Survival ofEscherichia coli in great salt lake water

Survival ofEscherichia coli was studied in water from the Great Salt Lake, a highly saline lake with an ionic composition much like sea water. Samples used were from the most concentrated north arm (343.1 g l^?1 solids) and the less concentrated south arm (about 113 g l^?1 solids). At temperatures from 20°C to 9°C the bacterial death rate (k) for the north arm was ?0.17 log day^?1 and the south arm and 1:3 dilution ?0.28 log day^?1. Above 9°C the rate of death increased approximately exponentially and at 19°C the rate of death increased approximately exponentially and at 19°C the death rate was ?1.31 log day^?1 in the north arm and ?0.98 log day^?1 in the lower salinity water. These rates fall within those reported for sea water and are much higher than fresh water. Possible causes of death are discussed with the most likely being the high concentrations of minor elements or osmotic stress. The survival characteristics ofE. coli in waters with a sea water-like composition should require the same health concern as sea water regardless of the actual concentration of salt. High salt water of other ionic composition may behave differently, however.     

Survival of Escherichia coli donor,recipient, and transconjugant cells in soil

Survival of Escherichia coli donor, recipient and transconjugant cells was studied in sterile soil incubated under a variety of conditions. In soil not amended with additional nutrients, the absence of transconjugants indicated that R-plasmid transfer had not occurred. In soil supplemented with a low level of nutrient broth, both donor and recipient cells survived over a 48 hr period. In addition, transconjugant cells were detected in soil as early as two hr after the soil was inoculated with donor and recipient strains.     

Fate of Escherichia coli and Escherichia coli O157 in soils and drainage water following cattle slurry application at 3 sites in southern Scotland

Abstract. Slurry from farm animals may contaminate water supplies, rivers and bathing waters with faecal coliforms, such as Escherichia coli . Where animals harbour the O157 strain the hazard to human health is particularly high, but both the hazard level, and the low incidence and sporadic nature of the excretion of E. coli O157 make it difficult to study this strain under field conditions. The survival of total E. coli and of E. coli O157 were compared in the laboratory for two soils under controlled temperature and moisture. E. coli O157 die-off rate was the same as or quicker than for total E. coli . This result meant that field experiments studying the fate of total E. coli should give a satisfactory evaluation of the risk of water contamination by the O157 strain. In four field experiments at three sites, slurry containing total E. coli numbers of 2.2 × 10⁴ to 5.7 × 10⁵ colony forming units per mL (c.f.u. mL^–1) was applied to drained field plots. Field die-off was faster than expected from laboratory experiments, especially in one experiment where two weeks dry weather followed application. In all but this experiment, the first drain flow events after slurry application led to very high E. coli concentrations in the drains (10³ to 10⁴ c.f.u. mL^–1). E. coli O157 was present in the slurry used for two of the experiments (33 c.f.u. per 100 mL in each case). However the proportion of E.coli O157 was very low (about 1 in 10⁵) and it was not detected in the drainage water. After the first week E. coli drainage water numbers decreased rapidly but they were 1–10 c.f.u. mL^–1 for much of the sampling period after slurry application (1–3 months).     

Occurrence and survival of coliform bacteria, Escherichia coli and Salmonella in various manure and compost

pp. 865–874
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 10² to 10⁶ cfu g¹ dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria ( E. coli , E. vulneria , Pantoea sp. and Buttiauxella agrestis ) were identified whereas isolates of Serratia marcescens , not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost.     

Survival and Plasmid Transfer Ability of Escherichia Coli in Wastewater

No differences were observed in the survival of plasmid-bearingand plasmid-free Escherichia coli strains in the course of a long-term survival process in wastewater, under both illuminated and non-illuminated conditions. While the CFU counts and the number of active cells decreased, the numberof nucleoid-containing cells remained constant throughout the 30 days of experimentation. Visible light efficiently contributed to the reduction in culturability, and T₉₀ values were very different under illuminated and nonilluminatedconditions. In the latter case the time necessary to reduce theculturability of a bacterial population by 90% was 27 days, while in the former it was only 1 day. Plasmid transfer was abundant, while the survival of donor and recipient cells was extensive. After 24 hr of survival in wastewater, transfer frequency values ranged from 5.92 × 10^-5 to 1.12 × 10^-2, depending on mating conditions. In the absence of illumination, the potential transfer abilitiesremained for survival periods of at least 20 days. Transfer assays between free and adhered cells were carried out by means of dialysis bags and submerged membrane diffusion chambers. Transfer frequency for adhered cells was greater than for free cells (2.80 × 10^-2 as opposed to 2.39 × 10^-3).     

Inactivation of Escherichia coli in soil by solarization

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g⁻¹ dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces.     

Inactivation of Escherichia coli in soil by solarization

Abstract

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g^?1 dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces.     

井灌水稻区晒水池升温机理的研究

该文针对井灌区井水灌溉水温低的特点，生产实际中需设晒水池来提高水温，以便防止井灌水稻冷水害的发生。通过太阳辐射——水体——土壤系统，对静水状态和动水状态下晒水池升温机理进行了探讨，建立了静水情况下晒水池平衡水温的数学模型，并说明各参数的数值算法；利用平衡水温的数学模型，通过解非奇次方程的方法，建立了晒水池内任意时刻各点水温的预测模型，并利用建三江垦区七星农场的晒水池实测水温加以验证，最大相对误差在9.6%以内，结果表明预测精度符合生产实际要求，这一数学模型为井灌区井水升温提供了较为科学的依据。     

产L-乳酸的大肠杆菌基因工程菌的构建

含有质粒pKD46的菌株BW25113．在L-阿拉伯糖诱导后，表达λ噬菌体的3个重组蛋白Exo、Bet和Gain，宿主菌就具有了同源重组的能力。利用λ噬菌体的Red重组系统构建了D-（＋）-乳酸脱氢酶基因（1dhA）、乙醇脱氢酶基因（adhE）双突变的大肠杆菌BW25113C工程菌株和乙酸激酶基因（ackA）单突变的大肠杆菌BW25113工程菌株。将表达牛链球菌L-乳酸脱氢酶基因（1dhL）的pSE380质粒转化到BW25113C菌株中发酵培养35h，用HLPC检测产物，尚未发现L-乳酸。     

胸腺素家族多肽在大肠杆菌中的表达

为提高胸腺素(又名胸腺肽,thymosin)的体外表达效率,本研究采用设计融合标签的方法对胸腺素α1和β4(Tα1,Tβ4)进行体外重组表达,通过超高效液相色谱-飞行时间质谱联用(LC/MS)法,对融合蛋白的完整分子量进行验证,利用反相超高效液相色谱法对不同表达载体的单位表达量进行测定.结果 表明,A 18-KEKE、...     

重组犬IFN-γ在大肠杆菌中的高效表达

提取Concavadin A诱导培养的犬脾细胞总RNA，经RT-PCR扩增出犬IFN-γ，克隆到pMD18-T载体并测序鉴定，然后把IFN-γ基因克隆到原核表达载体PJLA605，构建pRL-Ca／IFN-γ表达质粒；应用M9培养基通过摇瓶发酵，确定诱导时机和诱导表达时间。结果表明，工程菌pRL-CaIFN-γ在30℃培养至OD600为1．5于42℃诱导4h，菌体收获量湿重达20．0gm，目标蛋白表达量约占菌体总蛋白的32％，外源基因在该基因工程菌中得到了高效表达。     

Environmental Controls on the Fate of Escherichia coli in Soil

An improved understanding of factors that influence the survival and/or growth of Escherichia coli (E. coli) in soil is essential to allow the formation of land management practices to control the spread of the pathogenic strains of the bacteria, whose transmission to fresh produce is a threat to food safety. Persistence of E. coli in soils held at different water potentials and with carbon additions then subjected to post-freezing incubation temperatures and in the presence of Klebsiella terrigena (K. terrigena) were investigated. Soil samples adjusted to different water potentials (?0.03, ?0.1 and ?1.5 MPa) were inoculated with a multi-antibiotic resistant strain of E. coli (E. coli 2+), which allowed recovery of the organism from soil samples. In addition to manipulation of water content, different C levels were added and samples were frozen for varying lengths of time, thawed and incubated. In freezing studies, initial soil moisture content significantly affected E. coil 2+ survival in soils following thawing, resulting in lower survival rate (k) at water potential of ?0.03 than at ?0.1 and ?1.5 MPa. The effect of length of freezing time was significant only at ?0.03 MPa. Glucose addition at 1.25 mg C g^?1 improved survival rate versus glucose at 0.125. The low level glucose increased die-off rate versus no addition, suggesting that unless amendments provide C above a certain threshold level, they might facilitate the death of the bacteria. E. coli 2+ survival improved in the presence of K. terrigena at 6°C but not at 23°C. Persistence of E. coli under the interactive influence of various environmental factors highlights the urgency and importance of understanding its potential for transmission to fresh produce and water bodies.     

pfl-act基因在大肠杆菌中的高效表达及其纯化

丙酮酸甲酸裂解酶是肠道细菌在厌氧代谢中十分关键的酶,丙酮酸甲酸裂解酶激活因子(pyruvateform ate lyase activator,PFL-A)在功能上具有重要的作用。为进一步研究PFL-A的激活机理,以大肠杆菌K-12的基因组为模板,通过G enB ank上公布的序列设计引物,扩增出目的基因,克隆到pM D 18-T载体,经测序,所扩增出的基因与p f l-act基因具有99%的同源性。将p f l-act连接到高效表达载体pET-22b( )中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,结果发现,p f l-act以包涵体形式表达。改变诱导剂、诱导剂量或培养温度,对包涵体的形成均没有明显的影响。     

小鼠表皮生长因子在Escherichia coli中重组表达

以大肠杆菌（Escherichia coli）BL21为表达宿主，重组表达小鼠表皮生长因子。以pET30a为出发载体，构建了包含mEGF编码序列的重组表达载体pETmEGF，采用低温诱导表达和无破胞程序的冻融法进行纯化，得到了浓度为17．24μg/mL和纯度为57％的mEGF融合蛋白，其活性相当于标准小鼠表皮生长因子的29．16％。     

Protozoan predation of Escherichia coli in hydroponic media of leafy vegetables

Multiple outbreaks of food poisoning associated with fresh vegetable consumptions have occurred in many countries. Numerous reports have described human pathogenic bacteria, such as Escherichia coli O157:H7 and Salmonella spp., that can internalize into fresh vegetables via root or leaf surfaces. While attempting to obtain the threshold concentration of internalization of E. coli inoculated into hydroponic medium during vegetable cultivation, we observed a rapid decrease in E. coli numbers. In the present study, we determined that the rapid decline in E. coli was not due to a physiological change into a viable but non-culturable (VNC) state. The population crash was instead caused by true bacterial death, as the rapid descent was also confirmed by micro-colony fluorescence in situ hybridization, a culture-independent method that can detect VNC cells. We next monitored the number of E. coli inoculated into intact or filter-sterilized hydroponic medium after cultivation of various types of plants. We found that the number of E. coli in intact hydroponic medium decreased markedly, whereas the level in filter-sterilized hydroponic medium was completely unchanged. This result suggests that biotic factors were present that could be eliminated by filtering. Robust predation of E. coli by protozoa (ciliates and flagellates) was observed using fluorescently labeled bacteria incorporated into the hydroponic medium. Finally, morphological identification of flagellates by scanning electron microscopy revealed the presence of a species of Stramenopiles. These findings suggest the importance of protozoa as bacterial feeders in hydroponic systems and hence the use of these organisms as potential control agents of human pathogenic bacteria.     

在大肠杆菌内高效表达大分子量拟蜘蛛牵丝蛋白

依据已报道的蜘蛛(Nephilaclavipes)牵丝蛋白部分cDNA序列,设计合成两种不同结构的拟蜘蛛牵丝蛋白基因单体,大小分别为360和390bp。多聚化得到8倍体和16倍体后,克隆到表达载体pET-30a,得到4种表达载体,转化大肠杆菌(Escherichiacoli)BL21(DE3)诱导表达。用自制的抗蜘蛛牵丝蛋白血清Westernblot检测,表达产物呈梯度排列,主带与预计大小一致。蜘蛛牵丝蛋白表达量最高为800mg/L。     

人乙醛脱氢酶2的原核表达及其条件优化

为实现人乙醛脱氢酶2（ALDH2）基因在原核生物中高效表达,将含有6×His标签和SUMO融合蛋白标签的人乙醛脱氢酶2基因的表达载体转化至宿主菌BL21（DE3）中。在异丙基硫代-β-D-半乳糖苷（IPTG）诱导下,目的基因在大肠杆菌内高效表达。通过对表达条件的优化,37℃使用终浓度0.3mmol/L的IPTG诱导3h,重组大肠杆菌的表达量可占全菌蛋白的16%。SUMO融合蛋白标签的加入以及较低的诱导温度（16℃）有利于提高人乙醛脱氢酶2基因在大肠杆菌内的可溶性表达。     

猪源抗菌肽Protegrin-1基因在大肠杆菌中的融合表达

为了使猪源抗菌肽Protegrin-1(PG-1)在原核表达载体中获得高效表达,在不改变PG-1氨基酸的基础上,根据大肠杆菌偏好密码子表,替换PG-1部分密码子,设计两段互补的引物,利用搭桥PCR技术扩增完整的PG-1成熟肽基因,重组至融合表达载体pGEX-4T-1中,构建成抗菌肽PG-1基因融合表达载体pGEX4T-PG-1,转化至大肠杆菌(Escherichia coli)BL21(DE3)plyS中,筛选得到阳性克隆,并用1.0 mmol/L的IPTG进行诱导表达.SDS-PAGE电泳图谱显示在28 kD处有特异性的蛋白条带出现,经Western blot检测表明重组子已经成功地在大肠杆菌中表达了融合蛋白.纯化得到的GST-PG-1用凝血酶切割后,经用亲合层析得到分子量约为2 kD抗菌肽PG-1蛋白1.38 mg/L.对大肠杆菌和金黄色葡萄球菌(Staphylococcus aureus)抑菌试验结果表明,重组抗菌肽PG-1具有明显的抑菌效果.     

甘蓝型油菜中黑芥子酶基因在大肠杆菌中重组质粒的构建和表达

实验通过RT-PCR程序从提取的油菜总RNA中扩增出硫代葡萄糖苷水解酶（又称黑芥子酶,EC3.2.1.147）基因,酶解后插入大肠杆菌表达载体（Escherichia coli）pGEX-4T-1,获得克隆菌株。基因测序在 GenBank中的登陆号为EF583560,翻译的氨基酸序列与已报道的黑芥子酶（GenBank中的登陆号为Q00326）中的一段有一个氨基酸不同,同源性达到99%。经过IPTG诱导表达,在91KDa左右处有表达量很高的一条带。