate metabolism;MAPK signal pathway-yeast;ubiquine and other terpenoid-quinore biosynthesis,protein processing in endoplasmic reticulum;as well as glutathione metabolism were suppressed by E.faecium NCIMB 11181 addition.Conclusion:Collectively,our data suggested that dietary E.faecium NCIBM 11181 supplementation could ameliorate S.Typhimurium infection-induced gut injury in broiler chickens.Our findings also suggest that E.faecium NCIMB 11181 may serve as an effective non-antibiotic feed additive for improving gut health and controlling Salmonella infection in broiler chickens.

Enterococcus durans产胞外多糖EPS-Ⅰ的分离纯化和结构分析

The rough crystal of exopolysaccharide produced by Enterococcus durans, a strain of lactic acid bacteria screened from the intestine of a cock in our laboratory, was purified by CM-cellulose column chromatography, DEAE-Sephadex A-25 ionexchange and Sephadex G-100 gel chromatography to give EPS-Ⅰ. The EPS-Ⅰ was eluted as a single peak in HPLC analysis, indicating the homogeneity of EPS-Ⅰ and free from low-molecular-weight polysaccharides. The molecular weight of the EPS-Ⅰ was determined as 42 000 by the light scattering method. The result of its elemental analysis was C 41.08% and H 7.23% without the elements of N, P and S. Monosaccharide analysis showed that it was composed of Glc and Man in a molar ratio of 4∶1. Sugar composition analysis, methylation analysis and 1H and 13C NMR spectroscopy revealed that the EPS-Ⅰ was composed of pentasaccharide repeating units. The sequence of sugar residues was determined by using two-dimensional NMR, including heteronuclear multiple-bond correlation(HMBC) and nuclear overhauser effect spectroscopy(NOESY). The structure of the pentasaccharide repeating unit of EPS-Ⅰ was given, a new excellular polysaccharide from lactic acid bacterium compared with other EPSs was reported.

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.

Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease

AIM: To determine the features of Enterococcus that contribute to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease (IBD). METHODS: Multiplex polymerase chain reaction (PCR) was applied to assess the presence of genes that encode virulence factors [surface aggregating protein (asa1), gelatinase (gelE), cytolysin (cylA), extracellular surface protein (esp) and hyaluronidase (hyl)] in the genomic DNA of 28 strains of Enterococcus isolated from the intestinal tissues of children with IBD (n =16) and of children without IBD (controls; n = 12). Additionally, strains with confirmed presence of the gelE gene were tested by PCR for the presence of quorum sensing genes (fsrA, fsrB, fsrC) that control the gelatinase production. Gelatinase activity was tested on agar plates containing 1.6% gelatin. We also analysed the ability of Enterococcus strains to release and decompose hydrogen peroxide (using Analytical Merckoquant peroxide test strips) and tested their ability to adhere to Caco-2 human gut epithelium cells and form biofilms in vitro. RESULTS: A comparison of the genomes of Enterococcus strains isolated from the inflamed mucosa of patients with IBD with those of the control group showed statistically significant differences in the frequency of theasa1 gene and thegelE gene. Furthermore, the cumulative occurrence of different virulence genes in the genome of a single strain ofEnterococcus isolated from the IBD patient group is greater than in a strain from the control group, although no significant difference was found. Statistically significant differences in the decomposition of hydrogen peroxide and adherence to the Caco-2 epithelial cell line between the strains from the patient group and control group were demonstrated. The results also showed that profuse biofilm production was more frequent amongEnterococcus strains isolated from children with IBD than in control strains. CONCLUSION: Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process.

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.

Enterococcus faecium NCIMB 10415 administration improves the intestinal health and immunity in neonatal piglets infected by enterotoxigenic Escherichia coli K88

Background:This study aimed to investigate the effects of oral administration of Enterococcus faecium NCIMB10415(E.faecium)on intestinal development,immunological parameters and gut microbiota of neonatal piglets challenged with enterotoxigenic Escherichia coli K88(ETEC).A total of 961-day-old sow-reared piglets were randomly assigned to 2 groups,with 48 piglets in each group.The piglets were from 16 litters(6 piglets each litter),and 3 piglets each litter were allocated to the E.faecium-supplemented(PRO)group,while the other 3 piglets were allocated to the control(CON)group.After colostrum intake,piglets in the PRO group were orally administrated with 3×10~9 CFU E.faecium per day for a period of one week.On day 8,one piglet per litter from each group was challenged(CON+ETEC,PRO+ETEC)or not(CON-ETEC,PRO-ETEC)with ETEC in a 2×2 factorial arrangement of treatments.On day 10(2 days after challenge),blood and tissue samples were obtained from piglets.Results:Before ETEC challenge,there were no significant differences for the average daily gain(ADG)and fecal score between the two groups of piglets.After ETEC challenge,the challenged piglets had greater fecal score compared to the non-challenged piglets,whereas E.faecium administration was able to decrease the fecal score.Piglets challenged with ETEC had shorter villous height,deeper crypt depth,and reduced number of goblet cells in the jejunum and decreased m RNA abundance of claudin-1 in the ileum,whereas increased the percentage of lymphocytes,concentrations of IL-1βin the plasma and TNF-αin the ileal mucosa,as well as increased the m RNA abundances of innate immunity-related genes in the ileum tissue.These deleterious effects caused by ETEC were partly alleviated by feeding E.faecium.In addition,piglets in PRO-ETEC group had decreased the percentage of CD8^+T cells of the peripheral blood when compared to those in CON-ETEC group.Moreover,E.faecium administration increased Verrucomicrobia at phylum level and decreased Bilophila at genus level.Conclusions:These results suggest that oral administration of E.faecium alleviated the intestinal injury and diarrhea severity of neonatal piglets challenged by ETEC,partly through improving the intestinal microbiota and immune response.This offers a potential strategy of dietary intervention against intestinal impairment by ETEC in neonatal piglets.

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.

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.

Effect of addition of inulin and fenugreek on the survival of microencapsulated Enterococcus durans 39C in alginate-psyllium polymeric blends in simulated digestive system and yogurt

The use of biopolymers for probiotic microencapsulation has been investigated in this paper.The objectives are to enhance its survival rate,colonic release,and stability of these probiotic cultures in digestive condition during storage time.Nine types of biopolymers(alginate-psyllium)blend with different concentration of prebiotic;(inulin or fenugreek)were used as candidate for microencapsulation matrix.One strain of probiotic candidates,namely;Enterococcus durans 39C was used in this study.The microencapsulation of this strain with the respective polymer blend was performed by using a simple extrusion method.All blend of formulations have recorded high encapsulation efficiency at value>98%.The survival rate of viable probiotic cells under simulated digestive conditions was also high with value above 47%as compared to non-microencapsulated cells.These nine gel formulations also displayed the high survival rate of viable probiotic cells during storage time(28 d).Their release occurred after 2 h in colonic condition and sustained until 12th h of incubation period.An increase of prebiotic effect value added was observed in incorporated inulin and fenugreek formulations.In short,this study revealed that a new herbal-based psyllium and fenugreek polymers have suitable potential as a matrix for probiotic microencapsulation.

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.

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.

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.

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。

Bloodstream Infections Caused by Enterococcus spp：A 10-year Retrospective Analysis at a Tertiary Hospital in China

In order to discover the risk factors for 30-day mortality in bloodstream infections（BSI） caused by Enterococcus spp.strains,we explored the clinical and therapeutic profile of patients with Enterococcus spp.BSI and the characteristics of this condition.A total of 64 patients with BSI caused by Enterococcus spp.who were treated in our hospital between 2006 and 2015 were included in the study.The clinical features of patients,microbiology,and 30-day mortality were collected from the electronic medical records database and analyzed.The results showed that there were 38 patients infected by Enterococcus faecalis（E.faecalis）,24 by Enterococcus faecium（E.faecium）,1 by Enterococcus casseliflavus（E.casseliflavus）,and 1 by Enterococcus gallinarum（E.gallinarum）.A Charlson comorbidity score ≥5,corticosteroid treatment,placement of catheters or other prosthetic devices and history of antibiotic use were found more frequently in E.faecium BSI patients than in E.faecalis patients（P=0.017,P=0.027,P=0.008 and P=0.027,respectively）.Furthermore,the univariate and multivariate analysis showed that corticosteroid treatment（OR=17.385,P=0.008）,hospital acquisition（OR=16.328,P=0.038）,and vascular catheter infection（OR=14.788,P=0.025） were all independently associated with 30-day mortality.Our results indicate that E.faecalis and E.faecium are two different pathogens with unique microbiologic characteristics,which cause different clinical features in BSI,and the empiric antimicrobial treatments are paramount for patients with enterococcal BSI.

Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.

Isolation of Enterococcus faecium with Feeding Attractant Function from Pacific White Shrimp (Litopenaeus vannamei) Intestine

In the present study,we isolated the lactic acid bacterium strain SC-01 from Pacific white shrimp(Litopenaeus vannamei)intestine.Using conventional and molecular methods,we identified the bacterium as Enterococcus faecium,and found it had the function of feeding attractant and could inhibit the development of Vibrio parahaemolyticus(zone of inhibition:14 mm).The attractant effect of its fermentation broth is significantly better than that of the chemical attractant trimethylamine oxide(TMAO)(P<0.05),and is equivalent to that of dimethyl-beta-propiothetin(DMPT)based on the feeding behavior of shrimp.High performance liquid chromatography(HPLC)analysis suggested that inosine-5’-monophosphate(IMP)may be a component of the attractant.A biosecurity evaluation revealed a negative result in hemolytic assays,and no shrimp mortality was resulted from SC-01 fermentation broth challenge.Feeding trials(60 days)indicated that the SC-01 fermentation broth(viable counts:5.7×109 cfu mL−1)could improve feed intake,weight gain rate(WGR)and specific growth rate(SGR),and decrease the count of Vibrio sp.in the intestine of shrimp.

Exogenous endophthalmitis caused by Enterococcus casseliflavus:A case report

BACKGROUND Endophthalmitis caused by Enterococci is rare,and cases involving vancomycinresistant enterococci are even rarer.We report the first case of Enterococcus casseliflavus endophthalmitis associated with injury caused by a pig.We also review reported cases of exogenous endophthalmitis caused by Enterococcus casseliflavus and discuss the clinical management and prognosis of this disease.CASE SUMMARY A 43-year-old man with no previous visual problems complained of endophthalmitis in his left eye following injury caused by a pig.Visual acuity was light perception and B-ultrasonography revealed vitreous opacities with retinal detachment.He was treated with intravitreal vancomycin and ceftazidime after refusing vitrectomy.However,the vitreous opacities and retinal detachment deteriorated and he underwent vitrectomy 5 d post-injury.Intraoperatively,advanced rhegmatogenous and tractional retinal detachment with proliferative vitreoretinopathy were observed.On postoperative day 5,vitreous cultures grew Gram-positive cocci identified as Enterococcus casseliflavus.A 2-wk course of intravenous ampicillin and dexamethasone was commenced.On postoperative day 14,visual acuity improved to hand movement.At 6 mo post-injury,visual acuity improved to 20/667,but optic atrophy was present.CONCLUSION Systemic administration of linezolid in the treatment of Enterococcus casseliflavus endophthalmitis can improve visual acuity.However,intravitreal amikacin should be considered despite concerns of toxicity when oral linezolid fails to prompt improvement.