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The Transport of Nonindigenous Microorganisms IntoCaves by Human Visitation: A Case Study at CarlsbadCaverns National ParkDale W. Griffin a , Michael A. Gray b , Mark B. Lyles c & Diana E. Northup da U.S. Geological Survey , Tallahassee , Florida , USAb U.S. Geological Survey , St. Petersburg , Florida , USAc U.S. Naval War College , Newport , Rhode Island , USAd Biology Department , University of New Mexico , Albuquerque , New Mexico , USAPublished online: 30 Jan 2014.

To cite this article: Dale W. Griffin , Michael A. Gray , Mark B. Lyles & Diana E. Northup (2014) The Transport ofNonindigenous Microorganisms Into Caves by Human Visitation: A Case Study at Carlsbad Caverns National Park,Geomicrobiology Journal, 31:3, 175-185, DOI: 10.1080/01490451.2013.815294

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Geomicrobiology Journal (2014) 31, 175–185Copyright C© Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451.2013.815294

The Transport of Nonindigenous Microorganisms IntoCaves by Human Visitation: A Case Study at CarlsbadCaverns National Park

DALE W. GRIFFIN1∗, MICHAEL A. GRAY2, MARK B. LYLES3, and DIANA E. NORTHUP4

1U.S. Geological Survey, Tallahassee, Florida, USA2U.S. Geological Survey, St. Petersburg, Florida, USA3U.S. Naval War College, Newport, Rhode Island, USA4Biology Department, University of New Mexico, Albuquerque, New Mexico, USA

Received January 2013, Accepted June 2013

A series of atmospheric investigations was conducted in Carlsbad Cavern to determine if human visitation is a possible cause forthe contamination of the cave system with non-indigenous microorganisms. In 2004, site-specific culture-based data demonstratedthat Staphylococcus spp. colony-forming units (CFUs) were the most prevalent members of the atmospheric community along thepaved visitor trail (avg. 18.8% of CFU), while Knoellia spp. CFUs dominated off-trail locations (40.1% of CFU). Fungal culture datarevealed that Penicillium and Aspergillus were prevalent in the Lunch Room where food is stored, sold, and consumed. Ubiquitousgenera such as Cladosporium and Alternaria were prevalent near the Natural Entrance of the cave, and the general trend was adecrease in fungal CFUs with progression into the cave system, except for the area near the Lunch Room. Management practices suchas prohibition of crumb-generating types of foods could be considered to protect cave health. In 2009, nonculture-based analysesdemonstrated that Enterobacteriaceae were the dominant microbiota at sites along the descent trail and within the Lunch Room.Dominance of Enterobacteriaceae has not been previously demonstrated in caves. Either they are naturally occurring indigenousmembers, or their presence is a marker of anthropogenic contamination.

Keywords: aerobiology, aeromicrobiology, atmosphere, cave

Introduction

From the entrance gate to Carlsbad Caverns National Park(CCNP) in White City, New Mexico, to the Visitor Centeris a winding 11.3-km road that scales an elevation change of∼230 m (from 1117 m to 1347 m above sea level). What islost to most visitors is that one is driving up and through anancient limestone reef that was primarily formed by spongesand algae in what was a shallow-marine basin ∼280 to 240million years ago during the Permian Period. The reef wasnamed the Capitan Reef and in structure was approximately600 m thick, 3 to 4 km wide, and 640 km long (King 1948;USNPS 2007).

A series of tectonic uplifts and tilts that occurred withinthe last ∼40 million years, coupled with sporadic acidic dia-genesis of the skeletal reef structure, resulted in what is known

∗Address correspondence to Dale W. Griffin, Public Healthand Environmental Microbiologist, U.S. Geological Survey, 6004th Street South, St. Petersburg, Florida 33701, USA; Email:dgriffin@usgs.govColor versions of one or more of the figures in the article can befound online at www.tandfonline.com/ugmb.

today as the Guadalupe Mountain range and Carlsbad Cav-ern and other caves in CCNP. Geological research indicatesthat sulfate-reducing bacteria within the oil and gas depositsof the region produced hydrogen sulfide that migrated upwardalong fissures and oxidized to sulfuric acid microbially andchemically when it reached the oxygenated zone.

The sulfuric acid is hypothesized to have driven the disso-lution of limestone and subsequent cave genesis (e.g., sulfuricacid-driven speleogenesis) (Engel et al. 2004; Jagnow et al.2000). Diagenesis of limestone in the region was profuse overtime, and the primary cave system known as Carlsbad Cavernthat most people visit is only one of about 117 known caveslocated within the CCNP boundaries. Analyses of alunite iso-topes indicate that the Big Room level of Carlsbad Cavernformed approximately 4.0 to 3.9 million years ago (Polyaket al. 1998). Phases of stable water-table levels resulted in longperiods of sulfuric-acid generation and exposure that createdthe larger sections of Carlsbad Cavern, such as the Big Room,Bat Cave, Kings Palace, and the Lower Cave.

Speleothem structures throughout Carlsbad Cavern arespectacular and include those composed of calcite (primaryspeleothems), gypsum, and aragonite. Bacteria isolated fromthe surface of speleothems were previously shown to facilitate

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carbonate-crystal formation, and microbial communities arenow known to contribute significantly to the formation ofspeleothems (Barton and Northup 2007; Cacchio et al. 2004;Canaveras et al. 2001; Ercole et al. 2001; Melim et al. 2001;Melim et al. 2008).

In addition to the known microorganisms, insects, rodents,and birds that inhabit Carlsbad Cavern, the cave systemis also home to a colony of Brazilian free-tail bats whoseresident population in 2005 ranged from ∼400,000 in thesummer to ∼793,000 in the spring and fall migration period(Barr and Reddell 1967; Northup et al. 1993; USNPS 2012).Visitor traffic averaged ∼413,888 per year from 2004 through2009, and a primary concern of the National Park Serviceis how to protect the unique and evolving speleothems andcave ecosystems from visitation damage (Burger and Pate2001).

The primary visitor trail is ∼4.0 km long, starts at thecave’s Natural Entrance, and descends down ∼230 m to andaround the Big Room. The Big Room portion of the trail is∼2.0 km, and there is an elevator that can be used for de-scent and ascent from the Big Room to the surface visitorcenter. Next to the elevator are restrooms, several kiosks thatsell gift-shop items and candy-type consumables, a cafeteriathat sells drinks, sandwiches and other consumables, and ad-jacent to the cafeteria is a large area of picnic tables. Effec-tive stewardship of the park to limit and prevent potentialdamage to the system has included the replacement of agedsewer lines (wastewater generation ∼38 × 106 L yr−1), con-struction designs to minimize pollutant transport from roadand parking-lot runoff (>5.3 × 106 L yr−1), and programs tolimit potential pollutants originating from spills and water-related cleaning of the park’s many facilities (Burger and Pate2001). Additionally, there is an active program to remove hu-man lint after it was discovered that deposition was coveringand discoloring speleothems located close to the visitor trail(Pate 1999). Between 1988 and 1992, 90.7 kg of litter and34.0 kg of lint were removed from Carlsbad Cavern from ar-eas within 1 m of the system’s trail edges (Jablonski et al.1993).

In addition to clothing lint, humans shed ∼612 hairs and∼16.5 × 106 epithelial cells per hour (Roberts and Marks1980; Wasko et al. 2008). At an average stay of ∼2 hourswithin Carlsbad Cavern (at the above average annual visitorrate of 413,888), the deposition rates could be as high as∼5.1 × 108 hairs and ∼1.4 × 1013 epithelial cells annually.When humans shed hair and cells, they also shed the bacteriaand fungi that reside on them. Analyses have demonstratedthat the communities of bacteria and fungi that live onhuman hair and skin cells are diverse. Approximately 205genera of bacteria were found in one human skin study(primarily dominated by Actinobacteria and Firmicutes), and14 genera of fungi were observed within the human toe webenvironment alone (Grice et al. 2009; Oyeka and Ugwu 2002).

Although shedding is certainly a concern in such an envi-ronment, another source of nonindigenous microorganisms isthose associated with the consumption of food, such as wouldoccur in the Big Room’s cafeteria. In this case, microorgan-isms may find nutrient sources on food crumbs as well as onthe surfaces of adjacent speleothems. These nonindigenous

microorganisms might adapt and survive in this subsurfaceenvironment and potentially damage the native ecosystemsthrough niche displacement or parasitizing native microbialcommunities.

Here we report the results of a microaerobiology study thatwas conducted to determine if the types and concentrationsof microorganisms within the cave’s atmosphere varied withdistance along the paved visitor trail (starting at the NaturalEntrance), in areas off the visitor trail, and in the Big Room’scafeteria where people most frequently congregated.

Material and Methods

Samples were collected on the 27th of September 2004, the23rd and 24th of September 2005, and the 1st of April 2009.Sample-sites are illustrated in Figure 1. Samples were col-lected at the described points along the paved visitor trail thatdescends from the Natural Entrance to and around the BigRoom. Additionally, samples were collected in less-frequentedoff-trail areas to contrast with the paved trail sites.

Sites referred to as off-trail (site not frequented by touristvisitation) include Sand Passage, Hall of the White Giant,New Mexico Room Overlook, and Left Hand Tunnel (beyondthe end of the tourist tour). All other sample sites, with theexception of Lunch Room C (which is near the Lunch Roombut cordoned off by a two-railed fence), were along the regularpaved visitor trail extending from the Natural Entrance, downto and around the Big Room and within the Lunch Roomarea.

2004 and 2005 Samples

Isolation and identification of culturable bacteria and fungiIn 2004 and 2005, all listed sites illustrated in Figure 1 wereutilized for sample sites. Paved visitor trail sites and the LunchRoom sites were collected between 0830 and 1630 (within vis-itation hours) and off-trail sites were collected between 1700and 2030 (after guided off-trail tour period). Air samples werecollected along the visitor trail and cafeteria using a previ-ously described portable membrane-filtration apparatus andmembrane/culture protocol (Griffin et al. 2011).

For off-visitor-trail sample sites where it was not possi-ble to utilize the trail apparatus, a ‘breakdown’ system com-posed of the same type pump, a ∼0.7-m-long piece of PVCthat mounted vertically atop the vertical intake line of thepump, a small 12-volt computer backup battery, and an in-verter were utilized. Flow rates through the pumps averagedapproximately 10.5 L min−1 over a period of ∼20.0 min. Sam-ples were plated on R2A medium (Reasoner and Geldreich1985) sample side up and were incubated in the cave cafeteriaarea for 24 h at ambient cave temperature (∼16◦C) and rela-tive humidity (∼95–100%). Temperature and humidity mea-sured with a Kestrel 3000 handheld anemometer (KestrelMe-ters.com, Birmingham, MI). After 24 h of incubation in thecave, the plates were brought to the surface and incubatedanother 24 h at room temperature (23◦C).

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Human Visitation Transporting Microorganisms Into Caves 177

Fig. 1. Sample sites located along the paved visitors trail and the sites in the cafeteria area. Black boxes are the trail sample sites Athrough L and Lunch Room A, Lunch Room B, and Lunch Room C, respectively. NMR = route to the New Mexico Overlook; SPG,HWG = route to Sand Passage and the Hall of the White Giant, respectively.

Bacterial and fungal colony-forming units (CFUs) wereenumerated at 48 h. Plates were transported from New Mex-ico to Florida for isolation of CFUs. For isolation, colonieswere picked and streaked onto fresh plates of R2A. Iso-lated colonies were grown in Tryptic Soy Broth overnightat room temperature on a tabletop rocker set at low speed.The following day, 0.5 ml of each culture was transferredto a sterile cryogenic storage tube containing 200 μl of ster-ile glycerol and stored at –70◦C. Bacterial and fungal CFUswere identified using universal 16S and 18S PCR and am-plicon sequencing, as previously described (Griffin et al.2006). Amplicon sequence reads for 16S were ∼450 bp and18S were ∼600 bp. Sequence quality was determined man-ually via chromatograms. Bacteria sequences were checkedfor orientation via Orientation Checker (www.bioinformatics-toolkit.org/Help/Topics/orientationChecker.html) and fun-gal sequences via GenBank sequence comparisons and Se-quencher v5.0.1 (Gene Codes Corporation, Ann Arbor,Michigan). Operational Taxonomic Units (OTUs) were de-termined at 97% similarity using Lasergene v10 SeqMan Pro(DNASTAR Inc., Madison, Wisconsin). GenBank BLAST

was used to identify representative nearest-neighbor sequences(Altschul et al. 1997).

2009 Samples

Isolation of bacteria, fungi, and viruses for culture-and nonculture-based assaysSamples were collected on 1 April 2009 between the hours of0920 and 1538 at five locations (Sites A, E, Lunch Room A, H,and Left Hand Tunnel). Sample sites A, E, H, Lunch RoomA and Left Hand Tunnel were utilized to obtain membrane-filtration samples. Sites E and Lunch Room A were used toobtain liquid-impinger samples. Membrane-filtration sampleswere collected as described previously, but at a flow rate of 8.7L min−1 for 40 min.

In addition to the membrane-filtration samples utilized todetermine culturable bacterial and fungal CFUs, samples weresimultaneously collected using the same flow rate and flowperiod to determine total bacterial and viral direct counts.Atmospheric direct counts were obtained using a previously

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published protocol (Griffin et al. 2001). Liquid-impinger sam-ples were collected at Sites E and Lunch Room A for culture,nonculture, and direct-count protocols using an OMNI 3000(Evogen, Inc., Kansas City, Missouri) and Evogen, Inc. sterile1× PBS (phosphate-buffered saline) 10-ml sample cartridges.Filtration-flow rates ranged from ∼233 to ∼268 L min−1 overa 30.0-min sample period. After filtration, the 1× PBS sam-ple cartridges were labeled, placed in Evogen, Inc. cartridgezip-lock bags, and transported to the surface for spread-plateanalyses and mounting for direct-count analyses. Approxi-mately 2,150 μl were removed from each 1× PBS 10 ml samplecartridge using a sterile 10-cc syringe and needle.

The aliquot was then dispensed into a sterile 15-ml cen-trifuge tube, and 100 μl were used for spread-plate techniqueon R2A medium. Samples were incubated in the dark atroom temperature (∼23◦C) and enumerated at ∼48 h. Theremaining 2.0 ml were fixed with filter-sterilized formaldehyde(4% fixation) for 30 min and filtered through 0.02-μm Anodiscglass-fiber filters. Excess fluid was removed from the filter sur-faces by wicking from the underside of each filter, and the fil-ters were then stored via refrigeration or on ice until processingthrough the direct-count protocol described above upon re-turning to the USGS Microbiology Laboratory in Tallahassee,Florida.

DNA sequences were obtained from the liquid-impingersamples via the QIAGEN, Inc. DNeasy Blood and Tissuekit and the QIAGEN, Inc. PCR Cloning Kit for in-houseuniversal 16S and 18S PCR and amplicon-vector ligationfollowed by transformation, clone selection, and sequenc-ing by the Genome Center at Washington University (St.Louis, Missouri) and Epoch Life Sciences (Missouri City,Texas). Sequence integrity was determined via Sequencherv5.0.1 and manually and then following the approach de-scribed for the 2004 and 2005 sequences. MEGA v5.05(http://www.megasoftware.net/) was used to produce thealignment (Clustal W) and phylogenic (Neighbor-Joining) treefor the 2009 bacteria data.

Particle-count dataParticle-count data were obtained using an IQAir ParticleScanPro (IQAir North America, Inc., Santa Fe Springs, CA). Thisinstrument has a particle-detection size range of 0.3 to 30.0 μmand provides particle-size distribution over six size ranges (≥0.3, 0.5, 0.7, 1.0, 3.0, and 5.0 μm). In 2004, the peak particlecounts were manually recorded for all size ranges over a periodof several minutes and reported as particles per liter of air. In2005, the particle counter was tethered to a laptop and particlecounts were taken every 6 s over a period of ∼10 min and theaverage reported as particles per liter of air. In 2009, particlecounts for each size range were taken three times over a periodof 2 to 3 min and averaged.

Genbank accession numbersAccession numbers for the bacterial and fungal OTU se-quences are JX096838 – JX096905 (2004 Bacteria), JX096906– JX096916 (2004 Fungi), JX096917 – JX096944 2009 Fungi,Lunch Room A and Site E), JX 104053 - JX 104081 (2009Bacteria, Lunch Room A) and JX394035 - JX394060 (2009Bacteria, Site E).

Results

2004 and 2005 Data

Figure 2 illustrates the particle-count and bacterial and fungalCFU data obtained from each sample site in 2004 and 2005.Bacterial CFUs along the visitor trail ranged from 240.0 to247.0 CFUs per cubic liter in 2004 and 5.0 to 300.0 CFU percubic liter in 2005. Off-trail bacterial CFUs ranged from 207.0to 976.0 CFU per cubic liter in 2004 and 130.0 to 1768.0 CFUper cubic liter in 2005. Table 1 lists the 2004 bacterial CFU16S identifications (order, family, or genus based on closestneighbor from BLAST) for sample sites A, B, I, Lunch RoomA, Lunch Room B, Lunch Room C, New Mexico Overlook,

Fig. 2. Total particle-count data per liter of air and total bacterial and fungal CFU per ∼210 L of air for each sample site in 2004 and2005. Left Y-axis = Bacterial and Fungal CFU. Right Y-axis = particle counts. Total particle size range ≥ 0.3 μm.

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Table 1. Identified bacterial CFUs for 2004 sample sites A, B, I, Lunch Room A, Lunch Room B, Lunch Room C, New MexicoOverlook, and Sand Passage

2004 Bacterial CFU Lunch Lunch Lunch New Mexico Sandisolate IDs Site A Site B Site I Room A Room B Room C Overlook Passage

Arthrobacter 3 2 2 16 12Streptomyces 2 1 1 5 4Knoellia 2 28 33Nocardioides 1∗ 1∗Brevibacterium 3 1Dietzia 3∗Nocardia 1 2 5Janibacter 1 5 3 1 14 2Brachybacterium 1 1Rhodococcus 4 1 1 2Microbacterium 2 2 2 2 3 5Bacillus 1 1Staphylococcus 1 4 1 6 4 1 3Agrococcus 2 1 1Serinicoccus 1∗Rathayibacter 1∗Rarobacter 1∗Terrabacter 4 5Promicromonospora 1∗Xylanimicrobium 1∗Micrococcus 1 3 1 4 1Xanthomonas 1∗Acinetobacter 1∗Deinococcus 1∗Sphingopyxis 2∗Chryseobacterium 1∗Hymenobacter 1∗Methylobacterium 1∗Micrococcaceae (Family) 1∗Bacillales (Order) 1∗% of total CFUs sequenced 23.8 86.4 55.3 50.0 69.6 5.1 52.9 63.8% Staphylococcus 20 26.7 3.8 37.5 25 1.2 4.1% Knoellia 34.6 46.5

∗Isolate only noted at that location.

and Sand Passage. (Isolates at the remaining stations were notidentified.)

Several isolates were only observed at one site. Rathayibac-ter spp., Deinococcus spp. and Micrococcaceae isolates wereonly identified at site A (nearest the Natural Entrance), theAcinetobacter sp. isolate at site B, the three Dietzia spp. iso-lates at site I, the Rarobacter sp. isolate at the Lunch Room Asite, the Hymenobacter sp. isolate at the Lunch Room B site,the Chryseobacterium sp. isolate at the Lunch Room C site, theSerinicoccus sp., Xylanimicrobium sp., Xanthomonas sp., andtwo Sphingomyxis spp. isolates at the New Mexico Overlooksite, and the Promicromonospora spp. and Methylobacteriumspp. isolates at the Sand Passage site. Knoellia spp. represented34.1 and 44.6% of the identified isolates at the off-trail NewMexico Overlook and Sand Passage sites, respectively, andwere only found along the visitor trail at site I at 7.7%. Generathat were only found at both of the off-trail locations includedthe two Nocardioides spp. and nine Terrabacter spp. isolates.

Staphylococcus spp. represented an average of 18.3% of identi-fied bacteria isolated from visitor trail/lunch-room sites versus2.7% at the off-trail sites.

Fungal CFUs for both years were generally highest atthe Natural Entrance (∼60 CFU per cubic liter of air) andtapered off with depth into the cave system with the exceptionof the Lunch Room sample sites where the peak countsoccurred. Fungal CFUs were rarely noted off-trail. FungalCFUs along the visitor trail ranged from 0.0–9.0 CFU per100 liters in 2004 and 0.0–95.0 CFU per cubic liter in 2005.Off-trail fungal CFUs ranged from 0.0 to 4.0 CFU per cubicliter in 2004 and 0.0–5.0 CFU per cubic liter in 2005. Figure 3illustrates the distribution of 18S identified fungal CFUs ateach location in 2004.

Fifty-eight fungi were identified from Sites A (8 isolates),B (12 isolates), I (10 isolates), Lunch Room A (13 isolates),Lunch Room B (10 isolates), and Lunch Room C (3 isolates).Isolates identified (GenBank megablast closest neighbor) as

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Fig. 3. Percent identified fungal CFU (18S sequencing) for sample sites A, B, I, Lunch Room A, Lunch Room B, and Lunch Room C,2004. General orientation of view is inside the cave looking out. Site A = inner ring, 8 colony forming units (CFU). Site B = secondring from center, 12 CFU. Site I = third ring from center, 10 CFU. Lunch Room A = fourth ring from center, 12 CFU. Lunch RoomB = fifth ring from center, 10 CFU. Lunch Room C = outer ring, 3 CFU.

belonging to the family Trichocomaceae (isolates only identi-fied at the family level; Penicillium and Aspergillus are generawithin this family) and genera Penicillium or Aspergillus rep-resented 76.9% of the identified isolates collected from theLunch Room sample sites.

Aspergillus spp. were only identified at the Lunch Room Asite. Penicillium spp. were not recovered from Site A, three wererecovered at Site B and none were recovered from Site I. Iso-lates whose closest GenBank neighbor were at the Trichoco-maceae family level were only observed at the Lunch Roomsites. Alternaria alternate was the dominant isolate at Site A(50.0%) followed by Cladosporium spp. (25.0%). Alternariaalternate was also the dominant isolate at Site B (41.7%),followed by Penicillium spp. (25.0%). Isolates identified as be-longing to the order Hypocreales dominated the Site I isolates(70.0%).

2009 Data

Table 2 lists the particle counts, CFUs, and direct-count datacollected at each sample site. As previously observed, the par-ticle counts are highest at the Natural Entrance and decreasewith descent into the system. The dominant particle size-distribution range was ≥0.3 and ≤0.5 μm at each site, andthis size fraction increased from the Natural Entrance to theLeft Hand Tunnel site. Percent concentration of this size frac-tion versus the five other larger fractions was 69.9% at site A,77.7% at site E, 78.4 % at the Lunch Room A site, 85.9% atSite H, and 91.1% at the Left Hand Tunnel site. The bacte-rial CFUs and direct-count data in Table 2 demonstrate thatthe culturable fraction of bacteria was less than 0.1% of whatwas enumerated with the direct-count assay whether using themembrane-filtration apparatus or the liquid impinger and that

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Table 2. Atmospheric data collected at each site, 1 April 2009

Researcher/ Total CFU m3 CFU m3 Direct count m3 Direct count m3

Relative Tourist Particles (Bact./Fung.) (Bact./Fung.) (Bact./Virus) (Bact./Virus)Humidity Count ≥0.3 m3 Memb. Filt. Liq. Imp. Memb. Filt. Liq. Imp.

A 17.1 27.9 47 2.1 × 107 5.9/11.8 nc 8.4 × 103/1.1 × 104 NcE 13.5 82.9 70 6.8 × 106 2.9/5.9 13.3/26.7 6.6 × 104/2.1 × 104 1.3 × 106/3.3 × 105

LunchRoom A

14.4 81.3 21 4.2 × 106 11.8/11.8 103.7/103.7 4.1 × 104/4.1 × 104 3.0 × 105/1.6 × 105

H 15.3 90.3 137 6.6 × 106 5.9/0 nc 7.3 × 104/6.4 × 104 NcLeft Hand

Tunnel17.1 92.8 3 7.4 × 105 0/0 nc 3.7 × 104/6.4 × 103 Nc

employment of the liquid impinger resulted in higher CFUsand direct counts versus the membrane-filtration approach(Griffin et al. 2011).

Viral direct counts listed in Table 2 ranged from 6.4 × 103

(low end of the membrane-filtration direct counts) to 3.3 × 105

(high end of the liquid-impinger direct counts) depending onthe site location and method used. As observed with the bac-terial direct counts, viral direct counts were higher using theliquid impinger than membrane filtration by approximatelyan order of magnitude. At the Lunch Room A site, using

direct DNA extraction from the liquid-impinger samples anduniversal 18S PCR, there were 51 usable fungal sequencesobtained from screening 196 clones that grouped into 13OTUs.

OTU groups based on closest GenBank neighbor werePleosporaceae (22 members), Cordycipitaceae (a family of theorder Hypocreales) (5), Penicillium griseofulvum (3), Phane-rochaete sp. (3), Arthrinium spp. (2), Exophiala spp. (withinthe order Chaetothyriales) (1), Schizophyllum commune (4),Peniophora nuda (1), Cladosporium spp. (3), Agaricomycotina

Fig. 4. Identified bacteria via universal 16S amplicon sequencing of liquid-impinger extracts for sample sites E and Lunch Room A,2009. Squares = Lunch Room A sequences. Circles = Site E sequences.

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(1), Candida catenulate (1), Mycosphaerellaceae (1), andPulcherricium caeruleum (1).

The Phanerochaete spp., Arthrinium spp., Schizophyllumcommune, Peniophora nuda, Candida catenulate, and Pulcherri-cium caeruleum sequences were only noted in the Lunch RoomA sample. From the Site E sample, 54 usable fungal sequenceswere obtained from 196 sequenced clones that grouped intonine OTUs. OTU groups were Pleosporaceae (25), Penicil-lium griseofulvum (5), Cladosporium spp. (5), Agaricomy-cotina (4), Mycosphaerellaceae (2), Phacidium lacerum (4),Chaetothyriales (3), Hypocreales (5), and Chaetomium globo-sum (1). Phacidium lacerum, and Chaetomium globosum se-quences were only noted in the Site E samples.

One hundred and sixty-six usable bacterial sequences wereobtained from 196 sequenced clones from the Lunch RoomA site. The sequences grouped into 25 OTUs that includedEnterobacteriaceae (84), Pseudomonas spp. (25), Enterobacterspp. (12), Vibrio metschnikovii (6), Halomonas spp. (5), Pan-toea ananatis (5), Streptococcus spp. (5) Janthinobacteriumspp. (4), Neisseria spp. (3), Chromohalobacter marismortui(2), Aeromonas veronii (1), Averyella spp. (1), Bacteriodetesspp. (1), Burkholderiaceae (1), Burkholderia spp. (1), Chry-seobacterium spp. (1), Massilia spp. (1), Microbacteriaceae (1),Pandoraea norimbergensis (1), Vibrio cincinnatiensis (1), Pro-pionibacterium spp. (1), Pseudomonas libanensis (1), Serratialiquifaciens (1), Serratia proteamaculans (1), and Staphylococ-cus saprophyticus (1).

One hundred and eighty usable bacterial sequences wereobtained from 196 sequenced clones from Site E (the site onthe visitor descent trail located near audio Marker #14). Thesesequences grouped into 25 OTUs that included two groups ofEnterobacteriaceae (89 and 5), Halomonas spp. (24), Cede-cea davisae (16), Janthinobacterium spp. (9), Streptococcusspp. (7), Pseudomonas spp. (7), Burkholderia spp. (3), Neis-seria spp.(2), Pandoraea norimbergensis (2), Pantoea spp. (2),Acidovorax spp. (1), Comamonas spp. (1), Chryseobacteriumspp. (1) Deltaproteobacterium (1), Firmicute (1), Gammapro-teobacterium (2 different), Legionella spp. (1), Pasteurella spp.(1), Propionibacteriaceae (1), Serratia spp. (1), Sphingomonasspp. (1), Vibrio metschnikovii (1), and an unknown (1). Fig-ure 4 is a phylogenetic tree showing the distribution of thesesequences between sample sites.

Discussion and Conclusion

In all study years, the highest concentrations of all airborne-particle size ranges occurred near the Natural Entrance andthen dropped dramatically with site progression down and intothe Big Room, as illustrated in Figure 2 and Table 2. It has beennoted that in the summer months, the atmosphere of CarlsbadCavern is stable, and in the winter months outside air is ableto penetrate into the system (Wilkening and Watkins 1976). Asimilar seasonal trend in air movement was observed in a studyof a Florida cave system and the influence of seasons on caveaerobiology has been previously demonstrated (Kowalczk andFroelich 2010; Mulec et al. 2012).

Although the 2009 study was conducted in April, which isone of the cooler months for the region (average high 26.1◦C),the overall concentrations of suspended particulates was notthat notably different than what was observed for the 2004and 2005 studies that were conducted during September ofeach year (average high 30.6◦C). It is possible that bird, bat,insect, rodent, and human activities in and around the NaturalEntrance mask changes in interior versus exterior seasonalatmospheric mixing patterns.

The bacterial CFU data for both 2004 and 2005 were fairlyconsistent along the main paved visitor trail. Off the touristtrail, the CFU counts were much greater due to the foot trafficof our research team. Because humidity levels were near 100%at all sites, on the off-trail sites of the New Mexico Room Over-look and Sand Passage sites, particulate matter and our breathwere visible in our headlamp beams. Even though we sampledon the forward side of our progression or slightly behind ourpath slightly off trail where the atmosphere appeared clear, thebacterial CFU numbers at these sites were greater than whatwas observed along the paved trails. More importantly, fungalCFU counts were on the lower range of what was observedalong the visitor trail.

What was distinct about the two off-trail sites listed in Ta-ble 1 was the dominance of Knoellia species. Outside of thesetwo off-trail sites, only a couple of Knoellia sp. CFUs wereidentified at Site I. As Table 1 displays, Staphylococcus spp.CFUs were the dominant culturable microbiota along the vis-itor trail in 2004, and this microorganism is a common skininhabitant of humans. The highest Staphylococcus CFU con-centration, 37.5% of cultured isolates, occurred at the LunchRoom A site. Staphylococcus was previously observed and be-lieved to be a marker of human visitation in Lechuguilla andMammoth Cave (Lavoie and Northup 2005; Northup et al.1994; Northup et al. 1997).

It is interesting to note that CFUs from genera observedin this study such as Acinetobacter, Arthrobacter, Methylobac-terium, Nocardioides, and Xanthomonas were previously ob-served in other cave studies (wall or floor samples) thatemployed nonculture-based methods to identify communitymembers and that with the exception of the lone Acinetobac-ter isolate (Site B), most of these CFUs were observed off trail(there were 7 Arthrobacter CFUs found on trail, but 28 wereobserved off trail) (Barton et al. 2004; Barton et al. 2007).

In regard to the fungal CFUs identified in 2004, membersof the family Trichocomaceae (genera include Penicillium andAspergillus) dominated at the Lunch Room sites. Isolates re-covered from the Lunch Room were primarily Penicillium orAspergillus spp. (76.9% of isolates), and Aspergillus isolateswere only found at the Lunch Room site. Penicillium and As-pergillus spp. are common bread molds and have been wellknown as such since the latter part of the 19th century (Carllet al. 1954).

Although these two genera have been identified as breadmolds, they have been identified in previous studies conductedin other cave systems such as Lechuguilla Cave that hadonly recently been opened for exploration (Northup et al.1994). Aspergillus and Penicillium spores were the mostprevalent observed (50% annual mean) within the Cave ofNerja (Southern Spain) in a four-year study identifying the

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temporal distribution of spore types (Docampo et al. 2011).The highest peaks (3 × 105 spores m−3) of these generaoccurred during periods when the cave was being used for afestival (Docampo et al. 2011).

Although the data indicated that other genera observedwithin the Nerja cave system originated from outside the cave,this observation was not noted with Aspergillus and Penicil-lium spores (Docampo et al. 2011). It is possible that thesegenera were conveyed into that cave system by humans andwere sustained by nonindigenous nutrient sources like anthro-pogenic detritus. In a study conducted within the Cave of Cas-tanar de Ibor (Western Spain), researchers noted Penicilliumspores in visited sections of the cave, but not in nonvisitedsections and furthermore that common genera such as Cla-dosporium were not part of the cave’s interior microbiota dueto the use of a double-door lock system to limit the transportof exterior microbiota into the system (Porca et al. 2011).

Within the Cave of Ardales (Spain), spore concentrationswere ∼100 times higher during visitor hours versus whenthe system was closed to visitation (Porca et al., 2011). Inour study at Sites A and B, which are the two sites at andjust within the Natural Entrance, Alternaria spp. were themost prevalent fungal CFUs. The two CFU of Cladosporiumsp. observed in this study were only found at Site A. BothAlternaria and Cladosporium are genera that are ubiquitous innature and are commonly observed in aeromicrobiology stud-ies (Griffin, 2007). It was interesting to note that Hypocrealeswere the only CFUs recovered at Site I (10 isolates) and wereonly recovered at this site. Hypocreales has previously beenobserved in cave studies (Matocec and Ozimec 2001).

As a whole the fungal CFU data demonstrate the pres-ence of ubiquitous fungal genera at the caves entrance anda decrease of CFU with progression into the cave system.The exception was the lunchroom area, the only site wheregenus Aspergillus was observed. These data indicate that theobserved fungal occurrence in the lunchroom area is due tohuman presence (congregation) and activity (consumption)as apposed to bat-related, where one would expect that sim-ilar fungal genera and concentrations should be recoverablealong their flight path (through the lunchroom and up to theentrance of the cave).

One of the most interesting results from the culture-independent work conducted in 2009 was the dominance ofEnterobacteriaceae sequences at both Lunch Room A andSite E (the paved visitor descent-trail site located near audiostation #14) at 59.0% and 62.7%, respectively. Although col-iform CFU presence had previously been observed and usedas a marker of human visitation in a number of other caves(Lechuguilla and Mammoth), their dominance in culturablespecies or in culture-independent work has not been previ-ously observed (Hunter et al. 2004; Lavoie and Northup 2005;Northup et al. 1997).

The Enterobacteriaceae dominance at Lunch Room A andSite E may be due to a number of possibilities. First, theymay dominate due to bat residency as species of the generaEnterobacter and Hafnia have been identified in guano (Ade-siyun et al. 2009; Di Bella et al. 2003; Konieczna et al. 2007).Furthermore, the bats that roost near Lake of Clouds at the

end of Left Hand Tunnel fly through the Lunch Room, up theMain Corridor and out the Natural Entrance.

Second, they may be due to human visitation as previouslyhypothesized, but their presence or absence would have tobe verified in lightly trafficked areas of Carlsbad Cavern orneighboring cave systems (Lavoie and Northup 2005; Northupet al. 1997). Anecdotal evidence from park rangers suggeststhat some visitors urinate and defecate on visitor trails whentraffic is light. Third, their presence may be an artifact of theonce leaking sewage lines that existed directly above CarlsbadCavern (Burger and Pate 2001).

It has previously been reported that species of gut mi-crobiota once deposited in soil may adapt and proliferatein soil environments and in this case would have permeatedthroughout the system from the overlying point of inoculum(Fujioka et al. 1988; Hardina and Fujioka 1991). Althoughmost of the site members were similarly distributed betweenthe two sites, Cedecea (16 sequences) or Enterobacter wereonly seen at sites E and Lunch Room A, respectively. Gram-positive bacteria only accounted for 4.8% (the Streptococcus,Staphlyococcus, Propionibacterium, and Microbacteriaceae se-quences) and 4.4% (the Streptococcus, Propionibacterium, andFirmicutes sequences) of the sequences obtained from LunchRoom A and Site E, respectively.

Previous research in Carlsbad Cavern reported the presenceof bacterial genera observed in this 2009 data set (Acidovoraxspp., Comamonas spp., Janthinobacterium spp., Massilia spp.,and Pseudomonas spp.), but Enterobacteriaceae were not de-tected (Barton et al. 2007). Overall these data indicate similardiversity between prokaryote communities in the lunchroomand at a mid-Main Corridor site. Determining if the Enter-obacteriaceae are of animal (human, bats, rodents) origin orare an established native member in this particular cave sys-tem would require further evaluation of off-trail environments.Whether the Enterobacteriaceae are native or invasive in na-ture, there dominance in atmosphere at these two sites is curi-ous and novel.

The fungal sequence data for the 2009 study showed a sim-ilar distribution of members among the sites. Sequences be-longing to the genera Arthrinium, Candida, Peniophora, Phae-rochaete, and Pulcherricium were only observed at the LunchRoom A site. Sequences specific to Site E included those be-longing to the genera Chaetomium and Phacidium. Sequencesbelonging to Hypocreales were observed at both locations incontrast to the 2004 culture-based work in which members ofthis order were only observed at Site I. Sequences belonging tothe genera Cladosporium and Penicillium were also observedat both sites.

The direct-count data (Table 2) demonstrated that cultur-able bacteria represented less than 0.1% of the total cell countand that viruses were present at considerable concentrationsat each site. Another observation of this study is that the liq-uid impinger was superior for culture and molecular assaysin contrast to that observed with the membrane-filtration unit(Griffin et al. 2011). Unfortunately, the particular high-volumeimpinger utilized in this study was a bit less usable due to sizein tightly constricted areas of the cave than the breakdownmembrane-filtration unit.

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184 Griffin et al.

We recommend use of a high-volume impinger in thesetypes of studies if transport throughout the system permits.Another observation worthy of comment were particle-countspikes as individuals traversed our sample sites. Obviouslywalking creates considerable atmospheric turbulence. Thesepassing and trailing turbulences may serve to convey microor-ganisms throughout the cave system whether they are indige-nous or not and regardless of their point of origin. Movementalong the visitor trail may be an important mechanism for thepenetration and transport of nonindigenous microbiota intoand throughout the cave system.

In summary, the 2004 and 2005 culture-based data demon-strated that Staphylococcus spp. CFUs were the most prevalentmembers of the atmospheric community along the paved vis-itor trail as Knoellia sp. CFU dominated off the trail. Thesedata, coupled with the fungal-culture data in which the breadmolds Penicillium and Aspergillus were prevalent members inthe Lunch Room while ubiquitous outdoor members like Cla-dosporium and Alternaria were prevalent members near theNatural Entrance, indicate that humans do convey nonindige-nous microbiota into the system.

Further, the distinct prevalence of fungal concentrationsand genera in the lunchroom area indicates that consumption(generation of potential nutrient sources, i.e., crumbs) maybe a source for sustenance for foreign microbiota. The re-moval of food and the prohibition of consumption of crumb-generating-types of foods could be considered to protectthe health of the cave system. The 2009 nonculture-basedEnterobacteriaceae-prevalence data at both sites is interestingyet perplexing. Their presence may be a marker of the residentmacrobiota, or they may be naturally occurring microbiotain this particular or type of ancient skeletal reef environment.The alternative is that their presence is a marker of anthro-pogenic contamination. Future studies in more remote areasof this system or in nearby systems will be needed to shed lighton this issue.

Acknowledgments and Disclaimer

We would like to thank the following National Park personnel,Stan Allison, Paul Burger and Dale Pate, and others, JessicaSnider, Ara Kooser, Morgan Griffin, Jenny Hathaway, andSam Waddell, who provided invaluable guidance and help onthis project. Any use of trade names is for descriptive purposesonly and does not imply endorsement by the U.S. Government.

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