屎肠球菌透明质酸酶全长基因的克隆表达和免疫原性

目的构建屎肠球菌透明质酸酶（hyaluronidase，hyl）基因重组表达质粒，在大肠埃希菌中表达获得重组蛋白，探讨Hy1蛋白经口服免疫小鼠后，机体产生的免疫应答。方法PCR扩增hyl基因片段，克隆至pQE-30质粒上，构建重组质粒pQE-hyl，转化E，coli DH5α，经异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达融合蛋白Hy1；Western印迹分析其免疫反应性。融合蛋白经镍离子柱纯化后，Hy1和屎肠球菌全菌蛋白TX0016分别给小鼠灌胃进行免疫，ELISA法检测小鼠血清IgG、IgA、小肠黏液sIgA和粪便sIgA。用TX0016进行腹腔攻击已免疫的小鼠，观察其免疫保护性。结果经测序hyl基因全长1662bp，为编码553个氨基酸残基的多肽。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）分析相对分子质量约为60000。可溶性表达占全菌的38％以上，经亲和层析后可获得纯度为92％以上的重组蛋白。Western印迹证实了其免疫反应性。灌胃免疫小鼠后，ELISA法检测结果Hy1组血清IgA、IgG、粪sIgA、肠黏液sIgA分别为0．365±0．048、0，431±0，064、0．743±0，056和1．112±0．113，对照组则分别为0，051±0．013、0．098±0．019、0．102±0，032和0，187±0，051，差异有统计学意义（P〈0．01）。用半数致死量（I，D50）的细菌量攻击后，Hy1免疫组小鼠的存活率为70％，而对照组为50％。结论融合蛋白Hy1经口服免疫可有效诱导黏膜免疫应答，产生高水平的sIgA，发挥局部黏膜免疫作用。     

屎肠球菌Hyl蛋白的免疫保护性研究

目的用基因工程方法获得屎肠球菌类透明质酸酶(Hyalronidase,Hyl)蛋白,经口服免疫小鼠后,探讨机体产生的免疫应答和抗感染保护作用。方法Hyl用Ni2+-NTA柱纯化后,作为抗原给小鼠灌胃进行免疫,ELISA检测小鼠血清IgG、IgA、小肠粘液sIgA和粪便sIgA。并用屎肠球菌TX0016腹腔攻击已免疫的小鼠,观察其免疫保护性。结果重组蛋白经Ni2+-NTA柱纯化后,灌胃免疫小鼠后。ELISA检测结果血清IgA、IgG,粪sIgA,肠粘液sIgA明显高于对照组(P<0.01)。通过细菌攻击后,小鼠的平均存活时间也明显长于对照组。结论重组蛋白Hyl经口服免疫可有效诱导粘膜免疫应答,产生高水平的sIgA,发挥局部粘膜免疫作用和抗菌保护作用。Hyl有望作为预防屎肠球菌口服疫苗的候选抗原。     

青藏高原牦牛携带屎肠球菌及其毒力基因和耐药性检测

目的 了解青藏高原牦牛携带屎肠球菌(Enterococcus faecium)情况,以及耐药性和毒力基因。方法 使用链球菌培养基分离细菌;使用生化反应和16s rDNA序列分析方法鉴定;采用PCR方法检测从牦牛粪便中分离的屎肠球菌携带细胞溶血素(cytolysin,cylA)、明胶酶E(gelatinase, gelE)、表面蛋白(enterococcal surface protein, esp)、胶质蛋白粘附素collagen-binding-adhesin of Enterococcal faecium,acm)、聚集物质(aggregation substance, asa1)及透明质酸酶(hyalronidase,hyl)6种毒力基因的情况;应用K-B纸片法对屎肠球菌分离株进行16种抗生素的敏感性分析;参照PulseNet 脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)实验方法对屎肠球菌分离株进行PFGE分型分析。结果 我们从320份牦牛粪便分离到33株屎肠球菌。这些菌株中毒力基因asa1的阳性率最高,为6.1%,acm和hyl次之,均为3.0%,其他毒力基因皆未检出。33株牦牛屎肠球菌中,有19株菌株具有抗生素耐药性,包括4株多重耐药菌株和15株单耐菌株。牦牛屎肠球菌对利福平耐药率最高,为48.5%;对其他抗生素的耐药率从高到低依次为青霉素G15.2%、四环素12.1%、强力霉素12.1%、红霉素9.1%、环丙沙星6.1%、氨苄西林6.1%、高浓度庆大霉素6.1%、磷霉素6.1%、左氧氟沙星6.1%。所有菌株对万古霉素、替拉考宁和氯霉素均敏感。细菌染色体DNA酶切片段的PFGE分析发现,33株屎肠球菌分离株共产生30种PFGE带型,可分为A-H 8个聚类群,耐药菌株分布在6个聚类群中。结论 青藏高原牦牛携带屎肠球菌,呈现高度遗传多态性,部分菌株携带毒力基因,对多种抗生素耐药。研究提示,屎肠球菌可能会通过牦牛相关食品传播。     

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。     

耐万古霉素肠球菌临床菌株毒力基因携带情况及差异

目的检测不同种类耐万古霉素肠球菌(VRE)毒力基因携带情况及其差异,为研究VRE致病机制提供基础。方法采用微量稀释法检测浙江地区490株屎肠球菌和862株粪肠球菌临床分离菌株对万古霉素的耐药性。采用PCR检测VRE菌株ace、asa1、cylA、efaA、esp、gelE、hyl七种毒力基因。结果 10%(49/490)屎肠球菌株和0.8%(7/862)粪肠球菌株为VRE。万古霉素耐药屎肠球菌株中,5株未检出任何毒力基因,其余44株仅可检出asa1、esp、gelE和hyl基因,其中esp基因(73.5%,36/49)和hyl基因(53.1%,26/49)为优势基因,以单或双基因为主要携带模式。万古霉素耐药粪肠球菌株中可检出除hyl基因以外其他6种毒力基因,且每株菌均同时携带3~6种致病基因。结论本地区VRE以屎肠球菌为主,耐万古霉素屎肠球菌和粪肠球菌毒力基因携带率、携带种类和模式均有明显差异。     

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.     

临床分离屎肠球菌对8种抗生素的耐药性分析

目的了解广东、河北、甘肃等地临床分离的39株屎肠球菌对8种抗生素的耐药性及产β-内酰胺酶的情况。方法采用纸片琼脂扩散法检测屎肠球菌耐药情况;用头孢硝噻吩法检测屎肠球菌产β-内酰胺酶情况。结果 39株屎肠球菌对红霉素的耐药率最高,达到94.87%,其次是高浓度庆大霉素和青霉素,分别为84.62%和79.48%,环丙沙星的耐药率也达到76.92%,米诺环素为38.46%,而氯霉素则体现出较好的敏感率,其耐药率为17.95%,尚未发现有耐万古霉素及呋喃妥因的菌株,两者耐药率均为0。39株屎肠球菌β-内酰胺酶阳性率为0。汕头地区分离屎肠球菌呈多重耐药性,对青霉素、红霉素、环丙沙星和高浓高度庆大霉素耐药率达100.00%。结论临床分离的屎肠球菌多重耐药性严重,尤其是汕头地区分离的细菌,临床应根据药敏结果合理选用抗感染药物。     

屎肠球菌感染致多器官功能障碍一例

患者男 ,2 0岁。受凉后出现发冷、发热 ,用磷霉素、清开灵和蒙药治疗无效。 4ｄ后有脐周阵发性绞痛 ,泻黑绿色稀水便 ,每日 6～ 10次 ,伴头晕、头痛、呕吐 ,2ｄ未缓解 ,遂收住入院。体检 :体温 40 .4°Ｃ ,脉搏 12 8次 /ｍｉｎ ,呼吸 30次 /ｍｉｎ ,血压 130 / 5 8ｍｍＨｇ(17.3/ 7.7ｋＰａ) ,急性热病容 ,神志清 ,咽部充血 ,颈软 ,双肺呼吸音粗 ,腹平软 ,脐周压痛( ) ,肠鸣音 8次 /ｍｉｎ。予氧氟沙星 0 .2 ｇ ,每日 2次 ,静脉滴注。当晚 6点患者表情淡漠 ,球结膜下出现散在出血点 ,血压 6 0 / 40ｍｍＨｇ(8/ 5 .3ｋＰａ)。血常规 :白细胞 …     

屎肠球菌磷霉素耐药基因的高通量测序筛选

目的 从对磷霉素耐药的屎肠球菌临床分离株中筛选介导屎肠球菌对磷霉素耐药的基因,并验证其作用。方法通过药物敏感测定及接合转移实验,了解磷霉素耐药屎肠球菌的耐药性及其可转移性。通过Solexa高通量测序筛选磷霉素耐药基因,并对该耐药基因进行克隆确认其基因功能。结果屎肠球菌临床株Efm-HS0661对糖肽类抗生素及磷霉素耐药,且可通过接合转移试验发生磷霉素耐药性的转移。Solexa高通量测序及比对筛选获得一2414 bp核苷酸序列,含有全长420 bp的fosB基因,与已知fosB基因氨基酸序列同源性达99.8％。含该基因的DH5α克隆转化子的磷霉素最低抑菌浓度较不含者明显升高。结论 Solexa高通量测序技术可用于临床菌株未知耐药基因的快速筛选,质粒介导的磷霉素耐药基因fosB可导致屎肠球菌对磷霉素耐药。     

一起由多重耐药屎肠球菌引起的人群感染暴发流行

1　临床资料　　 1998年 7月下旬至 9月初 ,江苏省如皋、海安、泰县等地区发生成批生猪发病 ,数以千计病猪死亡 ,并有 40人发病 ,12人死亡。生猪疫情为连村式或跳跃式 ,病猪表现为起病急骤、不思饮食、步态不稳、呼吸急促 ,体温升高(可达 42℃ )。病猪特征性的表现为眼球结膜、睑结膜充血 ,有分泌物 ,全身皮肤出现瘀斑、口鼻出血。病猪起病后多在 1～ 3天内死亡。人群发病者多有与病、死猪接触史 ,其中屠宰或参与屠宰猪者 18例 ,售肉或加工猪肉、皮者 6例 ,清洗生肉并食用者 7例 ,周围有病猪或病人或接触途径不明者 9例。 40例中有皮肤破损 …     

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS

Objective To examine the clonal diversity of vancomycin‐resistant enterococci (VRE). Methods A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed‐field gel electrophoresis (PFGE) typing. Results Forty‐nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC ≥ 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D_i = 0.93) and belonged to 24 PhP‐types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C→A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. Conclusion The isolated clinical VRE strains were highly diverse in Tehran.     

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.     

Linezolid in the treatment of vancomycin-resistant Enterococcus faecium in solid organ transplant recipients: report of a multicenter compassionate-use trial

Abstract: Vancomycin‐resistant Enterococcus faecium (VRE) is increasing in incidence in solid organ transplant recipients and has a high (up to 83%) associated mortality rate. Until recently, there have been no consistently effective antimicrobial therapies for VRE infection. Linezolid is a new antibiotic that belongs to the class of oxazolidinones approved by the FDA for the treatment of VRE infections, including those with bacteremia. Here, we report the experience with linezolid in an open‐label, compassionate‐use trial at 53 US centers for the treatment of documented VRE infections in patients with solid organ transplants. Eighty‐five patients with solid organ transplants and documented VRE infections were studied. Blood cultures were positive for VRE in 43 patients, while 42 patients had other, non‐rectal, sites of infection. Fifty‐three patients responded well to treatment, with clinical resolution of the infection (62.4% survival rate). Of these, 47 had documented negative cultures post therapy. The mean duration of therapy for cured patients was 23.5 days. Thirty‐two (37.6%) patients died, 28 due to sepsis and organ failure (32.9% failure rate), and 4 due to unrelated causes. Mortality rates for patients with bacteremia were comparable to mortality rates observed with patients who had positive cultures from other sites. Adverse reactions to linezolid included thrombocytopenia (4.7%), decreased leukocyte count (3.5%), and an increase in blood pressure (1.2%), none of which led to discontinuation of therapy. Linezolid appears to be a safe and effective treatment option for VRE, even in the presence of bacteremia, and may lead to decreased mortality in solid organ transplant recipients with VRE infection.     

一起由屎肠球菌引起的人、猪败血症暴发流行的相关性

目的 通过基因分析研究引起人、猪败血症暴发流行的屎肠球菌的相关性。方法 随机对患者和病猪的血培养分离菌进行16S核糖体RNA基因(16SrRNA基因或16SrDNA)测序，与基因库中菌种比较，从基因水平上确定细菌种类。分别将人、猪血液分离菌基因组经20U SamI酶消化后经脉冲场凝胶电泳(PFGE)，确定两者的相关性。结果 16SrRNA基因测序分析证明人、猪分离菌100％相同，与基因库中原型屎肠球菌仅相差一个核苷酸(999％相同)，两株细菌全基因组经Saml酶消化后PFGE谱完全一致。结论 此起暴发流行是由猪传播的屎肠球菌败血症所致。     

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium,June 2021

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.     

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。     

Bacteriophage Rescue Therapy of a Vancomycin-Resistant Enterococcus faecium Infection in a One-Year-Old Child following a Third Liver Transplantation

Phage therapy is an experimental therapeutic approach used to target multidrug-resistant bacterial infections. A lack of reliable data with regard to its efficacy and regulatory hurdles hinders a broad application. Here we report, for the first time, a case of vancomycin-resistant Enterococcus faecium abdominal infection in a one-year-old, critically ill, and three times liver transplanted girl, which was successfully treated with intravenous injections (twice per day for 20 days) of a magistral preparation containing two Enterococcus phages. This correlated with a reduction in baseline C-reactive protein (CRP), successful weaning from mechanical ventilation and without associated clinical adverse events. Prior to clinical use, phage genome was sequenced to confirm the absence of genetic determinants conferring lysogeny, virulence or antibiotic resistance, and thus their safety. Using a phage neutralization assay, no neutralizing anti-phage antibodies in the patient’s serum could be detected. Vancomycin-susceptible E. faecium isolates were identified in close relation to phage therapy and, by using whole-genome sequencing, it was demonstrated that vancomycin-susceptible E. faecium emerged from vancomycin-resistant progenitors. Covering a one year follow up, we provide further evidence for the feasibility of bacteriophage therapy that can serve as a basis for urgently needed controlled clinical trials.