1.0 ABSTRACT

1. B C A E F D BANG Collison 3-Jet Aerosol parameters MMAD (m) 0.54 1.0 GSD 1.2 1.4 Aerosol Dilution (Spray factor1) Starting Concentration (Culture) 1.0E+07 3.91E-06 1.19E-05 1.0E+08 7.07E-06 1.67E-05 1.0E+09 6.15E-06 1.11E-05 MEAN 5.71E-06 1.32E-05 (Flow Cytometry) 1.0E+07 4.15E-03 1.31E-03 1.0E+08 6.09E-03 4.20E-03 1.0E+09 9.11E-03 4.67E-03 MEAN 6.45E-03 3.39E-03 COMPARISON OF IMPACTION AND DEAD- SPACE NEBULIZERS ON THE BIOAEROSOL CHARACTERISTICS OF VEGETATIVE BACTERIA  Brittany Goodenow and Chad J. Roy Division of Toxinology & Aerobiology, Department of Aerobiology & Product Evaluation United States Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland, USA 4.0 RESULTS 1.0 ABSTRACT 3.0 MATERIALS AND METHODS (cont.) Aerosol generation of biological aerosols (bioaerosols) is an essential part of ongoing vaccine efficacy and agent pathogenesis research efforts at USAMRIID. In this study, two commerically-available nebulizers (Collison 3-jet and the BioAerosol Nebulizing Generator; BANG) were compared to assess the impact of aerosolization on the viability of the bacteria Pseudomonas aeruginosa. Operating procedures were kept constant between the nebulizers and aerosol samples were taken continuously during generation of the experimental atmospheres. Serial dilutions (107, 108, and 109 CFU/ml) of the P.aeruginosa were used as starting concentrations in each of the nebulizers. Aerosol samples were analyzed using both conventional culture and nonculture techniques such as fluorescent microscopy and flow cytometry. Particle analysis indicated smaller and less heterodisperse aerosols were generated by the BANG (MMAD=0.54;GSD=1.2) than the Collison (MMAD=1.0;GSD=1.4). Results of the culture analysis indicated the Collison generated slightly higher viable aerosol concentrations of P.aeruginosa than the BANG suggested by lower mean aerosol dilution factors (Collison;1.32E-05  3.0E-04, BANG;5.71E-061.6E-06). Flow cytometric analysis of total organism output was virtually identical from the nebulizers. In contrast to the culture data, Live/dead analysis by flow cytometry indicated a higher percentage of viable bacterial cells relative to the concentration of the starting material in the BANG output. The results of this preliminary study suggest that these nebulizers are comparable in total particle output. The Collison produced a slightly higher viable bioaerosol, although differential flow cytometric analysis of viability indicated the BANG produced biaoerosols with a higher viability ratio. This disparity of results may be explained by the “viable but nonculturable; VBNC” phenomena observed in vegetative bacteria exposed to unusual environmental stress during aerosolization. Bioaerosol Quantitation. The starting solution were seven-fold diluted in triplicate and 100 l of each dilution was plated on TSA. The AGI samples were diluted at 1:10, 1:100, and 1:1000 in triplicate and 100l of each dilution was plated on TSA. The plates were incubated at 37C and counted at 48 hours. Diluent PBS was also plated and total CFU were subtracted from colony counts from the experimental samples. Flow cytometry was performed on the starting material and AGI samples for total microorganism count. The preparation for flow cytometry has been previously described in the literature (Lange et al., 1997). A bacterial counting kit (Molecular Probes, Inc., Eugene, OR) was used for flow cytometry analysis. Briefly, one l of universal DNA stain was added to a one ml aliquot of either the starting solution or AGI sample. The solution was then vortexed, and allowed to incubate at 37 C for five minutes. A microsphere standard supplied in the kit was sonicated for 10 minutes before addition to the preparation. Exactly ten l of the microsphere standard was added, and the preparation again was vortexed. A live/dead bacterial viability kit (Molecular Probes, Inc., Eugene, OR) was used for flow cytometry analysis. A mixture of a red and green DNA stain were added at a 1:1 dilution with the starting solution or AGI sample. The solution was vortexed and incubated at room temperature for 30 minutes. All flow cytometric analysis was performed using a Becton Dickinson FACScan with a single excitation argon ion laser at a wavelength of 488 nanometers. Results were analyzed using a Multiple Document Interface program (Version 2.8, J. Trotter, Scripps Research Institute, CA). 4.0 RESULTS Figure 2. Dot plots of serial total aerosol samples for the BANG (A, B, C) and Collison 3-jet (D, E, F) at starting concentrations of 107, 108, 109, respectively. Aerosol samples analyzed for total organisms indicated minimal differences in the total particle output between nebulizers. This is evident in the dot plots above; the total bacteria (R2; green) increases as the starting concentration increases. Internal microsphere standards (R1; red) were 10 m PSL beads. 2.0 OBJECTIVE Figure 1 Figure 2 Figure 3. Live/dead flow cytometric analysis of aerosol samples. A trend of increased viability is observed in the aerosols that were performed with a higher concentration of P.aeruginosa in the nebulizer starting concentration. Higher viability counts relative to the total bacterial cells counted are observed in the aerosols generated by the BANG compared to the Collison 3-jet at the highest concentration attempted. The objective of this study was to compare the Collison and the BANG nebulizer using bacterial viability as the primary indicator of differences between the two devices. This was accomplished by organism enumeration by both culture and nonculture methods before and after aerosol generation. 3.0 MATERIALS AND METHODS Nebulizers and Exposure Chamber. The BANG, a low flow, dead space, single pass nebulizer, was specially-designed to incorporate 3 jets to approximate Collison output (CH Technologies, Westwood, NJ). The Collison 3-jet nebulizer was commerically-obtained (BGI Inc., Waltham, MA). HEPA-filtered, dried air at 25 psi was supplied to each nebulizer during the aerosol exposures. Appropriate operating pressure for the BANG has been previously performed and showed minimal differences in aerosol output at a supplied pressure higher than 20 psi (L. Chen, personal communication). Clean secondary air was also supplied in the aerosol mixing tube into the exposure chamber. The primary air flow used with the BANG was 7 LPM with the total airflow for the system being 10 LPM. The primary air flow used with the 3 jet Collison was 7.5 LPM with the total airfow for the system being 19.5 LPM. The exposure chamber used was a 0.01 m3 plexiglass box used in conjunction with ongoing rodent studies. The average relative humidity and temperature in the chamber were 35% and 72°F during aerosol runs.  Aerosols. For the nebulizer characterization, 1X fresh phosphate-buffered saline (PBS) was used (Gibco, Grand Island, NY). For the aerosols involving bacteria, Pseudomonas aeruginosa (ATCC, Rockville, MD) was grown in Tryptic soy broth (TSB; Difco, Detroit, MI) at 37 degrees C for 24 hours. Thereafter, the broth was centrifuged, the pellet washed, and resuspended in 1X PBS. Seven-fold dilution aliquots were plated in triplicate on Tryptic soy agar (TSA; Difco, Detriot, MI). The 24 hour counts yielded a stock concentration of 1.0E+10 CFU/ml. Dilutions of 1.0E+09, 1.0E+8, and 1.0E+07 CFU/ml were viable concentrations. Aerosol Characterization. Particle characterization was performed using an aerodynamic particle sizer (APS Model 3320, TSI Products, St. Paul, MN). APS samples were taken for one minute twice per aerosol run; each aerosol run lasted five minutes. Aerosol Sampling. 6 LPM all glass impingers (AGI) containing 10 ml of PBS (Ace Glass, Vineland, NJ) were used to sample the chamber atmospheres. The sampling efficiency of the AGI sampler in this bioaerosol system has been shown to be more than 97% efficient (L. Pitt, personal communication). 5.0 CONCLUSIONS Table 1. Aerosol Characteristics of the BANG and 3 Jet Collison 1The spray factor is a unitless measure of the ratio of starting concentration (cS) to aerosol concentration (cA) and can be expressed as: SF=cS (CFU/)/cA (CFU/) • Nebulizers produced equivalent particle output; the BANG nebulizer generated a smaller and less disperse aerosol • The process of aerosolization destroys a significant number of organisms relative to culturable organisms in the starting liquid regardless of nebuilzer used • The Collison nebulizer produced a higher culturable bioaerosol; the BANG returned a higher viable count at high (109) nebulizer starting concentrations • Vegetative bacteria undergo significant environmental stress when experimentally-aerosolized; this process may induce a “viable but nonculturable” state (Hiedelburg, 1997) Figure 1. Comparison of the BANG and 3-jet by culture and total organism count by flow cytometric analysis. Results show minimal differences between nebulizers regardless of analysis method, although the Collison 3-jet did produce slightly higher culturable counts than the BANG. A consistent 4-log difference between culturable and total organism aerosol concentration was detected independent of starting concentration in the nebulizers. 6.0 REFERENCES Hiedelburg, J.F., Shahamat, M., Levin, M., Rahman, I., Stelma, G., Grim, C., and Colwell, R.R. (1997). Effect of Aerosolization on Culturability and Viability of Gram-Negative Bacteria. Applied and Environmental Microbiology 63(9):3585-3588. Appl Environ Microbiol 1997 Apr;63(4):1557-63 Lange, J.L., Thorne, P.S., Lynch, N (1997). Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria63(4):1557-63.