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.     

Evaluation of four sampling devices for Burkholderia pseudomallei laboratory aerosol studies

Previous field and laboratory studies investigating airborne Burkholderia pseudomallei have used a variety of different aerosol samplers to detect and quantify concentrations of the bacteria in aerosols. However, the performance of aerosol samplers can vary in their ability to preserve the viability of collected microorganisms, depending on the resistance of the organisms to impaction, desiccation, or other stresses associated with the sampling process. Consequently, sampler selection is critical to maximizing the probability of detecting viable microorganisms in collected air samples in field studies and for accurate determination of aerosol concentrations in laboratory studies. To inform such decisions, the present study assessed the performance of four laboratory aerosol samplers, specifically the all-glass impinger (AGI), gelatin filter, midget impinger, and Mercer cascade impactor, for collecting aerosols containing B. pseudomallei generated from suspensions in two types of culture media. The results suggest that the relative performance of the sampling devices is dependent on the suspension medium utilized for aerosolization. Performance across the four samplers was similar for aerosols generated from suspensions supplemented with 4% glycerol. However, for aerosols generated from suspensions without glycerol, use of the filter sampler or an impactor resulted in significantly lower estimates of the viable aerosol concentration than those obtained with either the AGI or midget impinger. These results demonstrate that sampler selection has the potential to affect estimation of doses in inhalational animal models of melioidosis, as well as the likelihood of detection of viable B. pseudomallei in the environment, and will be useful to inform design of future laboratory and field studies.     

Measurement of the change in aerosol size distribution with bacterial growth in a pilot scale fermentor

A number of industrial processes require the addition of materials to the fermentation broth that are hazardous to health and environment. Agitation of broths inoculated with microorganisms can potentially release aerosols large enough to carry the microorganisms. The influence of agitation, air flow, and bacterial growth on aerosol size distribution, air flow, and bacterial growth on aerosol size distribution was investigated in an industrial pilot scale fermentor. A decrease in particle concentration was observed with increase in bacterial growth; this change was more pronounced in the size range above 2 mum. The aerosol size distribution was found to be practically independent of air flow rate and agitation rate for sizes less than 2 mum. However, for particles largar than 2 mum, the concentration was found to increase with an increase in air flow rate and agitation rate.     

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.     

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.     

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.     

Microbiological Evaluation of a Large-Volume Air Incinerator

Two semiportable metal air incinerators, each with a capacity of 1,000 to 2,200 standard ft(3) of air per min, were constructed to sterilize infectious aerosols created for investigative work in a microbiological laboratory. Each unit has about the same air-handling capacity as a conventional air incinerator with a brick stack but costs only about one-third as much. The units are unique in that the burner housing and combustion chamber are air-tight and utilize a portion of the contaminated air stream to support combustion of fuel oil. Operation is continuous. Aerosols of liquid and dry suspensions of Bacillus subtilis var. niger spores and dry vegetative cells of Serratia marcescens were disseminated into the two incinerators to determine the conditions required for sterilization of contaminated air. With the latter organisms (concentration 2.03 x 10(7) cells/ft(3) of air), a temperature of 525 F (274 C), measured at the firebox in front of the heat exchanger, was sufficient for sterilization. To sterilize 1.74 x 10(7) and 1.74 x 10(9) wet spores of B. subtilis per ft(3), the required temperature ranged from 525 to 675 F (274 to 357 C) and 625 to 700 F (329 to 371 C), respectively. Air-sterilization temperature varied with each incinerator. This was because of innate differences of fabrication, different spore concentrations, and use of one or two burners With dry B. subtilis spores (1.86 x 10(8)/ft(3)), a temperature of 700 F was required for sterilization. With dry spores, no difference was noted in the sterilization temperature for the two incinerators.     

Effects of bronchodilator particle size in asthmatic patients using monodisperse aerosols.

Aerosol particle size influences airway drug deposition. Current inhaler devices are inefficient, delivering a heterodisperse distribution of drug particle sizes where, at best, 20% reaches the lungs. Monodisperse aerosols are the appropriate research tools to investigate basic aerosol science concepts within the human airways. We hypothesized that engineering such aerosols of albuterol would identify the ideal bronchodilator particle size, thereby optimizing inhaled therapeutic drug delivery. Eighteen stable mildly to moderately asthmatic patients [mean forced expiratory volume in 1 s (FEV1) 74.3% of predicted] participated in a randomized, double-blind, crossover study design. A spinning-top aerosol generator was used to produce monodisperse albuterol aerosols that were 1.5, 3, and 6 microm in size, and also a placebo, which were inhaled at cumulative doses of 10, 20, 40, and 100 microg. Lung function changes and tolerability effects were determined. The larger particles, 6 and 3 microm, were significantly more potent bronchodilators than the 1.5-microm and placebo aerosols for FEV1 and for the forced expiratory flow between exhalation of 25 and 75% of forced vital capacity. A 20-microg dose of the 6- and 3-microm aerosols produced FEV1 bronchodilation comparable to that produced by 200 microg from a metered-dose inhaler. No adverse effects were observed in heart rate and plasma potassium. The data suggest that in mildly to moderately asthmatic patients there is more than one optimal beta2-agonist bronchodilator particle size and that these are larger particles in the higher part of the respirable range. Aerosols delivered in monodisperse form can enable large reductions of the inhaled dose without loss of clinical efficacy.     

Survival of mycobacterial species in aerosols generated from artificial saliva

Tuberculosis is transmitted primarily by the aerosol route and the aim of this study was to measure the ability of pathogenic mycobacteria to survive in aerosols generated from artificial saliva. Aerosols of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis were generated and maintained in air under controlled conditions using a Henderson apparatus and a rotating drum. There were no differences in aerosol survival between the three species, and all had a poor survival rate over a period of 1 h. These data confirm epidemiological studies that close and prolonged contact with a TB patient is required for transmission of infection.     

Role of Relative Humidity in the Survival of Airborne Mycoplasma pneumoniae

Aerosols of Mycoplasma pneumoniae were studied at several relative humidities at a controlled temperature of 27 C. Production of an experimentally reproducible aerosol required preatomization of the organism in its suspending fluid and was dependent on the type of fluid used in atomization as well as on the procedures used to produce an aerosol. The airborne particles studied were within the range of epidemiological significance, with most being 2 mum or less in diameter. Survival of the airborne mycoplasma in these particles was found to be best at very low and at very high humidities. The most lethal relative humidity levels were at 60 and 80%, at which levels fewer than 1% of the organisms survived over a 4-hr observation period. However, survival of the organism at most relative humidity levels was such that long-term infectivity could be expected from aerosols of M. pneumoniae. Because of the extreme sensitivity of M. pneumoniae at critical humidity levels, control of the airborne transmission of these organisms may be possible in selected spaces.     

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.     

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.     

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.     

An assessment of the Sartorius MD8 microbiological air sampler

Tests described in this paper show that gelatine membrane filters used in the MD8 microbiological air sampling system collected monodispersed aerosols between 0·7 and 1·0 μm containing viable Bacillus subtilis var. niger spores, with an efficiency of 99·9995%. Gelatine membrane filters linked to the MD8 control pump system were as effective as the well established Casella slit-to-agar device for collecting some viable bacteria, nebulized under controlled experimental conditions and naturally occurring airborne micro-organisms in a pharmaceutical plant. By using a long flexible hose connection to the control pump, the head could be positioned where sampling was required in locations remote from the pump exhaust, making it suitable for microbiological monitoring in critical locations such as laminar flow stations and isolators.     

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.     

A study on tracheobronchial deposition and 2-hour retention in rabbits.

Deposition of well-defined test aerosols of polystyrene particles was studied in rabbits. The deposition was estimated in a standardized part of the tracheobronchial tree following free dissection of the bronchial parts. Ten rabbits were exposed to a mixture of two test aerosols of about the same size(6-7 mum) tagged with 51-Cr and 46-Sc, respectively. There were interindividual differences in tracheobronchial deposition but a correlation (r equals 0.98) between 51-Cr and 46-Sc tagged particles. Eleven rabbits were first exposed to the 46-Sc tagged aerosol and 2 hours later to the 51-Cr tagged aerosol. In this experiment there was also a correlation (r equals 0.74) between the tracheobronchial deposition of 51-Cr tagged particles and the 2-hour retention of 46-Sc tagged particles. This result together with results from an earlier study indicate that the large interindividual differences in deposition are real and are not caused by differences in exposure technique, or in the aerosols.     

Survival of Airborne Pasteurella tularensis at Different Atmospheric Temperatures

The aerosol survival, recovery, and death rate of Pasteurella tularensis SCHU S5 disseminated in particle sizes of 1 to 5 mum were significantly affected by air temperature. The highest aerosol recovery of viable P. tularensis was observed within -7 and 3 C; the recovery decreased significantly below and above this temperature range. The death rate of airborne P. tularensis was not significantly influenced by an increase in temperature from -40 to 24 C. However, a progressive increase in atmospheric temperature from 24 to 35 C resulted in increased death rates; thus, a linear relationship appeared to be present between the temperature and death rates. At 49 C, the recoveries of viable airborne P. tularensis were significantly lower and the death rates were higher than at the other temperatures.     

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.     

Bees Scavenge Airborne Bacteria

Abstract An air conditioned wind tunnel system was designed, fabricated, and tested to determine whether tethered bees scavenge microbeads or Bacillus subtilis var. niger spores from aerosols. Tests showed that microbeads and spores were scavenged by bumblebees and honeybees, respectively. Five independent variables and their interactions were used in a stepwise multiple regression. Two of them, the cube root of the electrostatic charge on the honeybee and the dose of the spore aerosol, accounted for most of the statistically significant fit to the model's two dependent variables: the percentage of the dose adsorbed by honeybees and the number of spores adsorbed by the same bees. Both dependent variables increased directly so that an increase in electrostatic charge on the bee (i.e., cube root 32 pC) resulted in an increase (i.e., approximately 1%) in the spore dose adsorbed and the number of spores adsorbed by the bees. It was theorized that the spores were in an adsorption/desorption equilibrium that responded to the concentration ``pressure' of the spore aerosol. Further, the charge on the bee affected the adsorption force on the bee's surface, as well as increasing the effective aerosol volume accessible for the bee's scavenging. In short, relating these findings to bees scavenging bacteria from the ambient atmosphere, it appears that the spore exposure (where exposure means the product of the ambient concentration, the time the bee is exposed, and air volume through which the bee flies) controls the number of spores adsorbed by a bee, and the static charge on the bee controls the adsorption/desorption equilibrium and presumably the scavenging volume. Received: 22 November 1999; Accepted: 21 January 2000; Online Publication: 29 May 2000