Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology

Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimization, which greatly slows the advance of bioaerosol science. Here, we evaluated air filtration and liquid impingement for bioaerosol sampling across a range of environmental conditions. We also investigated the effect of sampling matrices, sample concentration strategies and sampling duration on DNA yield. Air filtration using polycarbonate filters gave the highest recovery, but due to the faster sampling rates possible with impingement, we recommend this method for fine ‐scale temporal/spatial ecological studies. To prevent bias for the recovery of Gram‐positive bacteria, we found that the matrix for impingement should be phosphate‐buffered saline. The optimal method for bioaerosol concentration from the liquid matrix was centrifugation. However, we also present a method using syringe filters for rapid in‐field recovery of bioaerosols from impingement samples, without compromising microbial diversity for high ‐throughput sequencing approaches. Finally, we provide a resource that enables molecular ecologists to select the most appropriate sampling strategy for their specific research question.     

Optical properties of Erwinia herbicola bacteria at 0.190-2.50 microm

We measure the complex index of refraction of Erwina herbicola (also known as Enterobacter agglomerans or Pantoea agglomerans) bacteria (ATTC 33243) over the spectral region from 0.190 to 2.50 microm (4000-52,632 cm(-1)). Transmission measurements are made on solid films of E. herbicola and on suspensions of the bacteria in water. These measurements, combined with spectral reflectance and Kramers-Kr?nig analysis, allow the determination of the real and imaginary parts over the entire wavelength interval. Accurate and consistent results are obtained for this complex and difficult to measure material. This is part of a continuing series of measurements of the optical constants of representative biological materials that are applicable to the development of methods for detection of airborne biological contaminants, where the material under study is used as a surrogate for a pathogenic agent.     

Generation of bioaerosols during manual mail unpacking and sorting

AIMS: The dynamics of bioaerosol generation in specific occupational environments where mail is manually unpacked and sorted was investigated. METHODS AND RESULTS: Total number of airborne particles was determined in four different size classes (0.3-0.5, 0.5-1, 1-5 and >5 microm) by laser particle counting. Time dependent formation of bioaerosols was monitored by culturing methods and by specific staining followed by flow cytometry. Besides handling of regular mail, specially prepared letters ('spiked letters') were added to the mailbags to deliberately release powdered materials from letters and to simulate high impact loads. These letters contained various dry powdered biological and nonbiological materials such as milk powder, mushrooms, herbs and cat litter. Regarding the four size classes, particulate aerosol composition before mail handling was determined as 83.2 +/- 1.0, 15.2 +/- 0.7, 1.7 +/- 0.4 and 0.04 +/- 0.02%, respectively, whereas the composition changed during sorting to 66.8 +/- 7.9, 22.3 +/- 3.6, 10.4 +/- 4.0 and 0.57 +/- 0.27%, respectively. Mail processing resulted in an increase in culturable airborne bacteria and fungi. Maximum concentrations of bacteria reached 450 CFU m(-3), whereas 270 CFU of fungi were detected. CONCLUSIONS: Indoor particle concentrations steadily increased during mail handling mostly associated with particles of diameters >1 microm. However, it was not possible to distinguish spiked letters from nonspiked by simple particle counting and CFU determinations. SIGNIFICANCE AND IMPACT OF STUDY: The dynamics of bioaerosol generation have to be addressed when monitoring specific occupational environments (such as mail sorting facilities) regarding the occurrence of biological particles.     

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

Diversity of aerosolized bacteria during land application of biosolids

AIMS: The purpose of this study was to determine the diversity of bacterial communities associated with bioaerosols generated during land application of biosolids using 16S ribosomal RNA (16S rRNA) PCR. METHODS AND RESULTS: Anaerobically digested Class B biosolids were land applied to an agricultural site located in South Central Arizona. Aerosol samples were collected downwind of the biosolids operations and were collected via the use of SKC Biosamplers and subsequently extracted for the presence of bacterial community DNA. All DNA was amplified using 16S rRNA primers, cloned and sequenced. All sequences were aligned and phylogenetic trees were developed to generate community profiles. The majority of aerosolized bacterial clone sequences belonged to the Actinobacteria and alpha- and beta-proteobacterial taxa. Aerosol samples collected downwind of soil aerosolization produced similar profiles. These profiles differed from upwind and background samples. CONCLUSIONS: No one clone sequence isolated from the aerosol samples could be solely attributed to biosolids; on the contrary, the majority appeared to have arisen from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that in dry, arid climates the majority of aerosols associated with biosolids land application appear to be associated with the onsite soil.     

The effect of surface charge, negative and bipolar ionization on the deposition of airborne bacteria

Aims:  A series of experiments were conducted to evaluate the effect of surface charge and air ionization on the deposition of airborne bacteria.
Methods and Results:  The interaction between surface electrostatic potential and the deposition of airborne bacteria in an indoor environment was investigated using settle plates charged with electric potentials of 0, ±2·5kV and ±5kV. Results showed that bacterial deposition on the plates increased proportionally with increased potential to over twice the gravitational sedimentation rate at +5kV. Experiments were repeated under similar conditions in the presence of either negative or bipolar air ionization. Bipolar air ionization resulted in reduction of bacterial deposition onto the charged surfaces to levels nearly equal to gravitational sedimentation. In contrast, diffusion charging appears to have occurred during negative air ionization, resulting in an even greater deposition onto the oppositely charged surface than observed without ionization.
Conclusions:  Static charges on fomitic surfaces may attract bacteria resulting in deposition in excess of that expected by gravitational sedimentation or simple diffusion. Implementation of bipolar ionization may result in reduction of bacterial deposition.
Significance and Impact of Study:  Fomitic surfaces are important vehicles for the transmission of infectious organisms. This study has demonstrated a simple strategy for minimizing charge related deposition of bacteria on surfaces.     

Dispersal in microbes: fungi in indoor air are dominated by outdoor air and show dispersal limitation at short distances

The indoor microbiome is a complex system that is thought to depend on dispersal from the outdoor biome and the occupants'' microbiome combined with selective pressures imposed by the occupants'' behaviors and the building itself. We set out to determine the pattern of fungal diversity and composition in indoor air on a local scale and to identify processes behind that pattern. We surveyed airborne fungal assemblages within 1-month time periods at two seasons, with high replication, indoors and outdoors, within and across standardized residences at a university housing facility. Fungal assemblages indoors were diverse and strongly determined by dispersal from outdoors, and no fungal taxa were found as indicators of indoor air. There was a seasonal effect on the fungi found in both indoor and outdoor air, and quantitatively more fungal biomass was detected outdoors than indoors. A strong signal of isolation by distance existed in both outdoor and indoor airborne fungal assemblages, despite the small geographic scale in which this study was undertaken (<500 m). Moreover, room and occupant behavior had no detectable effect on the fungi found in indoor air. These results show that at the local level, outdoor air fungi dominate the patterning of indoor air. More broadly, they provide additional support for the growing evidence that dispersal limitation, even on small geographic scales, is a key process in structuring the often-observed distance–decay biogeographic pattern in microbial communities.     

Procedures for the characterisation of bioaerosol particles. Part I: aerosolisation and recovery agent effects

The sampling and assay of bioaerosols are important ina number of industrial and health-care applications. Airborne microorganisms are notoriously difficult toenumerate accurately under such conditions and nosingle procedure is suitable for all applications. Problems are compounded by the differences in assaymethod or sampler type selected, making theinterpretation of results difficult.Understanding the airborne behaviour of microorganismsover a range of environmental conditions is vital ifprocedures are to be defined and recommended for theassessment of bioaerosols. Microorganisms that arerobust over a wide range of conditions are ideal astracer particles. Unfortunately, the large majorityof non-fungal bioaerosols are susceptible to damage. A predictable assessment procedure is required whichwill not affect the viability of the collectedsample.This paper examines how aerosolisation may affect the characteristics of two speciesof microorganism (Pseudomonas fluorescens andMS2 coliphage). It forms part of a larger programmeto develop standards for the assessment of biologicalparticles. The aim of the work was to develop procedures toexamine the effects of aerosolisation onmicroorganisms, with particular reference topre-aerosolisation protocol (spray suspension age) andpost-sampling handling protocol (aerosol age incollection solution). These procedures were then usedto examine the effect of recovery agents, addedto the spray suspension prior to aerosolisation, onthe culturability of E.coli.Aerosolisation reduces the culturability of P. fluorescensand the viability of viability of MS2coliphage. Pre-sampling and post-collection handlingand storage of these aerosolised microorganisms werealso found to have an effect. This and earlierstudies have shown that the culturable fraction ofmicroorganisms can be affected by the same factorsdescribed above. Of five microorganisms tested so farin the main programme, only Penicillium expansumspores were shown to be robust and stable with aconstant culturable fraction. Therefore, recommendinga particular microorganism (apart from P. expansum) as an airborne biological standard foraerosol studies is not advised. It is recommendedthat a microorganism, representative of the envisagedapplication, be characterised it in terms of theaerosolisation parameters, storage time and conditionsin the manner reported in this study. This can beachieved using the experimental equipment described.The addition of 0.1 mM concentrations of the sugarsinositol, trehalose and raffinose to spray suspensionsof Escherichia coli, prior to aerosolisation,made no significant difference to the culturablefraction of the aerosol.