Glucuronidase assay in a rapid MPN determination for recovery of Escherichia coli from selected foods

Glucuronidase is present in most strains of Escherichia coli but absent in most other enteric microorganisms; therefore, an assay for this enzyme is useful for determining the presence of the organism. The substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) is incorporated into either lauryl tryptose (LT) broth or EC medium; the inoculated tubes are then incubated under specified conditions and examined under longwave UV light for the presence of a fluorogenic glucuronidase end product. When compared with the 10-day most probable number (MPN) procedure of AOAC, the LT-MUG and the EC-MUG tests required 24 and 96 h, respectively, and gave comparable mean log MPN values for samples of crabmeat, sunflower kernels, and walnut pieces. However, false-positive and false-negative reactions were observed with foods tested by both of these rapid methods. Overall, method sensitivity was not compromised by using the LT-MUG rather than the EC-MUG method. Incorporation of 25 micrograms MUG/mL into LT broth resulted in diminished fluorescence of positive reactions, whereas MUG concentrations of 50 and 100 micrograms/mL provided decisive fluorogenic reactions.     

Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study

A collaborative study was conducted to compare a proposed LST-MUG method with the AOAC official method for Escherichia coli detection. E. coli produces an enzyme, beta-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-beta-D-glucuronide (MUG), to yield a fluorescent end product. Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a rapid quantitative method for screening E. coli, which is detected by fluorescence of the medium under longwave UV light. In this collaborative study, 5 food samples, 2 frozen (entree sauce/gravy and dairy topping) and 3 chilled (hamburger, pork sausage, and cheese), were tested for E. coli detection by 17 collaborating laboratories. Results indicate that the LST-MUG method is equal to or better than the current AOAC method for detecting E. coli. The LST-MUG method has been adopted official first action.     

Quantification of Escherichia coli O157:H7 in soils using an inhibitor-resistant NanoGene assay

Humic acids are ubiquitous and abundant in terrestrial environments; therefore, they are often co-extracted with nucleic acids and interfere with quantitative PCR (qPCR) assays. In this study a recently developed NanoGene assay that is resistant to interference by humic acids was evaluated for gene detection in soil samples. The NanoGene assay utilizes a combination of magnetic beads, dual quantum dots labels, and DNA hybridization in solution. Seven soil samples containing different amounts of organic matter were tested to compare NanoGene and qPCR assays for their respective ability to detect a bacterial pathogen. We spiked the soils with Escherichia coli O157:H7, extracted genomic DNA, and conducted NanoGene and qPCR assays targeting the E. coli O157:H7-specific eaeA gene. To prevent the inhibition of PCR that is common when using DNA extracted from soils, we used a range of template DNA concentrations and BSA addition in the qPCR assay. Compared to the qPCR assay the NanoGene assay was significantly more resistant to the inhibitory effect of humic acids, successfully quantifying the eaeA gene within a linear (R² = 0.99) range of 10⁵ through 10⁸ CFU/g soil for all seven soil samples tested. In contrast, the qPCR assay was significantly inhibited using the same template DNA isolated from soils containing a range of organic content (2.0%–12%). Interestingly, the qPCR assay was still inhibited despite additional purification steps, suggesting that humic acids were still associated with DNA at a level that was inhibitory to qPCR. This study demonstrated that the NanoGene assay is suitable for quantitative gene detection in diverse soil types and is not susceptible to inhibition by humic acids and other organic compounds that commonly lead to false negative results in qPCR assays.     

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。     

猪圆环病毒2型BF株ORF2基因在大肠杆菌中的表达

采用PCR从构建的猪圆环病毒2型／(PCV2)ORF2重组质粒(pGEM-T-ORF2）中扩增出大小为593bp的ORb2基因，克隆到表达载体pET-32a，经异丙基硫代半乳糖苷诱导，成功表达了ORF2基因编码的结构蛋白。表达的重组蛋白为融合蛋白，分子量40kD，表达量20％左右。经Western blot检测，重组蛋白可被PCV2阳性血清识别。     

犬白细胞介素-2 基因(IL-2 )的克隆与表达

根据GenBank上发表的犬(Canis familiaris)IL-2基因序列，设计了1对引物。采用RT-PCR技术，以ConA刺激的犬外周血淋巴细胞为材料，从总RNA中扩增出犬IL-2基因。琼脂糖凝胶电泳显示扩增片段约为550bp。分离纯化片段，克隆入pMD18-T载体，经酶切鉴定，测序结果显示，克隆的犬IL-2基因与GenBank上发表的序列一致，生物软件分析结果表明该序列与牛、羊和鹿的亲源性更近。构建表达质粒pET28-CaIL-2，转化大肠杆菌(Escherichia coli)BL21，IPTG诱导表达，SDS—PAGE电泳显示21．8kD左右的表达条带。MTT比色法测定活性结果表明：表达的重组犬IL-2蛋白具有极显著的体外增殖活化淋巴细胞的活性。     

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.     

Inactivation of Staphylococcus aureus and Escherichia coli in Water Using Photocatalysis with Fixed TiO2

Photocatalytic activity in titanium dioxide (TiO₂) has been extensively studied because of its potential use in sterilization, sanitation, and remediation applications. The aim of the study reported here was to assess the feasibility of “fixed” TiO₂ as the photocatalyst for inactivating pathogenic bacteria selected, Staphylococcus aureus and Escherichia coli, from a water stream. The investigation was undergone in a properly designed laboratory-scale evaluation. Using the system reported here, we obtained an effective bactericidal capability for E. coli and S. aureus with 90.0% and 98.0% after 30 and 10 min ultraviolet-A light irradiation with fixed TiO₂, respectively. Parameters such as the various initial bacteria concentration, TiO₂ concentrations, interruption of illumination, turbidity, and coexisted organic matters were examined to identify the removal efficiency in the photocatalytic reaction. Results indicated the negative effect by high bacteria concentration, coexisted organic matters, and turbidity on inactivation of bacteria, and positive effect on disinfection was associated with higher TiO₂ concentration. Furthermore, our results indicated that under the same experimental conditions, the removal efficiency of the system in synthetic water was performed better than that of crude water. This inferior removal capability in crude water is mainly caused by the negative effect from the unknown coexisted factors.     

猪TNNC2基因的克隆及其融合蛋白在大肠杆菌中的表达

根据GenBank上猪TNNC2（Fast skeletal muscle troponin C2）基因序列（GenBank accession No. DQ629177）设计一对引物,采用RT-PCR方法克隆得到617 bp TNNC2 cDNA片段（GenBank accession No. EF673726）,包括完整的开放阅读框（ORF）,与GenBank上公布的猪TNNC2基因（GenBank accession No. AY575058）的ORF核苷酸序列同源性达99 %,并发现开放阅读框内的5个点突变,319位点T→C,320 位点G→A,321位点C→T,导致氨基酸107位Ala（丙氨酸）→Met（蛋氨酸）,322位点A→G为同义突变,433位点A→T,导致氨基酸144位Glu（谷氨酸）→Asp（天冬氨酸）。根据已获得的TNNC2基因开放阅读框序列,重新设计引物扩增得到包含BamH I和EcoR I酶切位点的完整阅读框,将其首先克隆到pMD18-T载体中,经菌液PCR筛选和酶切鉴定后,用BamH I和EcoR I将目的片段切下,再克隆到原核表达载体pRSET A中构建重组表达质粒pRSET A-TNNC2。将重组质粒转化大肠杆菌BL21（DE3）,IPTG不同诱导条件诱导表达,经SDS-PAGE电泳和Western blot分析证实重组表达质粒pRSET A-TNNC2表达出24 kD左右的融合蛋白,最佳诱导时间为4 h,最佳的IPTG诱导浓度为0.6 mmol/L,表达产物以可溶性蛋白的形式存在。     

猪圆环病毒2型(PCV2)河南分离株进化分析及ORF2基因的表达

根据GenBank中猪圆环病毒2型(Porcine circovirus2,PCV2)的核苷酸序列设计2对引物,使用扩增全基因的引物对来源于河南省焦作、开封和滑县的3株PCV 2型JZ株、KF株和HX株的ORF2基因进行扩增,扩增片段克隆到pMD18 T上,获得重组质粒pMD18-T-ORF2,并对其进行测序,测序结果登录在GenBank上,登录号分别为EF028202、EF064149和EF467928.序列分析表明,PCV2河南分离株ORF2基因与其它PCV2 ORF2基因核苷酸序列同源性为92.4%～99.6%,氨基酸序列同源性为90.6%～97.9%,进化分析表明,河南分离株处于同一分支,与欧洲株亲缘关系较近.应用另一对引物从重组质粒pMD18-T-ORF2中PCR扩增出587 bp不包含核定位信号序列的ORF2基因,克隆到表达载体pET-32a,成功构建了重组质粒pET-32a-ORF2,经IPTG诱导表达了ORF2基因编码的结构蛋白,经SDS-PAGE和Western blotting检测,表达的重组蛋白分子量约为40 kD,可被PCV2阳性血清识别,说明PCV2 ORF2基因得到成功表达.     

Straw amendments did not induce high N2O emissions in non-frozen wintertime conditions: A study in northern Germany

An increasing area of oilseed rape cultivation in Europe is used to produce biodiesel. However, a large amount of straw residue is often left in the field in autumn. Straw mineralization provides both carbon (C) and nitrogen (N) sources for emission of soil nitrous oxide (N₂O), which is an important greenhouse gas with a high warming potential. Some studies have focused on soil N₂O emissions immediately post-harvest; however, straw mineralization could possibly last over winter. Most field studies in winter have focused on freeze-thaw cycles. It is still not clear how straw mineralization affects soil N₂O emissions in unfrozen wintertime conditions. We carried out a field experiment in northern Germany in winter 2014, adding straw and glucose as a source of C with three rates of N fertilizer (0, 30, and 60 kg N ha⁻¹). During the 26 days of observation, cumulative N₂O emission in treatments without C addition was negative at all N fertilizer levels. Straw addition produced –3.2, 11.2, and 5.0 mg N₂O-N m⁻² at 0, 30, and 60 kg N ha⁻¹, respectively. Addition of glucose surprisingly caused –1.5, 74.6, and 165 mg N₂O–N m⁻² at 0, 30, and 60 kg N ha⁻¹, respectively. This study demonstrates that oilseed rape straw does not cause high N₂O emissions in wintertime when no extreme precipitation or freeze-thaw cycles are involved, and soil organic C content is low. However, N₂O emission could be intensively stimulated, when both easily available organic C and nitrate are not limited and the soil temperature between 0 and 10°C. These results provide useful information on potential changes to N₂O emissions that may occur due to the increased use of oilseed rape for biodiesel combined with less severe winters in the northern hemisphere driven by global warming.     

鸡白介素-2基因在大肠杆菌中表达及多克隆抗体制备*

将编码鸡(Gallus gallus)白细胞介素-2(interleukin-2,IL-2)蛋白的基因亚克隆到大肠杆菌(Eschetichia coli)原核表达载体pPROEX^HT中，构建重组质粒并进行确证性序列测定。然后将重组质粒转化大肠杆菌DH5α并用IFTG于37℃诱导培养。SDS-PAGE和Western blot分析显示，表达的鸡IL-2融合蛋白分子量约为19kD。融合蛋白经薄层扫描发现目的蛋白表达量约占菌体蛋白的30％。包涵体被6mol／L，盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IL-2融合蛋白及其纯化产物免疫家兔，制备兔抗鸡IL-2多克隆抗血清，并用琼脂扩散实验对多克隆抗血清进行鉴定，表明其与纯化的重组鸡IL-2蛋白具有良好的反应性，而且该反应是特异的。     

猪圆环病毒2型ORF2基因片段的原核表达及间接ELISA诊断方法的初步建立

根据猪圆环病毒2型（PCV2）LC株序列,设计合成3对引物,通过PCR扩增PCV2的3个不同ORF2基因片段,分别将其克隆到pET-32a载体中,将重组表达质粒转化大肠杆菌BL21,表达并纯化了重组蛋白。Western-blot分析表明,重组蛋白可以与PCV2阳性血清反应,表明该蛋白具有良好的抗原性。以纯化的重组蛋白初步建立了间接酶联免疫吸附实验（ELISA）方法,经方阵滴定确定最佳包被浓度为0．24μg／mL,血清最佳的稀释度为1：40。     

Shrub encroachment does not reduce the activity of some soil enzymes in Mediterranean semiarid grasslands

Shrub encroachment is a worldwide phenomenon with implications for desertification and global change. We evaluated its effects on the activities of urease, phosphatase and β-glucosidase in Mediterranean semiarid grasslands dominated by Stipa tenacissima by sampling 12 sites with and without resprouting shrubs along a climatic gradient. The presence of shrubs affected the evaluated enzymes at different spatial scales. Soils under S. tenacissima tussocks and in bare ground areas devoid of vascular plants had higher values of phosphatase and urease when the shrubs were present. For the β-glucosidase, this effect was site-specific. At the scale of whole plots (30 m × 30 m), shrubs increased soil enzyme activities between 2% (β-glucosidase) and 22% (urease), albeit these differences were significant only in the later case. Our results indicate that shrub encroachment does not reduce the activity of extracellular soil enzymes in S. tenacissima grasslands.     

Increased microbial activity under elevated [CO2] does not enhance residue decomposition in a semi-arid cropping system in Australia

The association between the responses of microbial activity and residue decomposition to elevated atmospheric [CO₂] under field conditions in Australian cropping systems is unknown. We measured soil CO₂ emission and decomposition of wheat and field pea residues in a wheat cropping system in the field using the Australian Grains Free-Air CO₂ Enrichment (AGFACE) facility in Horsham, Victoria. Elevated [CO₂] (550 μmol mol⁻¹) increased soil CO₂ emission by 41%, but did not affect the percentage of the original mass or C remaining for either type of residue throughout the experimental period. Our findings suggest that the rates of residue decomposition and residue C mineralization in this semi-arid wheat cropping system were not affected by elevated [CO₂] despite higher microbial activity. This has major implication for the C sequestration potential of semi-arid cropping systems under future CO₂ climates.     

Phosphatase activity does not limit the microbial use of low molecular weight organic-P substrates in soil

Plant roots and soil microorganisms contain significant quantities of low molecular weight (MW) phosphorylated nucleosides and sugars. Consequently, upon death these can represent a significant input of organic-P to the soil. Some of these organic-P substrates must first be dephosphorylated by phosphatases before being assimilated by the soil microbial community while others can be taken up directly from soil solution. To determine whether sorption or phosphatase activity was limiting the bioavailability of low MW organic-P in soil we compared the microbial uptake and C mineralization of a range of ¹⁴C-labeled organic-P substrates [glucose-6-phosphate, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP)] to that of the parent compounds (adenosine and glucose). In a fertile grassland soil we showed that at low organic-P substrate concentrations (<0.5 mM) phosphatase activity did not limit microbial uptake or mineralization in comparison to their non-phosphorylated counterparts. However, at high substrate concentrations (1-10 mM) the mineralization of the organic-P compounds was significantly lower than that of the non-phosphorylated compounds suggesting that phosphatase activity or microbial transporter capacity limited bioavailability. Sorption to the solid phase followed the series glucose<adenosine<G-6-P<AMP<ADP=ATP. However, sorption of the organic-P compounds to the solid phase did not appear to greatly affect bioavailability. The high adenosine mineralization capacity of the microbial biomass suggests that nucleosides may represent a significant source of C and N to the soil microbial biomass. We conclude that at low organic-P substrate concentrations typical of those in soil, neither phosphatase activity nor sorption greatly limits their bioavailability.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Combined application of organic manure with urea does not alter the dominant biochemical pathway producing N2O from urea treated soil

Biology and Fertility of Soils - Combined application of organic amendment with synthetic fertilizer is an emerging management technique for maximized agronomic benefits without drastic soil health...     

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.