Field investigations on the survival of Escherichia coli and presence of other enteric micro-organisms in biosolids-amended agricultural soil

AIMS: To measure the survival of enteric micro-organisms in agricultural soil amended with conventional and enhanced treated biosolids in relation to environmental and edaphic conditions. METHODS AND RESULTS: Escherichia coli, Salmonella and F-specific RNA bacteriophage were enumerated in sludge and amended soil. Salmonella was not detected and only small numbers of bacteriophages were found in conventional, dewatered mesophilic anaerobically digested biosolids (DMAD). Neither organism was detected in soil. Escherichia coli numbers in soil increased with DMAD application compared with the unamended control, or soil receiving enhanced treated, thermally dried digested (TDD) and composted (CPT) biosolids. Empirical statistical models were developed summarizing the relationship between soil temperature, moisture content and time and E. coli populations. Background numbers of E. coli declined with increasing soil temperature and decreasing soil moisture responding to seasonal patterns in environmental conditions. Time following application was the only significant explanatory variable of E. coli numbers and decay in DMAD-amended soil. CONCLUSIONS: E. coli are an indigenous component of the microbial community in field soil and populations increased in cool, moist soil during autumn-winter and declined in warm, dryer soil during spring-summer. Enhanced treated biosolids were not a source of E. coli, but reduced the size of the indigenous population possibly by stimulating the activity of predatory and competing soil flora because of the organic substrate input from sludge. Conventionally treated biosolids increased E. coli numbers in soil. However, introduced bacteria declined rapidly and survival was limited to 3 months, irrespective of the timing of sludge application or environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide assurance that residual numbers of pathogens applied to soil in treated biosolids decay to background values well within cropping and harvesting restrictions imposed when sewage sludge is spread on farmland.     

A comparison of methods used to enumerate Escherichia coli in conventionally treated sewage sludge

AIMS: This study examined the suitability of three analytical methods for isolating and enumerating Escherichia coli from conventionally treated sewage sludge. METHODS AND RESULTS: Crude sewage, mesophilic anaerobic digested (MAD) sludge, and final product sludge samples were taken from six sewage treatment works for analysis. Two of the three methods tested were membrane filtration techniques, utilizing chromogenic E. coli/coliform (CEC) media and membrane-lactose glucuronide agar (MLGA); the third method was a most probable number (MPN) technique utilizing Colilert in Quantitray 2000 (Idexx). The methods were evaluated for variation, consistency, false-positive and false-negative results, as well as method correlation. The methods gave good and consistent recovery of E. coli for a range of conventionally treated sewage matrices. All of the methods had a false-positive rate of <3%, although MLGA had a high false-negative rate (35.5%) compared with Colilert (3.81%) and the CEC method (6.75%). This resulted in slightly lower presumptive counts but comparable numbers of confirmed counts. CONCLUSIONS: The three detection methods tested, chromogenic, MLGA and Colilert gave comparable recoveries, and did not vary by greater than one order of magnitude (1 log). SIGNIFICANCE AND IMPACT OF THE STUDY: Forthcoming revisions to the Use of Sludge in Agriculture Regulations (1989) will categorize sewage sludge as untreated, conventionally treated or enhanced treated in accordance to microbiological standards. The standard will be based upon numbers of E. coli removed through the sludge treatment process and the numbers remaining in the final product. It is recommended that the Colilert 2000 (Idexx, Westbrook, Maine) and CEC methods would be equally suitable to assess the reduction of indigenous E. coli in conventionally treated sludges, and that MLGA be used with follow-up confirmatory testing.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

不同土壤水分条件下华美新小杆线虫对枯草芽孢杆菌数量、活性及土壤氮素矿化的影响

采用悉生培养微缩体系,探讨了不同土壤含水量条件下,华美新小杆线虫(Caenorhabditis elegans)对枯草芽孢杆菌(Bacillus subtilis)数量和活性及土壤氮素矿化的影响.结果表明：在试验设置的不同含水量条件下,线虫对细菌的取食活动均促进了细菌的增殖 ,并明显提高了土壤呼吸强度; 不同含水量条件对细菌的增殖促进作用总体表现为23%含水量处理>17%含水量处理>28%含水量处理.线虫与细菌的相互作用显著提高了土壤铵态氮和矿质氮含量,促进了土壤氮的矿化.接种线虫对土壤氮素矿化的促进作用表现为23%含水量处理的矿质氮含量显著高于其它两个含水量处理.     

Survival of Escherichia coli and Salmonella spp. after application of sewage sludge to a Pinus radiata forest

AIMS: This study investigated the survival of Escherichia coli and Salmonella spp. in sewage sludge applied to young and old Pinus radiata forest in Spring and Autumn/Winter. METHODS AND RESULTS: Large numbers of E. coli were present in sludge applied to the forest blocks but Salmonella spp. numbers were low or nondetectable. In the mature stand in Spring, numbers of E. coli returned to back-ground after 3 weeks and die-off was significantly correlated with per cent solids of sludge. E. coli survived longer in mature and young stands in Autumn/Winter where numbers did not significantly decrease until weeks 5 and 13, respectively. Salmonella spp. was detectable in the mature stand until week 4 and in the young stand until week 11 in Autumn/Winter. CONCLUSIONS: Microbial die-off was related to desiccation of the sewage sludge, and was faster in warmer, drier conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: In many countries, environmental and health risks associated with the application of sewage sludge to land are minimized by 'best management practice' guidelines, where risks are managed by restriction of public access to these sites. This study provides supporting evidence that withholding periods of greater than 6 months are sufficient to reduce microbial contaminants to background levels.     

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Aims: To develop a duplex real‐time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2‐encoding genes which are pivotal to EHEC‐mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non‐E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H^?, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H^?, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real‐time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real‐time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real‐time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.     

提高大肠杆菌表达外源蛋白胞外含量的策略

大肠杆菌的蛋白质表达平台在工业和农业中得到了广泛应用,使目的蛋白质表达后释放至胞外更有利于大规模的生产。目前,已经研究出许多改善外源蛋白胞外含量的方法。本文从蛋白质分泌机制、菌株、信号肽、载体和培养条件的选择和优化改造、密码子的优化和蛋白质跨膜转运过程的改善等方面总结了提高大肠杆菌表达外源蛋白的胞外含量的各种策略,指出多因素协同作用才能更全面地提升蛋白质的胞外产量。     

An integrated metabolic modeling approach to describe the energy efficiency of Escherichia coli fermentations under oxygen-limited conditions: Cellular energetics, carbon flux, and acetate production

An integrated metabolic model for the production of acetate by growing Escherichia coli on glucose under aerobic conditions is presented. The model is based on parameters which are easily determined by experiments. Forming the basis for this integrated metabolic model are the 12 principal precursor metabolites for biosynthetic pathways, the Embden-Meyerhof-Parnas pathway, the pentose phosphate cycle, the tricarboxylic acid cycle and the anapleurotic reactions, the Crabtree effect, the Pasteur effect, and the details of bacterial respiration. The result can be used to explain phenomena often observed in industrial fermentations, i.e., increased acetate production which follows from high glucose uptake rate, a low oxygen concentration, a high specific growth rate, or a combination of these conditions. (c) 1993 John Wiley & Sons, Inc.     

湿润和干旱条件下喀斯特地区洼地表层土壤水分的空间变异性

在桂西北喀斯特洼地,用地统计学方法研究了旱季初期湿润和干旱条件下表层（0—5和5～10cm）土壤水分的空间结构及其分布特征．结果表明,表层土壤水分存在明显的空间异质性和各向异性,呈现差异显著的斑块状分布格局．湿润条件下,土壤水分具有中等和较强的空间相关性,变程分别为33．15n,和15．75m,其中0～5cm层具有明显的趋势效应;干旱条件下,土壤水分则呈现出强烈的空间相关性,而且相似斑块的空间尺度有所减小,变程最小仅为8．22m;在平均含水量较低时（干旱条件）其变异程度较大,实际应用中应根据平均含水量水平采取不同的取样设计．实验区表层土壤水分空间变异及其分布格局的显著差异,主要是受地貌、平均含水量（降水）和地形等因素的影响．     

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.     

Modeling Escherichia coli fate and transport in the Kabul River Basin using SWAT

Access to safe water is the primary goal of all development plans, yet population increase, urbanization lead to contamination of water resources. This paper focuses on microbial contamination and aims to analyze the fate and transport of Escherichia coli in the Kabul River Basin using SWAT model to evaluate the contribution of different sources. The SWAT is calibrated and validated for the monthly time step using observed E. coli concentrations for April 2013–July 2015. The model skill score; coefficients of determination (R²) equal 0.72 and 0.70, Nash–Sutcliffe efficiencies (NSE) equal 0.69 and 0.66, and percentages bias (PBIAS) equal 3.7 and 1.9 respond well for both calibration and validation, respectively. Regional measured and modeled concentrations are very high with peaks of up to 5.2 ¹⁰log cfu/100?ml in the wet season. Overall, point sources that are comprised of human feces from the big cities and livestock manure from animal sheds, contribute most (44%) to the E. coli concentrations. During peak discharge the non-point sources become the most important contributors due to wash-off from the land and diluted point sources. Allthough such studies are lacking in developing countries, they can be helpful for sanitation management by developing and accessing regional sanitation scenarios.     

耕作和覆盖对黄土高原果园土壤水分和温度的影响

研究了几种不同耕作方式（免耕、旋耕和翻耕）和覆盖措施（覆草、生草和覆膜）对黄土高原果园土壤水分和温度的影响.结果表明：5月不同耕作方式下0～1 m土层土壤水分差异极显著,表现为免耕（14.28%）>旋耕（14.13%）>翻耕（13.57%）,9月差异不明显;不同覆盖措施中,覆草处理的土壤水分最高,且与生草、覆膜、裸地处理间差异极显著;不同耕作方式和覆盖组合处理中以免耕覆草保水效果最好.不同覆盖处理白天土壤平均温度表现为覆膜>裸地>生草>覆草,土壤温度变幅表现为裸地>覆膜>生草>覆草.各覆盖处理的蓄水量与其土壤温度并不都呈负相关,而是由不同覆盖物的保水效果和保温性质共同决定.综上所述,黄土高原果园的保护性耕作体系应以免耕覆草为主.     

Environmental and nutritional responses of a Pinus radiata plantation to biosolids application

Since the mid-1990s, a Pinus radiata (D. Don) plantation growing on a sandy, low fertility soil at Rabbit Island near Nelson, New Zealand received aerobically digested liquid biosolids. An experimental research trial was established on the site to investigate the effects of biosolids applications on tree growth, nutrition, soil and ground water quality. Biosolids were applied to the trial site in 1997 and 2000, at three application rates: 0 (control), 300 (standard) and 600 kg N ha⁻¹ (high). Biosolids application significantly increased tree growth. This was mainly attributed to improved N supply, demonstrated by the enhanced N concentration in the tree foliage. Soil analysis indicated that biosolids application have not caused significant changes in concentrations of most nutrients. However, biosolids treatments significantly increased the available P (Olsen P). Of the heavy metals only total Cu concentrations in the soil increased after biosolids application. Groundwater quality, which was monitored quarterly, has not been affected by biosolids application. The concentrations of nitrate and heavy metals in groundwater were well below the maximum acceptable values in drinking water standards. Biological treatment of sewage and digestion of sewage sludge resulted in the enrichment of ¹⁵N in the biosolids (δ¹⁵N values between 5.0 and 8.7‰). Such enrichment was used as a tracer to study the fate of biosolids derived N. The elevated δ¹⁵N in biosolids treated pine foliage indicated that a considerable amount N was sourced from biosolids. Analysis of δ¹⁵N in understorey plants showed that both non-legume and legume understorey plants took up N from the biosolids, and acted as a N sink, reducing N availability for leaching. Our study showed that application of biosolids to a plantation forest can significantly improve tree nutrition and site productivity without resulting in any measurable adverse effect on the receiving environment.     

温度、水分对湿地土壤有机碳矿化的影响

采用密闭培养法,研究了小叶章(Deyeuxia angustifolia)湿地土壤有机碳的矿化动态,探讨了温度和水分条件对有机碳矿化的影响.结果表明:湿地土壤有机碳在培养初期(0～2 d)矿化速率较高,之后矿化速率逐渐降低;33 d培养期间,表层(0～10 cm)土壤的总矿化量为1.59～2.62 mg C·g-1,为下层(10～100 cm)的4～22倍;温度升高10℃使总矿化量分别增加60%～210%(75%WHC)和30%～200%(淹水);一级动力学方程能较好地描述湿地土壤有机碳矿化动态,其C0值随土壤深度呈指数递减变化,且C0和C0/SOC值均随温度的升高而升高;不同深度土壤Q10值分别变化为1.7～3.1(75% WHC)和1.2～3.0(淹水),且与土壤深度之间存在明显的二次抛物线相关;土壤深度、培养温度对湿地土壤有机碳矿化具有显著影响,而水分处理对有机碳矿化的影响不显著.     

土壤含水量对华北大黑鳃金龟生长发育的影响

室内试验研究土壤含水量对华北大黑鳃金龟Holotrichia oblita Faldermann生长发育的影响。结果表明,土壤含水量对华北大黑鳃金龟的影响因其发育阶段的不同而异。卵期适宜土壤含水量为10%～20%,幼虫期、蛹期适宜土壤含水量分别为10%～15%和15%～20%。土壤含水量为10%～15%时,成虫性比接近1∶1。土壤含水量为15%时,世代存活率最高。     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

不同温度条件下土壤水分对羊草幼苗生长特性的影响

采用生长箱控制方法,研究了羊草幼苗生长对5个温度和5个水分梯度的响应。结果表明,不同温度改变了羊草生物量对土壤水分变化的响应类型。在20～26℃下羊草植株生物量对土壤水分的响应曲线呈三次多项式或抛物线变化规律,而在29～32℃下为指数方程。在温度较低或适度时,轻度乃至中度干旱有促进生长的作用,但在温度较高时轻度干旱及比其程度更高的干旱显著地影响羊草的生长,表明羊草在较高温度下对水分更加敏感。在适当的温度下(26℃),羊草根的贡献率或根冠比与土壤水分呈直线关系。高温加强了干旱对羊草幼苗生长的抑制作用,表明高温降低了羊草对干旱的适应能力。