Sampling Submicron T1 Bacteriophage Aerosols

Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 mu were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity.     

Face Mask Sampling for the Detection of Mycobacterium tuberculosis in Expelled Aerosols

Background

Although tuberculosis is transmitted by the airborne route, direct information on the natural output of bacilli into air by source cases is very limited. We sought to address this through sampling of expelled aerosols in face masks that were subsequently analyzed for mycobacterial contamination.

Methods

In series 1, 17 smear microscopy positive patients wore standard surgical face masks once or twice for periods between 10 minutes and 5 hours; mycobacterial contamination was detected using a bacteriophage assay. In series 2, 19 patients with suspected tuberculosis were studied in Leicester UK and 10 patients with at least one positive smear were studied in The Gambia. These subjects wore one FFP30 mask modified to contain a gelatin filter for one hour; this was subsequently analyzed by the Xpert MTB/RIF system.

Results

In series 1, the bacteriophage assay detected live mycobacteria in 11/17 patients with wearing times between 10 and 120 minutes. Variation was seen in mask positivity and the level of contamination detected in multiple samples from the same patient. Two patients had non-tuberculous mycobacterial infections. In series 2, 13/20 patients with pulmonary tuberculosis produced positive masks and 0/9 patients with extrapulmonary or non-tuberculous diagnoses were mask positive. Overall, 65% of patients with confirmed pulmonary mycobacterial infection gave positive masks and this included 3/6 patients who received diagnostic bronchoalveolar lavages.

Conclusion

Mask sampling provides a simple means of assessing mycobacterial output in non-sputum expectorant. The approach shows potential for application to the study of airborne transmission and to diagnosis.     

Surface Display of Recombinant Proteins on Bacillus thuringiensis Spores

The insecticidal protoxin from Bacillus thuringiensis has been shown to be a major component of the spore coat. We have developed a novel surface display system using B. thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein. The expression vector with a sporulation-specific promoter was successfully used to display green fluorescent protein and a single-chain antibody (scFv) gene that encodes anti-4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (anti-phOx) antibody. The spores that carry the anti-phOx antibody can bind to phOx specifically.     

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.     

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (I_D), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the I_D of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the I_D of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its I_D values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the I_D values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited I_D values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples.     

Surface Reconstruction through Poisson Disk Sampling

This paper intends to generate the approximate Voronoi diagram in the geodesic metric for some unbiased samples selected from original points. The mesh model of seeds is then constructed on basis of the Voronoi diagram. Rather than constructing the Voronoi diagram for all original points, the proposed strategy is to run around the obstacle that the geodesic distances among neighboring points are sensitive to nearest neighbor definition. It is obvious that the reconstructed model is the level of detail of original points. Hence, our main motivation is to deal with the redundant scattered points. In implementation, Poisson disk sampling is taken to select seeds and helps to produce the Voronoi diagram. Adaptive reconstructions can be achieved by slightly changing the uniform strategy in selecting seeds. Behaviors of this method are investigated and accuracy evaluations are done. Experimental results show the proposed method is reliable and effective.     

Effect of Substratum Surface Chemistry and Surface Energy on Attachment of Marine Bacteria and Algal Spores

Two series of self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium Cobetia marina (formerly Halomonas marina) and zoospores of the alga Ulva linza (formerly Enteromorpha linza). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols. C. marina attached in increasing numbers to SAMs with decreasing advancing water contact angles (θ_AW), in accordance with equation-of-state models of colloidal attachment. Previous studies of Ulva zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum θ_AW and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of Ulva was significantly different, suggesting that a more complex model of interfacial energetics is required.     

枯草芽胞杆菌孢子表面展示外源蛋白的研究

枯草芽胞杆菌孢子表面展示技术是最近十几年新兴的一种外源蛋白固定方法,已在酶学、疫苗学、靶向药物制备、金属污染治理等领域获得了广泛应用。以孢子衣壳蛋白为载体蛋白,已经成功地把许多抗原、酶和其他蛋白展示在孢子外表面。枯草芽胞杆菌孢子衣壳由多种衣壳蛋白组成,但可用做载体蛋白的并不多,且它们的特性不同。综合介绍了枯草芽胞杆菌孢子表面展示外源蛋白这种新型技术的具体机理,及其在国内外各领域应用的研究进展。     

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m²) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m² for wet sampling and 100.5 ± 10.2 CFU/m² for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.     

枯草芽孢杆菌芽孢表面展示重组抗原疫苗研究进展

枯草芽孢杆菌芽孢所具有的独特的理化特性及生理特征,使之成为新型的蛋白或酶类药物载体而倍受关注。简介了枯草芽孢杆菌芽孢结构特征,以及诱导机体产生的免疫反应,重点阐述了利用芽孢表面展示技术研制重组外源抗原疫苗,最后对芽孢表面展示外源抗原疫苗的应用前景进行了展望。     

Surface Structure of Streptomycete Spores as Revealed by Negative Straining and Freeze-Etching

The application of a simple negative staining technique and freeze-etching has revealed detailed information on the species-specific surface structure of spores and aerial hyphae of streptomycetes.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm’s hemolymph due to both the resistant characters of spores and the addition of enterokinase site.     

Surface Display of Human Serum Albumin on Bacillus subtilis Spores for Oral Administration

Human serum albumin (HSA) is the major protein component of human plasma. To date, HSA for clinical uses is mostly produced by fractionation of human whole blood, which is accompanied by a lot of limitations. To obtain long-term bioactive albumin, we used hsa as a foreign gene and constructed a recombinant plasmid pJS700-HSA which carries a recombinant gene cotC-hsa under the control of cotC promoter. Plasmid pJS700-HSA was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spore formation, western blot and fluorescence immunoassay were used to monitor HSA surface expression on spores. We estimated that HSA displayed on the spore accounted for 0.135 % of the total spore proteins and about 0.023 fg HSA were exposed on the surface of each spore. Oral administration to mice with spores displaying HSA implied that the recombinant spores may have potential ability to increase the serum albumin level in vivo due to the resistant characters of spores.     

Aerosols

We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise – for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.     

以CotC为分子载体在枯草芽孢杆菌表面展示海藻糖合酶

海藻糖是自然界中普遍存在的一种非还原性双糖,是一种极好的天然干燥剂和保鲜剂。海藻糖合酶能够催化α,α-1,4-糖苷键连接的麦芽糖直接转化为α,α-1,1-糖苷键连接的海藻糖,是生产海藻糖的首选。为获得具有良好展示效果的海藻糖合酶,将其高效稳定的展示于枯草芽孢杆菌芽孢表面,实验同时分别选取增强型绿色荧光蛋白(EGFP)和海藻糖合酶(Tres)作为模型蛋白,以来自枯草芽孢杆菌的芽孢衣壳蛋白Cot C作为枯草芽杆菌表面展示的锚定蛋白进行表面展示研究。利用流式细胞仪分析EGFP在芽孢表面展示的情况,结果表明芽孢衣壳蛋白Cot C可以将EGFP固定在芽孢的表面。然后将荧光蛋白基因egfp通过酶切替换为海藻糖合酶基因tres,将重组菌株使用p H7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在50℃水浴条件下作用2h,反应产物利用HPLC检测,能够检测到海藻糖峰,通过计算得到的酶活为252U/ml。说明海藻糖合酶基因通过与芽孢衣壳蛋白Cot C融合后可被展示在芽孢的表面。     

Spores of microorganisms

The addition of penicillin (300–1,000 units/ml.) to a culture ofBacillus cereus during formation of the refractive prespores leads to lysis of the sporangia and to the release of spore components (calcium and dipicolinic acid) from the cells. Penicillin mildly raises the incorporation of amino acids, including diaminopimelic acid, into hot-TCA precipitate of cells, while chloramphenicol lowers it. In the later phases of penicillin inhibition, DAP-containing structures are also destroyed, including the fraction firmly bound to the envelope structures of the spore (in the control culture this fraction is not released until later, during digestion by enzymes localized in the envelope structures themselves). Penicillin inhibition of sporogenesis can be reversed by adapting the culture to penicillin or by simultaneously adding chloramphenicol. After the presporulation phase, sporogenesis is relatively resistant to chloramphenicol, but the whole process is considerably slowed down. Chloramphenicol also affects the morphology of the spores during their formation and inhibits their release from the sporangia until the late phase of sporulation.