Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

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Using flow cytometry and light-induced fluorescence to characterize the variability and characteristics of bioaerosols in springtime in Metro Atlanta, Georgia

Atmospheric Chemistry and Physics ◽

10.5194/acp-20-1817-2020 ◽

2020 ◽

Vol 20 (3) ◽

pp. 1817-1838 ◽

Cited By ~ 1

Author(s):

Arnaldo Negron ◽

Natasha DeLeon-Rodriguez ◽

Samantha M. Waters ◽

Luke D. Ziemba ◽

Bruce Anderson ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Aerosol Particles ◽

Fungal Spores ◽

Epifluorescence Microscopy ◽

Nucleic Acid Content ◽

Rain Event ◽

Bacterial Cells ◽

Biological Aerosol Particles

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation, and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA, over a 2-month period and showed clearly defined populations of nucleic-acid-containing particles: low nucleic acid-content particles above threshold (LNA-AT) and high nucleic acid-content particles (HNA) likely containing wet-ejected fungal spores and pollen. We find that the daily-average springtime PBAP concentration (1 to 5 µm diameter) ranged between 1.4×104 and 1.1×105 m−3. The LNA-AT population dominated PBAP during dry days (72±18 %); HNA dominated the PBAP during humid days and following rain events, where HNA comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that fluorescent biological aerosol particles (FBAP) and total FCM counts are similar; HNA (from FCM) moderately correlated with ABC-type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 µm diameter). However, the FCM LNA-AT population, possibly containing bacterial cells, did not correlate with any FBAP type. The lack of correlation of any WIBS FBAP type with the LNA-AT suggests that airborne bacterial cells may be more difficult to unambiguously detect with autofluorescence than currently thought. Identification of bacterial cells even in the FCM (LNA-AT population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed the highest concentration on a humid and warm day after a rain event (14 April 2015), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days and an LNA-AT population dominating the bioaerosol community during dry days.

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Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1775-1782.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1775-1782 ◽

Cited By ~ 241

Author(s):

Philippe Lebaron ◽

Pierre Servais ◽

Helene Agogué ◽

Claude Courties ◽

Fabien Joux

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Acid Content ◽

Fluorescent Dyes ◽

Specific Activity ◽

Aquatic Systems ◽

Nucleic Acid Content ◽

Leucine Incorporation ◽

Bacterial Cells ◽

Syto 13

ABSTRACT The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

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Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

Aquatic Microbial Ecology ◽

10.3354/ame028131 ◽

2002 ◽

Vol 28 ◽

pp. 131-140 ◽

Cited By ~ 104

Author(s):

P Lebaron ◽

P Servais ◽

AC Baudoux ◽

M Bourrain ◽

C Courties ◽

...

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Cell Size ◽

Acid Content ◽

Specific Activity ◽

Nucleic Acid Content

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Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

Gut Microbes ◽

10.1080/19490976.2021.1903289 ◽

2021 ◽

Vol 13 (1) ◽

pp. 1-15

Author(s):

Mariia Taguer ◽

B. Jesse Shapiro ◽

Corinne F. Maurice

Keyword(s):

Nucleic Acid ◽

Gut Microbiota ◽

Acid Content ◽

Nucleic Acid Content ◽

Bacterial Cells ◽

Human Gut ◽

Human Gut Microbiota ◽

Translational Activity

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Characteristics and Dynamics of Bacterial Populations during Postantibiotic Effect Determined by Flow Cytometry

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.5.1005 ◽

1998 ◽

Vol 42 (5) ◽

pp. 1005-1011 ◽

Cited By ~ 24

Author(s):

Magnús Gottfredsson ◽

Helga Erlendsdóttir ◽

Ásbjörn Sigfússon ◽

Sigurdur Gudmundsson

Keyword(s):

Flow Cytometry ◽

Nucleic Acid ◽

Fluorescence Microscopy ◽

Acid Content ◽

Quantitative Information ◽

Nucleic Acid Content ◽

Dependent Manner ◽

Postantibiotic Effect ◽

E Coli ◽

Drug Removal

ABSTRACT Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM). However, EM is qualitative and subject to individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid contents of Escherichia coli and Pseudomonas aeruginosa were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy. The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for E. coli, whereas for P. aeruginosa imipenem and ciprofloxacin were investigated. After exposure of E. coli to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy. These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry. For the β-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria. These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal. In contrast, the subpopulations induced by ciprofloxacin did not return to normal until >180 min after the end of the classically defined PAE. Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure. For P. aeruginosa an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem. These changes persisted past the PAE, as defined by viability counting. Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth. In summary, for β-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms. Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE.

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Activity and diversity of bacterial cells with high and low nucleic acid content

Aquatic Microbial Ecology ◽

10.3354/ame033041 ◽

2003 ◽

Vol 33 ◽

pp. 41-51 ◽

Cited By ~ 82

Author(s):

P Servais ◽

EO Casamayor ◽

C Courties ◽

P Catala ◽

N Parthuisot ◽

...

Keyword(s):

Nucleic Acid ◽

Acid Content ◽

Nucleic Acid Content ◽

Bacterial Cells

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Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

10.1101/2020.10.05.327163 ◽

2020 ◽

Author(s):

Mariia Taguer ◽

B. Jesse Shapiro ◽

Corinne F. Maurice

Keyword(s):

Nucleic Acid ◽

Gut Microbiota ◽

Gut Microbiome ◽

Acid Content ◽

Membrane Damage ◽

Bacterial Activity ◽

Nucleic Acid Content ◽

Bacterial Cells ◽

Human Gut ◽

Human Gut Microbiota

AbstractBackgroundChanges in bacterial diversity in the human gut microbiome, characterized primarily though DNA sequencing methods, have been associated with many different adverse health conditions. However, these changes do not always reflect changes in bacterial activity, and thus how the gut microbiome is implicated in disease is still not often understood. New methods that link together bacterial function to bacterial identity are needed to further explore the role of the gut microbiome in health and disease. We optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare and contrast to other methods of bulk community measurements of activity and viability: physiological staining of relative nucleic acid content and membrane damage. Relative nucleic acid content has previously been linked to metabolic activity, yet remains currently undefined for the human gut microbiota.ResultsTen healthy, unrelated individuals were sampled to determine the proportion and diversity of distinct physiological fractions of their gut microbiota. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content (HNA) bacteria also represent about half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community.ConclusionsBONCAT is a suitable method to probe the translationally active members of the human gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community, and so further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Taking into account physiologically distinct subsets of the gut microbiota may be more informative than relying on whole community profiling.

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