利用鸡胚浸出液制作细菌培养基的研究

利用鸡胚浸出液制作的肉汤和琼脂平板培养基均能适合副鸡嗜血杆菌、大肠杆菌和沙门氏菌的生长,用平板计数法、麦氏比浊法和离心称重法分别对副鸡嗜血杆菌、大肠杆菌和沙门氏菌的培养产物进行含菌量、菌泥湿重测定,结果表明,鸡胚浸出济培养基培养的沙门氏菌产量是普通培养基培养的沙门氏菌产量的20倍以上,培养的副鸡嗜血杆菌产量是普通培养的3倍以上,而鸡胚浸出液培养基与普通培养基对大肠杆菌的培养数量则无明显差异。     

大肠杆菌和肺炎克雷伯氏菌的共培养研究

为研究大肠杆菌(Escherichia coli)和肺炎克雷伯氏菌(Klebsiella pneumoniae)混菌体系,构建了基于抗生素筛选的2株重组菌。将携带卡那霉素抗性基因的载体pET-28a转化到肺炎克雷伯氏菌中,获得重组菌K.pneumoniae(pET-28a);将携带氯霉素抗性基因cm的重组载体pET-cm(卡那霉素和氯霉素双抗性载体)转化到大肠杆菌中,获得重组菌E.coli(pET-cm),在卡那霉素抗性培养基中单独培养及混合培养上述两株重组菌。结果发现:单独培养条件下,E.coli与K.pneumoniae的相对菌体密度为57.87%;混合培养条件下,E.coli与K.pneumoniae的相对菌体密度为1.94%;E.coli和K.pneumoniae相对于各自单独培养的存活率分别为2.57%和76.60%。上述结果表明,K.pneumoniae为混菌体系的优势菌,可强烈抑制E.coli的生长。     

构建伴有β-葡萄糖苷酶表达的乙醇发酵大肠杆菌

研究构建能够分泌表达纤维素酶的产乙醇菌株,实现降解木质纤维素生产乙醇的整合生物加工过程。文中通过克隆来自运动发酵单胞菌Zymomonas mobilis ZM4的丙酮酸脱羧酶基因pdc和乙醇脱氢酶基因adhB,并通过Red重组将二者整合到大肠杆菌Escherichia coli JM109基因组中,首先构建了一株可以利用葡萄糖进行乙醇发酵的重组菌E. coli P81。随后将来源于多粘芽胞杆菌Bacillus polymyxa1.794的β-葡萄糖苷酶基因bglB在E. coli P81中进行了分泌表达,得到了一株可以进行纤维二糖降解和乙醇发酵双重功能的重组菌E. coli P81(pUC19-bglB)。该菌胞外分泌β-糖苷酶活达到84.78 mU/mL菌液,纤维二糖酶活达到了32.32 mU/mL菌液。该重组菌E. coli P81(pUC19-bglB) 以纤维二糖为碳源进行乙醇发酵,乙醇得率达到了理论产率55.8％,而在葡萄糖和纤维二糖的共发酵中,其乙醇产量达到了理论产率46.5％。构建得到的此株整合生物加工大肠杆菌能够利用β-葡萄糖苷酶生产乙醇,为构建能利用木质纤维素分解产物生产燃料乙醇的高效、稳定生产用工程菌奠定了良好的基础。     

重组大肠杆菌表达铜绿假单胞菌溶血性磷脂酶C

[目的]构建产溶血性磷脂酶C (Hemolytic Phospholipase C,PLCH)的重组大肠杆菌(Escherich coli菌株,并初步优化其发酵条件.[方法]首先利用卵黄硼砂平板分离法筛选到一株产磷脂酶C(Phospholipase C,PLC)活性较高的菌株,命名为铜绿假单胞菌(Pseudomonas aeruginosa)41;进一步以P.aeruginosa 41基因组DNA为模板设计引物,PCR扩增获得溶血性磷脂酶C(PLCH)基因,构建重组大肠杆菌表达质粒并转化大肠杆菌E.coli BL21 (DE3);筛选转化子并检测PLC活性和溶血能力,并初步优化其发酵条件.[结果]成功构建了重组大肠杆菌E.coli BL21(DE3) /pET28a-plcH;在硼砂卵黄平板上对重组菌进行PLC活性测定,显示重组菌有明显的磷脂酶C活性;在哥伦比亚血琼脂平板上对重组菌进行溶血性试验,表明PLCH具有较强的溶血活性;初步优化摇瓶发酵条件为:5％转接量,37℃、200 r/min下培养4h添加IPTG至终浓度为0.9 mmol/L,转为25℃、150 r/min诱导培养14 h;优化后重组菌的酶活可达到722.89±0.47 U/mL.[结论]本文成功构建了一株产溶血性磷脂酶C活性较高的重组大肠杆菌菌株,并通过优化发酵条件使其酶活达到了722.89±0.47 U/mL,本实验在国内首次实现了铜绿假单胞菌来源的溶血性磷脂酶C基因在大肠杆菌的胞内表达,该研究为研究磷脂酶C产业化奠定了一定的基础.     

蛇毒抗菌肽OH-CATH对大肠杆菌引起家兔泌尿系感染的保护作用

为探讨蛇毒抗菌肽OH-CATH对大肠杆菌标准株和临床耐药株引起家兔泌尿系感染的保护作用,该文利用大肠杆菌标准株(E.coli ATCC 25922)和耐头孢菌素大肠杆菌临床耐药株建立雄性家兔泌尿系感染模型.通过膀胱直接给药的方式,分别给予动物感染模型生理盐水、蛇毒抗菌肽OH-CATH及头孢哌酮舒巴坦,并于给药后第1、5、l0及14天留取家兔中段尿用于尿液培养.将动物膀胱标本行H-E染色石蜡切片及透射电镜观察.结果显示:使用蛇毒抗菌肽OH-CATH的大肠杆菌标准株和临床耐药株不同组别中段尿培养阳性率明显降低;给予头孢哌酮舒巴坦可降低大肠杆菌标准株(E.coli ATCC 25922)引起感染的阳性率,但对耐头孢菌素大肠杆菌引起的感染的阳性率则无明显作用(P＜0.05);使用蛇毒抗菌肽OH-CATH组炎症细胞浸润、坏死及钙化组织较其他组为少.该结果提示蛇毒抗菌肽OH-CATH对大肠杆菌标准株(E.coli ATCC 25922)及耐头孢菌素大肠埃希菌临床耐药株有稳定活性,对其引起的家兔泌尿系感染有较好的保护作用,并为临床日益严重的耐药病原菌感染的治疗提供了新的思路和方向.     

肠外致病性大肠杆菌致病机制及公共卫生学意义

致病性大肠杆菌包括肠致病性大肠杆菌(intestinal pathogenic Escherichia coli, IPEC)和肠外致病性大肠杆菌(extraintestinalpathogenicE.coli,ExPEC),可引起人和动物多种感染性疾病。ExPEC主要在肠道外其他组织脏器定殖并导致感染,包括尿道致病性大肠杆菌(uropathogenicE.coli, UPEC)、新生儿脑膜炎大肠杆菌(newborn meningitis E. coli, NMEC)和禽致病性大肠杆菌(avian pathogenic E. coli, APEC)。人源ExPEC (UPEC和NMEC)主要引起人尿道感染、肾盂肾炎和新生儿脑膜炎,而APEC可导致禽类的大肠杆菌病,造成家禽业的巨大经济损失。另外,乳腺致病性大肠杆菌(mammary pathogenic E. coli, MPEC)和猪源ExPEC可导致奶牛乳房炎、猪的肺炎及急性败血症等病症。研究发现,ExPEC类菌株在基因组结构上很相似,与IPEC本质区别在于致病机制不同,ExPEC具有很多相同的毒力基因和耐药基因,而且动物源ExPEC...     

应用肠杆菌科四个属的诊断噬菌体快速诊断沙门氏菌

本文报告应用弗氏柠檬酸细萄噬菌体3组,大肠埃希氏菌噬菌体4组,阴沟肠杆菌噬菌体1组和沙门氏菌O-I噬菌体快速诊断沙门氏菌的结果。沙门氏菌0-I噬菌体可裂解沙门氏菌属地方株1393株中的1351株(97％)。柠檬酸细菌属噬菌体和共可裂解柠橡酸细菌属地方株381株中的362株(95％)。阴沟肠杆菌噬菌体Ent可裂解阴淘肠杆菌地方株l 50株中的133株(84．2％)。埃希氏菌属噬菌体E—1、E一2、E-3和E-4共可裂解埃希氏菌属地方株683株中的567株(83％)。由于E一1和E一2噬菌体的联合使用,可使o I噬菌体对埃希氏菌属地方株的误诊率从6．3％下降到0．6％。E一4噬菌体对沙门氏菌属地方株的误诊率可因与。一I噬菌体的联合使用而从0．36％下降到0．07％。     

粪便中大肠埃希菌的分离鉴定

2004年4月采集大连金州湾沿岸畜禽养殖场猪、牛、鸡和某中学人粪便样品。经改良的生化鉴定方法鉴定,得大肠埃希菌(Escherichia coli)猪源75株、牛源78株、鸡源69株、人源68株,占粪大肠菌比率分别为85.23%、92.86%、80.23%、80.00%。为验证改良的生化鉴定方法准确性,从中随机选取8株E.coli,扩增16S rRNA序列并与Genebank中E.coli16S序列比对,确定这8株细菌与E.coli同源相似率达99%以上,进而验证改良的生化鉴定方法的可行性。     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。     

四株儿茶酚类铁载体高产菌株产消化酶活性及其益生特性

【背景】儿茶酚类铁载体对胃肠道菌群的生长代谢具有重要作用,研究儿茶酚类铁载体高产菌株的消化酶活性,挖掘其潜在益生特性具有重要意义。【目的】分析4株分离自健康成人粪样的儿茶酚类铁载体高产菌的产酶特性,通过分析菌株耐酸耐胆盐能力、粘附定殖能力、抗生素耐受性和急性毒性研究其益生特性。【方法】测定4株菌的蛋白酶、淀粉酶、脂肪酶、纤维素酶、植酸酶、乳糖酶和β-葡萄糖苷酶的酶活性。4株菌经人工模拟胃、肠液连续培养后分别计算其活菌数;分析4株菌的自凝集率、黏蛋白粘附率和表面疏水率;对小鼠连续7 d灌胃不同剂量的4株高产菌,观察并记录小鼠的一般体征,计算小鼠脏器指数,进行阳性细菌移位试验。【结果】在试验所测的7种消化酶中,E. coli Gut 07、E. coli Gut 12无蛋白酶和脂肪酶活性,B. cereus Gut 16无乳糖酶活性,E. coli Gut 20无蛋白酶活性,其余均具有。4株菌经人工模拟胃液培养6h后存活率均大于60%,转移到人工模拟肠液培养24 h后活菌数均大于初始菌落数;该4株菌具备在胃肠道中粘附定殖的能力,对大多数抗生素敏感,在浓度低于4.5×10~(11)CFU/m L、灌胃剂量为20m L/kg-bw时对小鼠无急性毒性,无阳性菌株移位现象。【结论】4株儿茶酚类铁载体高产菌株可作为潜在益生菌进行进一步的安全性和功能性研究。     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7.

Twenty-four Escherichia coli strains producing standard colicins were evaluated for inhibitory activity against 27 diarrheagenic E. coli strains of serotypes O15:H-, O26:(H11, H-), and O111:(H8, H11, H-), including O157:H7, representing diarrheagenic E. coli clones, 3, 4, 8, 9, and 10. Overlay techniques were used to assess inhibition on Luria agar and Luria agar supplemented with 0.25 micrograms of mitomycin C per ml to induce colicin production. As a group, the A colicins (Col) E1 to E8, K, and N inhibited 23 to 25 (85.2 to 92.6%) of the 27 diarrheagenic strains on mitomycin C-containing agar, whereas the most active group B colicins, Col D and Ia, inhibited 9 and 12 (33.3 and 44.4%), of the diarrheagenic strains, respectively. Col G and H and Mcc B17 inhibited 22 to 27 (81.5 to 100%) of the diarrheagenic strains on Luria agar but were suppressed on mitomycin C-containing agar medium. All O157:H7 strains evaluated were sensitive to Col E1 to E8, K, and N on mitomycin C-containing agar and to Col G and H and Mcc B17 on Luria agar. Sensitivity to colicins of the selected set of diarrheagenic strains was in the order diarrheagenic E. coli clone 9 > 4 > 3 > 10 > 8 and was not restricted to strains of a single clone or serotype. Strain 8C from clone 8 was resistant to most test colicins. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrheagenic E. coli strains, including serotype O157:H7.     

Analytical limits of four beta-glucuronidase and beta-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert (Colilert), Readycult Coliforms 100 (Readycult), Chromocult Coliform agar ES (Chromocult), and MI agar (MI) are beta-galactosidase and beta-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect beta-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect beta-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect beta-glucuronidase production and MI the weakest to detect beta-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

磷酸盐对V族大肠杆菌素形成的影响

含ColV质粒的大肠杆菌在含有磷酸盐的牛肉膏蛋白胨培养基上形成的菌落,当覆盖敏感菌后可以形成较大的抑菌圈。说明磷酸盐对ColV质粒所编码的V族大肠杆菌素的形成有促进作用。在培养基中加入二价离子螯合剂——EDTA对大肠杆菌素形成同样有促进作用,而增加二价阳离子Ca++或Mg++却起到相反的作用。磷酸盐的这种促进作用是由于它降低了牛肉膏蛋白胨培养基中二价阳离子的浓度而引起的。因此,在培养基中添加磷酸盐有助于分离ColV质粒含有菌和对V族大肠杆菌素的研究。     

A Rapid and Direct Plate Method for Enumerating Escherichia coli biotype I in Food

Direct enumeration of Escherichia coli biotype 1 in foods within 24 h has been achieved by a development of the method of Delaney, McCarthy & Grasso (1962) which is based on the production of indole at 44°. Indole positive E. coli growing at this temperature on a cellulose acetate membrane overlaying tryptone bile agar can be demonstrated. Characterization of 555 indole positive colonies from 843 samples of food showed that 95% were E. coli biotype 1 and a further 3·4% were 'faecal coliforms'. Anaerogenic and non-lactose fermenting E. coli are detected and the significance of these strains is discussed.     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

Occurrence of p-fimbriae in enteropathogenic E coli (EPEC) and in E. coli strains isolated from patients with urinary tract infections]

The aim of this study was to gain knowledge of prevalence of P+ clones among EPEC strains isolated from children with diarrhoea and E. coli strains isolated from urine. Three hundred eighty four E. coli strains isolated from children with diarrhoea were tested. They belonged to 11 serotypes (018, 025, 026, 044, 055, 0111, 0114, 0119, 0124, 0125, and 0128). Nine hundred thirty colonies of E. coli from Mac Conkey's agar plated quantitatively with urine samples of 178 individuals suffering from urinary tract infections were also tested. All strains were assayed by mannose-resistant active haemagglutination test (MRHA) and by slide agglutination using self prepared latex reagent for detection of P fimbriae. Out of 384 E. coli strains tested 122 (31.8%) showed presence of adhesins detected by mannose-resistant active haemagglutination test (MRHA) and in 90 (23.3%) out of all tested strains the presence of P fimbriae was found. The highest percentage of P fimbriae prevalence was found in E. coli belonging to the following serotypes: 018 (in 68.9% strains), 025 (in 29.2% strains), and 0125 (in 25.0% strains). This type of fimbriae was also detected in serotypes 026 (9.1%), 044 (8.7%), 055 (5.6%), and 0119 (in 2 strains out of 5 isolated). Out of 933 colonies of E. coli, isolated from 178 urine samples, 434 (46.5%) colonies gave positive results in MRHA test, including 133 positive in latex test for P fimbriae. These studies showed that for MRHA adhesins, including P fimbriae, a parallel examination of higher number of E. coli was necessary.(ABSTRACT TRUNCATED AT 250 WORDS)     

IMPROVEMENT OF SELECTIVITY FOR ISOLATION OF ESCHERICHIA COLI O157:H7 FROM GENERIC ESCHERICHIA COLI

A modified selective medium was developed to increase selectivity for isolation of Escherichia coli O157 from generic E. coli based on the knowledge that E. coli O157:H7 has more resistance against HCl condition than E. coli. As a preliminary experiment, four strains of E. coli O157:H7 (ATCC 35150, 43889, 43890, and 43894) were tested to determine the maximum concentration of 6N HCl (from 0 to 250 μL) added to 50 mL of MacConkey agar medium (MAC). The maximum level was 125 μL/50 mL (6N HCl/MAC), which E. coli O157:H7 strains could tolerate against the HCl concentration. After determination, comparative growth of 15 isolates of E. coli O157 and generic E. coli were evaluated on modified selective medium (HCl-MAC; with the addition of 125 μL/50 mL) and conventional MAC, respectively. All tested strains of E. coli O157 were grown on both media, whereas 9 out of 15 generic E. coli (60% of tested strains) were strongly inhibited on HCl-MAC. For selective isolation of E. coli O157 from generic E. coli, HCl-MAC has an effective potential for an implemental use. This information can extend as a baseline for use of HCl to conventional medium for successful isolation E. coli O157 from generic E. coli.     

Congo red binding test in enteroinvasive and nonpathogenic Escherichia coli strains.

Out of 6 variants the appropriate media to perform Congo red binding test for enteroinvasive E. coli strains were established (trypto-soy agar Eiken, T.S.A.--Cantacuzino Institute and B.T.S.D.). 12 E. coli strains belonging to enteroinvasive O-serogroups formed on Congo red agar red-coloured, non-coloured colonies or both; cultures from 59 red colonies and 61 white colonies were inoculated in guinea pig eyes. The correlation between positive Congo red binding test and positive Sereny test was 91% (out of 59 red colonies, 47 evoked keratoconjunctivitis in both infected eyes and 7 in only one eye). The negative Congo red binding test corresponds (98.4%) to the failure to induce illness in the guinea pigs' eye (only one out of 61 Crb = colonies was Sereny positive, evoking keratoconjunctivitis in only one of the two infected eyes of a guinea pig). Comparing in vivo lack of pathogenicity in 44 E. coli strains isolated from human normal intestinal flora and negative Congo red binding test, a correlation of 72.73% on B.T.S.D. and 65.91% on T.S.A. medium was found. Developing an appropriate method based on Crb test about 70% of the nonpathogenic E. coli colonies could be eliminated from the laborious agglutination with enteroinvasive O-serogroups E. coli antisera.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.