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Non-spore forming eubacteria isolated at an altitude of 20,000 m in Earth's atmosphere: extended incubation periods needed for culture-based assays

On 13 August 2004, an atmospheric sample was collected at an altitude of 20,000 m along a west to east transect over the continental United States by NASA’s Stratospheric and Cosmic Dust Program. This sample was then shipped to the US Geological Survey’s Global Desert Dust program for microbiological analyses. This sample, which was plated on a low nutrient agar to determine if cultivable microorganisms were present, produced 590 small yellow to off-white colonies after approximately 7 weeks of incubation at room-temperature. Of 50 colonies selected for identification using 16S rRNA sequencing, 41 belonged to the family Micrococcaceae , seven to the family Microbacteriaceae , one to the genus Staphylococcus , and one to the genus Brevibacterium . All of the isolates identified were non-spore-forming pigmented bacteria, and their presence in this sample illustrate that it is not unusual to recover viable microbes at extreme altitudes. Additionally, the extended period required to initiate growth demonstrates the need for lengthy incubation periods when analyzing high-altitude samples for cultivable microorganisms.

Aerobiologia

Stratospheric microbiology at 20 km over the Pacific Ocean

An aerobiology sampling flight at 20 km was conducted on 28 April 2008 over the Pacific Ocean (36.5° N, 118–149° W), a period of time that coincided with the movement of Asian dust across the ocean. The aim of this study was to confirm the presence of viable bacteria and fungi within a transoceanic, atmospheric bridge and to improve the resolution of flight hardware processing techniques. Isolates of the microbial strains recovered were analyzed with ribosomal ribonucleic acid (rRNA) sequencing to identify bacterial species Bacillus sp., Bacillus subtilis, Bacillus endophyticus, and the fungal genus Penicillium. Satellite imagery and ground-based radiosonde observations were used to measure dust movement and characterize the high-altitude environment at the time of collection. Considering the atmospheric residency time (7–10 days), the extreme temperature regime of the environment (-75°C), and the absence of a mechanism that could sustain particulates at high altitude, it is unlikely that our samples indicate a permanent, stratospheric ecosystem. However, the presence of viable fungi and bacteria in transoceanic stratosphere remains relevant to understanding the distribution and extent of microbial life on Earth.

Aerobiologia

Observations on the use of membrane filtration and liquid impingement to collect airborne microorganisms in various atmospheric environments

The influence of sample-collection-time on the recovery of culturable airborne microorganisms using a low-flow-rate membrane-filtration unit and a high-flow-rate liquid impinger were investigated. Differences in recoveries were investigated in four different atmospheric environments, one mid-oceanic at an altitude of ~10.0 m, one on a mountain top at an altitude of ~3,000.0 m, one at ~1.0 m altitude in Tallahassee, Florida, and one at ~1.0 m above ground in a subterranean-cave. Regarding use of membrane filtration, a common trend was observed: the shorter the collection period, the higher the recovery of culturable bacteria and fungi. These data also demonstrated that lower culturable counts were common in the more remote mid-oceanic and mountain-top atmospheric environments with bacteria, fungi, and total numbers averaging (by sample time or method categories) <3.0 colony-forming units (CFU) m −3 . At the Florida and subterranean sites, the lowest average count noted was 3.5 bacteria CFU m −3 , and the highest averaged 140.4 total CFU m −3 . When atmospheric temperature allowed use, the high-volume liquid impinger utilized in this study resulted in much higher recoveries, as much as 10× greater in a number of the categories (bacterial, fungal, and total CFU). Together, these data illustrated that (1) the high-volume liquid impinger is clearly superior to membrane filtration for aeromicrobiology studies if start-up costs are not an issue and temperature permits use; (2) although membrane filtration is more cost friendly and has a ‘typically’ wider operational range, its limits include loss of cell viability with increased sample time and issues with effectively extracting nucleic acids for community-based analyses; (3) the ability to recover culturable microorganisms is limited in ‘extreme’ atmospheric environments and thus the use of a ‘limited’ methodology in these environments must be taken into account; and (4) the atmosphere culls, i.e., everything is not everywhere.

Aerobiologia

Microbial survival in the stratosphere and implications for global dispersal

Spores of Bacillus subtilis were exposed to a series of stratosphere simulations. In total, five distinct treatments measured the effect of reduced pressure, low temperature, high desiccation, and intense ultraviolet (UV) irradiation on stratosphereisolated and ground-isolated B. subtilis strains. Environmental conditions were based on springtime data from a mid-latitude region of the lower stratosphere (20 km). Experimentally, each treatment consisted of the following independent or combined conditions: -70 &deg;C, 56 mb, 10-12%relative humidity and 0.00421, 5.11, and 54.64 W/m 2 of UVC (200-280 nm), UVB (280-315 nm), UVA (315-400 nm), respectively. Bacteria were deposited on metal coupon surfaces in monolayers of ~1 x 10 6 spores and prepared with palagonite (particle size< 20 &mu;m). After 6 h of exposure to the stratosphere environment, 99.9% of B. subtilis spores were killed due to UV irradiation. In contrast, temperature, desiccation, and pressure simulations without UV had no effect on spore viability up through 96 h. There were no differences in survival between the stratosphere-isolated versus ground-isolated B. subtilis strains. Inactivation of most bacteria in our simulation indicates that the stratosphere can be a critical barrier to long-distance microbial dispersal and that survival in the upper atmosphere may be constrained by UV irradiation.

Aerobiologia

Presence of amphibian chytrid fungus (Batrachochytrium dendrobatidis) in rainwater suggests aerial dispersal is possible

Abstract Global spread of the pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) may involve dispersal mechanisms not previously explored. Weather systems accompanied by strong wind and rainfall have been known to assist the dispersal of microbes pathogenic to plants and animals, and we considered a similar phenomenon might occur with Bd. We investigated this concept by sampling rainwater from 20 precipitation events for the presence of Bd in Cusuco National Park, Honduras: a site where high Bd prevalence was previously detected in stream-associated amphibians. Quantitative PCR analysis confirmed the presence of Bd in rainwater in one (5 %) of the weather events sampled, although viability cannot be ascertained from molecular presence alone. The source of the Bd and distance that the contaminated rainwater traveled could not be determined; however, this collection site was located approximately 600 m from the nearest observed perennial river by straight-line aerial distance. Although our results suggest atmospheric Bd dispersal is uncommon and unpredictable, even occasional short-distance aerial transport could considerably expand the taxonomic diversity of amphibians vulnerable to exposure and at risk of decline, including terrestrial and arboreal species that are not associated with permanent water bodies.

Aerobiologia

Science questions and knowledge gaps to study microbial transport and survival in Asian and African dust plumes reaching North America

The Sahara in North Africa and the Gobi and Taklamakan deserts in Asia are the primary sources of mobilized dust in the atmosphere, with regional or global airborne transport estimated at 2 to 5 billion tonnes per year. Annual Asian dust plumes take about 7 to 10 d to cross the Pacific Ocean, and often reach the northwest USA between late February and May. In contrast, the peak season for the movement of African dust storms to the southeastern USA is typically June to August, and dust plumes take about 5 to 7 d to reach Florida. Although studies have documented that a wide range of bacteria, fungi, archaea, and viruses in dust plumes reach the USA each year, little is known about temporal and spatial variability in the microbial biodiversity in transoceanic dust plumes, or the effect on the deposition environments. A scoping study (called the Transoceanic Aerobiology Biodiversity Study) was conducted to develop field-based campaigns centered on examining the abundance, diversity, survival, and impact of microorganisms in transoceanic dust plumes arriving in the continental USA from Asia and Africa. This effort identified Science Questions (SQs) and Knowledge Gaps (KGs) that are highly relevant toward an understanding of the microbial diversity, transport, survival, and dispersal in transoceanic dusts. Science Questions were defined as broad science topics in transoceanic dust plume microbiology that were underexplored by the aerobiology community. Knowledge Gaps were defined as specific project-level research questions for each SQ that represented important topics in the study of transoceanic aerobiology.

Aerobiologia

Efficiency of bioaerosol samplers: A comparison study

Bioaerosols, including bacteria and fungi, are ubiquitous and have been shown to impact various organisms as well as biogeochemical cycles and human health. However, sample collection poses a challenge for aeromicrobiologists and can determine the success of a study. Establishing a standard collection procedure for bioaerosol sampling could help advance the field. We tested the efficiency (number of organisms collected and DNA yield per unit time) of three sampling devices: a membrane filtration device, a liquid impinger, and a portable electrostatic precipitator bioaerosol collector. We compared the efficiency of these three devices for both culture-dependent studies, by enumerating colony forming units (CFUs), and culture-independent studies, by extracting and quantifying total DNA. Our results show that the electrostatic precipitator collected microorganisms significantly more efficiently than the membrane filtration and liquid impingement in both types of studies over the same time interval. This is due to the high flow rate of the device. This work is important and timely because aeromicrobiology is currently restricted by long sampling times and risk of evaporation, desiccation, or freezing during sample, which increases with sampling times. Fieldwork convenience and portability of instruments are an additional challenge for sampling. Using a sampler that can overcome these technical hurdles can accelerate the advancement of the field, and the use of a lightweight, battery-powered, inexpensive, and portable bioaerosol collection device could address these limitations.

Aerobiologia

Initial comparison of pollen counting methods using precipitation and ambient air samples and automated artificial intelligence to support national monitoring objectives

Given the endemic nature of pollen throughout the environment, the impact upon human health, and the need for more extensive and better measurements of pollen in the USA, a preliminary project within the National Atmospheric Deposition Program’s (NADP) National Trends Network (NTN) was developed. Pollen was measured in ambient air by several methods and in precipitation wet deposition samples at three monitoring sites in the NTN. A method for counting pollen on filters was developed and provided pollen counts for NADP atmospheric wet-deposition samples and high-volume ambient air samplers (HVAS) for comparison with co-located traditional National Allergy Bureau microscopy samples and a commercially available pollen sensor (PS) counting method during the 2021 pollen season. The goals of this project were to test the potential of available air-monitoring infrastructures to obtain improved spatial measurements of aeroallergens, compare pollen counting results from the various methods, and to determine the suitability of using wet deposition samples for pollen collection. The onset and senescence of pollen seasons for general categories of genera compared favorably for each method at each site, indicating that pollen monitoring using wet-deposition and ambient air sampling filters could provide useful information to inform scientific studies, but not likely for public health objectives. Pollen counts were log transformed for Pearson product moment correlation. Tree pollen counts were correlated at all sites for daily PS data and traditional counting data ( R = 0.69–0.84), but statistical correlations between methods for grass and weed pollen were weak (0.40 < R < 0.60) or considered not correlated ( R < 0.40). Total pollen counts in NADP precipitation samples were correlated with traditional and PS counts at only one of three sites. Pollen counts for the weekly HVAS filter samples were correlated with PS counts for trees ( R = 0.62) and with NAB counts for trees ( R = 0.68) and weeds ( R = 0.72). Correlations in the data between methods suggest that, given further methods development, a variety of techniques could be integrated to expand and enhance existing pollen monitoring networks. Improved ambient air and atmospheric deposition sampling methods specifically targeted for pollen capture and analysis could support the collection of accurate and efficient meaningful aeroallergen data from existing atmospheric monitoring networks.

Aerobiologia

African desert dust in the Caribbean atmosphere: Microbiology and public health

Air samples collected on St. John in the U.S. Virgin Islands were screened for the presence of viable bacteria and fungi to determine if the number of cultivatable microbes in the atmosphere differed between "clear atmospheric conditions" and "African dust-events." Results indicate that during "African dust-events," the numbers of cultivatable airborne microorganisms can be 2 to 3 times that found during "clear atmospheric conditions." Direct microbial counts of air samples using an epifluorescent microscopy assay demonstrated that during an "African dust-event," bacteria-like and virus-like particle counts were approximately one log greater than during "clear atmospheric conditions." Bacteria-like particles exhibiting autofluoresence, a trait of phototrophs, were only detected during an "African dust-event.".

Aerobiologia

Atmospheric microbiology in the northern Caribbean during African dust events

Between July 2000 and August 2001 forty-three air samples were collected in the northern Caribbean: Twenty-six in the US Virgin Islands, and 17 samples aboard ship during two 1-week cruises. Samples were collected during African dust events and non-dust conditions and screened for the presence of culturable bacteria and fungi. A total of 3,652 liters of air were collected during non-dust conditions, with 19 bacteria and 28 fungi being recovered. During dust conditions a total of 2,369 liters of air were screened resulting in the recovery of 171 bacteria and 76 fungi. A statistically significant difference was found between the two data sets. These results support previous African dust research and further demonstrate that dust particles can serve as a vessel for the global dispersion of bacteria and fungi. Dustborne microorganisms may play a significant role in the ecology and health of downwind ecosystems.

Aerobiologia

Characterization of aerosolized bacteria and fungi from desert dust events in Mali, West Africa

Millions of metric tons of African desert dust blow across the Atlantic Ocean each year, blanketing the Caribbean and southeastern United States. Previous work in the Caribbean has shown that atmospheric samples collected during dust events contain living microbes, including plant and opportunistic human pathogens. To better understand the potential downwind public health and ecosystem effects of the dust microbes, it is important to characterize the source population. We describe 19 genera of bacteria and 3 genera of fungi isolated from air samples collected in Mali, a known source region for dust storms, and over which large dust storms travel.

Aerobiologia

Terrestrial microorganisms at an altitude of 20,000 m in Earth's atmosphere

A joint effort between the U.S. Geological Survey's (USGS) Global Desert Dust and NASA's Stratospheric and Cosmic Dust Programs identified culturable microbes from an air sample collected at an altitude of 20,000 m. A total of 4 fungal (Penicillium sp.) and 71 bacteria colonyforming units (70 colonies of Bacillus luciferensis believed to have originated from a single cell collected at altitude and one colony of Bacillus sphaericus) were enumerated, isolated and identified using a morphological key and 16S rDNA sequencing respectively. All of the isolates identified were sporeforming pigmented fungi or bacteria of terrestrial origin and demonstrate that the presence of viable microorganisms in Earth's upper atmosphere may not be uncommon.

Aerobiologia

The relationship between gorgonian coral (Cnidaria: Gorgonacea) diseases and African dust storms

The number of reports of coral diseases has increased throughout the world in the last 20 years. Aspergillosis, which primarily affects Gorgonia ventalina and G. flabellum, is one of the few diseases to be characterized. This disease is caused by Aspergillus sydowii, a terrestrial fungus with a worldwide distribution. Upon infection, colonies may lose tissue, and ultimately, mortality may occur if the infection is not sequestered. The spores of A. sydowii are <5 ??m, small enough to be easily picked up by winds and dispersed over great distances. Aspergillosis is prevalent in the Caribbean, and it appears that this primarily terrestrial fungus has adapted to a marine environment. It has been proposed that dust storms originating in Africa may be one way in which potential coral pathogens are distributed and deposited into the marine environments of the Caribbean. To test the hypothesis that African dust storms transport and deposit pathogens, we collected air samples from both dust storms and periods of nondust in St. John, U.S. Virgin Islands. Because we focused on fungal pathogens and used A. sydowii as a model, we isolated and cultured fungi on various types of media. Fungi including Aspergillus spp. were isolated from air samples taken from dust events and non-dust events. Twenty-three separate cultures and seven genera were isolated from dust event samples whereas eight cultures from five genera were isolated from non-dust air samples. Three isolates from the Virgin Islands dust event samples morphologically identified as Aspergillus spp. produced signs of aspergillosis in seafans, and the original pathogens were re-isolated from those diseased seafans fulfilling Koch's Postulates. This research supports the hypothesis that African dust storms transport across the Atlantic Ocean and deposit potential coral pathogens in the Caribbean.

Aerobiologia