The response of a co-culture lung model to fine and ultrafine particles of incinerator fly ash at the air-liquid interface

Elevated concentrations of particulate matter in the environmental atmosphere constitute a potential risk to human health. In vitro cell-based assays are therefore necessary to evaluate the toxicological potential of inhaled particulate emissions. In this study, the exposure of a co-culture cell model at the air-liquid interface was used to evaluate the dose-dependent biological effects of a test aerosol. The CULTEX system was used to expose human cells to an environmentally-relevant aerosol, generated from fly ash collected in a commercial municipal waste incinerator and resuspended in filtered air. Human bronchial epithelial cells, BEAS-2B, co-cultured with differentiated THP-1 macrophages growing on Transwell inserts, were employed in the bioassay. Analyses of cell viability, interleukin-8 (IL-8) release, intracellular glutathione, and haeme oxygenase-1 enzyme expression were performed. Transportation of the cells and exposure to humidified filtered air or the test aerosol, at 100 ml/min for 1 to 6 hours, were well tolerated by the cells and had no effect on their viability. Levels of IL-8 release and haeme oxygenase-1 expression were elevated by exposure to fly ash aerosol as a function of time, but not by exposure to clean air. For IL-8 release, a dose-dependent effect was demonstrated with the assumption that the deposited mass of the particles correlated with exposure time. Exposure to the test aerosol did not affect the intracellular glutathione concentration. This in vitro approach simulates particle deposition in the human lung more realistically than does submerged exposure, and it preserves the inherent properties of the particles. It shows promise for use to detect particulate emissions which are potentially detrimental to human health.     

Aerosol stability of infectious and potentially infectious reovirus particles.

The aerosol stability of two particle forms, infectious and potentially infectious, of reovirus were examined under static conditions for a range of relative humidities at 21 and 24 degrees C. Virus aerosolization efficiency was determined for two methods of dissemination: Collison nebulizer and Chicago atomizer. Suspensions of Bacillus subtilis var. niger spores were added to reovirus preparations that included both particle forms and disseminated into a dynamic aerosol toroid to estimate the physical decay of the aerosols. At 90 to 100% relative humidity, both reovirus particle forms showed less than 10-fold loss of infectivity after 12 h of aging. At lower relative humidities the aerosol decay curve showed rapid initial decay followed by a markedly lower decay rate. Our findings reveal that reovirus particles are relatively stable in the airborne state.     

Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets

Shower heads and hot-water faucets containing Legionella pneumophila were evaluated for aerosolization of the organism with a multistage cascade impaction air sampler. Air was collected above two shower doors and from the same rooms approximately 3 ft (91 cm) from the shower doors while the hot water was running. Low numbers (3 to 5 CFU/15 ft3 [0.43 m3] of air) of L. pneumophila were recovered above both shower doors, but none was recovered from the air in either room outside the shower door. Approximately 90% (7 of 8 CFU) of the L. pneumophila recovered were trapped in aerosol particles between 1 and 5 micron in diameter. Air was collected 1 to 3 ft (30 to 91 cm) from 14 sinks while the hot water was running. Low numbers (1 to 5 CFU/15 ft3 of air) were recovered from 6 of 19 air samples obtained. Approximately 50% (6 of 13 CFU) of the organisms recovered were trapped in aerosol particles between 1 and 8 microns in diameter. Shower heads and hot-water taps containing L. pneumophila can aerosolize low numbers of the organism during routine use. The aerosol particle size is small enough to penetrate to the lower human respiratory system. Thus, these sites may be implicated as a means of transmission of L. pneumophila from potable water to the patient.     

Aerosols containing Legionella pneumophila generated by shower heads and hot-water faucets.

Shower heads and hot-water faucets containing Legionella pneumophila were evaluated for aerosolization of the organism with a multistage cascade impaction air sampler. Air was collected above two shower doors and from the same rooms approximately 3 ft (91 cm) from the shower doors while the hot water was running. Low numbers (3 to 5 CFU/15 ft3 [0.43 m3] of air) of L. pneumophila were recovered above both shower doors, but none was recovered from the air in either room outside the shower door. Approximately 90% (7 of 8 CFU) of the L. pneumophila recovered were trapped in aerosol particles between 1 and 5 micron in diameter. Air was collected 1 to 3 ft (30 to 91 cm) from 14 sinks while the hot water was running. Low numbers (1 to 5 CFU/15 ft3 of air) were recovered from 6 of 19 air samples obtained. Approximately 50% (6 of 13 CFU) of the organisms recovered were trapped in aerosol particles between 1 and 8 microns in diameter. Shower heads and hot-water taps containing L. pneumophila can aerosolize low numbers of the organism during routine use. The aerosol particle size is small enough to penetrate to the lower human respiratory system. Thus, these sites may be implicated as a means of transmission of L. pneumophila from potable water to the patient.     

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.     

Experimental Technique for Studying Aerosols of Lyophilized Bacteria

An experimental technique is presented for studying aerosols generated from lyophilized bacteria by using Escherichia coli B, Bacillus subtilis var. niger, Enterobacter aerogenes, and Pasteurella tularensis. An aerosol generator capable of creating fine particle aerosols of small quantities (10 mg) of lyophilized powder under controlled conditions of exposure to the atmosphere is described. The physical properties of the aerosols are investigated as to the distribution of number of aerosol particles with particle size as well as to the distribution of number of bacteria with particle size. Biologically unstable vegetative cells were quantitated physically by using ¹⁴C and Europium chelate stain as tracers, whereas the stable heat-shocked B. subtilis spores were assayed biologically. The physical persistence of the lyophilized B. subtilis aerosol is investigated as a function of size of spore-containing particles. The experimental result that physical persistence of the aerosol in a closed aerosol chamber increases as particle size is decreased is satisfactorily explained on the bases of electrostatic, gravitational, inertial, and diffusion forces operating to remove particles from the particular aerosol system. The net effect of these various forces is to provide, after a short time interval in the system (about 2 min), an aerosol of fine particles with enhanced physical stability. The dependence of physical stability of the aerosol on the species of organism and the nature of the suspending medium for lyophilization is indicated. Also, limitations and general applicability of both the technique and results are discussed.     

Human occupancy as a source of indoor airborne bacteria

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM(10) and PM(2.5) size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM(10). On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments.     

Effect of Surface Coating with Magnesium Stearate via Mechanical Dry Powder Coating Approach on the Aerosol Performance of Micronized Drug Powders from Dry Powder Inhalers

The objective of this study was to investigate the effect of particle surface coating with magnesium stearate on the aerosolization of dry powder inhaler formulations. Micronized salbutamol sulphate as a model drug was dry coated with magnesium stearate using a mechanofusion technique. The coating quality was characterized by X-ray photoelectron spectroscopy. Powder bulk and flow properties were assessed by bulk densities and shear cell measurements. The aerosol performance was studied by laser diffraction and supported by a twin-stage impinger. High degrees of coating coverage were achieved after mechanofusion, as measured by X-ray photoelectron spectroscopy. Concomitant significant increases occurred in powder bulk densities and in aerosol performance after coating. The apparent optimum performance corresponded with using 2% w/w magnesium stearate. In contrast, traditional blending resulted in no significant changes in either bulk or aerosolization behaviour compared to the untreated sample. It is believed that conventional low-shear blending provides insufficient energy levels to expose host micronized particle surfaces from agglomerates and to distribute guest coating material effectively for coating. A simple ultra-high-shear mechanical dry powder coating step was shown as highly effective in producing ultra-thin coatings on micronized powders and to substantially improve the powder aerosolization efficiency.     

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.     

Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method

Conventional parenteral injection of vaccines is limited in its ability to induce locally-produced immune responses in the respiratory tract, and has logistical disadvantages in widespread vaccine administration. Recent studies suggest that intranasal delivery or vaccination in the respiratory tract with recombinant viral vectors can enhance immunogenicity and protection against respiratory diseases such as influenza and tuberculosis, and can offer more broad-based generalized protection by eliciting durable mucosal immune responses. Controlled aerosolization is a method to minimize vaccine particle size and ensure delivery to the lower respiratory tract. Here, we characterize the dynamics of aerosolization and show the effects of vaccine concentration on particle size, vector viability, and the actual delivered dose of an aerosolized adenoviral vector. In addition, we demonstrate that aerosol delivery of a recombinant adenoviral vaccine encoding H1N1 hemagglutinin is immunogenic and protects ferrets against homologous viral challenge. Overall, aerosol delivery offers comparable protection to intramuscular injection, and represents an attractive vaccine delivery method for broad-based immunization campaigns.     

Direct Method for Determining the Viability of a Freshly Generated Mixed Bacterial Aerosol

A direct method was developed to determine the viability of a freshly generated mixed bacterial aerosol. A mixed suspension of (32)P-labeled Staphylococcus aureus and (35)S-labeled Proteus mirabilis was nebulized, and the aerosol was collected and separated according to particle size with an Andersen sampler. Quantitative and qualitative bacteriological and radioisotopic techniques were used to obtain ratios of bacterial to radioactive counts for each organism in samples of the nebulizer suspension and aerosol. Loss of viability was calculated from the change that occurred between the ratio of the nebulizer suspension and the ratio of the aerosol. The viability of S. aureus was unaffected by aerosolization, whereas the viability of P. mirabilis declined by 20 to 60% and was inversely proportional to particle size. The advantages of this method over present indirect methods, as well as potential applications of the method, are discussed.     

The cell membrane as a major site of damage during aerosolization of Escherichia coli

This study aimed to provide data on the survival and site of damage of Escherichia coli cells following aerosolization using two different techniques, nebulization and flow focusing. Four metabolic stains were assessed for their ability to detect respiratory activities and membrane homeostasis in aerosolized E. coli cells. The degree of sublethal injury increased significantly over the 10-min period of aerosolization in E. coli cells aerosolized by using the Collison nebulizer, reaching up to 99.9% of the population. In contrast, a significantly lower proportion of the population was sublethally damaged during aerosolization using the flow-focusing aerosol generator (FFAG). Concomitantly, loss of membrane homeostasis increased at a higher rate in nebulized cells (68 to 71%) than in those aerosolized by using the FFAG (32 to 34%). The activities of respiratory enzymes decreased at increased rates in nebulized cells (27 to 37%) compared to the rates of decrease in cells aerosolized by using the FFAG (59 to 61%). The results indicate that the physiology of an aerosolized bacterium is linked to the method of aerosol generation and may affect the interpretation of a range of aerobiological phenomenon.     

Naval Biomedical Research Laboratory, Programmed Environment, Aerosol Facility

Mathematical considerations of the behavior of aerosolized particles in a rotating drum are presented, and the rotating drum as an aerosol-holding device is compared with a stirred settling chamber. The basic overall design elements of a facility employing eight rotating drums are presented. This facility provides an environment in which temperature can be maintained within 0.5 F (0.25 C) of any set point over a range of 50 to 120 F (10 to 49 C); concomitantly the relative humidity within any selected drum may be controlled in a nominal range of 0 to 90%. Some of the major technical aspects of operating this facility are also presented, including handling of air support systems, aerosol production, animal exposure, aerosol monitoring, and sampling.     

A two-component theory of aerosol deposition in lung airways

The deposition of aerosol particles in the human lung airways is due to two distinct mechanisms. One is by direct deposition resulting from diffusion, sedimentation and impaction as the aerosol moves in and out of the lung. The other is an indirect mechanism by which particles are transported mechanically from the tidal air to the residential air and eventually captured by the airways due to intrinsic particle motion. This last mechanism is not well understood at present. Using a trumpet airway model constructed from Weibel's data, a two-component theory is developed. In this theory, the particle concentrations in the airways and the alveoli at a given airway depth are considered to be quantitatively different. This difference in concentrations will cause a net mixing between the tidal and residential aerosol as the aerosol is breathed in and out. A distribution parameter is then introduced to account for the distribution of ventilation. The effect of intrinsic particle motion on the aerosol mixing is also included. From this theory, total and regional deposition in the lung at the steady mouth breathing without pause is calculated for several different respiratory cycles. The results agree reasonably well with the experimental data.     

Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models

Aims: We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis. Methods and results: The viability and infectivity of the challenge suspension was determined in relation to aerosolization time, concentration, method of preparation and M. tuberculosis strain. Challenge stocks generated from frozen aliquots of M. tuberculosis were shown to undergo a 1 log₁₀ CFU ml^?1 decrease in viability during the first 10 min of aerosolization. This correlated with a decrease in surface lung lesions developing in guinea pigs challenged during this time. The phenomenon of decreased viability in vitro was not observed when using freshly grown, nonfrozen cells of M. tuberculosis. The viability of aerosolized bacilli at the point of inhalation relative to the point of aerosolization always remained constant. Conclusion: Based on these findings, we have developed an improved strategy by which to reproducibly deliver aerosol infection doses to individually challenged animals and separately challenged groups of animals. Significance and Impact of the Study: Study of the aerobiological characteristics of micro‐organisms is a critical step in the validation of methodology for aerosol infection animal models, particularly where large numbers of animals and nonhuman primates are used.     

Development of an Analytical Method for the Metaproteomic Investigation of Bioaerosol from Work Environments

The metaproteomic analysis of air particulate matter provides valuable information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters is developed. Different protein extraction procedures are tested to select the best extraction protocol based on protein recovery. The optimized method is tested for the extraction of proteins from spores of ubiquitous bacteria species and used for the metaproteomic characterization of filters from three work environments. In particular, ambient aerosol samples are collected in a composting plant, in a wastewater treatment plant, and in an agricultural holding. A total of 179, 15, 205, and 444 proteins are identified in composting plant, wastewater treatment plant, and agricultural holding, (cow stable and blending plant), respectively. In agreement with the major categories of primary biological aerosol particles, all identified proteins originated primarily from fungi, bacteria, and plants. The paper is the first metaproteomic study applied to bioaerosol samples collected in occupationally relevant environmental sites and, even though not aimed at monitoring the risk exposure of workers, it provides information on the possible exposure in the working environmental sites.     

Using selective media to assess aerosolization damage and ultraviolet germicidal irradiation susceptibility of Serratia marcescens

This study determined whether selective media, McConkey agar (MC) and minimal salt glucose agar (MA) are suitable for monitoring aerosolization damage of airborne Serratia marcescens in our laboratory aerosol exposure system and assessed the relationship between bacterial culturability in these media and ultraviolet germicidal irradiation (UVGI) susceptibility of the bacteria. Two types of bacterial cultures were prepared. The first culture was taken from bacteria growing on Tryptic soy agar (TSA) as complete medium (fresh culture), which provided nearly 100% of MC/MA tolerant bacteria, while the second one was prepared from freezing the fresh culture (frozen culture), which produced 55 and 81% of MC and MA tolerant bacteria, respectively. We monitored bacterial culturability in TSA, MC and MA from these cultures in the nebulizer reservoir and bioaerosls collected on a six-stage Andersen cascade bio-impactor. The results indicated that both concentration and percentage of MC/MA tolerant bacteria maintained at a similar level during nebulization. For the bioaerosols, although the concentration recovered in the media from the fresh culture was higher than that from the frozen culture, the percentage of MC/MA tolerant bacteria was similar to that before aerosolization. We concluded that MC and MA are not suitable for monitoring aerosolization damage of the bacteria. Moreover, culturability of the bacteria in MC and MA has no effect on their survival after aerosolization. With respect to the bacterial susceptibility to UVGI, MC/MA sensitive and tolerant population as well as the fresh and frozen cultures showed the same susceptibility.     

Generation of a nonequlibrium plasma in heterophase atmospheric-pressure gas-liquid media and demonstration of its sterilization ability

Results are presented from experiments on the generation of a low-temperature nonequilibrium plasma in atmospheric-pressure heterophase gas-liquid media of different compositions: (i) a liquid with air bubbles and (ii) air with liquid aerosol. To illustrate possible application of a low-temperature plasma in a heterophase medium, experiments on the inactivation of some microorganisms by a low-temperature plasma have been performed.     

Hazard Inherent in Microbial Tracers: Reduction of Risk by the Use of Bacillus stearothermophilus Spores in Aerobiology

The use of a „biological tracer” forms an essential part of many aerobiological experiments. Where release of such tracers is likely to result in deliberate or inadvertent human exposure, safety becomes a primary consideration in the selection of the tracer organism. Of the three most commonly used organisms, namely Bacillus subtilis, Escherichia coli, and Serratia marcescens, only the first comes near to satisfying the need for nonpathogenicity and even it has been incriminated as a cause of human infection, sometimes with a fatal outcome. The relevant characteristics of B. stearothermophilus were, therefore, investigated. Because it can grow only at elevated temperatures (minimum 41 C; optimum 56 C), it should not pose a threat to human health and this view is supported by experimental evidence to be presented. It is extremely easy to grow and maintain in the laboratory, and spore suspensions are easily prepared and stored. It withstands the stresses of aerosolization and sampling and its stability in the aerosol state compares favorably with that of B. subtilis var. niger.     

Lidars: a key component of urban biodefense

A biological aerosol attack in a city could infect tens of thousands of people. In the absence of announcements by the attacker or detection by present point detection systems, victims would be unaware of their exposure prior to developing symptoms. Since infections are most effectively countered before the onset of symptoms, detection technologies that provide early awareness of an attack should be given high priority. Current biological point detection (BPD) systems collect environmental air samples and then analyze them in laboratories so as to permit detection within 12-36 hours of an attack. Improvements in the pipeline may reduce this lag time to as little as a few hours. However, BPD systems have inherent weaknesses when used to detect and respond to an aerosol attack. The likelihood of a limited number of BPD systems intercepting an aerosol plume in a vast attack space may be low. Moreover, BPD systems do not provide critical information needed for response, such as the source location, precise time, and geographic reach of an attack. The missing information would invaluably guide prophylaxis distribution, identification of contaminated areas, and criminal apprehension. This article describes how lidars used for real-time observation of aerosol plumes could complement BPD systems by providing fine-scale spatial and temporal information. A lidar system also could be used to corroborate positive BPD system results, to improve reaction to positive results, and/or to provide an independent basis for low-regret protective steps. Lidar systems can resolve key biodefense challenges, and this article describes three compatible concepts of operations. Leveraging lessons from a lidar system now operating at the Pentagon, a test of an expanded lidar network would provide immediate protection for key Washington, DC, assets, demonstrate the synergy of BPD systems and lidars, and provide a test bed for research to improve lidar's shortcomings.