Development of an enzyme assay for rapid assessment of Escherichia coli in seawaters

AIMS: An analytical protocol has been developed and applied for the detection of glucuronidase activity in marine waters as a rapid alternative approach to assess the microbiological quality of seawaters. METHODS AND RESULTS: The fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide is cleaved to a fluorescent product, methylumbelliferone, by the enzyme beta-glucuronidase, specific to Escherichia coli and closely related enterobacterial species (Shigella). The results suggest that this test is related to E. coli numbers, as estimated by immunofluorescence, more significantly than to faecal coliform numbers, obtained from culture media. CONCLUSIONS: The determination of the potential rate of glucuronidase activity may be used as a diagnostic tool for the indirect estimation of the presence of E. coli in seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be particularly useful in the early warning of seawater pollution, allowing the screening of coastal areas with different contamination levels in reduced time.     

Growth and in situ detection of a pathogenic Escherichia coli in biofilms of a heterotrophic water-bacterium by use of 16S- and 23S-rRNA- directed fluorescent oligonucleotide probes

Abstract The colonization of biofilms of a benzoate-degrading Gram-positive water bacterium, strain B4, by a pathogenic Escherichia coli was studied in a continuous flow reactor. E. coli added to a fixed bed reactor colonized by B4, was able to grow in the biofilms and subsequently re-enter the free water phase in high numbers. Mixed biofilms of strain B4 and E. coli were also grown on glass slides for detailed examination of the spatial order of the mixed population biofilm. Individual cells as well as microcolonies of E. coli were detected in the biofilms by hybridization with a fluorescently labeled 23S rRNA-targeted oligonucleotide probe. The spatial distribution of E. coli could be analyzed in all layers of even thick biofilms.     

Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81_kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.     

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.     

Escherichia coli detection using mTEC agar and fluorescent antibody direct viable counting on coastal recreational water samples

Aims:  Escherichia coli is the faecal indicator species recommended by the US Environmental Protection Agency (USEPA) for monitoring fresh recreational water. Viable but nonculturable (VBNC) E. coli are living cells that are dormant and not culturable using standard microbiological cultivation methods. This study reports a comparison between the mTEC culture method recommended by USEPA for E. coli enumeration and a fluorescent antibody-direct viable count (FA-DVC) method to visualize living E. coli cells with a microscope.
Methods and Results:  Escherichia coli , faecal coliforms and Enterococcus were detected using standard methods recommended by the USEPA. VBNC E. coli was visualized with FA-DVC. Results were analysed with standard statistical methods (Pearson correlation; paired-sample t -test). Significantly higher numbers of E. coli were detected using the FA-DVC method than using the mTEC method. Escherichia coli results were also compared with faecal coliform (mFC broth) and Enterococcus (mEI agar) counts in the same samples.
Conclusions:  The results of this comparative study demonstrate that E. coli can be present in higher numbers than what are detected with standard culture methods.
Significance and Impact of the Study:  This study re-emphasizes the need for a rapid, accurate and precise method for detecting health risks to humans who use recreational waters.     

重组基因表达对大肠杆菌生理的影响

重组基因在表达外源蛋白质时常常会耗用大量的宿主细胞资源,从而对宿主造成代谢负荷,代谢负荷使得宿主的生化和生理产生很大的变化,甚至损害宿主正常的代谢功能。而过重的代谢负荷会影响目标蛋白的表达量和表达质量。综述了产生代谢负荷的原因,宿主细胞对代谢负荷的应激反应、以及减轻代谢负荷的策略。     

Influence of DNA supercoiling on the loss of culturability of Escherichia coli cells incubated in seawater

The relationship between the loss of culturability of Escherichia coli cells in seawater and the DNA supercoiling level of a reporter plasmid (pUC8) have been studied under different experimental conditions. Transfer to seawater of cells grown at low osmolarity decreased their ability to grow without apparent modification of the plasmid supercoiling. We found that E. coli cells could be protected against seawater-induced loss of culturability by increasing their DNA-negative supercoiling in response to environmental factors: either a growth at high osmolarity before the transfer to seawater, or addition of organic matter (50-mg/l peptone) in seawater. We further found conditions where a DNA-induced relaxation was accompanied by an increase in seawater sensitivity. Indeed, inactivation of either one of the subunits A and B of DNA gyrase, which leads to important DNA relaxation, was accompanied in both cases by an increased loss of culturability of conditional mutants after transfer to seawater which could not be explained uniquely by the increase in the temperature required to inactivate the gyrase. Similarly, a strain harbouring a mutation in topoisomerase I, compensated by another mutation in subunit B of the gyrase, was more sensitive to seawater than the isogenic wild-type cell and this greater sensitivity was correlated to a relaxation of plasmid DNA. Again, in these different cases, a previous growth at high osmolarity protected against this seawater sensitivity. We thus propose that the ability of E. coli cells to survive in seawater and maintain their ability to grow on culture media could be linked, at least in part, to the topological state of their DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种用质粒DNA转化大肠杆菌感受态细胞的实用操作技巧

目的是建立一种简化、实用的用质粒DNA转化大肠杆菌的操作方法.采用氯化钙法制备大肠杆菌感受态细胞.以质粒pUC18,pCSN44,pAN52-1Not,pETts,pANth和植物双元表达栽体pCAMBIA1301分别转化用于质粒扩增与保存的常用大肠杆菌菌株Top10和DH5α以及用于原核表达的常用大肠杆菌菌株BL21(DE3)和TB1.质粒与感受态细胞的混合液置冰上作用一定时间后,直接涂布含有筛选抗生素的LB平板,于37℃培养12～16h.结果表明,用不同大小的质粒DNA转化不同的大肠杆菌菌株,都可以获得满足实验要求,转化效率可高达103～4阳性克隆/μg.该方法较标准的转化流程更加简便、省时、实用.     

抗菌肽OH-CATH对大肠杆菌Escherichia coli的作用(英文）

OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽.它在1%NaCI存在的条件下对多种细菌都有较强的抗菌活性,同时,在高浓度下对人红细胞无溶血活性.OH-CATH足开发新型抗菌药物的优良模板.蜊明OH-CATH的作用机理及其对微生物的选择性,对研发以OH-CATH为先导结构的药物研发有十分重要的意义.本文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌一大肠杆菌ATCC 25922相互作用的效应研究.结果揭示:OH-CATH对大肠杆菌的作用涉及到3个步骤.首先,OH-CATH借助其带正电的氨基酸残基附着到细菌带负电荷的细胞壁:然后,附着的OH-CATH在达剑一定浓度后发生聚集,以孔道彤成的方式破坏细菌的膜结构;最终,由十细菌膜的损坏,膜的渗透性被破坏,胞内内含物释放造成细菌死亡.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

大肠杆菌染色体上严谨型启动子的构建

【背景】启动子的渗漏表达是代谢工程和合成生物学较为关注的问题,探索严谨型启动子使之能像开关一样控制基因的表达有助于解决这一问题。【目的】为避免在质粒上研究启动子带来的弊端,本研究将在染色体上对严谨型启动子进行构建和评价。【方法】基于4种调控元件四环素tetO、乳糖lacO、阿拉伯糖araC和鼠李糖rhaR的序列,以及2种来源的启动子PL和Plac序列,设计和组合构建了6个启动子PtetO2、PtetO3、PlacO2、PlacO3、PlacO+ara和PlacO+rha。应用CRISPR/Cas9系统将这6个启动子序列整合到大肠杆菌ATCC 8739染色体上,利用绿色荧光蛋白(Green fluorescent protein,GFP)的表达,分析这6个启动子的相对表达强度和严谨型控制情况。【结果】GFP表达分析显示,启动子PlacO+rha为最佳严谨型启动子,在无诱导剂时表达为0.02,有诱导剂时最大表达强度为lac Z基因启动子的12倍,相对控制范围为600倍。【结论】研究结果将为代谢工程和合成生物学中的精确调控基因表达奠定良好的应用基础。     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Identification of cognate ligands for the Escherichia coli phnD protein product and engineering of a reagentless fluorescent biosensor for phosphonates

The Escherichia coli phnD gene is hypothesized to code for the periplasmic binding component of a phosphonate uptake system. Here we report the characterization of the phosphonate-binding properties of the phnD protein product. We find that PhnD exhibits high affinity for 2-aminoethylphosphonate (5 nM), the most commonly occurring natural phosphonate produced by lower eukaryotes, but also binds several other phosphonates with micromolar affinities. A significant number of man-made phosphonates, such as insecticides and chemical warfare agents, are chemical threats and environmental pollutants. Consequently, there is an interest in developing methods for the detection and bioremediation of phosphonates. Bacterial periplasmic-binding proteins have been utilized for developing reagentless biosensors that report analytes by coupling ligand-binding events to changes in the emission properties of a covalently conjugated environmentally-sensitive fluorophore. Several PhnD conjugates described here show large changes in fluorescence upon binding to methylphosphonate (MP), with two conjugates exhibiting up to 50% decrease in emission intensity. Since MP is the final degradation product of many nerve agents, these PhnD conjugates can function as components in a biosensor system for chemical warfare agents.     

Rapid assessment of physiological status in Escherichia coli using fluorescent probes

Rapid and direct viability assessment of Escherichia coli in filtered, sterile lake water was possible using multiparameter flow cytometry. Fluorescent dyes were used as probes for different cellular functions (membrane potential, membrane integrity and intracellular enzyme activity), which were correlated with the ability of the cells to respond to nutrient addition while in a stressed state. Measurement of several criteria circumvented limitations imposed by other methods, and provided extensive evidence for the validity of the methods for monitoring cell viability during adoption of a viable-but-non-culturable state in starved E, coli. Macromolecular staining was concomitantly used to monitor changes in cellular protein, RNA and DNA as additional indicators of physiological status during starvation/stress.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Allelic subtyping of the intimin locus (eae) of pathogenic Escherichia coli by fluorescent RFLP

Intimin is a highly polymorphic protein encoded by the eae gene and plays a crucial role in the attaching-effacing phenotype of diarrheagenic Escherichia coli and related pathogens. We have developed a method to quickly and accurately uncover allelic variation at the eae locus through the use of fluorescent RFLP (fRFLP). Application of fRFLP to 151 eae -positive strains (including the newly described Escherichia albertii ) revealed 26 different fRFLP types that correspond to 20 of the 28 previously described eae alleles. Two sequence variants of the γ, ι, κ, and ζ alleles and three variants of ɛ were also observed. In addition to being reliable and accurate, the method can be easily adapted to accommodate new eae allelic sequences, as they become known.     

Elevation of rat intestinal permeability by enterotoxigenic Escherichia coli in comparison to non-toxigenic E. coli and Salmonella typhimurium. Application of fluorescent dextran 3000 as permeability probe

Abstract The effect of bacterial enterotoxins on rat intestinal permeability properties was studied by comparing the effect of toxin-positive and toxin-negative Escherichia coli and Salmonella typhimurium inoculated into a segment of rat small intestine. Fluoresceinated dextran 3000 (FITC-D3; M _r 3000) was applied as permeability marker. The E. coli strain C922a-1 producing heat-labile (LT) and heat-stable (ST) enterotoxins and colonising factor CFA/II increased the transmural passage of the dextran probe into portal blood. In contrast, its plasmid-negative variant, a non-toxin producer lacking CFA, caused permeability changes indistinguishable from the bacteria-free nutrient broth control. Another pair of enterotoxigenic E. coli strains, 1628–14 (LT⁺, ST⁺, CFA/I⁺) and 1628–15 (LT⁺, ST⁻ and CFA/I⁻) both increased the intestinal permeability. The observations indicate that the LT⁺-only E. coli strain 1628–15 has the ability to promote permeability of rat intestine. The toxin-negative, rough S. typhimurium 395MR10 bacteria had a very small effect on the permeability, which was also achieved with culture filtrate only.
It is concluded that enterotoxigenic E. coli (ETEC) can alter the properties of the mucosal barrier towards intermediate-sized molecules that could be of antigenic significance, or which could play a crucial role in the nutritional status of the host organism.