一株羊源致病性屎肠球菌的分离鉴定及耐药性分析

试验从甘肃肃南县某羊场死亡羊中分离出疑似某球菌菌株XCP,为进一步确定该致病菌株及其耐药性情况,进行了病原菌分离、革兰氏染色、生化试验、16S rRNA PCR扩增鉴定、小鼠致病力试验、药敏试验、毒力基因和耐药基因扩增试验研究。结果发现,该菌革兰氏染色阳性,高盐、蜜二糖、蔗糖等生化反应阳性,VP、马尿酸盐、阿拉伯糖等生化反应阴性;16S rRNA基因PCR扩增后,进行BLAST比对,结果与GenBank中的屎肠球菌16S rRNA基因序列同源性为100%;小鼠致病力试验发现,该菌株具有很强的致病性;该菌株对β-内酰胺类抗生素苯唑西林、青霉素G,喹诺酮类抗生素诺氟沙星、环丙沙星、左氟沙星,以及四环素等高度耐药,对红霉素、万古霉素、克林霉素等抗生素敏感;毒力基因Asal、cylA、acm和耐药基因TetM、ant（6）-Ⅰ、aac（6'）-aph（2"）、ermB扩增均为阳性。结果表明,该菌为屎肠球菌（Enterococcus faecium）,具有较强的致病性和耐药性,可能与毒力基因和耐药基因的高阳性率有关。     

猪肠球菌溶血素cylA基因原核表达载体的构建

以猪致病性粪肠球菌染色体DNA为模板,PCR扩增溶血素cylA基因,相应酶切后,克隆到原核表达载体pET32a中,构建pET32a-cylA重组质粒,并将pET32a-cylA质粒转化入大肠杆菌BL21(DE3)。经HindⅢ、XhoI双酶切及测序鉴定,并与已发表的肠球菌溶血素cylA基因序列比较,同源性达99.8%。表明成功构建猪肠球菌溶血素cylA基因的重组表达质粒,为制备单抗、开发疫苗及其致病机制研究奠定了基础。     

应用16SrRNA基因序列分析鉴定猪源肠球菌

对河南省不同地区病猪分离的11株疑似肠球菌提取基因组DNA,PCR扩增16SrRNA基因片段,经克隆后测序,并与GenBank中注册的相关肠球菌菌株的16SrRNA基因序列进行比较、绘制系统发育树,进行鉴定。结果表明,4株临床分离株鉴定为粪肠球菌（E.faecalis）,7株为屎肠球菌（E.faecium）。首次从分子水平上证实了猪肠球菌在河南省的存在,为临床诊断及分子鉴定提供依据。     

水稻根分泌物及其与粪产碱菌的相互作用

水稻植株通过根系向根际分泌多种有机酸、糖、氨基酸和植物激素。平均每克鲜根分泌有机酸2.25mg,柠檬酸含量较高,其次是苹果酸、琥珀酸、乳酸;氨基酸2.61mg,大约有15种,碱性氨基酸含量最高;糖12.6mg,以蔗糖、葡萄糖、果糖为主;植物激素仅测到赤霉酸（GA#-3）。不同水稻品种的根分泌物在组成及含量上都有差异。粪产碱菌在纯培养条件下能分泌9种氨基酸,一种有机酸即苹果酸以及植物激素如生长素（IAA）、赤霉酸（GA#-3）,但不分泌糖。接种粪产碱菌能刺激水稻根的分泌作用,并对根分泌物的组成和含量有所影响。2.63%的水稻光合产物以根分泌物形式释放到根区,接种粪产碱菌后分泌量增至3.52%,并且大部分为粪产碱菌吸收利用。水稻的光合作用及根分泌能力影响根际固氮活性。具有较强分泌能力的植株,其根际固氮活性也较高。     

The Influence of Shock Treatment on Survival Rate of Intestinal Probiotics during Freezing

Probiotics which can provide potential health benefits for consumers and prevent disease, is the most important research field for functional food in the future. It is the key point that how much the count of bacteria and how long it can preserve. Freeze-drying skill is one of the best preservation methods, but it's defect that damage to biological systems can be attributed to changes in the physical state of membrane lipids or/and changes in the structure of sensitive proteins and the decreased survival rate. This work used pretreatment methods-cold shock and hot shock to reduce the damage to bacteria during freeze-drying. Using unpretreated samples as control, aspects of cold shock or heat shock. The response of Enterococcous faecalis A31 was investigated during lag phase, the middle of exponential growth phase and the terminal of exponential growth phase. The results suggested that when cold shock, the shock protein concentration produced by treated sample within 2 h during the middle of exponential growth phase was higher than 4, 8, 24 h, The concentration of shock protein produced by treated sample at 10 ℃ was higher than 20℃, The concentration of sample protein at the middle of exponential growth phase was higher than that at the terminal of exponential growth phase. After one-month storage, the survival rate at 10℃ was better than 20 ℃ and 4 ℃ comparatively. The survival rate at (6 h) 4 ℃/24 h (lag period) was higher than (10 h) 4 ℃/24 h (the middle of exponential growth phase), and the effect of 10 ℃ /8 h shock treatments was best during the middle of exponential growth phase, when hot shock, The concentration of heat shock proteins produced was not obvious, and survival rate was higher at 45℃, 30 min than at 45 ℃, 60 min.     

粪肠球菌对吉富罗非鱼的生长、体组成、消化酶活性及血液生理生化指标的影响

为研究饲料中添加不同浓度的粪肠球菌对吉富罗非鱼生长、体组成、消化能力及血液生理生化指标的影响,实验选用初始体质量为(50.59±0.59)g的吉富罗非鱼300尾,随机分成5组,每组设3个重复,每个重复20尾鱼,养殖实验期60 d。分别投喂实测含1.3×10~2(对照组)、1.4×10~5、1.7×10~6、1.5×10~7和1.8×10~8 CFU/g粪肠球菌的5种等氮(36%)等脂(6.75%)的实验饲料。结果显示:(1)罗非鱼的末均重(FBW)、增重率(WGR)、特定生长率(SGR)和尾均摄食量(FI)均在1.5×10~7 CFU/g组达到最大且显著高于对照组,而饲料系数(FCR)显著低于对照组。以FBW、WGR和SGR为评价指标,通过二次回归分析得出,罗非鱼饲料的粪肠球菌适宜添加浓度范围为7.5×10~7~1.1×10~8 CFU/g。(2)各添加组的全鱼粗蛋白含量均显著高于对照组,1.5×10~7 CFU/g组的粗脂肪含量显著高于对照组,各组间全鱼水分和粗灰分含量无显著差异。1.5×10~7 CFU/g组的干物质、蛋白质和脂肪沉积率均达到最大值且显著高于对照组。(3)1.5×10~7和1.8×10~8 CFU/g组的肠脂肪酶活性均显著高于对照组,而1.8×10~8 CFU/g组的肠道蛋白酶活性显著低于对照组;各组间肠道淀粉酶活性无显著差异。(4)1.5×10~7 CFU/g组的平均红细胞体积、血红蛋白浓度和血小板数均显著低于对照组,各组间红细胞数无显著差异。(5)1.8×10~8 CFU/g组的血清中胆固醇、葡萄糖和丙二醛含量显著低于对照组;1.5×10~7 CFU/g组血清中碱性磷酸酶活性显著高于对照组,而谷丙转氨酶和谷草转氨酶活性显著低于对照组。综上所述,吉富罗非鱼(50~210 g)饲料中粪肠球菌的适宜添加浓度范围为7.5×10~7~1.1×10~8 CFU/g,但添加粪肠球菌对血液载氧能力有负面影响。     

Dietary supplement of Enterococcus faecalis on digestive enzyme activities,short‐chain fatty acid production,immune system response and disease resistance of Javanese carp (Puntius gonionotus,Bleeker 1850)

This study investigated the effects of Enterococcus faecalis on digestive enzyme activities and short‐chain fatty acid production in fish intestine, resistance against Aeromonas hydrophila and humoral immunity response by 3 experiments on Javanes carp (Puntius gonionotus). The experiment 1 revealed that diet supplemented with E. faecalis significantly (P < 0.05) increased protease and lipase activities compared to control fed fish. Moreover, E. faecalis supplementation significantly enhanced the production of propionic and butyric acid in the intestine, while no significant difference (P > 0.05) in acetic acid production was observed. In the challenge study (experiment 2), fish were injected (intraperitoneal) with 10⁷ A. hydrophila per ml and survival was significantly improved when fish were fed diet supplemented with E. faecalis compared to control fish. In experiment 3, dietary E. faecalis affected immune system response as fish fed the probiont and exposed to 10⁶ A. hydrophila per ml displayed significantly elevated antibody levels compared to control fed fish. Fish fed diet supplemented with E. faecalis but not exposed to the pathogen revealed significantly higher antibody level than control fish (P < 0.05). Therefore, E. faecalis can be used as a probiotic in Javanese carp farming.     

2株猪源益生性肠球菌对酸和胆盐及热的耐受性研究

为了筛选动物肠道中的有益优势菌,研究了2株分离自猪肠道的益生性屎肠球菌(E1,E7)的耐受特性。本试验设pH 2,2.5,3,4的4个处理组,以pH 7为对照组;设胆盐浓度分别为0.2%,0.4%,0.6%,0.8%,1%的5个处理组,以不含胆盐的培养液为对照组;温度设50,80,90℃,以37℃为对照,分别作用5,10,15,20 min,经该3种方法处理后各取100μL培养液用细菌稀释计数法测定活菌数。结果表明:E1,E7都有较强的耐酸能力,在pH 2时,作用2 h后仍能检测到活菌。胆盐浓度达到0.4%时,E1,E7仍有一定的耐受能力,随胆盐浓度升高至0.6%时,活菌数由108.2CFU/mL降到102.5CFU/mL。2菌株在不同温度下经不同作用时间处理后呈不同的生长态势。     

Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune response

The aim of this study was to analyze the effect of a probiotic bacterium, Enterococcus faecium ZJ4 on growth performances and immune responses of tilapia (Oreochromis niloticus). The tilapias were treated with E. faecium ZJ4 at a final concentration of 1 × 10⁷ cfu ml^− 1 in aquaria water every 4 days. Six aquaria with three replicates for treated and controls were used. After 40 days, the tilapias supplemented with the probiotic showed significantly better final weight and daily weight gain (DWG) than those fed the basal diet (Control) (P < 0.05). There was no remarkable difference (P > 0.05) in the total serum protein, albumin content, globulin concentration and A/G ratio between the treated and control tilapias. The result of lysozyme activity assay was similar to these biochemical indexes. However, the complement component 3 content, myeloperoxidase (MPO) activity and the respiratory burst activity of blood phagocytes were higher (P < 0.05) in E. faecium treated tilapias (trial 1) than the controls.     

养殖废水中异养硝化细菌的分离筛选和鉴定

为了获得脱氮功能强的异养硝化菌株用于养殖废水的脱氮处理,通过富集、分离和纯化等步骤,并结合格利斯试剂检验菌株硝化能力的方法,从某养猪场污水处理池污泥中分离筛选了4株具异养硝化功能的菌株,分别标号为79、84、L116、L117,通过16SrDNA序列分析和美国全自动微生物分析仪Biolog鉴定,4株菌均为粪产碱杆菌（Alcaligenesfaecalis）,并验证了这4株菌的硝化能力。结果表明,当液体培养基初始氨氮浓度为90mg.L-1左右时,在振荡培养48h内,菌株79、84、L116、L117培养基中氨氮和总氮均快速下降,氨氮去除率分别达到44.4%、47.9%、61.3%和56.4%,总氮（除菌）去除率达到39.9%、38.5%、43.4%和40.7%。