Extraction,Purification and Determination of Capsular Polysaccharide in Enterococcus faecium

In order to extract and purify the capsular polysaccharide of Enterococcus faecium and measure contents of polysaccharide, high pressure crushing methods and enzyme decomposition methods (RNase A (25 μg/mL), DNase (0.5 mg/mL), lysozyme (0.25 mg/mL) and protease K (1.25 mg/mL)) were used to remove the nucleic acids and proteins to obtain crude capsular polysaccharide, and centrifugal ultrafiltration with 30 and 100 ku ultrafiltration centrifuge tubes and DEAE Sepharose Fast Flow column chromatography were also adopted to obtain the purified capsular polysaccharides of Enterococcus faecium. Then the content of polysaccharide was determined by the phenol-sulfuric acid method. The results showed the capsular polysaccharide content was 75.21%. The results revealed that capsular polysaccharide ofEnterococcus faecium was extracted successfully. The method of extracting capsular polysaccharide in Enterococcus faecium was established initially.     

东北黑熊源粪肠球菌EfaA基因的克隆表达及生物信息学分析

为检测熊源粪肠球菌心内膜炎抗原（EfaA）基因,用于快速检测东北黑熊粪肠球菌感染病例,本试验根据GenBank登录的粪肠球菌EfaA基因序列（登录号：U03756.1）合成了1对PCR引物,通过PCR法扩增合成长度为689 bp的目的片段。将PCR产物克隆于pMD19-T载体,之后将其转入大肠杆菌DH5α感受态细胞筛选阳性克隆。提取扩增后的重组质粒pMD19-T-EfaA并进行PCR、酶切鉴定、序列测定及蛋白质结构预测。结果显示,本试验成功克隆出熊源粪肠球菌EfaA基因,与GenBank登录的ATCC 29212菌株同源性为100.0%。本试验成功构建了重组克隆载体pMD19-T-EfaA,并对测序结果进行了蛋白质结构的预测,为进一步建立黑熊粪肠球菌病的诊断方法提供了理论依据,同时也为黑熊疾病的防制奠定了基础。     

猪、鸡源分离粪肠球菌的核糖体分型及耐药性分析

对上海地区1个规模化养猪场和1个规模化养鸡场分离鉴定的40株粪肠球菌,采用微量肉汤稀释法对12种抗菌药物进行药敏试验,采用Riboprinter^®微生物基因指纹鉴定系统检测分离菌株的基因指纹图谱,分别检测分离菌株中的核糖体型和耐药性。结果表明,在40株粪肠球菌中,80%以上为多重耐药菌株,有4株猪源粪肠球菌对万古霉素耐药,其中有2株表现为对万古霉素高度耐药（MIC>64 mg/L）,且对链霉素和庆大霉素同时表现为高度耐药（MIC>2 048 mg/L）。从耐药表型和核糖体分型共同分析粪肠球菌的耐药性,发现多重耐药性严重,同一核糖体型菌株的耐药表型并非完全一致。     

pH对粪肠球菌高产双乙酰的影响

将分离自内蒙古农家酸马奶(Koumiss)的粪肠球菌KLDS 4.1004经NTG诱变获得诱变株KLDS 4.1003,后者的LDH、DR酶活力下降,且ALD酶失活。对两者进行8个pH点条件下的酶活、生长情况与代谢物测定,确定KLDS 4.1003在pH 6.5、7.0时双乙酰产量最高。在6个时间点分别检测两个pH点的菌数与双乙酰产量变化,发现发酵16 h两项指标均最高,最终确定最高产双乙酰条件为pH 7.0、16 h,最大产量为1.29 g.L-1。结果表明,诱变株KLDS 4.1003产双乙酰性能优良,可进一步开发成产香型益生发酵剂菌种。     

粪肠球菌对产蛋后期蛋鸡生产性能、蛋品质及肠道组织形态的影响

试验旨在研究添加不同剂量的粪肠球菌对产蛋后期蛋鸡生产性能、蛋品质及肠道形态结构的影响。选取70周龄产蛋率相近的海兰褐蛋鸡270只,随机分为3个处理,每个处理6个重复,每个重复15只鸡。试验设对照组（饲喂基础日粮）、试验Ⅰ组（基础日粮+3.75 mg/kg粪肠球菌）和试验Ⅱ组（基础日粮+7.5 mg/kg粪肠球菌）,预试期7 d,正试期50 d。结果表明,与对照组相比,试验Ⅰ、Ⅱ组产蛋后期蛋鸡产蛋率、产蛋量极显著提高（P<0.01）,料蛋比极显著降低（P<0.01）;与对照组相比,试验Ⅰ、Ⅱ组蛋鸡的蛋壳厚度、蛋壳强度、蛋形指数、蛋白高度、哈夫单位、蛋黄相对重、蛋壳相对重均无显著变化（P>0.05）;与对照组相比,试验Ⅰ、Ⅱ组产蛋后期蛋鸡十二指肠、回肠的黏膜厚度极显著增加（P<0.01）,回肠绒毛高度显著提高（P<0.05）。综上所述,在产蛋后期蛋鸡日粮中添加适宜剂量的粪肠球菌可提高蛋鸡生产性能,改善肠道形态结构。     

毛皮动物源粪肠球菌的分离鉴定

为获得毛皮动物源粪肠球菌,并评价其益生特性,本研究从健康成年毛皮动物（水貂、狐狸、貉）粪便中分离粪肠球菌,通过形态学观察、生化试验和16S rRNA序列分析等方法进行种属鉴定。测定分离菌株生长曲线、产酸能力、抗菌药敏感性,并挑取部分菌株测定其对于温度、人工胃液、人工胆盐的耐受能力。结果表明,分离菌株中共5株为革兰氏阳性菌,生化特性与粪肠球菌标准株基本相符,且经16S rRNA序列分析鉴定为粪肠球菌。5株菌均于培养后2 h进入对数期,8~10 h进入稳定期,且具有弱产酸能力。分离菌株对四环素、左氟沙星耐药性强,耐药率为100%,其次为青霉素（80%）、红霉素（80%）、庆大霉素（80%）、氯霉素（40%）,对氨苄西林和万古霉素敏感。水貂、狐狸和貉源的粪肠球菌对于60 ℃以下温度处理、pH>3.0的人工胃液、0.3%~0.5%浓度的胆盐耐受能力较强,对70 ℃以上高温和pH<3.0的人工胃液耐受性差。综上所述,本研究共获得5株毛皮动物源（水貂、狐狸、貉）粪肠球菌,分离菌株繁殖较快,适合在毛皮动物肠道中定植并发挥益生作用,且具有较好的益生特性和抗逆性,可作为动物用微生态制剂的候选菌种进一步研究。     

甘肃省乳房炎奶牛乳源肠球菌的分离、药敏试验及毒力基因原核表达载体的构建

为分离临床型奶牛乳房炎中的肠球菌,检测其耐药性和携带毒力基因的情况,本试验采集了甘肃省东、中和西部3个地区41头临床型乳房炎奶牛的奶样93份,并构建了肠球菌毒力基因的原核表达载体。试验使用选择性培养基分离纯化肠球菌,16S rRNA和生化试验结合的方法鉴定所分离菌株的种;选取16种抗菌药进行药敏试验;常规PCR方法检测11种毒力基因的携带情况,最后对具有免疫原性的毒力基因进行原核表达载体的构建。鉴定结果显示,93份乳样中18株为肠球菌,并分为9种。药敏结果显示,分离株多数为多重耐药菌（multiple resistant bacteria,MDR）,占88.89%,未发现耐万古霉素菌株（vancomycin-resistant enterococcus,VRE）,万古霉素敏感率94.12%,所有分离株至少对一种抗菌药耐药。PCR检测结果表明,11种毒力基因均有检出,cob基因检出率最高（44.44%）,efaA、hyl、ccf和esp基因检出率分别为33.33%、27.78%、27.78%和22.22%,Asa1、cylA、EF3314和gelE基因检出率均为16.67%,Ace和cylM基因检出最低（11.11%）;毒力基因组合因菌种不同而存在差异,选择具有免疫原性的Ace和gelE基因成功构建出原核表达载体pET32a-Ace和pET32a-gelE。本试验为后续绘制3个地区奶牛乳房炎流行病学区域谱以及制备相应抗体和亚单位疫苗提供了基础数据及生物材料。     

Positive effects of dietary supplementation of three probiotics on milk yield,milk composition and intestinal flora in Sannan dairy goats varied in kind of probiotics

In this study, we investigated the effects of Saccharomyces cerevisiae (SC), Bacillus subtilis (BS) and Enterococcus faecalis (EF), singly and in combination, on the dry matter intake (DMI), milk production and composition, and faecal microflora of Saanen dairy goats. Fifty goats were randomly divided into five groups: (a) basal diet (control); (b) basal diet + SC; (c) basal diet + BS; (d) basal diet + EF; and (e) basal diet + mixed probiotics. Each treated animal received 5 g/d of probiotics for a total administration of 5 × 1,011 CFU/goat per day. The inclusion of B. subtilis and E. faecalis in the diet of lactating Saanen goats increased DMI (p < .05). Enhanced milk yield was observed with BS and EF. Milk fat percentage was significantly increased by feeding mixed probiotics compared with the control (p < .05); supplying SC, BS and mixed probiotics enhanced the protein percentage (p < .05). The milk lactose percentage in the SC and BS groups was higher than in the control (p < .05). The amount of milk total solids was higher after feeding EF or mixed probiotics than in the control group (p < .05). Non-fat solids showed no notable differences among groups (p > .05). There was no significant influence on gut bacterial abundance and diversity from adding these three probiotics, singly or in combination. Bacteroidales, Escherichia–Shigella and Christensenellaceae abundances were decreased by supplying these probiotics but Succinivibrionaceae increased. In conclusion, there were positive influences of probiotic feed supplementation on intake, milk performance and intestinal microecology.     

产enterocin E5细菌素粪肠球菌发酵条件的优化

采用琼脂扩散法测定粪肠球菌发酵上清液对大肠埃希菌K88的抑菌活性,从而确定产enterocin E5细菌素粪肠球菌的发酵条件。结果表明,粪肠球菌E5产细菌素的发酵培养基为MRS培养基,最适温度为37℃,最适起始pH值6.5,最佳接种量2%,种龄14 h,发酵时间16 h,最佳培养基组分氮源为1%胰蛋白胨、0.5%酵母浸粉,最佳碳源为1%葡萄糖、0.5%蔗糖,0.1%Tween-80有利于enterocin E5的产生。这是分离自北京优良商品猪黑六产enterocin E5粪肠球菌的首次报道。     

3种不同水源粪肠球菌耐药情况

为了解水环境中粪肠球菌对常用抗生素的耐药情况,从某校鱼塘、污水沟和模型池塘中共分离出99株粪肠球菌,采用美国临床实验室标准委员会（NCCLS）推荐的微量肉汤稀释法进行药敏试验.结果显示：污水沟分离菌的耐药率最高,其中红霉素、土霉素、环丙沙星、氯霉素和氨苄西林的耐药率分别为89.3%、89.3%、82.1%、46.4%和32.1%,且多数对3-5种抗生素多重耐药;其次为鱼塘分离菌,耐药谱多为对1-2种抗生素同时耐药;模型池塘分离敏感菌较多;3处均未分离出万古霉素耐药粪肠球菌.提示不同水源粪肠球菌耐药情况与水源受抗生素污染程度存在联系,表明细菌耐药性监测及环境保护的重要性.