Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.     

Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Analysis of proteolytic degradation of a crude protein mixture using a surface acoustic wave sensor

Degradation of a crude protein mixture by proteases with pH optima from acidic to basic was followed in real time using a surface acoustic wave biosensor in Love-wave geometry. Proteases EC 3.4.23.18 from Aspergillus saitoi, EC 3.4.21.62 from Bacillus licheniformis, and Novozyme from Bacillus sp. have been used. Kinetic constants extracted from the curves resulted for comparable protease concentrations in maximal degradation rates between 1.1 x 10(-2) and 1.5 x 10(-2)s(-1). For the three proteases investigated, similar amounts of up to about two-thirds of the estimated 28 ng/cm2 bound molecules were proteolyzed. The residual material not degraded by the proteases was removed from the surface with 0.5% SDS. The analysis of the sensor signal allows: (1) estimation of the total mass of protein bound to the sensor surface and of the degradable fraction; (2) extraction of the pure mass signal; and (3) kinetic evaluation.     

Bacteria detection using disposable optical leaky waveguide sensors

Novel disposable absorbing material clad leaky waveguide sensor devices (LWD) have been developed for the detection of pathogenic particles such as bacteria. These chips are tailored to give the maximum extension of the evanescent field at the sensor surface in order to place the entire volume of the bacteria captured by immobilized antibodies on the chip surface within this field. This in turn increases the interaction of the light with the bacteria's bulk volume. Disposable LWD chips were fabricated at room temperature and without the use of expensive fabrication equipment. These LWDs have been characterised by detecting refractive index (RI) changes, scattering and fluorescence from bacterial spores at the sensor surface when illuminated at the coupling angle. The detection limit of Bacillus subtilis var. niger (BG) bacterial spores was 10(4) spores/ml and the illumination intensity of the spores was found to be three times greater than the illumination intensity generated using the surface plasmon resonance (SPR).     

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta.

Toxicity of Bacillus thuringiensis spores to the tobacco hornworm, Manduca sexta, is described. The numbers of larvae killed were in relation to spore dry weight. At a surface application of 6.8 ng/cm2, there was an 85 percent survival, but less than 50 percent survived at 68.2 ng/cm2. Striking similarity of spores to parasporal crystals is revealed by slope of mortality curves, inhibition of stadial growth, and 50 percent lethal dose values based on protein content.     

Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples.

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.     

Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.     

Novel technology for rapid species-specific detection of Bacillus spores

There is an urgent need for a small, inexpensive sensor that can rapidly detect bio-warfare agents with high specificity. Bacillus anthracis, the causative agent of anthrax, would be a perilous disease-causing organism in the event of a release. Currently, most anthrax detection research is based on nucleic acid detection, immunoassays and mass spectrometry, with few detection levels reported below 10(5) spores. Here, we show the ability to distinguish Bacillus spores to a level approaching 10(3) spores, below the reported median infectious dose of B. anthracis, using pyrolysis--micromachined differential mobility spectrometry and novel pattern recognition algorithms that combine lead cluster mapping with genetic algorithms.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

Inactivation of spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by chlorination

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.     

Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light.

The inactivation of Bacillus thuringiensis spores and spores treated with two protectants, one proteinaceous and the other a commercial product, Shade, at wavelengths of the near-ultraviolet and visible spectra and at 254 nm is described. Determination of the inactivating wavelengths may be used to establish an efficient sunlight protective system for B. thuringiensis when used as a microbial insecticide.     

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subsp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki. The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis.     

Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.     

An improved enzyme-linked immunoassay for the detection and quantification of the entomocidal parasporal crystal proteins of Bacillus thuringiensis subspp. kurstaki and israelensis

An improved and simplified enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of parasporal crystalline toxins from Bacillus thuringiensis subsp. kurstaki . The improved procedure involved pretreatment of the polystyrene cuvettes with glutaraldehyde before antibody coating. A direct comparison of treated and untreated cuvettes is provided. ELISAs were then used for the analysis of the entomocidal crystalline proteins in commercial and experimental formulations of B. thuringiensis subspp. kurstaki and israelensis .     

An improved system for the surface immobilisation of proteins on Bacillus thuringiensis vegetative cells and spores through a new spore cortex-lytic enzyme anchor

An improved surface-immobilisation system was engineered to target heterologous proteins onto vegetative cells and spores of Bacillus thuringiensis plasmid-free recipient strain BMB171. The sporulation-dependent spore cortex-lytic enzyme from B. thuringiensis YBT-1520, SceA, was expressed in vegetative cells and used as the surface anchoring motif. Green fluorescent protein (GFP) and a Bacillus endo-β-1,3-1,4-glucanase (BglS) were used as the fusion partners to test the binding efficiency and the functional activities of immobilised surface proteins. The surface localisation of the SceA-GFP fusion protein on vegetative cells and spores was confirmed by Western blot, immunofluorescence microscopy and flow cytometry. The GFP fluorescence intensity from both vegetative cells and spores was measured and compared to a previously characterised surface display system using a peptidoglycan hydrolase anchor (Mbg). Results demonstrated comparable efficiency of SceA- and Mbg-mediated immobilisation on vegetative cells but a more efficient immobilisation on spores using the SceA anchor, suggesting SceA has greater potential for spore-based applications. The SceA protein was then applied to target BglS onto vegetative cells and spores, and the surface immobilisation was verified by the substantial whole-cell enzymatic activity and enhanced whole-spore enzymatic activity compared to vegetative cells. A dually active B. thuringiensis vegetative cell and spore display system could prove especially valuable for the development of regenerable and heat-stable biocatalysts that function under adverse environmental conditions, for example, an effective feed additive for improved digestion and nutrient absorption by livestock.     

Estimation of the spectrum of the aerial dispersion of Bacillus thuringiensis

The present paper estimates the number of viable spores of Bacillus thuringiensis per droplet and reveals the importance of this data to improve B. thuringiensis treatments. For a given diameter, droplets from Futura formula contained two times more spores than droplets from the formulas used to date. It is preferable to have B. thuringiensis droplets larger than those of chemical insecticides. A Grumman AgCat aircraft calibrated to give the desired larger droplets resulted in successful B. thuringiensis treatments.     

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300 spores/mL

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3x10(6) spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 5+/-5 Hz (n=3), 92+/-7 Hz (n=3), 500+/-10 Hz (n=3), 1030+/-10 Hz (n=2), and 2696+/-6 Hz (n=2) corresponding to 0, 3x10(2), 3x10(3), 3x10(4), and 3x10(6) spores/mL, respectively. The reduction in resonant frequency is proportional to the change in cantilever mass, and thus the observed changes are due to the attachment of spores on the sensor surface. Selectivity of the antibody-functionalized sensor was determined with samples of BA (3x10(6)/mL) mixed with Bacillus thuringiensis (BT; 1.5x10(9)/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:125, 1:250, 1:500 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10(9)/mL and no BA) gave a steady state frequency decrease of 10 Hz, which is within noise level of the sensor, indicating excellent selectivity. The observed binding rate constant for the pure BA and BT-containing samples ranged from 0.105 to 0.043 min-1 in the spore concentration range 300 to 3x10(6)/mL. These results show that detection of B. anthracis spore at a very low concentration (300 spores/mL) and with high selectivity in presence of another Bacillus spore (BT) can be accomplished using piezoelectric-excited millimeter-sized cantilever sensors.     

Optical microchip array biosensor for multiplexed detection of bio-hazardous agents

An optical waveguide array biosensor suitable for rapid detection of multiple bio-hazardous agents is presented. SpectroSens? optical microchip sensors contain multiple spatially-separated waveguide channels with integral high-precision Bragg gratings sensitive to changes in refractive-index; selective surface-functionalisation of discrete sensing channels with different antibodies as bio-recognition elements enables selective multi-analyte biological detection. Interactions between target antigens in the test sample and respective surface-immobilised antibodies result in localised changes in refractive-index; the biosensor response manifests as increases in wavelength of light reflected from specific sensing channels. Multiplexed, label-free detection of 8 different biological agents, encompassing bacterial spores, vegetative cells, viruses and proteinaceous toxins has been demonstrated in real-time. Selective detection of Bacillus atrophaeus (BG) spores, Escherichia coli cells, MS2 viruses and ovalbumin (OVA) protein (simulant bio-hazardous agents) was first demonstrated as proof-of-concept; subsequently, detection of Bacillus anthracis (BA) spores (UM23CL2 strain), Franciscella tularensis (FT) cells (live vaccine strain), Vaccinia viruses (heat-killed) and ricin toxin (bio-hazardous agents) was proven. Two optical microchip sensors, each comprising 8 sensing channels were packaged into a single disposable cartridge allowing simultaneous 16-channel data acquisition. The specific antibody deposition sequence used in this study enabled detection of either 4 simulants or 4 bio-hazardous agents using a single consumable. The final device, a culmination of the multidisciplinary convergence of the fields of biology, chemistry, optoelectronics and microfluidics, is man-portable and inherently robust. The performance characteristics of the SpectroSens? technology platform highlight its potential for exploitation as a ‘detect to warn/treat’ biodetector in security and defence operations.