Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775

Abstract Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h⁻¹ to 0.5 h⁻¹, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.     

粪肠球菌Fsr群体感应系统的感染调控

群体感应(quorum sensing,QS)是细菌间的一种通讯方式,是控制微生物信号传递的重要机制,它使细菌能够感知周围环境中其他细菌的存在,并对其密度的变化做出快速反应,在调控基因表达和生物膜形成过程中起着重要作用。作为粪肠球菌(Enterococcus faecalis,E. faecalis)特有的Fsr QS系统,通过调控E. faecalis毒力因子及生物膜等的表达,从而引起机体的各种感染。本综述通过阐述E. faecalis Fsr QS系统在介导毒力因子的表达及生物膜的形成中的作用,为E. faecalis所引起的感染提供理论基础。     

耐利奈唑胺粪肠球菌感染的危险因素分析

分析深圳市南山区人民医院粪肠球菌感染患者的临床资料,探讨引起感染的危险因素,为防治耐利奈唑胺粪肠球菌感染提供临床参考。选取2010年1月-2015年9月在深圳市南山区人民医院住院的165例粪肠球菌感染患者,根据药敏结果分为利奈唑胺敏感组(103例)和利奈唑胺中介/耐药组(62例)。165例粪肠球菌主要来源于中段尿培养,占53.94%,其次为伤口分泌物培养(21.82%)、血培养(6.06%);科室分布以泌尿外科和肝胆外科为主,分别占35.76%和9.70%。单因素分析显示,碳青霉烯类抗生素暴露、留置尿管与感染相关。Logistic回归分析进一步明确碳青霉烯类抗生素暴露、留置尿管为耐利奈唑胺粪肠球菌感染的危险因素,提示应严格掌握碳青霉烯类抗生素的适应证,加强医院内感染的控制管理。     

牦牛肠道粪肠球菌的分离和鉴定

试验为获得1株与牦牛肠道共生的粪肠球菌,通过对健康牦牛粪样中的肠球菌进行分离培养、革兰染色、生化试验、16S rRNA基因测序比对等生物学鉴定,初步证实该分离菌株为粪肠球菌。此试验为进一步分析牦牛肠道菌群结构,探索牦牛耐饥、耐渴、耐粗饲、对高山草原极强适应性与其肠道菌群的关系,进一步研究与牦牛共生的粪肠球菌的生物学特性、生理特性、对宿主危害性等提供一些实验依据。     

Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Abstract: Enterococcus faecalis was grown under anaerobic conditions in chemostat cultures on energy sources with different degress of reduction (i.e. mannitol, glucose, pyruvate) at various culture pH values. Intracellular NADH/NAD ratios were measured and were found to be influenced both by the nature of the energy source and by the culture pH value. Highest ratios were found with mannitol as energy source and with high culture pH values. A role for the redox potential of the NADH/NAD couple as a regulatory effector is suggested by a correlation of the redox potential with the in vivo distribution of the carbon flux between pyruvate formate lyase and the pyruvate dehydrogenase complex.     

不同分离源粪肠球菌的毒力基因比较

【背景】粪肠球菌作为一种重要的乳酸菌在食品和医药领域应用广泛。由于很多粪肠球菌为条件致病菌,因此充分了解粪肠球菌基因组中毒力基因(Virulence genes)的携带情况对合理利用该菌种有重要的意义,但目前还没有研究专门报道不同分离源粪肠球菌基因组中毒力基因的携带情况。【目的】了解不同分离源粪肠球菌毒力基因的携带情况,评估分离自自然发酵乳制品中的粪肠球菌的安全性。【方法】利用比较基因组学方法确定107株分离自乳源、血液、尿液、粪便和水源中的粪肠球菌携带毒力基因情况,使用主成分分析比较不同分离源菌株毒力基因的差异,通过卡方检验筛查出环境特异性毒力基因。【结果】在107株粪肠球菌基因组共找到88种编码不同功能蛋白的毒力基因,其中与粘附相关的毒力基因最多。同时发现乳源分离株与其他环境分离株所携带的毒力基因没有显著差异。【结论】乳源分离株中携带的毒力基因与其他环境分离株无显著差异,表明分离自自然发酵乳制品中的粪肠球菌可能同样存在致病风险,因此在食品工业中使用粪肠球菌时一定要对菌株的安全性做全面的评估。     

重庆市1264株粪肠球菌和屎肠球菌耐药性监测

【摘 要】 目的 了解2011年中国重庆市主要7所教学医院临床分离粪肠球菌和屎肠球菌对各类抗菌药物的耐药性。方法 重庆市主要7所教学医院(6所综合性医院,1所儿童医院)按统一方案、采用统一的材料、方法和判断标准(CLSI 2011年版)进行粪肠球菌和屎肠球菌的耐药性监测。数据用WHONET 5.5软件按照CLSI 2011年版折点进行分析。结果 共分离到非重复粪肠球菌589株、屎肠球菌675株,对利奈唑胺、万古霉素、替考拉宁仍极敏感,耐药率<2%,万古霉素耐药粪肠球菌和屎肠球菌检出率分别为0.3%、0.7%。粪肠球菌对青霉素、氨苄西林、呋喃妥因的耐药率较低,分别为14.8%、8.6%和5.1%,对高浓度庆大霉素的耐药率分别为46.9%;屎肠球菌耐药性明显高于粪肠球菌,对青霉素和氨苄西林耐药率接都在90％左右。儿童和成人耐药率存在一定差别。结论 本市医院肠球菌感染以屎肠球菌为主, 粪肠球菌次之,两者耐药性明显不同, 监测其耐药情况对指导临床用药具有重要意义。     

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

对产自乳酸菌Enterococcuze fecalis 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。SDSPAGE和凝胶层析分子量分别为30ku及69ku。纯化酶最适作用温度为30℃,最适作用PH为7．5～8．0,在pH6．0～9．5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Km和Vmax分别为0．098％和72mg／（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。     

水溶性蜂胶对感染根管内粪肠球菌的体外抑菌试验

目的 研究水溶性蜂胶对感染根管内常见菌粪肠球菌的体外抑菌活性.方法 采用液体稀释法测定水溶性蜂胶对粪肠球菌的最低杀菌浓度(Minimal bactericidal concentrations,MBC).结果 蜂胶对粪肠球菌的MBC为0.156%.结论 蜂胶对感染根管内粪肠球菌具有良好的抑菌活性,将其用于感染根管消毒具...     

BTK在粪肠球菌介导的炎症环境中的破骨作用

目的 在粪肠球菌脂磷壁酸（LTA）作用的炎症环境下,研究布鲁顿酪氨酸激酶（BTK）在破骨细胞中的作用,从而为根尖周炎的治疗提供实验依据。方法 PCR检测粪肠球菌LTA刺激破骨细胞后BTK基因水平的表达情况。以浓度300 ng/mL的人重组蛋白BTK（recombinant human BTK,rhBTK）刺激破骨细胞,用CCK8法检测破骨细胞增殖和RT-PCR检测破骨细胞分化标志因子TRAP基因水平表达情况。结果 破骨前体细胞5 d诱导成功。PCR结果发现粪肠球菌LTA刺激后BTK和TRAP的mRNA表达量明显增高;免疫荧光可见LTA刺激后BTK在破骨细胞中的定位情况;300 ng/mL rhBTK组可以促进破骨细胞增殖;PCR结果显示,加入rhBTK后,破骨细胞分化标志因子TRAP的mRNA水平升高。结论 在粪肠球菌LTA作用的炎症环境下,BTK表达升高;增高BTK后,可以促进破骨细胞的增殖及分化。研究发现BTK参与了破骨细胞的炎症反应进程。     

粪肠球菌对泰利霉素药物敏感性与耐药基因erm的分布

目的 了解粪肠球菌对泰利霉素和其他常用抗菌药物的耐药性,以及泰利霉素耐药与红霉素耐药相关基因ermA、ermB、ermC之间的关系。方法 对本院2010‒2016年从各种临床标本收集鉴定的320株粪肠球菌,用微量肉汤稀释法测定这些菌株对泰利霉素及8种临床常用抗菌药物的最小抑菌浓度,并用PCR法检测耐药基因ermA、ermB、ermC的分布。结果 320株粪肠球菌对泰利霉素中介耐药26株,耐药138株,耐药率达51.3%;对红霉素耐药率达95.6%,泰利霉素抗粪肠球菌效果优于红霉素。对利奈唑胺、万古霉素、呋喃妥因和氨苄西林耐药率分别为15.6%、0.6%、2.2%和0.6%。共10株（3.1%）携带ermA基因,207株（64.7%）携带ermB基因,对泰利霉素中介组中有23株ermB基因阳性,耐药组有131株ermB基因阳性,仅1株（0.3%）ermC基因阳性,该菌同时携带ermB基因。结论 粪肠球菌对泰利霉素已有较高耐药率。粪肠球菌对泰利霉素MIC值改变与ermB基因密切相关,与ermA、ermC基因无明显相关性。     

RNA-Seq探索低水平利奈唑胺耐药粪肠球菌机制

为了探索低水平利奈唑胺耐药粪肠球菌机制,本研究采用Illumina HiSeq 4000高通量转录组测序技术对低水平利奈唑胺耐药粪肠球菌(P10748 MIC:8 mg/L)和利奈唑胺敏感粪肠球菌(3138 MIC:2 mg/L)进行测序及生物信息学分析,以标准菌株ATCC29212作为质量评估参考。利用GO和KEGG数据库对差异表达基因进行注释与富集分析,并利用荧光定量PCR对测序中的差异表达基因进行验证。结果显示,测序共获得了3.57 Gb有效数据,通过De novo拼接获得1 920条unigenes,总长度为2 122 210 bp,平均长度为1 105 bp。差异基因模式聚类分析显示共有150个显著性差异表达基因(FDR≤0.001,|log2 Ratio|≥1),其中141个上调,9个下调。其中生物膜形成和外排泵相关基因esp、optrA、fexA在耐药菌中显著上调。GO功能注释结果显示,催化活性、代谢过程和细胞是注释最多的条目;Pathway富集分析发现,肽聚糖生物合成、缬氨酸亮氨酸和异亮氨酸的降解和硫代谢为显著性富集通路(p<0.05)。荧光定量PCR结果与测序结果基本一致。推测膜转运蛋白和生物膜形成可能在耐药机制中具有重要作用,为低水平利奈唑胺耐药肠球菌机制提供了可参考的耐药靶标。     

粪肠球菌精氨酸脱亚胺酶酶学性质研究

经硫酸铵分级沉淀、Q-Sepharose Fast Flow阴离子交换层析、SephadexG-75凝胶柱层析从NJ402自溶细胞超声破碎液中提纯得到精氨酸脱亚胺酶(ADI), 纯化倍数为34.5, 活力回收率为31.4％, 经SDS-PAGE以及Native-PAGE测定结果表明, ADI亚基分子量约为46 kD, 该酶非变性情况下的分子量约为190 kD左右, 该酶为同四聚体结构。酶学性质研究结果表明：ADI催化最适温度和最适pH分别为50℃和6.5, 在45℃以下和pH 5~8之间有很好的稳定性。ADI是L-型脱亚胺酶, 具有严格的光学选择性, 适当浓度的Mn2+、Mg2+、Co2+对ADI催化活力的促进作用较大, 高浓度的Zn2+和Co2+对酶有一定程度的抑制作用, L-瓜氨酸对酶无抑制作用而L-鸟氨酸却表现出较强的抑制作用。ADI在最佳催化条件下作用于L-精氨酸的米氏常数为3.2686 mmol/L, 最大反应速度为2.44 μmol/min。     

粪肠球菌精氨酸脱亚胺酶酶学性质研究

经硫酸铵分级沉淀、Q-Sepharose Fast Flow阴离子交换层析、SephadexG-75凝胶柱层析从NJ402自溶细胞超声破碎液中提纯得到精氨酸脱亚胺酶(ADI),纯化倍数为34.5,活力回收率为31.4%,经SDS-PAGE以及Native-PAGE测定结果表明,ADI亚基分子量约为46 kD,该酶非变性情况下的分子量约为190 kD左右,该酶为同四聚体结构.酶学,胜质研究结果表明:ADI催化最适温度和最适pH分别为50℃和6.5,在45℃以下和pH 5～8之间有很好的稳定性.ADI是L-型脱亚胺酶,具有严格的光学选择性,适当浓度的Mn2 、Mg2 、Co2 对ADI催化活力的促进作用较大,高浓度的Zn2 和Co2 对酶有一定程度的抑制作用,L-瓜氨酸对酶无抑制作用而L-鸟氨酸却表现出较强的抑制作用.ADI在最佳催化条件下作用于L-精氨酸的米氏常数为3.2686 mmol/L,最大反应速度为2.44 μmol/min.     

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: Implications for their activity in vivo

Abstract The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecelis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.     

Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin

The mature forms of the extracellular muramidase-2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active-site-containing N-terminal domain fused to a multiple-repeat C-terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.     

Insertional inactivation by Tn3701 of pIP964 hemolysin expression in Enterococcus faecalis

Abstract Transposition of the conjugative mobile element (Tn 3701 ) of Etreptococcus pyogenes strain A454 onto the Enterococcus feacalis hemolysin plasmid pIP964 modified the expression of the genes involved in hemolysin production. By hemolysis complementation tests the genes coding for two components required for the hemolytic activity expression were found to be located on the Eco RI E and G fragments of pIP964.     

Conjugal transfer of plasmid-borne bacteriocin production in Enterococcus faecalis 226 NWC

Enterococcus faecalis 226 NWC, isolated from natural whey cultures utilized as starter in water-buffalo Mozzarella cheese manufacture, produces a bacteriocin, designated Enterocin 226 NWC, which is inhibitory to Listeria monocytogenes. Plasmid analysis of E. faecalis 226 NWC showed a single 5.2-kb plasmid, pEF226. In conjugation experiments, pEF226 was transferred into a plasmid-free strain of E. faecalis JH2-2. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The identity of conjugation was confirmed by digestion with SmaI restriction endonuclease and subsequent pulsed-field gel electrophoresis (PFGE) of the genomic DNA of E. faecalis 226, E. faecalis JH2-2 and of the isolates after the mating. The data indicate that the ability of E. faecalis 226 NWC to produce the bacteriocin is linked to the 5.2-kb conjugative plasmid pEF226.     

血流感染粪肠球菌和屎肠球菌的临床分布及耐药研究

回顾性分析上海市某三甲医院血培养阳性标本中粪肠球菌和屎肠球菌的临床分布及对抗菌药物的耐药特征,为临床治疗其所致感染奠定基础。收集上海市某三甲医院2012年2月—2016年9月血流感染患者血液标本中的粪肠球菌和屎肠球菌,采用法国生物梅里埃公司的VITEK 2Compact全自动细菌鉴定和药敏分析系统进行细菌鉴定及药敏测定,研究细菌临床分布特点及对常用抗菌药物的耐药特征。共分离获得30株粪肠球菌和17株屎肠球菌。粪肠球菌样本主要来自泌尿科、消化科和血液科,所占比例分别为13.33%、16.67%和10.00%。粪肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星、四环素和红霉素的耐药率分别为13.33%、10.00%、36.67%、33.33%、66.67%和60.00%。屎肠球菌样本主要来自消化科(29.41%),其对以上抗菌药物的耐药率分别为88.24%、82.35%、88.24%、76.47%、23.53%和70.59%。屎肠球菌对青霉素、氨苄西林、环丙沙星和左氧氟沙星的耐药率显著高于粪肠球菌,而对四环素的耐药率显著低于粪肠球菌。两者均对替加环素、利奈唑胺和万古霉素敏感,但万古霉素对屎肠球菌的最低抑菌浓度显著低于粪肠球菌。结果提示,屎肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星的耐药率高于屎肠球菌,对万古霉素敏感,且其万古霉素最低抑菌浓度低于粪肠球菌。本研究为治疗这两种细菌所致感染的经验性用药提供了数据支持。