Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released Pseudomonas syringae. The upper limits of the impactor samplers were exceeded at a spray concentration of 10⁷ CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.     

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.     

Comparison of two biological aerosol sampling methods.

Two biological aerosol samplers, the Andersen two-stage microbial impactor and the May three-stage glass impinger, were examined to determine the benefits and effectiveness of the May sampler compared with the Andersen sampler, one of the most widely accepted samplers. Side-by-side samples were collected during simulated wastewater spray irrigation dispersion studies. Escherichia coli colony counts and air concentrations were statistically treated to determine the dependability of the May results with respect to the Andersen results. After data pairs containing potentially overloaded Andersen counts were eliminated, a linear regression of the remaining data was performed. It indicates that although the May sampler reports 82% of the Andersen sampler value, the correlation between the two samplers is good with an r2 value of 0.84. This comparison indicates that although there are differences between the two samplers, they do give comparable results and that when both are used in a sampling program, they tend to complement each other.     

Comparison of two biological aerosol sampling methods

Two biological aerosol samplers, the Andersen two-stage microbial impactor and the May three-stage glass impinger, were examined to determine the benefits and effectiveness of the May sampler compared with the Andersen sampler, one of the most widely accepted samplers. Side-by-side samples were collected during simulated wastewater spray irrigation dispersion studies. Escherichia coli colony counts and air concentrations were statistically treated to determine the dependability of the May results with respect to the Andersen results. After data pairs containing potentially overloaded Andersen counts were eliminated, a linear regression of the remaining data was performed. It indicates that although the May sampler reports 82% of the Andersen sampler value, the correlation between the two samplers is good with an r2 value of 0.84. This comparison indicates that although there are differences between the two samplers, they do give comparable results and that when both are used in a sampling program, they tend to complement each other.     

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km² area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.     

Comparison of methods for quantitative determinations of airborne bacteria and evaluation of total viable counts.

Three different methods of estimating airborne bacteria were compared. An Anderson sampler, a slit sampler, an impinger, and filter samplers with gelatine filters or membrane filters were tested for collection efficiency. The comparisons were made in laboratory experiments with an aerosol of Staphylococcus epidermidis or Serratia marcescens, in field experiments in two different industries, i.e., cotton mill and sewage plant, and in experiments with skin fragment sampling. Experiments were also performed estimating the total number of viable microorganisms on the airborne particles. The Andersen sampler gave the highest bacterial counts in all environments tested. The slit sampler gave statistically lower counts only in the aerosol experiments and cotton mill experiments, where the size of the majority of the particles carrying visible bacteria was 2 to 6 micrometers or smaller. In the sewage plant and skin fragment experiments, where the particles were mainly 5 micrometers or larger, the difference was not significant. The filters were efficient in sampling in skin fragment experiments only. With the agar impingement method, the total viable cell count showed a rising index value with increasing particle size. A mean of 13 bacteria was found per particle in the cotton mill, a mean of 24 in the sewage plant, and a mean of 147 in skin fragment experiments.     

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.     

Oropharyngeal Aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c Mice

Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.     

Precision of the all-glass impinger and the andersen microbial impactor for air sampling in solid-waste handling facilities.

A method was devised to determine the precision of the all-glass impinger and the Andersen six-stage microbial impactor over a wide range of aerosol concentrations like those found in facilities which process solid waste. Simultaneous samples were collected inside a municipal solid-waste recovery system, and the data were treated statistically to estimate the precision of each air-sampling device. All-glass impingers yielded colony counts which indicated a linear relationship between samplers over an observed aerosol concentration of 1.1 X 10(3) to 2.8 X 10(7) colony-forming units per m3 of air. Impactors also yielded colony counts which indicated a linear relationship over an observed aerosol concentration range of 3.9 X 10(3) to 1.9 X 10(5) colony-forming units per m3 of air. The coefficients of variation for the all-glass impinger and the six-stage impactor in an environment with a high and variable dust level were determined to be 0.38 and 0.23, respectively.     

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.     

Novel Multi-Slit Large-Volume Air Sampler

Scientific investigators who are interested in the various facets of airborne transmission of disease in research laboratories and hospitals need a simple, continuous, high-volume sampling device that will recover a high percentage of viable microorganisms from the atmosphere. Such a device must sample a large quantity of air. It should effect direct transfer of the air into an all-purpose liquid medium in order to collect bacteria, viruses, rickettsia, and fungi, and it should be easy to use. A simple multi-slit impinger sampler that fulfills these requirements has been developed. It operates at an air-sampling rate of 500 liters/min, has a high collection efficiency, functions at a low pressure drop, and, in contrast to some earlier instruments, does not depend upon electrostatic precipitation at high voltages. When compared to the all-glass impinger, the multi-slit impinger sampler collected microbial aerosols of Serratia marcescens at 82% efficiency, and aerosols of Bacillus subtilis var. niger at 78% efficiency.     

Medical Applications of Dust-free Rooms. III. Use in an Animal Care Laboratory

Bacterial air sampling in an animal care laboratory showed that dense aerosols are generated during cage changing and cage cleaning. Reyniers and Andersen sampling showed that the airborne bacteria numbered 50 to 200 colony-forming units (CFU)/ft³ of air. Of the viable particles collected by Andersen samplers, 78.5% were larger than 5.5 μm. A low velocity laminar air flow system composed of high-efficiency particulate air (HEPA) filters and a ceiling distribution system maintained the number of airborne viable particles at low levels, generally less than 2 CFU/ft³. Vertical air flow of 15 ft/min significantly reduced the rate of airborne infection by a strain of Proteus mirabilis. Other factors shown to influence airborne infection included type of cage utilized, the use of bedding, the distance between cages, and the number of animals per cage.     

A Monitor for Airborne Bacteria

A unique agar drum sampler is described which indicates, continuously, the number of viable, bacterial particles per unit volume of air at the time and point of sampling. By selection of the timer and the sampling rate the sampler is suitable for quite a wide range of concentration and time. An impaction line of 484 in. greatly increases the capacity of this device over slit samplers and other instruments designed to give time-concentration data for viable airborne particles. This sampler should prove useful for: (i) monitoring airborne bacteria in hospitals, public places, and food and industrial plants; (ii) decay rate studies of bacterial aerosols; (iii) evaluation of aerial germicides; (iv) determination of effectiveness of air conditioning systems in removing airborne bacteria; and (v) many other studies in aerobiology.     

Dispersal of Pseudocercosporella herpotrichoides Spores from Infected Wheat Straw

Evidence from spore samples collected amongst infected straw spread on fallow ground supported the conclusion that spores of Pseudocercosporella herpotrichoides are dispersed mostly by rainsplash. Most spores travelled a short distance in the larger ballistic splash droplets, although some may have travelled further in smaller airborne droplets. Weekly spore counts from microscope slides under rainshields, a funnel and an impinger, evaluated as samplers for spores of P. herpotrichoides, showed a similar seasonal pattern. The funnel, as the largest sampler, generally collected most spores, but the impinger collected more spores per unit area of sampling surface. Slides sometimes collected spores when none was recovered from other samplers. Young wheat plants, exposed with the samplers and changed weekly, subsequently developed eyespot symptoms for most of the season.     

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.     

Field studies measuring the aerosolization of endotoxin during the land application of Class B biosolids

Endotoxins are a component of Gram-negative bacteria cell walls and are known to be present in biosolids. Endotoxins have been shown to be potent stimulators of the innate immune response causing airway irritation and shortness of breath. Class B biosolids are routinely applied to agricultural lands to enhance soil properties and can be used as an alternative to chemical fertilizers. This study investigated the aerosolized endotoxin dispersed during the land application of Class B biosolids on agricultural land and a concrete surface at two sites in Colorado, USA. Aerosolized endotoxin was captured using HiVol samplers fitted with glass fiber filters, polycarbonate filter cassettes (both open and closed) and BioSampler impinger air samplers. Endotoxins were also measured in the biosolids to allow for correlating bulk biosolids concentrations with aerosol emission rates. Endotoxin concentrations in biosolids, impinger solutions and filter extracts were determined using the kinetic Limulus amebocyte lysate assay. Aerosolized endotoxin concentration was detected from all sites with levels ranging from 0.5 to 642 EU/m³. The four types of sampling apparatus were compared, and the HiVol and open-faced cassette samplers produced higher time-weighted average (TWA) measurements (EU/m³) than the impinger and closed cassette samplers. Ambient wind speed was found to be the variable best describing the observed results with optimal wind speed for highest deposition estimated at 5 m s^?1. It is argued that HiVol air samplers are a particularly reliable approach and subsequent analyses relating TWA measurements to wind speed and biosolids characteristics were based on the measurements collected with those samplers.     

Impaction onto a Glass Slide or Agar versus Impingement into a Liquid for the Collection and Recovery of Airborne Microorganisms

To study impaction versus impingement for the collection and recovery of viable airborne microorganisms, three new bioaerosol samplers have been designed and built. They differ from each other by the medium onto which the bioaerosol particles are collected (glass, agar, and liquid) but have the same inlet and collection geometries and the same sampling flow rate. The bioaerosol concentrations recorded by three different collection techniques have been compared with each other: impaction onto a glass slide, impaction onto an agar medium, and impingement into a liquid. It was found that the particle collection efficiency of agar slide impaction depends on the concentration of agar in the collection medium and on the sampling time, when samples are collected on a nonmoving agar slide. Impingement into a liquid showed anomalous behavior with respect to the sampling flow rate. Optimal sampling conditions in which all three new samplers exhibit the same overall sampling efficiency for nonbiological particles have been established. Inlet and collection efficiencies of about 100% have been achieved for all three devices at a sampling flow rate of 10 liters/min. The new agar slide impactor and the new impinger were then used to study the biological factors affecting the overall sampling efficiency. Laboratory experiments on the total recovery of a typical environmental microorganism, Pseudomonas fluorescens ATCC 13525, showed that both sampling methods, impaction and impingement, provided essentially the same total recovery when relatively nonstressed microorganisms were sampled under optimal sampling conditions. Comparison tests of the newly developed bioaerosol samplers with those commercially available showed that the incorporation of our research findings into the design of the new samplers yields better performance data than data from currently available samplers.     

Methods for Sampling of Airborne Viruses

Summary: To better understand the underlying mechanisms of aerovirology, accurate sampling of airborne viruses is fundamental. The sampling instruments commonly used in aerobiology have also been used to recover viruses suspended in the air. We reviewed over 100 papers to evaluate the methods currently used for viral aerosol sampling. Differentiating infections caused by direct contact from those caused by airborne dissemination can be a very demanding task given the wide variety of sources of viral aerosols. While epidemiological data can help to determine the source of the contamination, direct data obtained from air samples can provide very useful information for risk assessment purposes. Many types of samplers have been used over the years, including liquid impingers, solid impactors, filters, electrostatic precipitators, and many others. The efficiencies of these samplers depend on a variety of environmental and methodological factors that can affect the integrity of the virus structure. The aerodynamic size distribution of the aerosol also has a direct effect on sampler efficiency. Viral aerosols can be studied under controlled laboratory conditions, using biological or nonbiological tracers and surrogate viruses, which are also discussed in this review. Lastly, general recommendations are made regarding future studies on the sampling of airborne viruses.     

Characterization and Deposition of Respirable Large- and Small-Particle Bioaerosols

The deposition patterns of large-particle microbiological aerosols within the respiratory tract are not well characterized. A novel system (the flow-focusing aerosol generator [FFAG]) which enables the generation of large (>10-μm) aerosol particles containing microorganisms under laboratory conditions was characterized to permit determination of deposition profiles within the murine respiratory tract. Unlike other systems for generating large aerosol particles, the FFAG is compatible with microbiological containment and the inhalational challenge of animals. By use of entrapped Escherichia coli cells, Bacillus atrophaeus spores, or FluoSphere beads, the properties of aerosols generated by the FFAG were compared with the properties of aerosols generated using the commonly available Collison nebulizer, which preferentially generates small (1- to 3-μm) aerosol particles. More entrapped particulates (15.9- to 19.2-fold) were incorporated into 9- to 17-μm particles generated by the FFAG than by the Collison nebulizer. The 1- to 3-μm particles generated by the Collison nebulizer were more likely to contain a particulate than those generated by the FFAG. E. coli cells aerosolized using the FFAG survived better than those aerosolized using the Collison nebulizer. Aerosols generated by the Collison nebulizer and the FFAG preferentially deposited in the lungs and nasal passages of the murine respiratory tract, respectively. However, significant deposition of material also occurred in the gastrointestinal tract after inhalation of both the small (89.7%)- and large (61.5%)-particle aerosols. The aerosols generated by the Collison nebulizer and the FFAG differ with respect to mass distribution, distribution of the entrapped particulates, bacterial survival, and deposition within the murine respiratory tract.     

Comparison of coliphage and bacterial aerosols at a wastewater spray irrigation site.

Microbiological aerosols were measured on a spray irrigation site at Fort Huachuca, Ariz. Indigenous bacteria and tracer bacteriophage were sampled from sprays of chlorinated and unchlorinated secondary-treatment wastewaters during day and night periods. Aerosol dispersal and downwind migration were determined. Bacterial and coliphage f2 aerosols were sampled by using Andersen viable type stacked-sieve and high-volume electrostatic precipitator samplers. Bacterial standard plate counts averaged 2.4 x 10(5) colony-forming units per ml in unchlorinated effluents. Bacterial aerosols reached 500 bacteria per m3 at 152 m downwind and 10,500 bacteria per m3 at 46m. Seeded coliphage f2 averaged 4.0 x 10(5) plaque-forming units per ml in the effluent and were detected 563 m downwind. Downwind microbial aerosol levels were somewhat enhanced by nighttime conditions. The median aerodynamic particle size of the microbial aerosols was approximately 5.0 micrometer. Chlorination reduced wastewater bacterial levels 99.97% and reduced aerosol concentrations to near background levels; coliphage f2 was reduced only 95.4% in the chlorinated effluent and was readily measured 137 m downwind. Microbiological source strength an meteorological data were used in conjunction with a dispersion model to generate mathematical predictions of aerosol strength at various sampler locations. The mean calculated survival of aerosolized bacteria (standard plate count) in the range 46 to 76 m downwind was 5.2%, and that of coliphage f2 was 4.3 %.