Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.     

Quantification of airborne Aspergillus fumigatus spores in rabbit houses by real-time polymerase chain reaction

To accurately quantify airborne Aspergillus fumigatus (A. fumigatus) spores in rabbit houses, the real-time polymerase chain reaction (real-time PCR) and culture-based counting method (CCM) were employed to determine the airborne A. fumigatus spore concentrations. The results showed that, of the three rabbit houses (A, B, and C), the average concentrations of airborne A. fumigatus spores determined by real-time PCR were 3.0 × 10³, 3.3 × 10³, and 1.5 × 10³ spores/m³ air, respectively, while those determined by CCM were 2.5 × 10², 2.8 × 10², and 1.1 × 10² colony-forming unit/m³ air (CFU/m³ air), respectively, i.e., the former concentration was 12–14 times higher than the latter one. Therefore, the conventional CCM underestimated the concentrations of airborne fungal spores, and it is insufficient to determine the microbial aerosol concentration and evaluate the health risk only using CCM.     

In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.     

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.     

Airborne movement of anthrax spores from carcass sites in the Etosha National Park, Namibia

Tests for airborne movement of anthrax spores downwind from three heavily contaminated carcass sites were carried out under a range of wind conditions. Anthrax spores were detected in just three of 43 cyclone or gelatin filter air samples taken at distances of 6, 12 and 18 m from the sites. In addition, nine positives resulted during sampling sessions in which the site was mechanically disturbed, with a further five positives being found in sessions subsequent to those in which the site had been disturbed. The three positive samples not related to man-made disturbance were associated with the highest winds experienced during the study. Despite colony counts exceeding 100 on the culture plates in three instances, calculations showed that these represented very low worst case probable spore inhalation rates for animals or humans exposed to such levels. The low number of positives, the clear pattern of rapidly declining numbers of anthrax spores with distance downwind from the centres of the sites apparent on settle plates, and the persisting levels of contamination despite wind and rain, collectively suggest that the anthrax spores were associated with fairly heavy particles, although this was not seen by electron microscopy on soil samples from the sites. Overall, the findings are interpreted as indicating that it is very unlikely that Etosha animals contract anthrax by the inhalation route while simply in transit near or across a carcass site. The significance of the observations in relation to weather conditions in the Etosha, other studies on particulate aerosols in the region, and reports of long-distance airborne movement of microbes, is discussed.     

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.     

Microbial imprinted polypyrrole/poly(3-methylthiophene) composite films for the detection of Bacillus endospores

The fabrication of Bacillus subtilis endospore imprinted conducting polymer films and subsequent electrochemical detection of bound spores is reported. Imprinted films were prepared by absorbing spores on the surface of glassy carbon electrodes upon which a polypyrrole, followed by a poly(3-methylthiophene), layer were electrochemically deposited. Spore template release was achieved through soaking the modified electrode in DMSO. Binding of endospores to imprinted films could be detected via impedance spectroscopy by monitoring changes in Y' (susceptance) using Mn(II)Cl2 (0.5M pH 3) as the supporting electrolyte. Here, the change in Y' could be correlated to spore densities between 10(4) and 10(7)cfu/ml. More sensitive detection of absorbed spores was achieved by following endospore germination via changes in film charge as measured using cyclic voltammetry. Here, imprinted films were submerged in spore suspensions to permit absorption, heat activated at 70 degrees C for 10 min prior to transferring to an electrochemical cell containing germination activators. By using the assay format it was possible to detect 10(2)cfu/ml. The observed changes in film charge could be attributed to the interaction of the supporting conducting polymer with dipicolinic acid (DPA) and other constituents released from the core in the course of germination. In all cases, it was not possible to regenerate the imprinted films without losing electrode response. In summary, the study has provided proof-of-concept for fabricating microbial imprinted films using conducting polymers.     

Activation of subtilisin Carlsberg in hexane by lyophilization in the presence of fumed silica

Subtilisin Carlsberg (SC) was lyophilized from an aqueous buffer solution containing different amounts of unmodified commercial fumed silica. The activity of the enzyme/fumed silica preparation in hexane was compared to pure freeze-dried enzyme, and to a freeze-dried preparation reported in the literature with potassium chloride as additive. A sharp increase in enzyme activity was found to correlate with an increasing amount of fumed silica added to the enzyme solution prior to freeze-drying. A weight-ratio of 98.5 wt.% fumed silica relative to the mass of the final enzyme/fumed silica preparation led to about 130-fold increased activity of SC in hexane (when compared to pure lyophilized SC in hexane). This is about twice the activation effect compared to including potassium chloride in the buffer solution before freeze-drying [1]. When freezing at −20 °C instead of in liquid nitrogen, even better activation was observed with fumed silica. We hypothesize that the activation of SC in hexane by immobilization of the enzyme on fumed silica is likely due to the distribution of the enzyme on the large surface area of fumed silica. This alleviates mass transfer limitations.     

Dispersal of fungal spores from three types of air handling system duct material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min–1. The duct air velocity was approximately 2.8 m sec–1. The airborne concentration of culturable P. chrysogenum spores (CFU m–3), total P. chrysogenum spores (spores m–3), and total P. chrysogenum-sized particles (particles m–3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m–3; 105 spores m–3; 104 particles m–3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.     

Detection of anthrax spores in endemic regions of northern Canada

AIMS: To determine the level of anthrax spore contamination in endemic regions of northern Canada between outbreaks. METHODS AND RESULTS: Bacterial endospores were extracted from specimens via flotation and cultured on selective PLET medium. Of 588 environmental specimens collected, 11 (1.9%) contained viable anthrax spores. CONCLUSION: High environmental concentrations of anthrax spores in northern Canada appear limited to scavenger faeces and anthrax carcass sites. Burial and cremation appear equally effective at removing anthrax spores from the immediate environment, though cremation may be improved by re-burning cremation sites containing unburned animal hair. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes an effective anthrax spore detection system. It provides the first bacteriological evidence that mammalian scavengers can disseminate anthrax spores in northern Canada, and its results may be compared with future environmental studies of untreated anthrax carcass sites to help improve government response plans.     

Immunomagnetic-Electrochemiluminescent Detection of Bacillus anthracis Spores in Soil Matrices

Rapid (<=1.5-h) detection of anthrax spores in soil suspensions was accomplished by an immunomagnetic electrochemiluminescence method. Strain-dependent detection limits in the range of 10(sup2) to 10(sup5) spores were achieved in buffer. The rank order of sensitivity for the assay in buffer was Sterne > Ames > Vollum 1B. Detection was up to 3 orders of magnitude less sensitive in soil suspensions, and the rank order of sensitivity was altered.     

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.     

Methods for integrated air sampling and dna analysis for detection of airborne fungal spores

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.     

Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism‐related micro‐organisms

Aims: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. Methods and Results: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10‐μl sample volume onto the sample support was 10⁶ particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 μl, the detection limit could be reduced to 10⁵ particles per ml for spores and to 5 × 10⁴ particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. Conclusions: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. Significance and Impact of the Study: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.     

Defensive and simultaneous actions of glycoconjugates during spore decontamination

An estimated $1 billion was lost in decontaminating areas exposed to anthrax in the 2001 attacks. To counter the threat of biological attacks, an effective 'green' decontaminant is vital to minimize the consequences of such attacks. The objective of our research was to study the ability of glycoconjugate ligands to decontaminate Bacillus cereus spores on hard surfaces. Polyvalent glycoconjugates (also known as neoglycoconjugates) were tested during decontamination of B. cereus spores. Resulting colony forming units (CFU) of viable spores were a direct evidence of glycoconjugate decontamination efficacy. Our results indicate a substantial, decreasing CFU count due to defensive and simultaneous actions of glycoconjugates compared to spores only used as controls. Decontamination of B. cereus spores was most efficiently and consistently achieved using Galalpha1-->3GalNAcbeta-PAA-flu glycoconjugate under both defensive and simultaneous conditions. Atomic force microscopy (AFM) allowed us to visualize decontamination at the nanoscale level using glycoconjugates. AFM reveals the size of glycoconjugate agglomerates (clusters) and a noticeably different morphology of glycoconjugate-treated spores during decontamination. Morphological features of untreated spores disappear under a thin layer of glycoconjugate solution. This thin layer is formed due to the defensive action of glycoconjugates. Simultaneous action has shown agglomeration of glycoconjugates in solution with B. cereus spores in glycoconjugate suspensions. Glycoconjugates might be useful for the development of an environment-friendly decontaminant of bacterial spores.     

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.     

炭疽灭活和裂解芽胞抗原免疫家兔后血清中的IgG抗体应答

炭疽杆菌芽胞在炭疽免疫中发挥基本作用。实验中以炭疽活芽胞疫苗为原形,建立了制备灭活和裂解炭疽芽胞的方法,研究了各种灭活和裂解炭疽芽胞疫苗不同浓度、不同剂次免疫家兔的抗芽胞和毒素IgG应答,总结分析了各种灭活和裂解炭疽芽胞疫苗用于新疫苗成分之一的可能性。甲醛灭活炭疽芽胞疫苗设芽胞浓度2.5×108剂量组、5×108剂量组、1×109剂量组,于0、4、8周时3次免疫。在3剂免疫后血清抗炭疽芽胞IgG水平持续升高,首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度可达到600～16000。裂解炭疽芽胞疫苗的制备和动物免疫中,只采取了2.5×108芽胞浓度,两剂免疫,免疫时间为0、4周。在首次免疫后4、8、12周时家兔血清中抗芽胞IgG几何平均滴度分别为362、776和388。各时间点采集的家兔血清未能测出或只测出极微量的抗炭疽毒素IgG。通过上述研究认为,以裂解炭疽芽胞抗原作为炭疽疫苗成分之一,其抗原性和免疫原性是适宜的;免疫剂量可以设定为2.5×108芽胞浓度上下;免疫次数可定为2剂间隔1个月。     

Exploitation of immunofluorescence for the quantification and characterization of small numbers of Pasteuria endospores

The Pasteuria group of endospore-forming bacteria has been studied as a biocontrol agent of plant-parasitic nematodes. Techniques have been developed for its detection and quantification in soil samples, and these mainly focus on observations of endospore attachment to nematodes. Characterization of Pasteuria populations has recently been performed with DNA-based techniques, which usually require the extraction of large numbers of spores. We describe a simple immunological method for the quantification and characterization of Pasteuria populations. Bayesian statistics were used to determine an extraction efficiency of 43% and a threshold of detection of 210 endospores g(-1) sand. This provided a robust means of estimating numbers of endospores in small-volume samples from a natural system. Based on visual assessment of endospore fluorescence, a quantitative method was developed to characterize endospore populations, which were shown to vary according to their host.