检测病毒气溶胶存活的方法学研究

以钝齿棒状杆菌噬菌体B271血清型为病毒等小颗粒生物粒子的模拟剂,建立了一种适合这类小颗粒生物粒子气溶胶存活研究的方法。本文从该噬菌体耐气溶胶化特性、气溶胶粒谱、用气溶胶示踪剂求算物理衰亡的方法和气溶胶采样回收技术等方面探讨了病毒气溶胶存活研究中的几个关键技术问题,为病毒气溶胶存活研究提供了参考。     

Comparison of aerosol and bioaerosol collection on air filters

Air filters efficiency is usually determined by non-biological test aerosols, such as potassium chloride particles, Arizona dust or di-ethyl-hexyl-sebacate (DEHS) oily liquid. This research was undertaken to asses, if application of non-biological aerosols reflects air filters capacity to collect particles of biological origin. The collection efficiency for non-biological aerosol was tested with the PALAS set and ISO Fine Test Dust. Flow rate during the filtration process was 720 l/h, and particles size ranged 0.246–17.165 μm. The upstream and downstream concentration of the aerosol was measured with a laser particle counter PCS-2010. Tested bioaerosol contained 4 bacterial strains of different shape and size: Micrococcus luteus, Micrococcus varians, Pseudomonas putida and Bacillus subtilis. Number of the biological particles was estimated with a culture-based method. Results obtained with bioaerosol did not confirmed 100% filters efficiency noted for the mineral test dust of the same aerodynamic diameter. Maximum efficiency tested with bacterial cells was 99.8%. Additionally, cells reemission from filters into air was also studied. Bioaerosol contained 3 bacterial strains: Micrococcus varians, Pseudomonas putida and Bacillus subtilis. It was proved that the highest intensity of the reemission process was during the first 5 min. and reached maximum 0.63% of total number of bacteria retained in filters. Spherical cells adhered stronger to the filter fibres than cylindrical ones. It was concluded that non-biological aerosol containing particles of the same shape and surface characteristics (like DEHS spherical particles) can not give representative results for all particles present in the filtered air.     

Improved Large-Volume Sampler for the Collection of Bacterial Cells from Aerosol

A modified large-volume sampler was demonstrated to be an efficient device for the collection of mono-disperse aerosols of rhodamine B and poly-disperse aerosols of bacterial cells. Absolute efficiency for collection of rhodamine B varied from 100% with 5-μm particles to about 70% with 0.5-μm particles. The sampler concentrated the particles from 950 liters of air into a flow of between 1 and 2 ml of collecting fluid per min. Spores of Bacillus subtilis var. niger were collected at an efficiency of about 82% compared to the collection in the standard AGI-30 sampler. In the most desirable collecting fluids tested, aerosolized cells of Serratia marcescens, Escherichia coli, and Aerobacter aerogenes were collected at comparative efficiencies of approximately 90, 80, and 90%, respectively. The modified sampler has practical application in the study of aerosol transmission of respiratory pathogens.     

NaCl-amendment assay targeting airborne bacteria in tropospheric bioaerosols transported by westerly wind over Noto Peninsula

Bioaerosol particles including bacteria, fungi, and virus are originated from marine and terrestrial environments. The airborne microorganisms are transported for long distance through the free troposphere and are thought to influence the downwind ecosystems and human life. However, microbial communities in the free troposphere have not been understood in detail because the direct sampling of microbial cells at high altitude requires sophisticated sampling techniques. In this study, for the investigation of microbial species compositions in the free troposphere, air sampling using an aircraft was performed over the Noto Peninsula in Japan, where the tropospheric winds carry aerosol particles from continental areas. Two air samples were collected at 3,000 m on March 27, 2010, when air mass was carried from the Gobi Desert to Japan area. Microorganisms from one air sample grew in culture media containing up to 15 % NaCl, suggesting that halotolerant bacteria maintain their viabilities in the free troposphere. DGGE analysis revealed that the amended cultures were dominated by Bacillus subtilis, and the isolates obtained from the amended cultures were identical to B. subtilis. Furthermore, the 16S rDNA clone library (culture-independent survey) of the other air sample grew was composed of three phylotypes belonging to Firmicutes, Bacteroidetes, and Proteobacteria with the sequences of Firmicutes phylotype corresponding to that of the cultured B. subtilis sequence. Microscopic observation using FISH method indicated that B. subtilis particles occupied 80 % of total eubacterial particles on the mineral particles. The halotolerant bacteria identical to B. subtilis would dominate at high altitudes over Noto Peninsula where the prevailing westerly wind was blowing.     

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter < 200 nm and a geometric standard deviation σ_g < 2.5 ⁵. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size ⁶, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria ⁵.A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO₂ inhalation toxicology studies. Critical components: 1) novel nano-TiO₂ aerosol generator; 2) 0.5 m³ whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO₂ aerosols generated from bulk dry nano-TiO₂ powders (primary diameter of 21 nm, bulk density of 3.8 g/cm³) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m³). The mass (M) of the collected particles was determined as M = (M_post-M_pre), where M_preand M_post are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m³/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O₂ and CO₂ concentrations were monitored and controlled continuously. Nano-TiO₂ aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.     

Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size,Culturability, and Effect of Surgical Masks

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza.We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (“coarse”>5 µm, “fine”≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus.Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples.Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans.     

Enhancing Bioaerosol Sampling by Andersen Impactors Using Mineral-Oil-Spread Agar Plate

As a bioaerosol sampling standard, Andersen type impactor is widely used since its invention in 1950s, including the investigation of the anthrax attacks in the United States in 2001. However, its related problems such as impaction and desiccation stress as well as particle bounce have not been solved. Here, we improved its biological collection efficiencies by plating a mineral oil layer (100 µL) onto the agar plate. An Andersen six-stage sampler and a BioStage impactor were tested with mineral-oil-spread agar plates in collecting indoor and outdoor bacterial and fungal aerosols. The effects of sampling times (5, 10 and 20 min) were also studied using the BioStage impactor when sampling environmental bioaerosols as well as aerosolized Bacillus subtilis (G+) and Escherichia coli (G-). In addition, particle bounce reduction by mineral-oil-plate was also investigated using an optical particle counter (OPC). Experimental results revealed that use of mineral-oil-spread agar plate can substantially enhance culturable bioaerosol recoveries by Andersen type impactors (p-values<0.05). The recovery enhancement was shown to depend on bioaerosol size, type, sampling time and environment. In general, more enhancements (extra 20%) were observed for last stage of the Andersen six-stage samplers compared to the BioStage impactor for 10 min sampling. When sampling aerosolized B. subtilis, E. coli and environmental aerosols, the enhancement was shown to increase with increasing sampling time, ranging from 50% increase at 5 min to ∼100% at 20 min. OPC results indicated that use of mineral oil can effectively reduce the particle bounce with an average of 66% for 10 min sampling. Our work suggests that enhancements for fungal aerosols were primarily attributed to the reduced impaction stress, while for bacterial aerosols reduced impaction, desiccation and particle bounce played major roles. The developed technology can readily enhance the agar-based techniques including those high volume portable samplers for bioaerosol monitoring.     

Evaluation of a New Bactericidal Spray Effective against Staphylococcus aureus

A new germicidal spray consisting of an active ingredient, 2-chloro-4-phenylphenol, solvent, and propellant was evaluated against aerosols of Staphylococcus aureus and spores of Bacillus subtilis. The method of evaluation consisted of determining the decay rate of the bacterial aerosols in a small chamber, with and without the aerial germicide present. The method is unique in that the effect of very short exposures of airborne bacteria to aerial germicides can be measured accurately. By use of the method outlined, extremely potent aerial germicides may be evaluated against highly sensitive organisms. The 2-chloro-4-phenylphenol spray was found to be extremely effective against S. aureus but only moderately effective against spores of B. subtilis.     

Theophylline Cocrystals Prepared by Spray Drying: Physicochemical Properties and Aerosolization Performance

The purpose of this work was to characterize theophylline (THF) cocrystals prepared by spray drying in terms of the physicochemical properties and inhalation performance when aerosolized from a dry powder inhaler. Cocrystals of theophylline with urea (THF-URE), saccharin (THF-SAC) and nicotinamide (THF-NIC) were prepared by spray drying. Milled THF and THF-SAC cocrystals were also used for comparison. The physical purity, particle size, particle morphology and surface energy of the materials were determined. The in vitro aerosol performance of the spray-dried cocrystals, drug-alone and a drug-carrier aerosol, was assessed. The spray-dried particles had different size distributions, morphologies and surface energies. The milled samples had higher surface energy than those prepared by spray drying. Good agreement was observed between multi-stage liquid impinger and next-generation impactor in terms of assessing spray-dried THF particles. The fine particle fractions of both formulations were similar for THF, but drug-alone formulations outperformed drug-carrier formulations for the THF cocrystals. The aerosolization performance of different THF cocrystals was within the following rank order as obtained from both drug-alone and drug-carrier formulations: THF-NIC > THF-URE > THF-SAC. It was proposed that micromeritic properties dominate over particle surface energy in terms of determining the aerosol performance of THF cocrystals. Spray drying could be a potential technique for preparing cocrystals with modified physical properties.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9883-3) contains supplementary material, which is available to authorized users.Key words: aerodynamic diameter, cocrystal, spray drying, surface energy, theophylline     

A novel method for measuring the charge distribution of airborne microbes

A novel method for measuring bioaerosol charge distribution was investigated using electrostatic sampling and quantitative polymerase chain reaction (qPCR). Here, Bacillus subtilis var niger and Pseudomonas fluorescens were aerosolized and precipitated into different regions of two 96-well plates placed inside an electrostatic sampler. The concentrations of negatively charged bacteria in the air samples collected were quantified using qPCR, and the relevant bioaerosol charge levels were calculated using an equation developed in this study. Experimental results showed that B. subtilis var niger bioaerosols had wider charge distribution than P. fluorescens. For B. subtilis var niger, 93% of them carried a charge level of 13–42 units, 2% carried a charge level of 8–13 or lower, and 5% carried a charge level of 42–195 or higher. While for P. fluorescens, 99% of P. fluorescens aerosols carried a charge level of 14–34 elementary units. The indicative charge levels of bioaerosols collected at a certain point in the electrostatic sampling line largely depends on the particle size, bacterial species, electrostatic field strength, and the sampling flow rates for a given electrostatic sampler. In general, the trends for the charge levels here were similar to those obtained in previous studies. Use of qPCR technology in this study can distinguish between biological and non-biological particles collected. Thus, this avoided counting the non-biological particles in traditional bioaerosol charge measurements in which an optical particle counter is used. The developed technique here can be also used to analyze the charge levels for other types of bioaerosols.     

Stability of St. Louis Encephalitis Virus in the Airborne State

The aerosol stability of St. Louis encephalitis (SLE) virus was studied over a 6-hr period at a temperature of 21 C and relative humidity values of 23, 46, 60, and 80%. Aerosols were generated from and collected in 0.75% bovine albumin-buffered saline, and spores of Bacillus subtilis var. niger were used as the tracer to determine the physical decay of the aerosols. Aerosol samples were titrated in BHK-21 cell monolayers for surviving SLE virus. The results of this study indicated that, under the test conditions employed, relative humidity had no influence on the stability of SLE virus in the airborne state.     

Micrococcus lysodeikticus bacterial walls as a substrate specific for the autolytic glycosidase of Bacillus subtilis

The Bacillus subtilis 168 autolytic glycosidase degrades Micrococcus lysodeikticus cells or cell walls, whereas the B. subtilis autolytic amidase does not. The criteria used to establish this fact included: the determination of chemical bonds broken, heat-inactivated kinetics, pH dependence curves, and the physical separation of glycosidase from amidase. The physical separation involved LiCl elution from two different ion-exchange materials, walls from B. subtilis 168 strain βAO, and walls from mutant strain βA173 derived from strain βAO. No evidence was obtained for B. subtilis vegetative bacteria making any more autolysins than one autolytic amidase and one autolytic glycosidase.     

Effect of Particle Shape on Dry Particle Inhalation: Study of Flowability, Aerosolization, and Deposition Properties

The shape effects of dry particles on flowability, aerosolization, and deposition properties in inhalation drug delivery are studied. The properties are compared with similar size range particles of different shapes such as sphere, needle, cube, plate, and pollen. Flowability of the particles is characterized by Carr’s compressibility index and angle of slide (θ) method. The aerosolization and deposition properties of the particles are studied in vitro using an eight-stage Anderson cascade impactor with a Rotahaler®. Pollen-shaped particles are found to exhibit better flowability, higher emitted dose, and higher fine particle fraction than particles of other shapes in similar size range. They showed minimum θ of 35° and maximum emitted dose of 87% and fine particle fraction of 16%. The use of pollen-shaped particles can be a potential improvement in dry particle inhalation.     

Determining the basic characteristics of aerosols suitable for studies of deposition in the respiratory tract

Studies of aerosol particle deposition in the respiratory tract requires experimental inhalation of artificial model aerosols. The paper formulates some of the most important requirements for the properties of such aerosols. Several suitable fractions were prepared as part of a research project dealing with the use of microporous polymers for diagnostic purposes. 5 fractions of the polymer designated G-gel 60 with the particle size as stated by the manufacturer, ranging from 3 to 7 micron were evaluated using a 16-channel particle dispersity analyzer HIAC/ROYCO MT 3210 with the sensor 1200 and operated by a microprocessor, the equipment being coupled to an APPLE IIe computer. G-gel 60 particles introduced into the aerosol were characterized by the parameters CMAD, MMAD and sg both numerically and graphically. The measurement procedure was found to be very sensitive with respect to all fractions in evaluating the subtile differences between different lot numbers of the aerosol. G-gel 60 fractions characterized both numerically and graphically were compared with the known aerosols from paraffin oil and atmospheric air. The equipment MT 3210 enables prompt determination of the percentages of aerosol particles distribution by size class. The authors conclude that the procedure, both in its numerical and graphical versions, is particularly suitable for the diagnosis of aerosol particles deposition in the respiratory tract, offering a new application for HIAC/ROYCO in the field of medicine. In evaluating atmospheric aerosol in exhaled air, the number of particles was found to be below that in inhaled air, the difference being dependent on the choice of investigation methods. Percentual distribution of deposited particles following one minute ventilation proved to be at its maximum, as regards atmospheric aerosol, in the 0.30-0.50 micron range. The deposition curve was similar to already published curves, being characterized by an S-shaped pattern with maximum deposition in the greater size classes. An analysis of inhaled, exhaled and deposited aerosol suggested that deposited aerosol is more polydisperse and has particles of greater sizes than inhaled aerosol. Investigation of the effect of apnoe on deposition indicated that deposition increased as a function of apnoeic pause.     

Particle Size Distribution of Serratia marcescens Aerosols Created During Common Laboratory Procedures and Simulated Laboratory Accidents

Andersen air samplers were used to determine the particle size distribution of Serratia marcescens aerosols created during several common laboratory procedures and simulated laboratory accidents. Over 1,600 viable particles per cubic foot of air sampled were aerosolized during blending operations. More than 98% of these particles were less than 5 mu in size. In contrast, 80% of the viable particles aerosolized by handling lyophilized cultures were larger than 5 mu. Harvesting infected eggs, sonic treatment, centrifugation, mixing cultures, and dropping infectious material produced aerosols composed primarily of particles in the 1.0- to 7.5-mu size range.     

Influence of Serum and Glucose Additives on Survival of Actinobacillus pleuropneumoniae Aerosolized from the Freeze-Dried State

Serum and/or glucose added to Actinobacillus pleuropneumoniae suspensions before freeze-drying significantly increased survival rates of bacteria in aerosols. Aerosols with predictable numbers of viable bacteria can be made as required in an aerosol infection model. Sucrose supplementation of impinger fluids increased recovery of viable A. pleuropneumoniae.     

Characterization of Two Bacillus Probiotics

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.     

Modified Spinning Top Homogeneous Spray Apparatus for Use in Experimental Respiratory Disease Studies

The May spinning top generator was adapted to a modified Henderson tube for producing large aerosol particles (>4 mum) to obtain almost exclusive upper respiratory tract deposition of infectious aerosols in exposed mice. The system was installed in a biological safety cabinet to permit experimentation with pathogens. A novel mechanism utilizing parts from a machinists micrometer and the mechanical stage from a light microscope was developed for the spinning top generator as a means for precisely positioning the liquid feed needle. Aerosol light-scatter properties were continuously analyzed to provide relative measures of particle size distribution and aerosol concentration. When mice were exposed to influenza virus aerosols in which none of the virus was contained in particles with aerodynamic diameters <4 mum, essentially all of the virus was deposited in the upper respiratory tract tissues.     

Microbial validation of vent filters

The Upjohn Company uses filtration to remove microorganisms and particulates from air and other gases which may come in contact with sterile products. To validate the microbial retentivity of these filters, they were challenged with an aerosol of Bacillus subtilis var niger spores. An aerosol challenge was used because it more closely simulated the use for which these filters were designed. The test apparatus was constructed of autoclavable components using a jet-type nebulizer and heated air mixing tube. Characterization of the aerosol particle size distribution with a particle size analyzer demonstrated that 80% of the particles had a diameter of x 3.0 times;m and that the particles had a mean mass diameter of 1.9 times;m with a geometric standard deviation of 1.8 times;m. Studies conducted with aerosols of Bacillus subtilis var niger spores demonstrated that the test apparatus could recover ca. 50% of the spores that were aerosolized. Hydrophobic filters from various manufactures were challenged with an aerosol of at least 10(8) spores of Bacillus subtilis. All filters tested could retain at least 10(9) spores when physical integrity of the filter was verfield.