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Aerobiologia (2006) 22:211–226 DOI 10.1007/s10453-006-9033-z ORIGINAL PAPER Airborne microorganisms in the African desert dust corridor over the mid-Atlantic ridge, Ocean Drilling Program, Leg 209 Dale W. Griffin Æ Douglas L. Westphal Æ Michael A. Gray Received: 9 March 2005 / Accepted: 24 May 2006 / Published online: 6 September 2006 Springer Science+Business Media B.V. 2006 Abstract The objective of this study was to enhance our understanding of the fate and transAtlantic transport of dustborne microorganisms from Northern Africa to the Caribbean and Americas, and more specifically to determine if culturable populations could be detected at a mid-ocean site, closer to the source of dust relative to land-based Caribbean sites, during the early summer months of May and June. Between the dates of 22 May and 30 June 2003, daily air samples were collected and evaluated for the presence of culturable bacterial and fungal colony-forming units (CFU). Here we report a statistically significant correlation between daily atmospheric CFU counts at a mid-ocean research site (~15N, 45W) and daily desert dust concentrations as determined by the U.S. Navy’s Naval Aerosol Analysis and Prediction System (NAAPS) Global Aerosol Model (Honrath et al. (2004). Journal of Geophysical Research, 109; Johnson et al. (2003). Global Biogeochemical Cycles, 17, 1063; Reid et al. (2004). Geophysical D. W. Griffin (&) Æ M. A. Gray U.S. Geological Survey, 600, 4th St. South, St. Petersburg, FL 33701, USA e-mail: [email protected] D. L. Westphal Naval Research Laboratory, Aerosol and Radiation Section, 7 Grace Hopper Avenue, Stop 2, Monterey, CA 93943, USA Research Letters, 31; Schollaert, Yoder, Westphal, & O’Reilly (2003). Journal of Geophysical Research, 108, 3191). Keywords Aerobiology Æ Microbiology Æ Atmospheric microbiology Æ Atmosphere Æ Methods Æ Survival Æ Oceanic 1 Introduction The phenomena known as ‘desert-dust storms’ moves an estimated 2.2 · 109 metric tons (2.2 · 1015 grams) of soil and dried sediment through the Earth’s atmosphere each year (Goudie & Middleton, 2001). The largest of these events are capable of dispersing large quantities of dust across oceans and continents (Husar et al., 2001; Moulin, Lambert, Dulac, & Dayan, 1997). Since a gram of desert soil may contain as many as 1 · 109 bacterial cells (Whitman, Coleman, & Wiebe, 1998), the presence of airborne desert dust should correspond with increased concentrations of airborne microorganisms. Research projects undertaken to address the movement of microorganisms in association with dust clouds emanating from the Gobi and Takla Makan deserts of China, have documented elevated aerial concentrations at downwind locations (Korea and Taiwan) when the regions are impacted by airborne desert dust (Wu, Tsai, Li, 123 212 Lung, & Su, 2004; Yeo & Kim, 2002). Research has also demonstrated an association between African dust events and airborne microorganisms in Africa and the Caribbean (Griffin et al., 2003; Kellogg et al., 2004). Using a combination of satellite imagery and visual assessment of impaired atmospheric visibility in the U.S. Virgin Islands, elevated concentrations of bacteria and fungi above background levels were only noted during African dust events in late July and August of 2000 and 2001 (no association noted during events in May, June, and early July) (Griffin et al., 2003). This research project was undertaken to enhance our understanding of the fate and transAtlantic transport of dustborne microorganisms from Northern Africa to the Caribbean and Americas, and more specifically to determine if Fig. 1 Panel A = Heavy African desert dust activity forms an atmospheric bridge across the Atlantic Ocean. Marked research site (~15 N, 45 W). This 26 May 2003 image is courtesy of the SeaWiFS Project, NASA/Goddard Space Flight Center and ORBIMAGE. Panel B = NAAPS dust optical depth for 26 May 2003. Contoured at unitless values of 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 123 Aerobiologia (2006) 22:211–226 culturable populations could be detected at a mid-ocean site, closer to the source of dust relative to land-based Caribbean sites, during the early summer months of May and June. 2 Methods 2.1 Research site The drill sites for Ocean Drilling Program (ODP) Leg 209 were located along the mid-Atlantic Ridge (~45W) between 1443¢ N and 1544¢ N (Fig. 1) (Keleman et al., 2004). While on station, the Joint Oceanographic Institutions for Deep Earth Sampling (JOIDES) vessel Resolution was positioned with its bow to the east (into the trade Aerobiologia (2006) 22:211–226 winds). Samples were collected from the bridge deck located near the bow of the JOIDES Resolution. 2.2 Atmospheric conditions Table 1 lists daily measured atmospheric parameters including wind speed, wind direction, air temperature, and percent relative humidity. These parameters were measured to account for any extreme atmospheric conditions and for comparison with future or other studies. African dust transport across the Atlantic was monitored daily aboard ship using satellite imagery provided by NASA’s SeaWiFS Project (Fig. 1a). 2.3 Air samples for isolation of culturable bacteria and fungi Air samples were collected from the bow of the ship atop the right deck located in front of the bridge (~10 m above sea level) using a portable membrane-filtration apparatus (110 V vacuum pump, Fisher Scientific, PVC two-place-manifold and housing, assembled in house) between 0630 and 1845 h (local time). Presterilized filter housings containing 47-mm-diameter, 0.2-lm pore-size cellulose acetate filter membranes were used to collect all air samples (Fisher Scientific, Atlanta, GA). The filter housings were removed from their respective sterile bags and placed on an analytical filter manifold. The housing lid was removed and vacuum applied using a vacuum pump. Two to three air samples (small-volume replicates, smallvolume replicates plus a large-volume sample, or one small- and one large-volume sample, see Table 1) were collected each day. Filtration-flow rates ranged from 1.9 to 17.4 l min–1, and the total volume of air filtered ranged from 89.3 l to 12,179.9 l (avg. ~2,270 l). The low end flow rates utilized from 22 May through 30 May, and 25 June through 29 June, were due to a pump line restriction and a decrease in pump diaphragm efficiency respectively. Use of the two-placemanifold resulted in a normal flow rate of 8.7 l min–1 when simultaneously collecting two samples or 17.4 l min–1 when collecting a single sample. To control for handling contamination (once each day), an additional filter housing was 213 removed from its bag, placed on the manifold, and allowed to sit for 1 min without removing the lid or turning on the vacuum. After filtration, the housings and lids were sealed with parafilm, replaced in their respective bags, sealed with tape, and immediately transported to the microbiology laboratory for processing. R2A medium (Fisher Scientific, Atlanta, GA) (Reasoner & Geldreich, 1985), was utilized in the following manner for microbial culture. The sample filters were placed whole on R2A medium plates, sample side up, and were incubated in the dark at room temperature (~23C). Bacterial and fungal CFU were enumerated at 48 h, 96 h, and between 96 h and 336 h of incubation and separated by isolation streaking onto fresh plates of R2A. Isolated colonies were grown in Tryptic Soy Broth overnight at room temperature on a tabletop rocker set at low speed (Fisher Scientific, Atlanta, GA). The following day, 0.5 ml of each culture was transferred to a sterile cryogenic storage tube containing 200 ll of sterile glycerol and stored at –70C. 2.4 Evaluation of R2A medium for use in aeromicrobiology studies R2A medium was selected for use in the simultaneous recovery of potentially stressed (UVdamage, desiccation, temperature, etc.) airborne bacteria and fungi due to the need for an effective and efficient (cost and time) sampling assay. In the UV-induced stress protocols outlined below, a higher UV dose was selected for the fungi due to the protective nature of their spores relative to the bacteria (non-spore forming fraction). Airflow rates between the fungi and bacteria studies differed due to the number of samples simultaneously collected. To determine if R2A could be used to effectively recover airborne fungi (in comparison to traditional fungal substrates), 20 sets of samples (three samples each set) were collected in a courtyard in St. Petersburg, Florida. Flow rates through each sample/filter were 5.8 l min–1 over a 20 min sample time for a total volume of 116 l sample–1. Each filter was then cut in half using sterile scissors and each half was plated on Potato Dextrose Agar (PDA, Becton Dickinson, Sparks, MD), Sabouraud Agar 123 123 A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B C 828 828 743 743 746 746 751 751 729 729 727 727 737 737 723 723 716 716 721 721 736 736 718 718 734 734 746 746 754 754 729 729 749 749 743 743 1039 43 43 41 41 41 41 40 40 48 48 44 44 40 40 40 40 40 40 51 51 46 46 43 43 40 40 41 41 48 48 40 40 43 43 50 50 176 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 2.8 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 17.4 120.4 120.4 114.8 114.8 114.8 114.8 112 112 134.4 134.4 123.2 123.2 112 112 112 112 112 112 443.7 443.7 400.2 400.2 374.1 374.1 348 348 356.7 356.7 417.6 417.6 348 348 374.1 374.1 435 435 3062.4 5.8 5.8 6.4 6.4 8.2 8.2 5.6 5.6 5.1 5.1 5.3 5.3 5.1 5.1 5.6 5.6 2.2 2.2 3.1 3.1 6.7 6.7 6.1 6.1 6.1 6.1 7.8 7.8 5.4 5.4 5 5 6.2 6.2 5.7 5.7 5.7 113 113 112 112 82 82 102 102 97 97 87 87 90 90 58 58 48 48 60 60 110 110 110 110 67 67 78 78 70 70 75 75 70 70 70 70 70 27.3 27.3 26.1 26.1 25.8 25.8 25.1 25.1 25.8 25.8 25.8 25.8 25.4 25.4 25.2 25.2 25.5 25.5 24.9 24.9 25.4 25.4 26.1 26.1 25.6 25.6 25.6 25.6 25.6 25.6 25.5 25.5 26.4 26.4 25.6 25.6 25.6 70.7 70.7 76.2 76.2 70.9 70.9 81.8 81.8 68.1 68.1 75.5 75.5 73.6 73.6 77.5 77.5 74.8 74.8 75.3 75.3 69.5 69.5 65.7 65.7 68.3 68.3 68.2 68.2 68.8 68.8 79.4 79.4 73.9 73.9 71.9 71.9 71.9 0 0 0 0 0 0 2 1 1 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 2 0 0 0 0 0 0 44.7 17.8 14.9 7.4 8.1 0 17.8 0 0 0 8.9 0 0 0 2.5 0 0 0 0 0 0 0 0 0 5.7 0 0 0 0 0 0.7 8.2 5.5 4.9 47.4 110.9 76.6 65.3 25.5 6.6 5.7 8.8 8.2 3.5 2.0 2.1 3.4 3.9 3.4 0 0 0 31.3 11.2 4.1 8.9 0 4.5 0 1.3 0 0 0 0 2.9 0 0.2 Avg. total NAAPS dust Start End Total time Flow rate Total Wind Wind Temp. % Hum. Bacteria Fungi Total time time (min) (l/min) volume (l) speed direction (C) CFU CFU CFU/m3 CFU/m3/day at 10 m/day of air (lg/m3) (local time) (m/s) degrees 5/22/03 745 745 5/23/03 702 702 5/24/03 705 705 5/25/03 711 711 5/26/03 641 641 5/27/03 643 643 5/28/03 657 657 5/29/03 643 643 5/30/03 636 636 5/31/03 630 630 6/1/03 650 650 6/2/03 635 635 6/3/03 654 654 6/4/03 705 705 6/5/03 706 706 6/6/03 649 649 6/7/03 706 706 6/8/03 653 653 743 ID Date Table 1 Mid-Atlantic aeromicrobiology, atmospheric parameters and NAAPS dust data for the study period 22 May through 30 June 2004 214 Aerobiologia (2006) 22:211–226 6/25/03 6/24/03 6/23/03 6/22/03 6/21/03 6/20/03 6/19/03 6/18/03 6/17/03 6/16/03 6/15/03 6/14/03 6/13/03 6/12/03 6/11/03 6/10/03 737 737 1417 736 736 1350 734 734 733 733 741 741 1641 715 715 1705 751 1835 900 1655 739 1838 734 1845 735 1650 854 1705 725 1830 724 1710 725 1820 751 1821 739 1746 40 40 396 52 52 374 40 40 36 36 49 49 540 42 42 595 36 680 43 518 66 725 49 617 47 602 48 539 47 712 49 643 49 704 54 684 57 607 8.7 8.7 17.4 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 8.7 17.4 8.7 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 8.7 17.4 3.9 3.9 348 348 6890 452.4 452.4 6507.6 348 348 313.2 313.2 426.3 426.3 9396 365.4 365.4 10353 313.2 11518.8 374.1 8639.1 574.2 12040.5 426.3 10309.5 408.9 10065.9 417.6 8961 408.9 12179.9 426.3 11361.9 426.3 11823.3 469.8 11431.8 222.3 2367.3 5 5 5 4.5 4.5 4.5 6.2 6.2 6.7 6.7 5.8 5.8 5.8 6.3 6.3 6.3 6.1 6.1 5.4 5.4 4.7 4.7 4.9 4.9 3.9 3.9 4.8 4.8 6.7 6.7 4.9 4.9 6.6 6.6 4.6 4.6 6.5 6.5 110 110 110 76 76 76 67 67 78 78 82 82 82 95 95 95 78 78 87 87 105 105 70 70 65 65 65 65 65 65 85 85 90 90 75 75 62 62 25.3 25.3 25.3 25.9 25.9 25.9 25.9 25.9 25.2 25.2 26.2 26.2 26.2 25.6 25.6 25.6 25.8 25.8 26.2 26.2 25.7 25.7 25.9 25.9 25.9 25.9 29.6 29.6 26.1 26.1 26 26 25.9 25.9 26 26 25.8 25.8 71.4 71.4 71.4 71.4 71.4 71.4 78.3 78.3 82.8 82.8 68.8 68.8 68.8 70.9 70.9 70.9 68.1 68.1 75.4 75.4 75 75 72.9 72.9 79.6 79.6 70.7 70.7 74.6 74.6 74.3 74.3 76.4 76.4 75 75 67.4 67.4 0 0 0 0 0 1 0 0 0 3 0 0 0 0 0 0 0 2 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 5 0 1 0 0 0 0 0 0 0 0 0 11 0 11 1 7 0 6 2 7 0 3 0 1 0 2 0 0 0 0 0 0 0 0 0 0 0 0.9 0 2.8 0 9.6 0 0 0 0 0 0 0 1.1 0 1.3 1.7 1.2 0 0.6 4.9 0.7 0 0.3 0 0.1 0 0.2 0 0 0 0.1 0 0 0 0.1 0 0.1 0.1 0.2 2.8 0.3 1.5 0.7 0.6 0 0 4.8 1.4 0.3 0 6.3 7.4 7.2 5.3 14.7 20.1 32.9 23.7 21.4 15.3 11.9 5.0 5.7 15.5 17.2 7.4 5.1 657 657 741 644 644 736 654 654 657 657 652 652 741 633 633 716 715 715 817 817 633 633 648 648 648 648 806 806 638 638 633 633 636 636 657 657 636 739 A B C A B C A B A B A B C A B C A B A B A B A B A B A B A B A B A B A B A B 6/9/03 Avg. total NAAPS dust Start End Total time Flow rate Total Wind Wind Temp. % Hum. Bacteria Fungi Total time (min) (l/min) volume (l) speed direction (C) CFU CFU CFU/m3 CFU/m3/day at 10 m/day time of air (lg/m3) (local time) (m/s) degrees ID Date Table 1 continued Aerobiologia (2006) 22:211–226 215 123 123 10.1 24.4 45.3 21.8 17.3 0.6 0.3 15.9 5.7 0 0 1.2 0 0.5 28.5 3.2 11.2 0.2 0 0 0 3 0 1 1 4 1 2 0 0 0 0 0 0 5 0 0 0 0 0 79.2 79.2 81.2 81.2 76.4 76.4 67.6 67.6 74.8 74.8 24.8 24.8 24.8 24.8 26 26 26.6 26.6 25.8 25.8 110 110 77 77 78 78 75 75 75 75 6.5 6.5 5.1 5.1 6.5 6.5 5.3 5.3 3.9 3.9 119.7 2403.6 144.4 2108.6 210.6 1250.2 89.3 9338.5 417.6 11623.2 1.9 3.9 1.9 3.9 3.9 1.9 1.9 17.4 8.7 17.4 63 647 76 570 54 658 47 645 48 692 831 1815 832 1646 735 1835 757 1755 726 1810 6/26/03 728 728 6/27/03 716 716 6/28/03 641 737 6/29/03 710 710 6/30/03 638 638 A B A B A B A B A B ID Date Avg. total NAAPS dust Start End Total time Flow rate Total Wind Wind Temp. % Hum. Bacteria Fungi Total time time (min) (l/min) volume speed direction (C) CFU CFU CFU/m3 CFU/m3/day at 10 m/day of air (lg/m3) (local time) (l) (m/s) degrees Aerobiologia (2006) 22:211–226 Table 1 continued 216 (SA, Becton Dickinson, Sparks, MD), or R2A. One set of the plated filter halves were then exposed to UV-C light for 5 min (exposure rate = 95.5 lW cm–1), to simulate atmospheric UV induced stress. All filters were then incubated in the dark at room temperature (~23C) and fungal CFU enumerated at 96 and 144 h. To determine if R2A could be effectively used to recover airborne bacteria in comparison to nutrient-rich substrates, 20 sets of samples (two samples each set) were collected in a courtyard in St. Petersburg, Florida. Flow rates through each sample/filter were 8.7 l min–1 over a 20 min sample time for a total volume of 174 l sample–1. Each filter was then cut in half using sterile scissors and each half was then plated on Blood Agar (Sheep’s Blood Agar Base with 5% Sheep’s Blood), and R2A, respectively. One set of the plated filter halves were then exposed to UV-C light for 5 min (exposure rate = 49.5 lW cm–1), to simulate atmospheric UV induced stress. All samples were then incubated in the dark at room temperature (~23C) and bacterial CFU enumerated at 72 and 144 h. 2.5 Evaluation of air volume filtered on recovery of airborne bacterial and fungal CFU To determine the effect of air volume assayed when using membrane filtration to collect airborne bacteria and fungi, 22 air samples were collected using a flow rate of 6.3 l min–1 over sample times that ranged from 56 to 2165 min (356.7–13,791.1 l air). To account for temporal flux in airborne microbial concentrations small to medium volume air samples ( < 6,000 l) were collected at different times of the day. Large volume air samples (>6,000 l) were collected over a 24 h period starting and ending at approximately 0900 h. All sample filters were then plated on R2A, incubated in the dark at room temperature (~23C) and bacterial and fungal CFU enumerated at 96 h. 2.6 Genetic identification of bacteria Genetic identification of bacterial and fungal isolates was performed post-cruise. The polymerase Aerobiologia (2006) 22:211–226 chain reaction (PCR) was used to amplify a 1538base pair segment of the 16S rRNA gene using a universal prokaryote primer set (Upstream = 5¢CCGAATTCGTCGACAA CAGAGTTTGAT CCTGGCTCAG-3¢, Downstream = 5¢-CCCGG GATCCAAGCTTACGGCT ACCTTGTTACG ACTT-3¢) (Grasby et al., 20 03). Three-prime ends of primers correspond to Escherichia coli 16S rDNA nucleotide numbers 27 and 1492, respectively. For DNA extraction, bacterial isolates were recovered from cryogenic storage via streaking onto R2A followed by 48 h of incubation at room temperature. A sterile pipette tip was then used to pick and inoculate cells into 180 ll of lysis buffer recommended for extraction of DNA from Gram positive bacteria in a DNeasy Tissue kit (Qiagen Inc., Valencia, CA). The DNeasy Tissue kit protocol was followed, and purified DNA was eluted in 100 ll of the kit elution buffer. Ten microliters of purified DNA were used for PCR. The PCR master mix recipe per reaction was: 25 ll of HotStarTaq Master Mix (Qiagen Inc., Valencia, CA, catalog #203445), 1 ll each of 10-nM upstream and downstream primer (synthesized by Operon Technologies, Inc., Alameda, CA), and 13 ll of HotStarTaq Master Mix H2O. The Hot-Start PCR amplification profile was: one step for 15 min at 95C, 40 cycles of (30 s at 94C, 30 s at 45C, 2 min at 72C), one step for 10 min at 72C and hold at 4C. PCR amplicons were cleaned and directly sequenced using the listed downstream primer (one strand, one reaction) by Northwoods DNA, Inc. (Becida, MN). Sequence lengths ranged from 441 to 751 base pairs. GenBank Blast search (http://www.ncbi.nlm.nih. gov/BLAST/) was used for sequence/isolate identification. 217 REDExtract-N-Amp Plant PCR kit (SigmaAldrich Corp. St. Louis, MO) DNA-extraction solution in a 1.5-ml microfuge tube. A Freeze/ thaw technique was then used to enhance lysis of the fungi cells. This process was repeated 10 times using a crushed dry-ice/ethanol and boiling-water bath. After completion of the freeze/thaw protocol, the sample was processed as outlined in REDExtract-N-Amp kit protocol. The 20 ll PCR master mix recipe was: 10 ll of REDExtract-N-Amp PCR mix, 1 ll each of 10 nM upstream and downstream primer (synthesized by Operon Technologies, Inc., Alameda, CA), 4 ll of extracted DNA, and 4 ll of 0.02-lm filter sterilized and autoclaved Milli-Q water (Millipore, Bedford, MA). PCR reaction conditions as listed in the REDExtract-N-Amp Plant PCR kit protocol were followed using an annealing temperature of 45C. Amplicons were visualized using gel electrophoresis stained with ethidium bromide. For efficient sequencing reactions, amplicons were cloned into a plasmid vector using the QIAGEN PCR Cloning Plus kit (Qiagen, Valencia, CA). Overnight ligation of amplicon and plasmid vector was used. PCR plasmids were cleaned and sequenced (sequence length ranged from 207 to 716 bp) using the vector primer M13R (one strand, one reaction) by Northwoods DNA, Inc. (Becida, MN). GenBank Blast search (http://www.ncbi.nlm.nih. gov/BLAST/) was used for sequence/isolate identification. 2.8 Genbank accession numbers Accession numbers for the bacterial and fungal DNA sequences included in this report are AY857646 through AY857737. 2.7 Identification of fungi Fungi were identified to the genus level using PCR. Universal fungal primers (EF3 and EF4, 1556bp amplicon) were utilized for amplification (Smit, Leeflang, Glandorg, Elsas, & Wernars, 1999). For DNA extraction, cryogenically stored fungal isolates were streaked onto R2A followed by 7 days of incubation at room temperature. A sterile pipette tip was used to harvest < 3 mg of mycelia/spores that was inoculated into 100 ll of 2.9 NAAPS model dust concentration estimates Daily atmospheric dust concentrations reported as lg m–3 at 10 m above sea level were provided by the Naval Research Laboratory (Monterey, CA) post-cruise (see Table 1 and Fig. 1b). The concentration values listed in Table 1 are the averages of three daily estimates (600, 1200, 1800 h). The NAAPS model uses a first principles 123 218 Aerobiologia (2006) 22:211–226 approach to predict the distribution of mineraldust aerosol. The modeled processes include mobilization, vertical and horizontal mixing, advection, wet removal, and dry deposition. The model carries a bulk-dust component with properties that are representative of dust particles subject to long-range transport, i.e., particles with radii less than 5 lm. The dry-deposition velocity depends on the surface-layer turbulence, ocean roughness, and the mean particle-sedimentation velocity (Honrath et al., 2004; Johnson et al., 2003; Reid et al., 2004; Schollaert, Yoder, Westphal, & O’Reilly, 2003). Additional NAAPS information can be found at http://www.nrlmry. navy.mil/aerosol/Docs/globaer_model.html. 2.10 Statistical analysis SPSS 13.0 for Windows (SPSS Inc., Chicago, IL) was used for statistical analyses. Mid-Atlantic airborne microbial population size and NAAPS dust concentration data were tested for normality using a One-Sample Kolmogorov-Smirnov Test. (Dytham, 1999), which demonstrated that the data were not normally distributed (N = 40, a = 0.01, 2-tailed P < 0.001, 0.019, for microbial and dust data, respectively). Whereas a log transformation of the dust concentration values normalized the distribution of the data, it did not normalize the distribution of the values for microbial concentration. The correlation between the microbiology data and the NAAPS dust concentration data was then evaluated using Spearman’s rank-order correlation. (Dytham, 1999). In the study to compare recovery efficiency of the different culture media, population sizes of fungi recovered on R2A, PDA, and SA media (N = 20 set–1) were normally distributed (P range = 0.088–0.881). Hence, population sizes on R2A were compared with each of the other substrates using an Independent t-test. For bacterial population sizes, Independent t-tests were utilized when the data were normally distributed and the Mann–Whitney test when they were not. The membrane filtration air-volumeCFU-recovery data were evaluated using Pearson’s correlation a = 0.01 (2-tailed) as the data set was normally distributed. 3 Results 3.1 Evaluation of R2A and membrane filtration sample volumes When R2A was compared to PDA and SA for recovery of fungi (Table 2), analysis of the data sets (96 h of incubation—no UV, 96 h—UV, 144 h—no UV, and 144 h—UV) demonstrated Table 2 Evaluation of different culture media for use in cultivation of non-stressed and ultraviolet-C stressed airborne fungi and bacteria Fungal CFU counts Medium 96 h incubation— no UV treatment R2A 9.7 (5.3) PDA 9.6 (4.2) SA 8.2 (3.9) Bacterial CFU counts Medium 72 h—no UV R2A 4.7 (6.1) Blood Agar 3.6 (3.1) 96 h incubation— UV treatment 4.0 (2.8) 4.3 (2.2) 4.0 (2.3) 144 h incubation— no UV treatment 9.9 (3.6) 10.1 (4.8) 9.5 (4.5) 144 h incubation— UV treatment 4.8 (2.3) 4.4 (2.3) 4.6 (1.9) 72 h—UV 3.0 (1.9)* 1.5 (1.2) 144 h—no UV 5.6 (5.7) 4.8 (4.7) (N = 16) 144 h—UV 3.6 (2.2) 2.7 (1.6) (N = 19) Values represent the mean number of colony forming units (CFU) growing on cellulose acetate filters (20 media–1) through which outdoor air was filtered using a portable membrane filtration apparatus for 20 min at a flow rate of 5.8 l min–1 for fungi and 8.7 l min–1 for bacteria. After sampling, half of the filters were exposed to UV light for 5 min (95.5 lW cm–1 for fungi and 49.5lW cm–1 for bacteria) prior to plating on media R2A = Reasoner’s 2 Agar, PDA = Potato Dextrose Agar, SA = Sabouraud Agar, (##) = Standard deviation, * = Statistically significant difference, Mann–Whitney test, P = 0.009, UV = Ultraviolet-C light. (N = #) = number of plates used for analysis due to the loss of the remaining plates to overgrowth of fungi 123 Aerobiologia (2006) 22:211–226 that none of the data sets were significantly different (P range—0.581 to 1.0). When the R2A versus Blood Agar CFU count experiment was conducted (Table 2), analysis of the data sets (72 h of incubation—no UV, 72 h—UV, 144 h—no UV, and 144 h—UV) demonstrated that R2A at all time points produced higher CFU counts and significantly higher counts at 72 h of incubation with UV exposure (P = 0.009). For evaluating the influence of sample volume on membrane filtration CFU recovery rate, CFU count data were normalized by dividing the counts by the total liters of air sampled. Data were then converted and expressed at CFU m–3 (Fig. 2). Bacterial counts ranged from 1.5 to 48.9 CFU m–3 (avg. 16.4), fungal counts ranged from 15.7 to 100.9 CFU m–3 (avg. 35.0), and total counts ranged from 17.6 to 131.8 CFU m–3 (avg. 51.4). Data analysis demonstrated significant negative correlations (microbial concentrations versus air volume sampled) for total and bacterial CFU counts (r = –0.578, P = 0.005, and r = –0.776, P < 0.001, respectively), and fungal CFU counts showed a negative correlation that was not significant (r = –0.280, P = 0.206). 219 3.2 SeaWiFS image data The complete SeaWiFS image data set for the study period showed an almost continuous transmission of dust off the northwest coast of Africa toward the research site. Figure 1 panel A, a 26 May 2003 SeaWiFS image, shows an atmospheric bridge of African dust spanning the Atlantic Ocean, which coincided with the heaviest daily concentration of dust at the research site as determined by the NAAPS Global Aerosol Model (Fig. 3). The averaged NAAPS dust concentrations ranged from 2.0 to 110.9 lg m–3 with an average of 18.3 lg m–3day–1. 3.3 Mid-Atlantic data Wind direction and speed measurements for the duration of the aeromicrobiology study ranged from 48.0 to 113.0 relative to North (avg. 81.7) and 2.2 to 8.2 m s–1 (avg. 5.6 m s–1), (Table 1). Relative humidity and temperature, two factors that can influence airborne microbial survival (Choi et al., 1997; Gloster & Alexandersen, 2004; Peccia & Hernandez, 2001), Fig. 2 Determination of recovery efficiency of bacterial and fungal colony forming units (CFU) on R2A medium relative to air volume sampled. Panel A, fungal CFU. Panel B, bacterial CFU 123 220 Aerobiologia (2006) 22:211–226 Fig. 3 Mid-Atlantic airborne dust and total bacterial and fungalcolony-forming unit concentrations (normalized data for concentrations m–3 air) for each sample date, 22 May through 30 June 2003 averaged 73.6% (range 65.7–81.8%) and 25.8C (range 24.8–29.6). A total of 26 bacterial and 83 fungal CFU were recovered on 24 of 40 sample dates (no CFU noted on 16 of the sample dates). Total (bacterial and fungal) concentrations ranged from 0.1– 31.3 CFU m–3. All reported CFU were recovered between 96 and 336 h of incubation (no CFU noted at 48 h of incubation). Of the 26 bacterial strains isolated, 25 were recovered from cryogenic storage (1 of the stored strains was no longer viable) and identified by 16S rDNA sequencing, only two were Proteobacteria (Pseudomonas sp. and Novosphingobium subarticum. GenBank Blast nearest neighbor. Table 3). Of the 23 Grampositive bacteria, 12 were high G+C content (species of the genera Brevibacterium, Cellulomonas, Frigoribacterium, Gordonia, Kocuria, Lechevalieria, Leifsonia, and Lentzea), seven were spore-forming low G+C content (Bacillus sp.), and the remaining four were non-sporeforming low G+C content (Staphylococcus sp.). Of the 83 fungal strains collected, 62 were recovered from cryogenic storage and 18S rDNA sequences obtained. Seventeen different genera were identified from this group of sequences and included species of Alternaria, Aureobasidium, Cladosporium, Dendryphion, Emericella, Lojkania, Lithothelium, Massaria, Myriangium, Neotestudina, Penicillium, Phoma, Preussia, Setosphaeria, Stachybotrys, Trichophyton, and Ulocladium (Table 3). Statistical analysis 123 demonstrated a statistically significant correlation between the NAAPS dust and total microbialCFU-concentration data (rs = 0.608, P < 0.001), i.e., higher model-predicted dust concentrations correlated with higher CFU concentrations. 4 Discussion In all cases R2A produced higher average counts than those observed with Blood Agar use and significantly higher counts when recovering UV stressed bacteria at 72 h of incubation. Another side benefit of R2A is that the low nutrient concentrations in this medium limited overgrowth by fast growing fungi which allows simultaneous recovery of fungal and bacterial CFU on a single substrate and filter. Additionally, if utilizing spore morphology to identify fungal isolates, low nutrient agars enhance sporulation due to the growing fungal colonies quickly exhausting required nutrients, a condition known to induce sporulation. (Morton, 1961). Although the data was not statistically significant, R2A outperformed PDA and SA in 2 of the 4 fungal CFU recovery study parameters (96 h—no UV, and 144 h—UV) and preformed as well or better than at least one of the substrates in the other 2 data sets. The influence of sample volume on membrane filtration CFU recoveries demonstrated that longer sample times (larger volumes) at an air flow rate of 6.3 l min–3 resulted in lower 1 1 1 1 1 1 10 1 1 1 1 5 Bacteria 3 3 2 1 1 1 3 1 1 1 1 1 1 1 2 1 1 Fungi 1 # of isolates Lithothelium septemseptatum Massaria platani Myriangium duriaei Myrothecium sp., Letendraea helminthicola, Cucurbidothis pityophila, Paraphaeosphaeria sp. (pilleata or michotii) 98 (555/565) Alternaria brassicae, Embellisia sp., Pezizomycotina sp. Alternaria dauci Alternaria sp. Aureobasidium pullulans or Discosphaerina fagi Cladosporium sp. Cladosporium sp. or Trimmatostroma macowanii Dendryphion sp. Emericella nidulans Lojkania enalia 96 (597/616) 99 (577/580) 99 (373/375), (568/570), (574/575), (574/576), (594/596), 98 (613/625), (666,674), (694/704), 698/704), (575/576) 98 (579/586) 97 (508/523) 98 (511/521) 99 (318/321) 99 (309/310) 99 (667/671) 100 (584/584), (438/438), 99 (597/600, (671/676), 98 (695/704) 100 (436/436) 99 (617/623) 100 (353/353), 99 (633/634), 98 (704/716) 99 (640/643), (666/668), (667/668) 98 (685/696), 98 (625/633) 98 (606/615) 97 (655/669) 98 (621/629) 100 (685/685), 99 (668/669), (670/673) 100 (628/628) 97 (615/634) 98 (588/595) 97 (229/235) 95 (479/503) 99 (728/735) 98 (710/724) 100 (609/609), 99 (568/570) 100 (542/542) 100 (612/612) % Homology (sequence length) Bacillus aminovorans Bacillus benzoevorans Bacillus sp. Brevibacterium casei Cellulomonas terrae Frigoribacterium sp. Gordonia terrae Kocuria rosea Lechevalieria fradiae Leifsonia xyli or L. poae Lentzea sp. Novosphingobium sp. Pseudomonas sp. Staphylococcus epidermidis Staphylococcus pasteuri Staphylococcus sp. Unknown GenBank closest relative AY584662 AF164363 AY016347 AB195634 AY382475 AB008403 AY016346 (·10) AF548071 AY251121 AY741243*t AY741246*t AY030322, AY137509 (·4) AY741243*t AY211147*m, AY211154 (·2)*m AY043085 (·3) AY21131*m, DQ129237 X76564 AY884570 AF157479 AY771333 (·3) AY211167*m AY114175 AE016822 DQ008601 SSU37347 AJ575816 AJ717377 AB009944 (·2) DQ113448 AY345421 (Actinomyces) GenBank closest relative #(s) Table 3 Bacterial and fungal tropical mid-Atlantic (~15 N, 45 W) atmospheric isolates collected between 22 May and 30 June 2003 Aerobiologia (2006) 22:211–226 221 123 123 Neotestudina rosatii Penicillium chrysogenum Penicillium glabrum Penicillium sp. or Talaromyces leycettanus Penicillium sp., Uscovopsis sp., Thysanophora sp., Chromocleista malachitea, Talaromycis leycettanus, or Eupencillium sp. Phoma herbarum Pleosporaceae sp. Preussia terricola Setosphaeria monoceras, Pleospora herbarum, or Embellisia sp. Setosphaeria monoceras, S. rostrata, or Cochliobolus sativus Setosphaeria rostrata Stachybotrys kampalensis Trichophyton mentagrophytes, or T. rubrum Ulocladium botrytis Ulocladium botrytis, Clathrospora diplospora, or Alternaria sp. 8 1 1 1 6 1 4 99 (573/576), (660/667) 99 (581/583) 99 (436/438) 99 (620/622) 97 (397/409) 99 (571/576) 99 (371/373), 99 (578/583), (616/623), (617/621), (619/622) AY864822 (·5) 97 (399/409), (472/483)( · 2), (472/484), (489/504), (567/584) 99 (571/576), (576/580), (580/583), (643/649), (656/662), (674/679) AF548106 (·2) AF548106 SRU42487*f AF548099*c AY083226 AY016352 AB195634 (·2), AY642526 (·3), AF212309 AY544726 AF212309 AY640998 AF290082 AF548090 AY526487 L76623 GenBank closest relative #(s) 99 (437/438) 99 (561/566), (562/567), (565/567), (565/568), (612/615), (646/649), (647/650), 97 (561/565) 98 (496/503) 98 (556/562) 99 (637/643) % Homology (sequence length) Where multiple genus/species are listed only the first GenBank Blast # match is listed. *m = Closest GenBank relative a Bamako, Mali air isolate, *t = Closest GenBank relative an Erdemli, Turkey air isolate, *f = species recognized as a Florida plant/tree pathogen. *c = Closest GenBank relative a Caribbean air isolate 2 1 1 1 1 1 6 1 GenBank closest relative # of isolates Table 3 continued 222 Aerobiologia (2006) 22:211–226 Aerobiologia (2006) 22:211–226 recovery rates. These data also demonstrated that the fungi (spore formers) are less susceptible to desiccation stress than non-spore forming bacteria. While no single culture medium will result the growth of all viable cells in any environmental setting (soil, air water, serum, etc.), R2A is an efficient medium for utilization in single medium studies. R2A was selected for use in this and previous studies primarily because of its ability to simultaneously recover stressed and non-stressed bacteria and fungi using a single filter (Griffin et al., 2003; Kellogg et al., 2004). While UV light may be attenuated up to 50% in large dust plumes (Herman, Krotkov, Celarier, Larko, & Labow, 1999), a typical dust cloud transit (across the Atlantic) time of 5–7 days can result in significant microbial UV exposure. Desiccation stress from wind transport (Lighthart, 1997), which averaged 5.6 m s–1 for this study period, and collection via membrane filtration as demonstrated can further limit CFU recovery. Employment of a low nutrient substrate such as R2A limits nutrient-shock which may be experienced by stressed cells plated on a nutrient rich substrate and numerous studies have shown that R2A gives superior recovery of CFU in comparison to high nutrient substrates in many different settings (body fluids, water, etc.) (Guerrero, 1987; Horgan, Matheson, McLoughlin-Borlace, & Dart, 1999; Lesne, Berthet, Binard, Rouxel, & Humbert, 2000; Massa, Caruso, Trovatelli, & Tosques, 1998; Reasoner, 2004; Reasoner & Geldreich, 1985; van der Linde, Lim, Rondeel, Antonissen, & de Jong, 1999). A number of studies have successfully utilized R2A to recover bacteria from soils, the lower atmosphere, and extreme atmospheric altitudes (Bodour, Drees, & Maier, 2003; Gentry, Newby, Josephson, & Pepper, 2001; Griffin, 2004; Griffin et al., 2003; Kellogg et al., 2004; Ovreas & Torsvik, 1998). Membrane filtration was chosen for collection of culturable bacterial and fungal CFU due to cost, ease of use, and the ability of membranes to efficiently capture cells. To limit viability loss due to filtration desiccation, low flow rates and amount of air volumes filtered were selected. The pump air flow rate over the filter membrane was 0.4 m s–1 in contrast to the prevailing wind speed 223 which averaged 5.6 m s–1 and turbulence. Given this high-wind and thus desiccating environment, we believed that additional cell stress due to filtration will have minimal impact on surviving culturable populations relative to losses due to other types of collection stressors such as those seen with impaction (Stewart et al., 1995). The large volume air samples utilized after 6 June 2003 were chosen to increase the probability of recovering microbial CFU after a noted drop off in the recovery of CFU with the small volume samples following the elevated dust period of 25–29 May, 2003. It should be noted that the filtration volume CFU recovery data presented demonstrated statistically significant lower bacterial recovery with elevated volumes filtered and any bacterial CFU reported in Table 1 for the large volume samples should be considered conservative in nature. These experiments demonstrate that choice of methodology does make a difference in rates of recovery of microorganisms and this is well known in the various fields of microbiology. Of the identified bacteria recovered in this current study, 2 were Proteobacteria. Proteobacteria (Gram-negative bacteria) are the most prevalent bacterial group in marine waters (Sherr & Sherr, 2000), and during periods of sea-spray generation (severe weather, high surface activity, etc.), these organisms may become the dominant air isolate in marine environments, as previously observed (Griffin et al., 2003). All of the nonspore-forming bacteria recovered were pigmented. Pigmentation, high G+C content, and spores impart resistance to UV inactivation and as a result enhance atmospheric survival (Riesenman & Nicholson, 2000; Sundin & Jacobs, 1999). Although 16S rDNA sequences alone do not provide the resolution needed to determine relatedness (Stackebrandt & Goebel, 1994) or point of origin, two of the isolates (one Bacillus aminovorans, 353/353 bp, and Kocuria rosea, 628/ 628 bp) matched at 100% sequence similarity to two dust-borne isolates previously collected from the atmosphere in Bamako, Mali, Africa (Kellogg et al., 2004). Kocuria rosea has also been isolated from the atmosphere in the U.S. Virgin Islands when African desert dust was present, on several occasions, and is a known human pathogen (Altuntas et al., 2004; Griffin, Garrison, Herman, 123 224 & Shinn, 2001; Griffin et al., 2003). The remaining two isolates of B. aminovorans and the Bacillus sp. isolate listed in Table 3 also matched (GenBank closest relative) to Bamako, Mali isolates at a sequence identity range of 98–99% (633–704 bp). Three of the isolates were identified as Gordonia terra, and this species is capable of causing disease in humans (Lesens et al., 2000). Gordonia terrae, in addition to various species of Bacillus and Staphlyococcus, have also been identified in atmospheric samples collected in Bamako, Mali, Africa (Kellogg et al., 2004). The most prevalent fungal species isolated were 10 colonies of Lojikania enalia. Two of the currently available isolates of Lojikania enalia (American Type Culture Collection, ATTC 16472 and 16473) were isolated in Liberia, Africa, from an aerial root and a piece of driftwood, respectively. A 569 bp alignment of all the Lojikania enalia isolates listed in Table 3 with the ATCC 16473 Lojikania enalia isolate resulted in a sequence identity range of 99.3–99.8%. Twenty percent of the fungal isolates identified at the species level are recognized pathogens to humans, animals, plants, and trees (Alternaria dauci, Massaria platani, four isolates of Neotestudina rosatii and Ulocladium botrytis). The three Alternaria sp. isolates listed in Table 3 all matched to isolates (each matched to a previous GenBank submission as listed in Table 3) we have recovered from atmospheric samples in Erdemli, Turkey which is a dominant dust-borne microorganism at that coastal location (unpublished data). Spores of various species of Alternaria are potent allergens. Given that ~50% of the particles in Florida’s atmosphere each summer are African in origin (Prospero, 1999), it is interesting to note that Massaria platani (aka Splanchnonema platani), a causative agent of Florida Sycamore (Plantanus occidentalis) canker (Alfieri, Langdon, Wehlburg, & Kimbrough, 1984), and Alternaria dauci, an identified pathogen of Florida carrots (Dugdale, Mortimer, Isaac, & Collin, 2000), were isolated during this project. It is tempting to speculate that transatlantic transport of dust could be a vector to renew reservoirs of some plant and animal pathogens in North America and could also be a source of new diseases. 123 Aerobiologia (2006) 22:211–226 As Fig. 3 illustrates, the majority of bacterial and fungal CFU was recovered during periods of elevated dust concentration. The three highest total CFU counts relative to air volume assayed occurred on 25 May 2003, 26 May 2003, and 6 June 2003 (31.3, 11.2, and 15.9 CFU m–3, respectively). These counts corresponded with the two highest periods of dust activity as depicted by the NAAPS model (47.4–110.9 lg m–3, 25–28 May 2003, and 45.3 lg m–3, respectively). Figure 3 also illustrates that the highest recovery of culturable microorganisms occurred on 25 May 2003, one day before the model derived dust concentration peak on 26 May 2003. This could be due to the lighter dust particles moving out ahead of heavier dust cloud particulates, due to preferential wind-transport. Dust-borne aeromicrobiology research conducted in Bamako, Mali, Africa (February 3, 2001 to March 1, 2002) demonstrated bacterial and fungal population sizes of 3,548 and 129 CFU m–3, respectively (Kellogg et al., 2004). In this mid-Atlantic study, bacterial and fungal populations averaged (all samples) between 0.1 and 0.4 CFU m–3. These data indicate a dust-borne reduction of culturable bacterial and fungal CFU m–3 of ~104 and 102, respectively, from the continent of Africa to this mid-Atlantic research site during the early Northern Hemispheres trans-Atlantic dust season. This noted difference is probably due to the synergistic effects of particle fall-out during transport, atmospheric dilution, and cell stress (desiccation, UV exposure, temperature, humidity, etc.). It should be emphasized that microbial ecology studies have shown that what is typically cultured from a given volume of soil represents < 1.0% of the actual total prokaryote population present (Torsvik, Salte, Sorheim, & Goksoyr, 1990). Since African dust moving through the atmosphere is a component of African soil, the dust-associated CFU counts presented in this study may only represent a small fraction of the prokaryote community that was actually present in any of the given atmospheric samples. This study presents evidence of early summer survival and transport of microorganisms from North Africa to a mid-Atlantic research site. Additionally, the statistical correlation between the Aerobiologia (2006) 22:211–226 NAAPS model dust concentrations and microbial CFU counts observed demonstrates the potential for using models to address the global atmospheric dispersion of microorganisms (nonpathogenic and pathogenic) and other soil-associated constituents, since they pertain to ecological evolution and ecosystem and human health. Acknowledgements This research was supported by the U.S. Geological Survey (2090-OJ503) and the Ocean Drilling Program. Thanks to Norman Kuring and Gene Feldman of NASA’s SeaWiFS Project for providing daily satellite imagery while aboard ship, and Cindy Morris of INRA Avignon, Montfavet, France for several thorough edits and suggestions. Also, special thanks to ODP Leg 209 shipboard scientific party and crew. 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