水环境中大肠杆菌多特征抗原及抗体的制备研究

为了建立用于水环境中大肠杆菌的酶联免疫快速检测方法,针对水中大肠杆菌属多种血清型的特征,制备了水环境样品中大肠杆菌的多特征抗原,包括全菌体抗原、破碎全菌体抗原、菌体抗原、鞭毛抗原和菌毛抗原;采用这5种大肠杆菌抗原分别免疫新西兰大白兔,获得了5种大肠杆菌多克隆抗体,抗体效价高、纯度好,具有较强且稳定的特异性结合抗原的功能;间接ELISA方法表明,全菌体抗体和破碎全菌体抗体的效价均大于1×10⁵,性能优良.基于上述抗体,建立了水中大肠杆菌间接ELISA检测方法,实际水样的检测结果表明,检测限可达10⁴个/L.     

MTT法测定大肠杆菌活菌数实验研究

实验分析MTT比色法中的工作波长、反应时间、MTT用量、菌龄、二甲基亚砜（DMSO）等因素对大肠杆菌活细菌计数的影响.同时,通过与平板计数对比,确定了MTT细菌计数的可行性.结果表明,当细菌数在107～109cell·mL-1范围内时,双波长MTT法可用于检测大肠杆菌的活菌数量,最佳反应时间为60min,MTT溶液用量...     

圆孔多孔板水力空化杀灭大肠杆菌的实验研究

在以圆形孔口多孔板为空化空蚀发生器的水力空化装置中,对含有大肠杆菌的水样进行灭菌处理.通过检测大肠杆菌的灭菌率,研究了水力空化对水中大肠杆菌的灭活效果.分析了大肠杆菌初始浓度,水流空化数,孔口流速,孔口排布,孔口数量,孔口大小,空化空蚀作用时间等参数对灭菌效果的影响.试验结果表明：提高流速,降低空化数,选取适当的初始浓度,延长空化空蚀作用时间,增加孔口数量,减小孔口大小以及改进孔口排布可以进一步提高大肠杆菌的杀灭率.水力空化的空化空蚀作用能够杀灭水中的大肠杆菌,是一种饮用水消毒的新技术.     

Flow cytometric assessment of the effects of chlorine,chloramine, and UV on bacteria by using nucleic acid stains and 5-cyano-2,3-ditolyltetrazolium chloride

Flow cytometry (FCM) has been widely used in multi-parametric assessment of cells in various research fields, especially in environmental sciences. This study detected the metabolic activity of Escherichia coli and Staphylococcus aureus by using an FCM method based on 5-cyano-2,3-ditolyltetrazolium chloride (CTC); the accuracy of this method was enhanced by adding SYTO 9 and 10%R2A broth. The disinfection effects of chlorine, chloramine, and UV were subsequently evaluated by FCM methods. Chlorine demonstrated stronger and faster destructive effects on cytomembrane than chloramine, and nucleic acids decomposed afterwards. The metabolic activity of the bacteria persisted after the cytomembranewas damaged as detected using CTC. Low-pressure (LP) UV or medium-pressure (MP) UV treatments exerted no significant effects on membrane permeability. The metabolic activity of the bacteria decreased with increasing UV dosage, and MP-UV was a stronger inhibitor of metabolic activity than LP-UV. Furthermore, the membrane of Gram-positive S. aureus was more resistant to chlorine/chloramine than that of Gram-negative E. coli. In addition, S. aureus showed higher resistance to UV irradiation than E. coli.

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Synthesis and in vitro biological evaluation of 3, 4-annelated coumarin-pyrimido-pyrimidine systems

3,4-Annelated coumarin-pyrimido-pyrimidine systems (7-imino-8-mercapto-7H-5-oxa-7a,9,12-triaza-benzo[a]anthracen-6-one 2 and 8-hydroxy-7-imino-10-methyl-7H-5-oxa-7a,9,12-triaza-benzo[a]anthracen-6-one 3) were synthesized by the action of 4-amino-2-mercapto-pyrimidine, 2-amino-4-hydroxy-6-methylpyrimidine, respectively, on 4-chlorocoumarin-3-carbonitrile 1. The synthesized compounds were examined for in vitro antibacterial activity against 4 bacterial cultures: Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, and Aeromonas Salmonicida. The antibacterial activity of synthesized compounds 2 and 3 was compared to antibacterial activity of novobiocin as a standard drug.     

Characteristics of two transferable aminoglycoside resistance plasmids in Escherichia coli isolated from pig and chicken manure

pRKZ3 is a non-conjugative IncQ plasmid, while pKANJ7 is a conjugative IncX plasmid. The optimal mating time of pKANJ7 varied under different conditions. Both of the two transferable ARPs had little impact on the growth of their hosts. A relatively high level of fitness cost was observed for pKANJ7. The fitness cost of ARPs depended on their hosts. Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.     

磺胺和四环素类抗生素对大肠杆菌联合突变效应的研究

抗生素的滥用造成的环境安全问题已不可忽视,关于抗生素联合毒性效应研究较多,但联合突变效应研究较少。因此,本文以大肠杆菌为受试生物,研究了2种磺胺类抗生素(磺胺氯哒嗪(SCP)、磺胺二甲嘧啶(SMZ))和3种四环素类抗生素(二甲胺四环素(MH)、盐酸四环素(TH)和盐酸强力霉素(DH))单一及联合暴露时对大肠杆菌的突变效应。结果表明:在单一暴露下,磺胺类抗生素会促进大肠杆菌的突变效应,四环素类抗生素没有明显的促进作用;联合暴露下,磺胺类抗生素对大肠杆菌突变效应为相加,磺胺和四环素类抗生素对大肠杆菌突变效应为拮抗。本研究初步探索了抗生素对大肠杆菌的联合致突变风险,为今后环境中抗生素混合暴露的生态风险评价和抗生素污染控制标准制定提供一种理论支撑。     

环境中基因工程微生物的监测方法

介绍国外有关环境基因工程微生物的监测原理与方法。即环境ＧＥＭ监测标记系统的基本要求;几种环境ＧＥＭ监测常用的标记系统;抗性标记系统,发色标记系统和发光基因标记系统等;环境ＧＥＭ的检测方法;平皿培养计数法、免疫学法、遗传学法、生物发光法和流动细胞计数器对环境ＧＥＭ监测。     

一种工程菌的高酶活及其固定化细胞对农药的降解

将抗性尖音库蚊五带亚种的执有机磷农药基因—酯酶基因克隆到质粒pRL－439中．经检测,重组质粒中的酯酶基因已得到表达,获得高酶活的工程菌．将工程菌固定化后对两类难降解农药（有机氯、菊酯类）进行降解,结果表明,固定化细胞在1h内对上两类农药可降解90％以上,具有很高的降解效力．     

Sterilization of Escherichia coli cells by the application of pulsed magnetic field

The inactivation of microorganisms by pulsed magnetic field was studied. It was improved that the application of electromagnetic pulses evidently causes a lethal effect on E. coil cells suspended in phosphate buffer solution Na2HPO4/NaH2 PO4 (0.334/0.867mmol/L). Experimental results indicated that the survivability( N/N0 ; where N0 and N are the number of cells survived per milliliter before and after electromagnetic pulses application, respectively) of E. coil decreased with magnetic field intensity B and treatment time t. It was also found that the medium temperatures, the frequencies of pulse f, and the initial bacterial cell concentrations have determinate influences in destruction of E. coil cells by the application of magnetic pulses. The application of an magnetic intensity B = 160 mT at pulses frequency f= 62 kHz and treatment time t = 16 h result in a considerable destruction levels of E. coil cells ( N/N0=10^-4). Possible mechanisms involved in sterilization of the magnetic field treatment were discussed. In order to shorten the treatment time, many groups of parallel inductive coil were used. The practicability test showed that the treatment time was shortened to 4 h with the application of three groups of parallel coil when the survivability of E. coli cells was less than 0.01%; and the power consumption was about 0.2 kWh/m^3 .