Application of Flow Cytometry for the Assessment of Preservation and Recovery Efficiency of Bioaerosol Samplers Spiked with <i>Pantoea agglomerans</i>

Rule, Ana M.; Kesavan, Jana; Schwab, Kellogg J.; Buckley, Timothy J.

doi:10.1021/es062394l

Cited by 50 publications

(37 citation statements)

References 24 publications

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“…It also could be interesting to study several strains of the same bacterial species to specifically address strain-specific aerobiological stability characteristics. The ambition for the future also involves adopting additional characterization methods, including flow cytometry in combination with membrane integrity and metabolic cell staining (20,45,46) and quantitative PCR in combination with ethidium or propidium monoazide live-dead discrimination techniques (47)(48)(49)(50), as well as the study of multiple strains of the same bacterial species to investigate strain-specific aerobiological stability characteristics.…”

Section: Single-cell Particles and Cell Clusters Produced From Differmentioning

confidence: 99%

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

Dybwad

Skogan

2017

Appl Environ Microbiol

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The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H 2 O, phosphate-buffered saline [PBS], spent culture medium [SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research.IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gramnegative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.

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Section: Single-cell Particles and Cell Clusters Produced From Differmentioning

confidence: 99%

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

Dybwad

Skogan

2017

Appl Environ Microbiol

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“…Bacteria have been proposed to enter the viable-but-nonculturable (VBNC) state as a result of both aerosolization and impingement. VBNC cells represent a physiological state where bacteria cannot be cultured on nonselective agar yet retain certain vital processes indicative of life, such as active respiratory enzymes or maintenance of membrane integrity (12,31). These phenomena have importance in both experimental animal inhalational studies and natural respiratory transmission events, because the physiological status of the aerosolized bacteria will directly influence the potential to cause infection.…”

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confidence: 99%

The Cell Membrane as a Major Site of Damage during Aerosolization of Escherichia coli

Thomas

Webber

Hopkins

et al. 2011

Appl Environ Microbiol

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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.Aerosolization of bacteria has importance from medical, agricultural, and biodefense perspectives. Bacterial infections of the respiratory tract are transmitted by aerosols produced by coughing and sneezing (26,35,36,37). Insecticidal bacteria, such as Bacillus thuringiensis, may be disseminated by aerosols to control agricultural pests (3). Dissemination of biowarfare agents can occur by the production of an aerosol, and an understanding of survival is important for hazard management (32). In all these examples, for the aerosolized bacteria to exert an effect, they must remain viable in the aerosolized state for a period of time.The physiological status of bacteria changes with time spent in the aerosolized state. Loss of culturability on plating media is often biphasic, being defined by an initial rapid decline over the first few minutes followed by further decline at a lower rate (13,39,41,48). The initial process of aerosolization, for example, nebulization, will impart device-dependent mechanical stress, such as physical shear and wall impaction, resulting from operation of the pressurized system (33,34,38). After aerosolization, a number of parameters will contribute to loss of viability in the airborne bacteria, including temperature, particle size, desiccation (relative humidity), UV radiation, oxidative shock, and toxic pollutants (carbon monoxide, sulfur dioxide, nitrogen dioxide, nitrogen oxide, ozone, and "open air factor") (9,10,13,16,20,36,42,43,46,48). Furthermore, bacteriological factors, such as growth phase or sporulation state, will influence survival (8, 9). Bacteria have been proposed to enter the viable-but-nonculturable (VBNC) state as a result of both aerosolization and impingement. VBNC cells represent a physiological state where bacteria cannot be cultured on nonselective agar yet retain certain...

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“…Reliable methods for collecting and testing aerosols and even the reported units of measurement are debated (Agranovski 2007;Chinivasagam and Blackall 2005;Dabisch et al 2012;Park et al 2014). In addition, aerosolization and collection methods impose stress, resulting in damaged or viable but non-culturable (VBNC) bacteria (Rule et al 2007;Terzieva et al 1996). The handful of studies tested Fig.…”

Section: Discussionmentioning

confidence: 99%

Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

Orlofsky

Benami

Gross

et al. 2015

Water Air Soil Pollut

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A sensitive, high-throughput, and costeffective method for screening bacterial pathogens in the environment was developed. A variety of environmental samples, including aerosols, soil of various types (sand, sand/clay mix, and clay), wastewater, and vegetable surface (modeled by tomato), were concomitantly spiked with Salmonella enterica and/or Pseudomonas aeruginosa to determine recovery rates and limits of detection. The various matrices were first enriched with a general pre-enrichment broth in a dilution series and then enumerated by most probable number (MPN) estimation using quantitative PCR for rapid screening of amplicon presence. Soil and aerosols were then tested in non-spiked environmental samples, as these matrices are prone to large experimental variation. Limit of detection in the various soil types was 1-3 colony-forming units (CFU) g −1 ; on vegetable surface, 5 CFU per tomato; in treated wastewater, 5 CFU L −1 ; and in aerosols, >300 CFU mL −1 . Our method accurately identified S. enterica in non-spiked environmental soil samples within a day, while traditional methods took 4 to 5 days and required sorting through biochemically and morphologically similar species. Likewise, our method successfully identified P. aeruginosa in non-spiked aerosols generated by a domestic wastewater treatment system. The obtained results suggest that the developed method presents a broad approach for the rapid, efficient, and reliable detection of relatively low densities of pathogenic organisms in challenging environmental samples.

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Application of Flow Cytometry for the Assessment of Preservation and Recovery Efficiency of Bioaerosol Samplers Spiked with Pantoea agglomerans

Cited by 50 publications

References 24 publications

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

The Cell Membrane as a Major Site of Damage during Aerosolization of Escherichia coli

Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

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