Urban Aerobiology

Project 1: Urban Aerosol Dynamics and Microbiological Diversity

Climate change is likely to affect the nature of airborne pathogens and air chemistry along with their their fate and transport.   Thus, future health risks due to respirable pathogens may be evolving towards a very different suite of conditions than we currently experience.  Two major challenges to our preparation for these changes exist; first, the relationships between climate and infectious disease are often highly dependent upon local-scale parameters and there are potential pitfalls in extrapolating climate and disease relationships from one spatial/temporal scale to another.  Second, and maybe more problematic, we do not currently understand the atmospheric microbiome.  The knowledge of the microbial populations that exist as a function of attitude and even land surface type and their variability is in its infancy.  How do we prepare for the future when we do not have an adequate baseline?

In response to this challenge, my group is part of a interdisciplinary team of faculty and students with specialties in Microbiology, Chemistry, Atmospheric Sciences, Botany, Tropical Medicine and Infectious Diseases, Environmental Sciences, and Genomics formed to characterize the dynamical nature of atmospheric particulate in urban zones.  Aerobiological samples are collected in Washington, DC and Beltsville, MD and culture-based and genomic methods are used to determine microbial diversity in the air as a function of season, synoptic conditions, and local activities that might influence bioaerosols.  For a limited subset of the samples, 16S rRNA sequencing is being used – not only to rapidly identify prokaryotes in culture, but also to detect them directly from the environment. This technique can help overcome some of the disadvantages and limitations encountered when using traditional microbiology identification and characterization methods.

We anticipate the creation of a microbial database that will enable distinction of relevant species fluctuations throughout the year along with the elucidation of environmental conditions favorable for the transportation of microorganisms in the Washington D.C. area, and establish a relationship with the presence of airborne microorganisms, atmospheric parameters, and human health. Knowing the biological background of the atmosphere has potential implications for national security. When this research is completed, the database will help researchers differentiate between normal airborne pathogens and suspicious fluctuations in the nation’s capital, which can help identify risk. This research can also help scientists refine air quality tests that identify disease-causing bacteria and improve air quality standards. 

In addition to urban centers in the US, the aerobiological background of arid regions of the Sahara and the Sahel are representative source regions for mineral dust storms and biomass burning aerosols that are transported across the Atlantic Ocean toward North America and into the Caribbean air space. Air samples have been collected from both urbanized and rural regions of Mali, Senegal, Nigeria, Sudan, Niger, and Ethiopia.  The specific protocols for sampling at each site are determined based on site characteristics, including but not limited to geography, microclimate, site security, and stable, clean electrical power.  Air samples will be collected using a bioaerosol cyclone impactor with gravimetric filters.  In the future, a high volume filter sampler may also be employed if more comprehensive analyses are required.  The six-stage cyclone impactor is capable of use with either gravimetric filters or with Petri dishes equipped with agar or appropriate nutrients for collection and isolation of fastidious bacteria.  Each stage is designed to capture airborne particles of a specific aerodynamic size range.  Thus, each bioaerosol sample in the cyclone impactor will also be size-segregated.  Knowledge of the distribution of bacteria as a function of aerosol size provides key information for modeling long-range transport efficiencies.  Both the filter and the Petri dish sampling methods allow for offline characterization and treatment of the microbiological properties of the airborne species.  

We are using both culture-dependent and genomic methods to characterize and identify fastidious pathogenic bacteria and the full diversity of bacteria present on the airborne aerosol.  This information will provide supporting data on the presence of pathogenic bacterial species African air masses and their potential effect on downwind ecosystems, and public health. This information could also be used in combination with the physical characteristics of the atmospheric dust to provide supporting data on the distribution of microorganisms in Saharan dust that as well assist in trajectory modeling for the determination of potential source of these microorganisms.

All samples are screened for the presence of viable pathogenic bacteria.  Phenotypic characteristics are determined for all representative bacterial colonies and classified into families.  16S rRNA sequences will be determined for representatives of each family group.   In addition, data corresponding to the atmospheric conditions of the area are correlated with those of microbial diversity to enhance the development of models capable of predicting aerobiological trends and climate response.

Specific Activities

Time-series and Seasonal Analysis of Airborne Microbes in Washington, DC… Atmospheric filter samples were collected over a contiguous 14-month period in 2007-2008.  These samples are being analyzed for the microbial background of the urban airspace.  These data are being combined with various other data sets to examine correlations between aerosol composition and microbial distribution, meteorological parameters, air mass history, and time of year, among other environmental factors.  We are gearing up for another long-term study in 2019.  One of the student posters from the 2007 study is given here: http://www.howard.edu/amgenscholars/archives/Abstracts08.htm#Kelly 

Optimization of Aerobiological Collection and Recovery Methods …Ongoing studies are being conducted to quantify and improve collection efficiencies of biological materials from air, to characterize size-specific dependencies, sampling biases, and sampling best practices.

Microbiological and Genomic Characterization of Aerobiological Samples…Both traditional culturing and genomic sequencing techniques are being employed to fully characterize the complete microbial biodiversity of an environment.  Emphasis is placed on utilizing higher throughput genomic methods to obtain the full suite of DNA in atmospheric samples.  The goals of this project are to determine the size-specific distributions of microbes, their viability, virulence, and dependence on the chemical environment of the aerosol carrier.

Viable Airborne Bacterial Populations in large scale dust events using Phenotypic and Phylogenetic Characterization

Phospholipid and Methyl Ester Fatty Acids as Biomarkers for the Rapid Identification of    Viable-Nonculturable Microbiota in Saharan Dust

The detection and taxonomic classification of potentially pathogenic Saharan dust bacteria has classically been conducted using enrichment, molecularly (16SrRNA sequencing) and biochemically (Analytical Profile Index 20E [API20E]).  However, these methods are laborious, expensive and reflect between 0.1-10 percent of culturable bacteria.  Undoubtedly, these approaches to microbial identification undervalue the plethora of data available through direct (soil) chemical analyses of cell components such as proteins and lipids.  In the current study, fatty acid methyl esters (FAME) and phospholipid fatty acids (PLFA) biomarkers will be derived from viable/culturable and viable/non-culturable Saharan dust microbiota, soil samples from the Caribbean, North America and Africa and type cultures such as Enterobacter aerogenes (ATCC 35028), Escherichia coli 0157:H7, Salmonella typhimurium (ATCC 14028), Pseudomonas fluorescens (ATCC 13525), Enterococcus faecium (ATCC 51559), Streptomyces spp., Micrococcus luteus, Bacillus subtilis, Staphylococcus spp., Streptococcus spp. and Proteus spp.  Methyl esters, generated using saponification, derivatization and extraction will be analyzed using gas chromatography/mass spectrometry (GC/MS) and or liquid chromatography-electrospray ionization mass spectroscopy (LC-ESI MS). Data obtained will be evaluated using the principal component analysis (PCA) in addition to other methods.  

Intact Cell Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry as a Tool for the Presumptive Identification of Saharan Dust Microbiota 

Classical microbiological methods such as Analytical Profile Index 20E (API20E) and 16SrRNA sequencing have been used extensively for the detection and enumeration of airborne microorganisms, especially pathogens. However, these methods are time-consuming (days to perform), very expensive and only indicate a 0.1-10% glimpse of the total viable and culturable microbiota present in airborne samples.  Thus, there is an urgent need for us to seek robust, rapid, reliable and cost effective methods for screening and/or identification of microbiota using the whole cell approach. In this study, matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in conjunction with the Rapid Microorganism Identification Database (RMIDb), the Shannon-Weaver Diversity Index and 16SrRNA sequencing will be used to evaluate the diversity (H) and evenness (EH) of the viable/culturable bacteria contained in transatlantic Saharan dust and soil samples from Africa, North America and the Caribbean.  A standard MALDI-TOF MS protocol will be developed for type cultures which include E. aerogenes (ATCC 35028), E. coli 0157:H7, S. typhimurium (ATCC 14028), P. fluorescens (ATCC 13525), E. faecium (ATCC 51559), Streptomyces spp., M. luteus and B. subtilis, and then applied to unknown isolates.  Data obtained from MALDI-TOF MS/RMIDb will be evaluated using the principal component analysis (PCA) and also compared to the classical methods (API 20E/16SrRNA sequencing).  

Students with backgrounds or interest in aerobiology, microbiology, metagenomics, or environmental biology are encouraged to participate.   Required skills are critical thinking, working knowledge of error analysis and statistical methods, knowledge of molecular techniques for DNA extraction, isolation, and separation. 

Publications

  1. M. A. Velez-Quinones, B. Eribo, K. E. Nelson, G. A. Nunez, and V. R. Morris Analysis of Viable Airborne Bacteria in Ambient Aerosols of Bamako, Mali: Potential Sources and Transport Patterns [Manuscript in Preparation]
  2. M. A. Fayissa, V. R. Morris, S. Melaku, and N. Greene Fine Aerosol Size Distributions and Deposition Fluxes in Washington, DC [Manuscript in Preparation] 
  3. A. D. Allen, B. Eribo, M. A. Velez-Quinones, V. R. Morris MALDI-TOF MA and 16SrRNA as Tools of the Evaluation of Bacterial Diversity in Soils from Sub-Saharan Africa and the Americas Aerobiologia 31:111-126, 2015
  4. S. Abegaz, N. Greene, V. Morris Spatio-Temporal Distributions of Particulate Matter Exposures in Washington, D.C. Journal of Natural and Environmental Sciences 2(1) 1 2011
  5. S.  Abegaz, D. Raghavan, C. Hosten, V. R. Morris Evaluation of Heavy Metal Variability in Ambient Air in Washington, D.C.  Environmental Pollution 155 (1) 88-98 2008. 
  6. N. Greene and V. R. Morris Susceptible Populations Via Air Quality An Environmental Risk Assessment of Public Health in Washington, DC The International Journal of Environmental Health Research 3(1) 86-97 2006  
  7. N. Greene, J. White, V. Morris, S. Roberts, K. L. Jones, C. Warrick Evidence for Environmental Contamination in Residential Neighborhoods Surrounding the Defense Depot of Memphis, Tennessee The International Journal of Environmental Health Research 3(3), 224, 2006  

Sample Presentation – Air-Climate-Health_Briefing_May20