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1.
Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time‐consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix‐assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home‐made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water). Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

2.
Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.  相似文献   

3.
《Electrophoresis》2018,39(4):645-652
Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram‐negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram‐negative pathogens and also validated whether our system can identify Gram‐negative pathogens with the cell‐free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram‐negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX‐M group 1 to identify the ESBL producing Gram‐negative pathogens. All six target‐specific peaks were clearly separated without any non‐specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram‐negative clinical isolates, all of them were successfully identified without any non‐specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis‐causing, drug‐resistant Gram‐negative pathogens and also the major ESBL producing Gram‐negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram‐negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.  相似文献   

4.
There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.  相似文献   

5.
Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 106 to 109 and ca. 107 to 109 cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.  相似文献   

6.
Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1–10 μm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.  相似文献   

7.
L. J. Yu  S. F. Y. Li 《Chromatographia》2005,62(7-8):401-407
Capillary electrophoresis approaches have been utilized for the study of bacteria under specific experimental conditions. The main objective within our research work was to study electrophoretic behaviors of Pseudomonas aeruginosa by means of capillary electrophoresis with UV and fluorescence detection. Edwardsiella tarda and Enteropathogenic escherichia coli were also included in the study. The results showed that proper pretreatment (vortexing or sonication) for each bacterial sample before injection was necessary to disperse the clusters of cells, which is helpful to observe the single peaks and better peak shape of bacteria during electrophoresis. Apart from this, it was found that ionic strength of buffer affected mobilities of Pseudomonas aeruginosa as a result of increasing of buffer concentration from 25 mM to 150 mM. Moreover, sharp and single peaks were still observed without significant increase of noise in the concentration range. Eventually, mixtures of bacteria were well separated under optimized separation conditions with UV and fluorescence detection. In the mean time, comparison of concentration sensitivities for Pseudomonas aeruginosa by UV and fluorescence detection was made. Blue light emitting diode induced fluorescence detection was found to be more sensitive (8.5-fold higher) than UV detection with home-made fluorescence detection system. Generally, proposed CE methods for the analysis of bacteria could be potentially valuable for the monitoring of bacteria contamination in real life.  相似文献   

8.
The aim of this study was to investigate phenolic acids and flavonoids in methanolic, dichloromethane, acetone and ethyl acetate extracts and fractions from inflorescences of Cirsium canum (L.). RP-HPLC analysis enabled identification of the following: chlorogenic acid, caffeic acid, p-coumaric acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, trans-cinnamic acid, luteolin-7-glucoside, apigenin-7-glucoside, kaempferol-3-glucoside, linarin, apigenin, rutoside, luteolin and kaempferol. The antimicrobial activity of tested extracts was determined in vitro against reference microorganisms, including bacteria or fungi, belonging to yeasts. Our data showed that the tested extracts had no influence on the growth of the reference strains of Gram-negative bacteria and yeasts belonging to Candida spp. Among them, the fractions possessed the highest activity against Gram-positive bacteria, especially Streptococcus aureus and Streptococcus pneumoniae belonging to pathogens and Streptococcus epidermidis, Bacillus cereus and Bacillus subtilis belonging to opportunistic microorganisms.  相似文献   

9.
Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.  相似文献   

10.
Diseases of the lung, e. g. chronic obstructive pulmonary disease (COPD), interstitial lung diseases, bronchiectasis or cystic fibrosis, often lead to recurrent severe respiratory infections that cause exacerbations of the underlying disease. These acute or chronic inflammatory processes can result in a progressive destruction of the lung and in an ongoing decline in lung function. Therefore longer inpatient stays for intravenous antibiotic treatment are necessary and the quality of life in these patients is severely limited. A rapid detection of infectious agents in human lungs is often crucial, because the choice of the appropriate therapeutic regime depends at first on the identification of the infecting species. Standard methods for detection and identification are either time consuming, of low sensitivity or expensive. It is known that bacteria, and also mitosporic fungi, produce volatile organic compounds (VOCs) that can be detected in exhaled breath by ion mobility spectrometry (IMS), were a distinct detection of a specific VOC is related to a “peak”. We investigated, whether the detection and characterisation of VOCs by Multi-capillary column coupled to IMS in exhaled breath of patients whose airways are either infected or colonized by Pseudomonas aeruginosa compared to healthy non-smoker controls is capable of identifying those infectious agents. To realize a non invasive identification of pathogens, the exhaled breath of 53 persons (24 patients suffering chronic or infectious on Pseudomonas and 29 healthy controls) was investigated using an ion mobility spectrometer type BioScout. In total 224 different signals were found. Actually, 21 different signals are able to differentiate the two groups, Control and Pseudomonas, with rank sum values less than 0.2. For all 224 signals Box-and-Wisker plots were realized. The peaks with the lowest rank sum values F (0,107) and PS0 (0,112) show rather good separation of both groups. Our preliminary results demonstrate that distinct patterns of a small number of IMS-peaks are sufficient for the identification of these infectious agents. Therefore MCC-IMS seems to be a promising method for the non-invasive identification of patients which are colonized or infected with bacteria such as Pseudomonas aeruginosa.  相似文献   

11.
The reliable detection of pathogenic bacteria in complex biological samples using simple assays or devices remains a major challenge. Herein, we report a simple colorimetric paper device capable of providing specific and sensitive detection of Helicobacter pylori (H. pylori), a pathogen strongly linked to gastric carcinoma, gastric ulcers, and duodenal ulcers, in stool samples. The sensor molecule, an RNA‐cleaving DNAzyme obtained through in vitro selection, is activated by a protein biomarker from H. pylori. The colorimetric paper sensor, designed on the basis of the RNA‐cleaving property of the DNAzyme, is capable of sensitive detection of H. pylori in human stool samples with minimal sample processing and provides results in minutes. It remains fully functional under storage at ambient temperature for at least 130 days. This work lays a foundation for developing DNAzyme‐enabled paper‐based point‐of‐care diagnostic devices for monitoring pathogens in complex samples.  相似文献   

12.
The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 103 cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D2O are also reported. Figure Light scattering apparatus used to detect bacteria  相似文献   

13.
Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin‐layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix‐assisted laser desorption/ionization orthogonal time‐of‐flight mass spectrometry (IR‐MALDI‐o‐TOF‐MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL‐specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria‐specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time‐consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo‐series neutral GSLs recognized by P‐fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR‐MALDI‐o‐TOF‐MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate‐specific pathogens involved in human infectious diseases. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

14.
In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.  相似文献   

15.
A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO2) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO2 were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO2, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 103 CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 103 CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.  相似文献   

16.
Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2–6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy‐dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600‐fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point‐of‐care diagnostics.  相似文献   

17.
Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi‐preparative high‐speed counter‐current chromatography in a highly polar solvent system: propan‐1‐ol/acetonitrile/(NH4)2SO4satd. soln/H2O (1.0:0.5:1.2:1.0, v/v/v/v) in tail‐to‐head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high‐resolution ion‐trap time‐of‐flight mass spectrometry detection technique for the molecular formulae and multi‐step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6′‐O‐formyl‐amaranthin, and 6′‐O‐malonyl‐amaranthin as well as their diastereomeric forms differing in the configuration of the C‐15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter‐current chromatographic and high‐performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high‐speed counter‐current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6′‐O‐formyl‐amaranthin and 6′‐O‐malonyl‐amaranthin was possible. Different elution profiles of the pigments observed in the counter‐current chromatographic system in comparison to high‐performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.  相似文献   

18.
Carotenoid complex from Deinococcus radiodurans bacteria, highly resistant to ionizing radiation, is of interest from the viewpoint of effective radioprotective drugs development. In the present study a simple procedure for separation and mass spectrometric analysis of D. radiodurans carotenoid complex is described. Other compounds, molecular weight of which according to mass spectrometry, corresponds to phytoene and astaxanthin are revealed in bacterial extracts as well as three more carotenoids, characterized by m/z of protonated molecules 521, 553 and 569, whose identification demands specification. It is shown that the compound with molecular mass 564 Da, that was recognized earlier as an artifact, appeared to be actually present in the bacteria extracts.  相似文献   

19.
Animal venoms are complex mixtures of more than 100 different compounds, including peptides, proteins, and nonprotein compounds such as lipids, carbohydrates, and metal ions. In addition, the existing compounds show a wide range of molecular weights and concentrations within these venoms, making separation and purification procedures quite tedious. Here, we analyzed for the first time by MS the advantages of using the OFFGEL technique in the separation of the venom components of the Egyptian Elapidae Walterinnesia aegyptia snake compared to two classical methods of separation, SEC and RP‐HPLC. We demonstrate that OFFGEL separates venom components over a larger scale of fractions, preserve respectable resolution with regard to the presence of a given compound in adjacent fractions and allows the identification of a greater number of ions by MS (102 over 134 total ions). We also conclude that applying several separating techniques (SEC and RP‐HPLC in addition to OFFGEL) provides complementary results in terms of ion detection (21 more for SEC and 22 more with RP‐HPLC). As a result, we provide a complete list of 134 ions present in the venom of W. aegyptia by using all these techniques combined.  相似文献   

20.
Salmonella bacteria are the major cause for the infection of 16 million people worldwide with typhoid fever each year. Antibiotic‐resistant Salmonella strains have been isolated from various food products. As a result, the development of ultrasensitive sensing technology for detection and new approaches for the treatment of infectious bacterial pathogens that do not rely on traditional therapeutic regimes is very urgent for public health, food safety, and the world economy. Driven by this need, we report herein a nanotechnology‐driven approach that uses antibody‐conjugated oval‐shaped gold nanoparticles to selectively target and destroy pathogenic bacteria. Our experiments have shown the use of a simple colorimetric assay, with an anti‐salmonella antibody conjugated to oval‐shaped gold nanoparticles, for the label‐free detection of S. typhimurium with an excellent detection limit (104 bacteria per mL) and high selectivity over other pathogens. When bacteria conjugated to oval‐shaped gold nanoparticles were exposed to near‐infrared radiation, a highly significant reduction in bacterial cell viability was observed due to photothermal lysis. Ideally, this nanotechnology‐based assay would have enormous potential for rapid, on‐site pathogen detection to avoid the distribution of contaminated foods.  相似文献   

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