Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli

A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20 nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1 h).     

Selective immobilization of Acinetobacter junii on the natural zeolitized tuff in municipal wastewater

The immobilization of desired bacteria onto material was usually performed in synthetic media. The aim of this study was to test the immobilization of phosphate (P)-accumulating bacteria Acinetobacter junii onto natural zeolitized tuff (NZ) in the raw or sterilized municipal wastewater containing the common bacteria Escherichia coli and Enterococcus faecalis and the performance of immobilized A. junii in the same type of wastewater. In the sterilized wastewater which contained the mixture of A. junii, E. coli and E. faecalis, the A. junii was selectively immobilized onto NZ in significantly higher numbers than E. coli and E. faecalis. The A. junii added in the form of bioparticles to the wastewater containing E. coli and E. faecalis, multiplied and removed P from wastewater. The P removal from wastewater was a function of biomass of P-accumulating bacteria and not the amount of NZ or bioparticles used. The performance of A. junii was significantly better in membrane filtered than in autoclaved wastewater. The experiments that were performed in raw non sterilized wastewater showed that A. junii can be successfully immobilized onto NZ in competition with natively present heterotrophic bacteria, retain its metabolic activity and successfully remove P from such water, which makes this technology feasible from biotechnological aspect.     

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

N-Methylimidazolium modified magnetic particles-assisted highly sensitive Escherichia coli detection based on polymerase chain reaction and capillary electrophoresis

Effective bacteria detection and quantification are essential prerequisite for the prevention and treatment of infectious diseases. Herein, we report a method for the detection and quantification of Escherichia coli (E. coli).N-Methylimidazolium modified magnetic particles (MIm-MPs) are synthesized successfully and used as an efficient magnetic material for the isolation and concentration of E. coli. The factors including pH of binding buffer, concentration of elution buffer and elution time which may affect the capture and elution efficiencies are optimized. The linear correlation between bacteria concentration and peak area of polymerase chain reaction (PCR) product analyzed by capillary electrophoresis (CE) is determined. Rapid preconcentration of trace amount of E. coli (10¹ cfu mL⁻¹) in large volume of aqueous sample (500 mL) is achieved, and the capture efficiency can reach 99%. The quantification of bacteria in large volume of spiked tap water and mineral water samples is realized. The recoveries for different concentrations of E. coli in tap and mineral water samples are in the range between 83% and 93%. The results demonstrate that this MIm-MPs-PCR-CE method can be applied to detect and quantify bacteria in real samples.     

Rapid detection of Escherichia coli contamination in packaged fresh spinach using hyperspectral imaging

A rapid method based on hyperspectral imaging for detection of Escherichia coli contamination in fresh vegetable was developed. E. coli K12 was inoculated into spinach with different initial concentrations. Samples were analyzed using a colony count and a hyperspectroscopic technique. A hyperspectral camera of 400-1000 nm, with a spectral resolution of 5 nm was employed to acquire hyperspectral images of packaged spinach. Reflectance spectra were obtained from various positions on the sample surface and pretreated using Sawitzky-Golay. Chemometrics including principal component analysis (PCA) and artificial neural network (ANN) were then used to analyze the pre-processed data. The PCA was implemented to remove redundant information of the hyperspectral data. The ANN was trained using Bayesian regularization and was capable of correlating hyperspectral data with number of E. coli. Once trained, the ANN was also used to construct a prediction map of all pixel spectra of an image to display the number of E. coli in the sample. The prediction map allowed a rapid and easy interpretation of the hyperspectral data. The results suggested that incorporation of hyperspectral imaging with chemometrics provided a rapid and innovative approach for the detection of E. coli contamination in packaged fresh spinach.     

Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir

Biosurfactant produced by Pseudomonas aeruginosa, Bacillus subtilis and Rhodococcus erythropolis that isolated from the formation water of Chinese petroleum reservoir has been compared in surface abilities and oil recovery. Maximum biosurfactant production reached to about 2.66 g/l and the surface tension of liquid decreased from 71.2 to 22.56 mN/m using P. aeruginosa. Three strains exhibited a good ability to emulsify the crude oil, and biosurfactant of P. aeruginosa attained an emulsion index of 80% for crude oil which was greater than other strains. Stability studies were carried out under the extreme environmental conditions, such as high temperature, pH, salinity and metal ions. Results showed an excellent resistance of all biosurfactants to retain their surface-active properties at extreme conditions. It was found that the biosurfactants from three isolated bacteria showed a good stability above pH of 5, but at lower pH (from 1 to 5) they will harmfully be affected. They were able to support the condition up to 20 g/l salinity. P. aeruginosa biosurfactant was even stable at the higher salinity. Regarding temperature, all produced biosurfactants demonstrated a good stability in the temperature up to 120 °C. But stability of three biosurfactants was affected by monovalent and trivalent ions. Oil recovery experiments in physical simulation showed 7.2-14.3% recovery of residual oil after water flooding when the biosurfactant of three strains was added. These results suggest that biosurfactants of these indigenous isolated strains are appropriate candidates for enhanced oil recovery with a preference to biosurfactant of P. aeruginosa.     

Evaluation of Anthocyanin Content,Antioxidant Potential and Antimicrobial Activity of Black,Purple and Blue Colored Wheat Flour and Wheat-Grass Juice against Common Human Pathogens

The present study aimed to analyze the antioxidant and antimicrobial activity of anthocyanins extracted from colored wheat flour and wheat-grass juice against human pathogens. The total anthocyanin content and antioxidant potential in colored wheat flour and wheat-grass juice extracts were significantly higher than white flour and wheat-grass juice extracts. Ultra-performance liquid chromatography showed the maximum number of anthocyanin peaks in black wheat, with delphinidin-3-o-galactoside chloride, delphinidin-3-o-glucoside chloride, and cyanindin-3-o-glucoside chloride as the major contributors. Among flour extracts, maximum zones of inhibition against Staphylococcus aureus (MTCC 1934), Pseudomonas aeruginosa (MTCC 1434), Escherichia coli, and Candida albicans (MTCC 227) were produced by black flour extract, having the highest anthocyanin content. It exhibited a minimum microbicidal concentration (MMC) of 200 mg/mL against E. coli and C. albicans; and 100 and 150 mg/mL against S. aureus and P. aeruginosa, respectively. Black and purple flour extracts exhibited a minimum inhibitory concentration (MIC) of 50 mg/mL against S. aureus and P. aeruginosa. White flour extracts did not show MMC against E. coli and C. albicans. Among wheat-grass juice extracts, black wheat-grass was most effective and showed an MIC of 100–150 mg/mL against all pathogens. It exhibited an MMC of 200 mg/mL against S. aureus and P. aeruginosa. Hence, anthocyanin-rich colored wheat could be of nutraceutical importance.     

Miniature biochip system for detection of Escherichia coli O157:H7 based on antibody-immobilized capillary reactors and enzyme-linked immunosorbent assay

In this work, we report Escherichia coli O157:H7 detection using antibody-immobilized capillary reactors, enzyme-linked immunosorbent assay (ELISA), and a biochip system. ELISA selective immunological method to detect pathogenic bacteria. ELISA is also directly adaptable to a miniature biochip system that utilizes conventional sample platforms such as polymer membranes and glass. The antibody-immobilized capillary reactor is a very attractive sample platform for ELISA because of its low cost, compactness, reuse, and ease of regeneration. Moreover, an array of capillary reactors can provide high-throughput ELISA. In this report, we describe the use of an array of antibody-immobilized capillary reactors for multiplex detection of E. coli O157:H7 in our miniature biochip system. Side-entry laser beam irradiation to an array of capillary reactors contributes significantly to miniaturized optical configuration for this biochip system. The detection limits of E. coli O157:H7 using the ELISA and Cy5 label-based immunoassays were determined to be 3 and 230 cells, respectively. This system shows capability to simultaneously monitor multifunctional immunoassay and high sensitive detection of E. coli O157:H7.     

Chemical Constitution and Antimicrobial Activity of Kombucha Fermented Beverage

Kombucha is a traditional beverage of sweetened black tea fermented with a symbiotic association of acetic acid bacteria and yeasts. In this study, kombucha fermented beverage (KFB) appeared to include nine chemical groups (alcohols, acids, lactones, condensed heterocyclic compounds, antibiotics, esters, aldehydes, fatty acids, and alkaloids) of many bioactive metabolites, as elucidated by gas chromatography–mass spectrometry (GC-MS) and IR spectra. The fermented metabolic components of KFB seem collectively to act in a synergistic action giving rise to the antimicrobial activity. Four types of kombucha preparations (fermented, neutralized, heat-treated and unfermented) were demonstrated with respect to their antimicrobial activity against some pathogenic bacterial and fungal strains using agar well diffusion assay. KFB exerted the strongest antimicrobial activities when compared with neutralized and heat-treated kombucha beverages (NKB and HKB). Staphylococcus aureus ATCC6538 (S. aureus) and Escherichia coli ATCC11229 (E. coli) were the organisms most susceptible to the antimicrobial activity of kombucha beverage preparations. Finally, the KFB preparation showed remarkable inhibitory activity against S. aureus and E. coli bacteria in a brain heart infusion broth and in some Egyptian fruit juices (apple, guava, strawberry, and tomato). These data reveal that kombucha is not only a prophylactic agent, but also appears to be promising as a safe alternative biopreservative, offering protection against pathogenic bacteria and fungi.     

Coupling chemometrics and electrochemical-based sensor for detection of bacterial population

This work describes a home-made microelectrode array, based on reticulated vitreous carbon, which has been used to record the normal pulse voltammograms with the aim of obtaining the growth curves of Escherichia coli ATCC 13706 and Pseudomonas aeruginosa ATCC 27853 chosen as test microorganisms. The electrochemical signal data have been analysed with partial least squares (PLS) regression in order to highlight the useful analytical information and correlate with the data obtained from the aerobic plate counting. The obtained PLS models generally had a low root mean square error of cross-validation (RMSECV), a cross validated explained variance percentage near 90%.     

Synthesis, characterization and pharmacological activities of 2-[4-cyano-(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-yloxy)-6-(fluoropiperazinyl)-s-triazines

A series of 2-[4-cyano-(3-trifluoromethyl)phenyl amino)]-4-(4-quinoline/coumarin-4-yloxy)-6-(fluoropiperazinyl)-s-triazines has been synthesized by a simple and efficient synthetic protocol. The antimicrobial activity of the compounds was studied against several bacteria (Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 619, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 741, Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 1771, Shigella flexneria MTCC 1457) and fungi (Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC 343, Aspergillus clavatus MTCC 1323, Candida albicans MTCC 183) using paper disc diffusion technique and agar streak dilution method. Newly synthesized compounds were also tested for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using BACTEC MGIT and Lowenstein-Jensen MIC method.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Cyclopeptides and polyketides from coral-associated fungus, Aspergillus versicolor LCJ-5-4

Three new cyclopentapeptides, versicoloritides A-C (1-3), a new orcinol tetramer, tetraorcinol A (4), and two new lactones, versicolactones A and B (5 and 6) together with three known metabolites, diorcinol, glyantrypine, and cordyol C were isolated from the fermentation broth of the coral-associated fungus Aspergillus versicolor LCJ-5-4. Their structures were elucidated by spectroscopic and chemical methods. The new compounds 1-4 were evaluated for their radical-scavenging activity and antimicrobial activity against Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Candida albicans and cytotoxicity against P388 and Hela cell lines. Compound 4 showed weak radical-scavenging activity against the DPPH radical with an IC₅₀ value of 67 μM.     

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Sol-gel luminescence biosensors: Encapsulation of recombinant E. coli reporters in thick silicate films

A class of sensing elements based on the encapsulation of genetically engineered bioluminescent Escherichia coli (E. coli) reporter strains in sol-gel derived silicates is described. We demonstrate the concept by the immobilization of these bacterial cells in thick silicate films. Heat shock, oxidative stress, fatty acids, peroxides, and genotoxicity reporting bacteria were incorporated in the sol-gel silicates and their luminescence response was compared to that of the non-immobilized culture. All the immobilized bacteria maintained viability and luminescence activity for several months. The bacteria-silicate hybrids can be used either as disposable sensors or in multiple use sensing test-kits, and they can be also integrated in early warning devices operated in continuous flow conditions.     

Metal complexes of the third generation quinolone antibacterial drug sparfloxacin: preparation,structure, and microbial evaluation

The interactions of yttrium chloride, zirconium chloride, and uranium nitrate with sparfloxacin (SPAR) in ethanol, methanol, and acetone were studied. The isolated solid complexes were characterized by elemental analysis, infrared, ¹H-NMR and electronic spectra, and thermogravimetric analysis. The results support the formation of [Y(SPAR)₂Cl₂]Cl ? 12H₂O, [ZrO(SPAR)₂Cl]Cl ? 15H₂O, and [UO₂(SPAR)₃](NO₃)₂ ? 5H₂O. Infrared spectra of the isolated solid complexes indicate that SPAR is bidentate through the ring carbonyl oxygen and one oxygen of carboxylate. The calculated bond length and force constant, F(U=O), in the uranyl complex are 1.747 Å and 655.29 Nm^?1, respectively. The antimicrobial activities of the ligand and metal complexes have been tested against bacteria Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) and fungi Penicillium rotatum (P. rotatum) and Trichoderma sp., showing that the complexes exhibit higher antibacterial activity than SPAR.     

Fourier transform infrared microspectroscopy as a bacterial source tracking tool to discriminate fecal E. coli strains

The aim of this work was to use the FT-IR microspectroscopy technique, a union of a FT-IR spectrometer with a microscope, to discriminate fecal Escherichia coli strains from cows, chickens and humans and to compare the efficiency of this method with the genomic fingerprinting method, BOX-PCR. The obtained BOX-PCR profiles were able to correctly discriminate 93.75% of the chicken strains, 80% of the cow strains and 65% of the human strains. An efficient PLS-DA model was developed, using orthogonal signal correction and the second derivate of the FT-IR spectra. This model allowed the correct discrimination, according to the animal source, of all the E. coli strains analyzed. The bands in the FT-IR spectra that were responsible for the strains discrimination were in the region between 2816 and 3026 cm⁻¹ wavenumber, described as fatty acids. It was demonstrated that FT-IR microspectroscopy can be a suitable tool for fecal E. coli discrimination, because it is fast, easy to carry out and presents a flexible discrimination power.     

Potential bioactive secondary metabolites of Actinomycetes sp. isolated from rocky soils of the heritage village Rijal Alma,Saudi Arabia

The present study was designed to discover novel secondary antibiotic metabolites from Actinomycetes species from the soil of Rijal Almaa, Saudi Arabia. A laboratory-scale benchtop fermentation was utilized for the demonstration of antibiotics from the soil actinomycetes. Fourier transform-infrared spectroscopy (FT-IR) spectroscopy analysis of the fermented product (FP) was carried out, which showed unique fingerprint regions indicating the presence of phenolic hydroxyl groups, aliphatic compounds, carboxylic groups, esters, isothiocyanate, etc. GC-MS analysis of the FP depicted the unique structures of secondary metabolites, such as cyclononasiloxane octadecamethyl, cercosporin, ethyl iso-allocholate, octadecane, 3-ethyl-5-(2-ethylbutyl), dasycarpidan-1-methanol (acetate), heptadecane, 9-hexyl-, phthalic acid-butyl, and octadecane, 3-ethyl-5-(2-ethylbutyl). The TGA analysis showed the thermal stability of FP and the initial weight loss in FP was observed at 277.29 °C. The ¹H NMR and ¹³C NMR spectra of FP analysis demonstrated the various characteristic peaks presence of secondary metabolites. The XRD analysis at 2θ revealed distinct particles based on specific diffraction peaks. A set of six human bacterial pathogens, namely, the Gram-positive bacteria Staphylococcus aureus (S. aureus), Streptococcus pyogenes (S. pyogenes), and Bacillus subtilis (B. subtilis) and Gram-negative bacteria Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumoniae (K. pneumoniae), were utilized for screening. The FP exhibited promising antibacterial effects against both Gram-positive and Gram-negative bacterial organisms. The antibacterial spectrum of activity was greater for E. coli and B. subtilis than for K. pneumoniae.