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.     

Impact of soil solarization on the ciliate community structure of a greenhouse soil

Soil solarization is an ecologically friendly method of controlling various plant pathogens and pests, but also affects non-pathogenic members of the soil biota. Here, we studied the impact of soil solarization on the community structure of soil ciliates using a culture-independent molecular approach, namely denaturing gradient gel electrophoresis (DGGE) of targeted 18S rRNA gene fragments. Greenhouse soil with added organic fertilizers was solarized for 33 days at an average temperature of 47–48°C. Solarization caused a drastic change in the ciliate community. The variation between replicates was large, which suggested that the distribution of ciliates was spatially heterogeneous in the soil, probably due to their decreased numbers. In contrast, non-solarized soil had a stable and homogeneous ciliate community during the experimental period. In solarized soil, most of the original ciliate community recovered 76 days after solarization. Sequence analysis of DGGE fragments indicated that both r-selected and K-selected species of ciliates were affected by solarization but recovered with time after solarization. Our results demonstrated both the vulnerability and resilience of the ciliate community to soil solarization and also the utility of using molecular-based analysis of ciliate communities as bioindicators of soil stress caused by solarization.     

Potential health risks associated with the persistence of Escherichia coli O157 in agricultural environments

Abstract. Escherichia coli serotype O157 is a virulent human pathogen the global incidence of which has increased. It has been demonstrated that cattle are the primary reservoir of this pathogen. This has serious implications for the land-based disposal of organic wastes such as cattle manure, cattle slurry and abattoir waste. Further, it also has serious ramifications for the protection of surface and groundwater drinking supplies and public access to pasture land. However, while soil and vegetation can be expected to directly influence the survival of this pathogen, there is a paucity of information concerning the behaviour and survival of E. coli O157 in agricultural environments. It appears that E. coli O157 presently contaminates between 1 to 15% of UK cattle herds, depending on region, and that faecal excretion of the bacterium shows a distinct seasonality which also reflects the incidence of human infections. E. coli O157 can remain viable in soil for greater than 4 months and appears to be a highly resilient pathogen possessing the capability to adapt easily to environmental stresses. While most human cases of E. coli O157 related food poisoning have been associated with the consumption of contaminated meat and dairy products, there is also evidence that human infection has occurred through the ingestion of contaminated soil, fruit and vegetables and drinking water. In this review the potential threat to human health posed by the application of contaminated organic wastes to soil and possible strategies for reducing the amount of pathogen entering the food chain are highlighted.     

Effect of soil solarization on subsequent nitrification activity at elevated temperatures

Soil solarization is a nonchemical method of soil disinfection achieved by covering the soil surface with sheets of vinyl plastic to generate elevated soil temperature, generally over 45°C. Such elevated temperatures may be detrimental to some nitrifying microorganisms and favorable to others. However, little information exists to indicate how nitrification activity in soil is affected after solarization. We performed several experiments to investigate the effects of soil solarization on nitrification activity. We found that: (1) if a soil was subjected to pretreatment of 45 or 50°C for as little as 1 d, nitrification activity in a subsequent incubation at 30°C was less than that of a soil that did not receive any high-temperature pretreatment. However, if a soil received pretreatments of 45 or 50°C for more than 7 d, nitrification activity in a subsequent incubation at 45 or 50°C was greater than that of soil that did not receive high temperature pretreatment. (2) Nitrification activity in three kinds of soil taken from 0–5 cm depth after solarization treatment was greater at 45°C than 30°C. (3) Nitrification activity at 45°C in soil that had received solarization in the preceding year was greater than that in soil that had not been subjected to solarization. This was consistent with the fact that the population densities of ammonia oxidizers were greater in soils that had been subjected to solarization. These results suggest that soil solarization induces nitrifying microorganisms that are more active at 45–50°C than they are at 30°C, and that the effect of solarization on nitrification persists until the next crop season.     

黑膜覆盖控制黄瓜根结线虫(Meloidogyne incognita)的效果

从土壤线虫含量、根结级别和黄瓜生长与产量等方面分析了黑膜覆盖控制黄瓜根结线虫的效果。结果显示，黑膜覆盖消毒并经过一个黄瓜生长季以后，土壤5、10、15、20、30cm线虫数量比对照减少26．9％、13．9％、2．9％、1．9％和0．9％。控制效果随土壤深度增加而下降，同时植物寄生线虫数量减少5％。根结线虫主要分布在0～30cm土层，占总量的97．7％～98．5％。从对根系的危害来看，覆黑膜后黄瓜根结线虫的发病程度比对照明显下降，为害为Ⅰ级，而对常规药剂消毒的植株根系为害达到Ⅳ级。黑膜覆盖消毒不仅使黄瓜产量提高6％，畸形瓜率下降(黑膜覆盖下畸形率为10％，对照为60％)，而且成本比药剂消毒低57％。尽管黑膜覆盖消毒对土壤线虫含量的控制效果不太理想，但从黄瓜生长和产量以及根结着生来看，其控制效果仍然优于药剂消毒。这说明黑膜覆盖主要以降低线虫活性和侵染能力、推迟侵染时间为特征。初步得出结论，经过40d黑膜覆盖消毒可有效控制黄瓜根结线虫的发生及危害。     

CpG-DNA对大肠杆菌诱导的大鼠实验性乳腺炎的影响

72只雌性大鼠随机分成对照组和实验组(n =36)。对照组产后0 h于左后腿胫骨前肌肌肉注射0.01 mol/L，pH 7.2 磷酸盐缓冲液(PBS)100 μL,实验组肌注CpG-DNA 200 μg/只，72 h后经乳头管灌注2×1012 cfu (colony forming unit/mL)/mL,大肠杆菌(Escherichia coli )100 μL/侧到第4对(两侧)乳腺内，分别于灌注前0 h，灌注后8，16，24，48和72 h(n = 6)颈静脉放血处死。组织学观察显示大鼠感染后16 h为乳腺腺泡嗜中性粒细胞(polymorphonuclear leukocytes，PMN)浸润高峰，实验组PMN浸润迅速， 72 h已无浸润。乳腺组织细菌数在16 h上升至最高，实验组大鼠感染后16、24和48 h乳腺组织细菌数较对照组有显著下降。CpG-DNA能极显著地提高感染前乳腺组织中IL-2水平。乳腺组织IL-6在不同时间点均有显著上升，在16 h达最高。CpG-DNA能显著地提高16 h乳腺组织IL-6水平。实验组乳腺组织N-acetyl-β-D-glucosaminidase (NAGase)无显著变化。表明CpG-DNA可加速炎症初期PMN的快速浸润，促进IL-2和IL-6的产生，减少细菌数量，减轻炎症介质对细胞的损伤并缩短炎症过程，提示CpG-DNA对大肠杆菌诱发的大鼠实验性乳腺炎有一定的预防作用。     

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).     

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、...     

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.     

大肠杆菌海藻糖磷酸合酶基因的克隆

根据报道的大肠杆菌（Escherichia coli)海藻糖－6－磷酸合酶基因（otsA)，设计引物，通过PCR技术从大肠杆菌XLI菌株的总DNA中扩增到一个1．4kb片段。经克隆、测序分析，该片段长1425bp并含一完整的开放读框（ORF）。在核苷酸水平上，该ORF与已报道的otsA基因具有99．86％的同源性。在氨基酸水平上，其推断性的编码产物蛋白与OtsA具有100％一致性。     

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。     

白颈长尾雉圈养种群大肠杆菌耐药性及其耐药基因

对白颈长尾雉圈养条件下的38个样品进行大肠杆菌分离及PCR检定,并采用肠杆菌基因间重复共有序列PCR指纹法（ERIC）剔除各个样品的重叠分离株,检测获得的170个大肠杆菌分离株对9种抗生素的耐药性、Ⅰ型整合子携带率及其可变区抗性基因,结果显示：（1）来自白颈长尾雉的分离株对实验用的9种抗生素的抗性比率和多重耐药性远高于环境源和人源者（来自白颈长尾雉的分离株100%耐受3种及以下的抗生素,而环境源者为50.7%,人源者66.7%）;（2）来自白颈长尾雉的分离株的Ⅰ型整合子携带率（92%）高于环境源（87%）和人源（78%）;（3）来自白颈长尾雉和来自人的大肠杆菌分离株的Ⅰ型整合子可变区抗生素抗性基因检出率相同（36%）,但高于环境源（24%）;（4）携带Ⅰ型整合子的分离株对实验用的抗生素的抗性百分率一般高于不携带者,只有个别种类抗生素这种差异为非显著性差异;（5）Ⅰ型整合子可变区基因盒的基因为3类,即aadA、dfrA和未知功能的orfF;aadA、dfrA的频率相同;3类基因均以基因盒形式存在,分别是dfrA17-aadA5、dfrA12-ofrF-aadA2、dfrA12-aadA2。     

大肠杆菌分子伴侣表达的辅助载体构建及应

异源蛋白在大肠杆菌不能正确折叠表达成包涵体,分子伴侣能在细胞内帮助异源蛋白折叠,改善蛋白的聚集。本研究以大肠杆菌(Escherichia coli)DH5α菌株DNA为模板,利用PCR技术扩增出6个分子伴侣编码基因groEL、groES、dnaK、dnaJ、grpE和clpB,分别将groEL、groES和grpE(Ⅰ组)插入pCDFDuet-1载体,将dnaK、dnaJ和clpB(Ⅱ组)基因插入pRSFDuet-1,构建辅助载体pR-GESP和pC-DJKL,每个重组载体中含有一个人工操纵子,每个基因上游含有T7启动子。SDS-PAGE结果显示,诱导后的重组大肠杆菌上清除了DnaJ外其它5个蛋白明显表达。SDS-PAGE分析了共表达分子伴侣对玉米(Zea mays)四吡咯分子合成的谷氨酸-1-半醛氨基转移酶、尿卟啉原Ⅲ脱羧酶和西罗叶绿三酸:铁螯合酶在大肠杆菌的折叠和聚集影响,结果显示,GroEL、GroES和GrpE能防止玉米西罗叶绿三酸:铁螯合酶在大肠杆菌的聚集,但DnaK、DnaJ和ClpB分子伴侣对该酶则没有作用;GroEL、GroES和GrpE能部分抑制玉米谷氨酸-1-半醛氨基转移酶在大肠杆菌的聚集...     

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.     

大肠杆菌yqhD基因的克隆与表达

以大肠杆菌E scherich ia coli K-12基因组DNA为模板,利用PCR技术扩增得到假定的氧化还原酶(pu tative ox idoreductase)基因yqhD,将它连接到克隆质粒pGEM-3zf(+)上,得到重组质粒pGEM-yqhD,对此重组质粒进行序列测定,对其DNA序列分析表明,yqhD基因全长为1 164 bp。再将yqhD基因插入表达载体pSE 380,构建成重组子pSE 380-yqhD,并在E.coli BL 21中获得表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在30°C下,以1.0 mm o l/L IPTG诱导12 h的酶活力达到3.13 U/mL,比对照菌株提高4.4倍。     

禽多杀性巴氏杆菌和大肠杆菌二联灭活油乳剂疫苗免疫保护试验

用禽多杀性巴氏杆菌和大肠杆菌本地分离菌株,分别液体培养、灭活,按一定比例混合,加入油佐剂制成二联灭活油乳剂疫苗。以0．5mL／只、1．0mL／只两个剂量分别肌肉注射免疫2月龄非免鸡30只,同条件设非免疫对照组8只,免疫后21d用两种分离菌株分别攻毒及两种菌混合攻毒。试验结果显示,对禽多杀性巴氏杆菌的保护率为80％,对大肠杆菌病的保护率为100％,对两种菌混合攻毒的保护率为80％。本试验结果说明研制的二联灭活油乳剂疫苗安全有效。     

克雷伯氏菌1,3-丙二醇氧化还原酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae)中扩增得到含有1,3-丙二醇氧化还原酶基因(dhaT)的DNA片段,将其定向连接到克隆质粒pGEM-3xf上,得到重组质粒pGEM-dhaT。将此重组质粒转化到受体菌Escherichia coli DH5α中,通过蓝白斑鉴定挑选出阳性菌株。DNA序列分析表明其基因全长为1185bp。将该片段插入表达载体pSE380,构建成重组子pSE-dhaT,并在E．coli JM109中获得表达,经SDS-PAGE检测,表达产物分子质量与天然纯1,3-丙二醇氧化还原酶(DHAT)相同,约为43ku。     

猫白介素-18基因的克隆、序列分析及其表达

用猫白介素18(interleukin,IL-18)基因特异性引物对刀豆蛋白(ConA)刺激后猫外周血单核细胞(PBMCf)总RNA进行了RT-PCR扩增,并将扩增产物纯化后克隆入pMD18-T中进行核苷酸序列测定。结果该基因全长579bp,编码192个氨基酸(GenBank登录号:DQ100372)。在推导的猫IL-18氨基酸序列中,无信号肽序列和潜在的N-联糖基化位点,但存在4个Cys残基。与不同物种IL-18相比,猫IL-18与犬、羊、牛和猪IL-18核苷酸序列有较高的同源性,分别为89.8%、88.6%、88.4%和88.1%,但与小鼠和鸡IL-18有明显的种属差异。将目的基因片段进一步亚克隆到大肠杆菌(Escherichia coli)表达载体pET28a中构建了重组质粒pETIL-18,转化大肠杆菌BL21(DE3),并用IPTG诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到分子量为27.5kD的重组目的蛋白。经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的13.6%。     

近年来大肠杆菌K88ac菌毛的变异和生物学特性

2000~2005年，从猪大肠杆菌病 (colibacillosis)中分离到一些表达K88菌毛的大肠杆菌(Escherichia coli )，这些分离株只与K88 a因子单抗反应，而不与K88 b、c和d因子单抗反应。分离的菌株 (13/16)以O149为常见血清型，且全部拥有STb毒素基因，通过K88常规血清交叉吸收试验、SDS-PAGE和Western印迹，表明这些菌株不仅与K88ac参考菌株C83907制备的c因子血清反应，而且与以分离株SEC586制备且经K88ab、K88ac和K88ad参考菌株吸收后的血清也反应，表明这些分离株仍为K88ac大肠杆菌。对新近分离的SEC464、SEC525、SEC586、SEC799和EC910株及80年代我国分离的TM128株的K88主要亚单位结构基因faeG进行克隆、测序，发现新近分离株的faeG基因由846对核苷酸组成，编码菌毛主要亚单位的261个氨基酸及21个氨基酸的信号肽，比国内外报道的K88ac FaeG亚单位 (262个氨基酸)少了一个氨基酸，比K88ab、K88ad (264个氨基酸)少了3个氨基酸。TM128株的FaeG氨基酸序列与K88ac(M29375)的同源性为100.0%，SEC464、SEC525、SEC586、SEC799和SEC910株的FaeG亚单位氨基酸序列的同源性为97.7%~99.6%，它们与K88ac的同源性为94.6%~96.6%；与K88ab的同源性为90.0%~91.6%；与K88ad的同源性为87.0%~88.9%。 结果表明新分离的K88ac大肠杆菌黏附素主要亚单位已发生了部分变异。     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India