临床分离粪肠球菌和屎肠球菌菌株的检测及耐药性研究

目的分析粪肠球菌、屎肠球菌对抗菌药物的耐药性及敏感性,为合理使用抗菌药物提供证据。方法运用Phonex 100全自动微生物鉴定/药敏系统对临床分离的菌株进行鉴定和药敏试验。结果 103株肠球菌耐药率最高的药物依次为红霉素（94.2%）、环丙沙星（85.4%）、利福平（81.6%）,对万古霉素（94.2%）和替考拉宁（93.2%）的敏感性最高。发现万古霉素耐药的粪肠球菌和屎肠球菌各3株。粪肠球菌和屎肠球菌对青霉素的耐药率分别为22%和71.7%,对氨苄西林的耐药率分别为16.7%和86.8%。结论 肠球菌属总体耐药率较高,红霉素、环丙沙星、利福平对肠球菌的敏感率较低,已检测发现VRE,临床上应加强监测,并根据药敏结果、感染严重程度合理选择适当的抗菌药物。     

粪肠球菌gyrA基因突变与氟喹诺酮类耐药性关系的研究

目的探讨粪肠球菌DNA促旋酶A亚单位（gyrA）基因突变与氟嚎诺酮类耐药的关系。方法琼脂稀释法测定粪肠球菌对各种氟喹诺酮类药物的耐药情况;聚合酶链反应（PCR）结合单链构象多态性（SSCP）分析检测gyrA中氟哇诺酮类耐药决定区（QRDR）突变情况。结果粪肠球菌对诺氟沙星耐药率为78．1％（50／64）,加替沙星为54．7％（35／64）。41株耐药菌的第87位氨基酸编码碱基发生突变（GAA→GGA）。结论加替沙星的耐药率较低,优于其他氟哇诺酮类药物;gyrA基因突变是粪肠球菌对氟喹诺酮类耐药的重要原因。     

粪肠球菌和屎肠球菌的医院感染分布及耐药性

肠球菌广泛存在于自然界,可寄生于人类的胃肠道和女性生殖道,是人和动物肠道正常菌群的一部分,是仅次于葡萄球菌的重要院内感染病原菌[1],较多出现在有严重基础疾病的老年人和免疫力低下的患者中,主要引起尿路、腹腔、盆腔感染,还可引起菌血症、心内膜炎、呼吸系统感染.由于其固有的耐药性,对头孢菌素类、克林霉素、磺胺甲噁唑/甲氧苄啶、低浓度的氨基糖苷类抗生素即使在体外实验可表现出敏感,但临床治疗往往无效[2],所致感染治疗困难.肠球菌属中粪肠球菌和屎肠球菌占临床分离的绝大多数[3].本文就医院感染标本中分离出的粪肠球菌和屎肠球菌的来源和耐药性进行分析,以期为临床治疗肠球菌医院感染提供参考依据.     

肠球菌耐药性的检测与分析

肠球菌是引起临床感染的常见病原菌之一，固有多种抗生素抗性，临床上常采用协同用药治疗肠球菌感染，例如采用青霉素、氨于青霉素或万古霉素与氨基糖贰类药物联合使用，但是当肠球菌获得了氨基糖式类活性酶后，会发展成高度氨基糖忒类耐药性（MIC，≥2000μg／ml）．而失去协同作用。为了解当地临床分离的肠球菌耐药情况．我们对认1993～1995年我院分离的48珠肠球菌对氨基糖咸类（由大霉素和链霉素）的高度耐药性以及青霉素、氨个青霉素、四环素、氟哌酸、万古霉素的敏感性进行了测定，并对其结果进行了初步分析，现报告如下。材料与方法…     

肠球菌生物学性状及耐药性的调查

目的　了解湖北地区肠球菌的耐药性 ,指导临床用药。方法　对 9所大型综合医院感染标本中分离的肠球菌进行鉴定和药敏试验 ,并以“WHONET4”软件分析。结果　 335株肠球菌中 ,粪肠球菌检出率居首位 (77 9% ) ,屎肠球菌次之(9 2 % )。氨苄青霉素耐药肠球菌 (ARE)分离率为 17 0 % ,氨基糖苷类高水平耐药肠球菌 (HLAR)为 38 2 % ,未检出耐万古霉素肠球菌 (VRE)。结论　湖北地区肠球菌耐药性呈上升趋势 ,应加强肠球菌的鉴定及耐药监测     

粪肠球菌心内膜炎抗原efaA蛋白的原核表达及纯化

目的原核表达纯化粪肠球菌心内膜炎抗原efaA蛋白,为粪肠球菌心内膜炎的致病机制研究及临床血清学诊断奠定基础。方法从粪肠球菌中扩增efaA基因,相应酶切后,克隆到原核表达载体pET30a中,构建pET30a-efaA重组质粒。经BamhI、XhoI酶切及测序鉴定,将pET30a-efaA质粒转化入BL21(DE3)。以IPTG诱导BL21(DE3)表达efaA融合蛋白,亲和层析纯化重组蛋白,SDS-PAGE、WesternBlot分析鉴定。结果PCR体外扩增efaA基因产物约943bp,重组表达质粒pET30a-efaA在大肠杆菌BL21(DE3)中表达,通过亲和层析获得纯化重组蛋白。SDS-PAGE、Western免疫印迹显示蛋白表达带的分子量约为34kd。结论粪肠球菌心内膜炎抗原efaA蛋白在大肠杆菌BL21(DE3)中成功表达并纯化。     

粪肠球菌主要毒力因子研究进展

粪肠球菌为条件性致病菌,是许多国家医院内感染的主要病原菌,也是一种新的人畜共患病病原体。粪肠球菌感染和致死主要是由其毒力因子引起的,本文概述了粪肠球菌主要毒力因子尤其是溶血素的研究进展,为粪肠球菌致病机制的研究奠定基础。     

院内感染肠球菌的同源性及耐药性分析

目的了解医院感染肠球菌的同源性及耐药状况,为临床控制肠球菌感染及合理使用抗生素提供依据。方法运用随机引物扩增(RAPD)对27株院内感染肠球菌进行基因分型与同源性分析,琼脂稀释法检测7种抗生素对肠球菌的最低抑菌浓度。结果 27株肠球菌经RAPD聚类分析分为9个基因型,27株肠球菌对青霉素、氨苄西林、左旋氧氟沙星、红霉素、多西环素及万古霉素的耐药率分别为85.2%、66.7%、51.9%、51.9%、37.0%和7.4%。结论肠球菌感染以内源性感染为主,同时存在小规模的医院内交叉感染。肠球菌对抗生素的耐药状况日趋严重,万古霉素对肠球菌具有良好抗菌作用。     

粪肠球菌心内膜炎抗原EfaA在生物膜形成中的作用

目的 探讨粪肠球菌心内膜炎抗原EfaA在生物膜形成中的作用。方法 微量滴定板法测定生物膜形成能力,将efaA⁺、efaA^- 粪肠球菌S₁₄、S₁₄-pAT28、S₁₄-pAT28-efaA分别接种到96孔板,37 ℃培养24 h形成生物膜,结晶紫染色,测定孔板底部生物膜的OD₅₇₀ 值;MTT法比较S₁₄、S₁₄-pAT28、S₁₄-pAT28-efaA所形成的生物膜的活菌含量OD₄₉₂;共聚焦激光扫描显微镜(CLSM)观察比较efaA⁺、efaA^-肠球菌形成的生物膜的平均厚度、最大厚度。结果 efaA⁺ 转化株在各培养时间点微量滴定板法测定的生物膜形成能力OD₅₇₀ 值、MTT法测定的OD₄₉₂活菌含量、CLSM观察的生物膜的平均厚度、最大厚度均大于野生株及空质粒转化株,P<0.05;空质粒转化株与野生株比较无显著性差别,P>0.05。结论 粪肠球菌心内膜炎抗原efaA基因有利于肠球菌生物膜的形成。     

粪肠球菌在酒精性肝病中的研究进展

酒精性肝病(ALD)是世界范围内最常见的慢性肝病之一,包括脂肪变性、脂肪性肝炎、纤维化和肝硬化不同阶段。粪肠球菌是医院常见获得性感染菌群,对于酒精性肝炎患者预后具有重要影响。本篇综述重点介绍了ALD的发病因素和粪肠球菌的致病机理,总结了粪肠球菌在ALD中的研究进展,简述了临床上对于粪肠球菌感染的检测和治疗方法。由于临床上感染溶细胞性粪肠球菌的ALD患者死亡率极高,因此深入认识粪肠球菌成为当下重要问题。     

利奈唑胺体外诱导粪肠球菌耐药株23S rRNA V区基因突变位点分析

目的阐明体外诱导利奈唑胺耐药粪肠球菌的核糖体23SrRNA V区基因位点变异特征。方法收集1株血流感染的粪肠球菌和1株粪肠球菌质控菌ATCC29212（编号分别为F3和F4菌株,均为利奈唑胺敏感株）,通过体外浓度倍增法诱导利奈唑胺耐药;挑取单克隆,经E-test条测定MIC值,获得各菌株的耐药浓度梯度;提取耐药菌株基因组DNA,PCR扩增23SrRNA V区基因,扩增产物经测序后与野生株比较,获得V区的突变位点。结果经体外多步法诱导利奈唑胺耐药的不同MIC值粪肠球菌共13株。PCR测序分析2株母株均无变异位点,23SrRNA V区的突变位点主要是G2576U,此外还有T2504A、G2505A、C2610A、C2424U。结论体外多步法可诱导粪肠球菌利奈唑胺耐药,耐药机制与23SrRNA V区位点突变密切相关,突变位点随着MIC值的增高而增多。     

sprE基因敲除粪肠球菌突变株的构建和功能的初步研究

目的构建丝氨酸蛋白酶同源基因(serine protease gene,sprE)敲除粪肠球菌突变株,来研究sprE基因的功能。方法用pTX4577质粒构建粪肠球菌sprE基因重组自杀质粒pCQ001,通过体内同源重组,筛选获得sprE基因的突变株,体外研究不同温度和氧化条件对突变株生长能力的影响。结果经同源重组,利用卡那霉素抗性筛选,PCR、脉冲场电泳和southern blot进行鉴定获得基因突变株#sprE,突变株在40℃的生长能力以及在氧化条件下的存活率均明显降低。结论sprE基因敲除粪肠球菌突变株#sprE构建成功,为进一步研究其功能打下基础。     

粪肠球菌感染抗菌药物联合应用的体外效应研究

目的　评价万古霉素、奈替米星、阿米卡星、环丙沙星、左氧氟沙星和加替沙星 6种抗菌药物分别与第一代头孢菌素头孢硫脒联合用药 ,对于临床分离的粪肠球菌 (Enterococcusfaecalis,Ef)的体外联合抗菌效应。方法　采用棋盘法设计 ,微量肉汤稀释法测定。测定不同浓度组合的 6组抗菌药物对 30株临床分离的革兰阳性球菌的最低抑菌浓度 ,并计算FIC指数。FIC≤ 0 .5为协同作用 ,0 .5 2为拮抗作用。结果　头孢硫脒对粪肠球菌的MIC50 为 2～ 8mg L ,与万古霉素、奈替米星、阿米卡星、环丙沙星、左氧氟沙星、加替沙星联合应用后 ,其MIC50 分别显著地降低至 0 .1 2 5 ,0 .2 5 ,0 .2 5 ,0 .2 5 ,0 .2 5 ,0 .2 5mg L。FIC指数分布 :FIC≤ 0 .5占 50 %～ 86 .7% ;0 .5 2为 0。结论　 6种抗菌药物与头孢硫脒分别联合用药后 ,对粪肠球菌基本表现为协同作用和相加作用 ,并以协同作用为主 ,无关作用较少 ,无拮抗作用     

粪肠球菌丝氨酸蛋白酶基因突变株的构建和致病性研究

目的 构建粪肠球菌丝氨酸蛋白酶基因（sprE）突变株，并研究sprE基因的功能。方法 用自杀质粒pTX4577构建粪肠球菌基因重组自杀质粒pCQ001，通过体内同源重组，筛选获得sprE基因的突变株，体外研究不同温度和氧化条件对突变株生长能力的影响，通过小鼠腹膜炎和兔心内膜炎模型来研究突变株的毒力下降情况。结果 经同源重组，利用卡那霉素抗性筛选，PCR、脉冲场电泳和Southern印迹进行鉴定获得sprE基因突变株，命名为＊sprE，突变株在40℃的生长能力及在氧化条件下的存活率均明显低于野生株。在小鼠腹膜炎模型中，存活率明显高于野生株，差异有统计学意义（P〈0．01），引起的兔心内膜炎也较野生株轻，差异有统计学意义（P〈0．01）。结论 sprE基因突变株＊sprEgg建成功，sprE基因在粪肠球菌致病中起重要作用，可能是粪肠球菌的毒力因子之一。     

Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts

OBJECTIVES: Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors. METHODS: We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells. RESULTS: Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed. CONCLUSION: The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.     

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.     

Efficacy of Ciprofloxacin,Metronidazole and Minocycline in Ordered Mesoporous Silica against Enterococcus faecalis for Dental Pulp Revascularization: An In-Vitro Study

Pulp revascularization of teeth with necrotic pulp has become an alternative treatment in cases with immature apex. Microbial control is essential to achieve a successful outcome and continued root development. Enterococcus faecalis (E. faecalis) is the most frequently isolated bacterial species in root canals of endodontically failed teeth. Our main goal was to compare the in-vitro antimicrobial efficacy of different antibiotic formulations delivered by ordered mesoporous silica (OMS) against E. faecalis. To determine antibiotic susceptibility, we tested OMS and triple antibiotic paste (TAP; ciprofloxacin:metronidazole:minocycline) with different reagents in different concentrations, using the Kirby–Bauer disk diffusion method. OMS and metronidazole showed no antibacterial activity against E. faecalis. Mixtures of OMS and antibiotics in proportions of 2:2:14 and 4:1:7 (mg/L of ciprofloxacin:metronidazole:minocycline, respectively) showed the lowest antibacterial activity. The antibacterial activity of the combined solutions of ciprofloxacin and metronidazole was significantly higher (p < 0.005). Combinations in different concentrations of minocycline, ciprofloxacin, and metronidazole in OMS have shown activity against E. faecalis, although the combined use of ciprofloxacin and metronidazole has shown the most effective results. This study demonstrates the efficacy of intracanal antibiotic combination paste activity against E. faecalis, avoiding the use of minocycline, whose undesirable effect of teeth staining is a common problem for patients and professionals in dental clinic.     

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin,and Cefotaxime

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin’s host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.