1 Title: Culture-Independent Characterization of Archaea Biodiversity in Swine 1 Confinement ...

28 Pages·2009·1.09 MB·English

AEM Accepts, published online ahead of print on 26 June 2009
Appl. Environ. Microbiol. doi:10.1128/AEM.00726-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Title: Culture-Independent Characterization of Archaea Biodiversity in Swine
2 Confinement Buildings Bioaerosols
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4 Running title: Airborne archaea in swine confinement buildings
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6 Authors: Benjamin Nehmé1,2,‡ , Yan Gilbert1,2, ‡, Valérie Létourneau1,2, Robert J. nlo
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7 Forster3, Marc Veillette1, Richard Villemur4, and Caroline Duchaine1,* d
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8 Authors’ affiliation: fr
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9 1 Centre de Recherche, Institut Universitaire de Cardiologie et de Pneumologie de h
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10 Québec, Québec, QC, Canada :/
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11 2 Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, m
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12 Université Laval, Québec, QC, Canada m
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13 3 Agriculture and Agri-Food Canada, Lethbridge, AB, Canada rg
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14 4 INRS-Institut Armand-Frappier, Laval, QC, Canada n

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15 Correspondent footnotes: Caroline Duchaine n
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16 Address: Centre de Recherche, Hôpital Laval y

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17 2725, Chemin Ste-Foy, ,
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18 Québec, QC, Canada, G1V 4G5
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19 Tel.: [phone] #5837
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20 Fax: [phone] s
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21 Email: [email protected]
22 Footnotes: ‡ B.N. and Y.G. contributed equally to this work.
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1 Abstract
2 It was previously demonstrated that microbial communities of pig manure were
3 composed of both bacteria and archaea. Recent studies have showed that bacteria were
4 aerosolized from pig manure, but none have ever focused on the airborne archaeal
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5 burden. This study aims to develop and apply molecular ecology approaches to
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6 thoroughly characterize airborne archaea from swine confinement buildings (SCB). Eight n
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7 swine operations were visited twice in winter and once during summer. IOM cassettes
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8 loaded with 25 mm gelatin filters were used to capture the inhalable microbial biomass. fr
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9 Total genomic DNA was extracted and used as a template for PCR amplification of the h
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10 archaeal 16S ribosomal RNA gene (rDNA). High concentrations of archaea were found :/
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11 in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. m
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12 Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were m
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13 closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of rg
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14 the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA n

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15 gene denaturing gradient gel electrophoresis (DGGE). This analysis showed similar n
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16 fingerprints in each SCB and confirmed the predominance of methanogenic archaea in y

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17 the bioaerosols. This study brings a new light on the nature of bioaerosols in SCB and ,
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18 suggests that archaea are also aerosolized from pig manure.
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1 Introduction
2 Over the last thirty years, swine production in Canada evolved from small size
3 family farms to industrial facilities. Pig producers have increased animal density, building
4 mechanization, and confinement in order to decrease working and feeding time and to
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5 optimize space, leading to an increased contamination of air by bioaerosols.
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6 Even though the SCB environment has been studied for several years, little is n
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7 known about the real concentration and nature of airborne microorganisms. Moreover,
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8 increasing confinement level in modern barns has raised bioaerosol levels, modifying the fr
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9 health risk of exposed workers. So far, using culture-dependent methods was the only h
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10 strategy developed and used to describe SCB bioaerosols content and levels (6, 7). :/
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11 However, it is well known that culture-independent approaches are more likely to reveal m
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12 the presence of microorganisms never suspected in most environments (2). In m
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13 aerobiology, there are only a few reports using culture-independent methods (4, 15). rg
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14 Nehme et al. (20) applied molecular approaches to quantify and describe the bacterial n

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15 aerosols in SCB, and reported as much as 108 bacteria per cubic meter of air, with n
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16 significantly higher concentrations during winter when the confinement is maximal. Data y

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17 obtained were also compared with recent biodiversity studies of swine manures (13, 22, ,
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18 25). Anaerobic Gram-positive bacteria, being the greater part of the microbiological
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19 aerosols, appeared to originate from the swine manure. Those manure biodiversity studies
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20 revealed the presence of methanogenic archaea in hog wastes (22, 25). Since bacteria s
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21 observed in the aerosols seem to originate from the manure, it is plausible that archaea
22 from pig slurries are also aerosolized.
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1 This paper reports the characterization of the archaeal community of SCB
2 bioaerosols by using cultivation-independent approaches. The phylogeny of airborne
3 archaeal was assessed using 16S rRNA gene sequences. Archaeal biodiversity profiles
4 were determined with PCR-DGGE, and the concentration of aerosolized archaea was
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5 evaluated by real-time PCR by quantifying archaeal 16S rRNA gene copies in the air
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6 samples. n
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8 Materials and Methods fr
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9 Sampling sites h
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10 Eight SCB from the Quebec City area (Eastern Canada) were visited on three :/
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11 occasions: twice in winter, and once in summer. Samples were all collected in m
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12 growing/finishing rooms, occupied by weight-market pigs (about 100 kg). Wastes were m
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13 collected as a liquid mixture in a pit located under a slatted floor (66% to 100% slatted). rg
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14 All pigs were fed with feeds from the COOP fédérée (Agricultural field, Montréal, QC, n

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15 Canada) or from on-farm mills. n
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17 Samples collection and preparation ,
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18 Airborne samples were collected over 4 h, in triplicate or quadruple, using IOM
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19 (Institute of Occupational Medicine) (SKC, Ancaster, ON, Canada) cassettes loaded with
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20 gelatin membranes 25 mm in diameter (SKC, Ancaster, ON, Canada). They were taken 1 s
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21 m above the floor, as far away as possible from doors, windows or other ventilation
22 sources. The IOM-gelatin membrane system was plugged in to a Gilair-5 pump (Levitt-
23 Sécurité Limitée, Dorval, QC, Canada) calibrated at 2 L min-1 using a DryCal 2 Flow
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1 meter (SKC, Ancaster, ON, Canada). After sampling, cassettes were put on ice and
2 brought to the laboratory. Replicate gelatin membranes were pooled in 0.9% NaCl
3 solution (5 mL/membrane) and dissolved by vortexing at maximum speed for 30 min at
4 room temperature with a Multi-Pulse Vortexer (Glas-Col®, Terre Haute, USA). The
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5 resulting solution was aliquoted to obtain an equivalent of 144 L of air in each tube, and
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6 centrifuged (10 min, 21 000 × g, room temperature). Pellets were kept at -20°C until their n
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7 use for total DNA extraction.
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8 fr
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9 Total DNA extraction h
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10 Isolation of total genomic DNA from pellets was performed using the QIAamp :/
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11 DNA Mini Kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s m
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12 instructions for tissue with the required modifications for bacteria. Total DNA was eluted m
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13 in 200 µl of DNAse/ RNAse-free water (Sigma, Oakville, ON, Canada). rg
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14 n

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15 Quantitative real-time PCR for total archaea n
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16 PCR amplifications were carried out with the DNA Engine Opticon 2 (Bio-Rad, y

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17 Mississauga, ON, Canada). Quantification of archaea was performed using ARC787F and ,
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18 ARC1059R primers and the TaqMan probe ARC915F (Table 1). The components for the
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19 16S rRNA gene quantification assay per 25 µl were as follows: 1 to 100 ng of DNA
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20 template, 0.5 µM of each primer, 0.15 µM of TaqMan probe, and 12.5 µl of 2× s
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21 QuantiTech Probe PCR kit (Qiagen, Mississauga, ON, Canada). The PCR program to
22 amplify a 272 bp product was as follows: one hold at 37°C for 10 min and one hold at
23 95°C for 15 min, then 45 cycles of 95°C for 15 s and 60°C for 60 s. A standard curve was
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1 built using serial dilutions of Methanosarcina mazei (ATCC BAA-159D) 16S rRNA gene
2 cloned in a pCR®4-TOPO® plasmid (Invitrogen, Carlsbad, CA, USA). Polymerase chain
3 reaction efficiencies and detection sensitivities of the Methanosarcina mazei 16S rRNA
4 gene preparation and the environmental samples were performed by serial 10-fold
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5 dilutions. Data were acquired with the computer software Opticon Monitor (version
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6 2.02.24; Bio-Rad, Mississauga, ON, Canada), and analyzed by linear regression of the n
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7 log (target copy number) = f (Threshold Cycle) function. PCR efficiencies were
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8 determined as follow: E = 10(–slope)-1. Negative controls and field blanks were also added fr
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9 to each PCR run to evaluate the PCR reagents contamination. h
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10 :/
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11 Denaturing gradient gel electrophoresis (DGGE) m
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12 A nested PCR was used to amplify the variable V3 region of 16S rRNA gene m
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13 sequences from archaea. A first step consisted of using Ar3F and 1492R primers to rg
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14 amplify the archaea community followed by an amplification of diluted PCR products n

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15 using PARCH340-GC and 519r primers (Table 1). For both steps, the components for the n
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16 16S rRNA gene amplification per 25 µl were: 1 to 100 ng of DNA template, 0.25 µM of y

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17 each primer, 1.5 mM MgCl2, 2% DMSO, 0.2 mM dNTP, 1× TaKaRa PCR buffer (10 , 2
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18 mM Tris-HCl pH 8.3, 50 mM KCl), 1.25 U TaKaRa Taq polymerase (Fisher Scientific,
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19 Ottawa, ON, Canada). PCR was performed with a DNA Engine DYADTM thermocycler y
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20 (Bio-Rad, Mississauga, ON, Canada). The first step of the nested PCR protocol was: 5 s
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21 min at 95°C, then 30 cycles of 60 s at 95°C, 60 s at 55°C, 60 s at 72°C and a final
22 extension step of 5 min at 72°C. The second step protocol was: 5 min at 95°C, then 30
23 cycles of 60 s at 95°C, 60 s at 53.5°C, 120 s at 72°C and a final extension step of 5 min at
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1 72°C. After gel electrophoresis [1.5% (wt/vol) agarose gel] of 5 µl subsamples of the
2 PCR products, the amount of amplified DNA was quantified by comparing band
3 intensities to standard curves obtained with an EZ Load Precision Molecular Mass Ruler
4 (Bio-Rad, Mississauga, ON, Canada). Band intensities were measured with GeneTools
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5 analysis software (SynGen, Cambridge, England).
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6 Profiles of the amplified 16S rRNA gene sequences were produced by DGGE as n
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7 described by Muyzer et al. (18) using the DCode apparatus (Bio-Rad, Mississauga, ON, d
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8 Canada). The PCR products were loaded (60 ng) onto an 8% polyacrylamide gel in 0.5× fr
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9 TAE buffer (20 mM Tris pH 8.0, 10 mM acetic acid, 0.5 mM EDTA) (Bio-Rad, h
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10 Mississauga, ON, Canada) with a denaturant gradient [100% denaturant was 7 M urea :/
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11 and 40% (v/v) deionized formamide]. The electrophoresis was carried out in 0.5× TAE m
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12 buffer at 60 V for 16 h at 60°C. The DNA fragments were stained for 15 min in 0.5× m
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13 TAE buffer with SYBR Gold (Molecular Probes, Eugene, OR, USA). The gels were rg
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14 washed in distilled water for 15 min. Images of the gels were obtained using an imaging n

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15 system ChemiGenius 2 (SynGen, Cambridge, England) and the imaging software n
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16 GeneSnap (SynGen, Cambridge, England). y

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17 DNA bands from the polyacrylamide gels were excised and incubated in 40 µl of ,
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18 sterile DNAse/RNAse-free water (Sigma, Oakville, ON, Canada) overnight at 4°C. Five
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19 microlitres of the DNA extract were used as the template in a PCR amplification with the
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20 PARCH340 (no GC clamp) and 519r primers as described above. The PCR products s
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21 were cloned with the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA). Three
22 clones from each DGGE band were randomly chosen, and digested with restriction
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1 enzymes HaeIII, HhaI or MspI (New England Biolabs, Pickering, ON, Canada) in order
2 to confirm the purity of the re-amplified DNA from the DGGE bands.
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4 16S rRNA gene libraries construction
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5 PCR runs were carried out on samples collected during winter 1, from barns D, G
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6 and H, with the Ar3F and 1492R primers as described in the previous section. Nested n
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7 PCR was then performed using primers Ar3F and Ar9R, corresponding to nucleotide 7–
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8 927 in Escherichia coli (Table 1). The PCR mixture was the same as above and the PCR fr
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9 conditions were: one hold at 95°C for 5 min, then 30 cycles at 95°C for 60 s, 55°C for 60 h
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10 s, 72°C for 60 s, followed by a final extension step of 5 min at 72°C. The PCR products :/
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11 were cloned into E. coli using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA) m
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12 to generate 16S rRNA gene libraries. Approximately 200 clones were randomly picked m
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13 from each library and grown overnight in LB medium with 100 mg ml-1 ampicillin rg
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14 (Sigma, Mississauga, ON, Canada) at 37°C. The insert from each clone was directly n

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15 amplified from E. coli culture using the M13 primers (TOPO-TA cloning kit). The final n
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16 PCR products were then digested with restriction enzymes HaeIII and HhaI, according to y

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17 the manufacturer’s specifications, and separated on a 2% agarose gel that was ,
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18 electrophoresed at 100V for at least 30 min. Restriction fragment length polymorphisms
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19 were grouped according to their riboprint patterns. The PCR products of all different
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20 riboprint patterns were sequenced on both strands (Plateforme de séquençage et de s
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21 génotypage, CHUL Research Center, Québec, QC, Canada). When two or more clones
22 had the same riboprint pattern, up to 10 clones were sequenced for confirmation.
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1 Phylogenetic analysis
2 Each cloned DNA sequence was compared with sequences available in databases,
3 using BLASTN (1) from the National Center of Biotechnology Information
4 (http://www.ncbi.nlm.nih.gov/BLAST/). Potential DNA chimerical structures were
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5 searched with the CHECK_CHIMERA program (RDP-II) (5). Then all the sequences and
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6 their closest relatives were aligned using the ClustalW software (23). The sequences were n
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7 screened for manual alignment correction with ARB package (14). Finally, the
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8 phylogenetic tree was constructed with ARB with the maximum likelihood method, using fr
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9 pairwise gap removal and a conservation filter of 50%. h
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10 :/
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11 Statistical analysis m
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12 The statistical method used to perform comparisons of airborne archaea m
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13 concentrations between summer and winter was a one-way ANOVA. Relationships rg
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14 between parameters were expressed using the Pearson’s correlation coefficient as the n

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15 linearity between these observed parameters and statistically analyzed to determine if it n
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16 was significant using a T-test. The results were considered significant if p-value was ≤ ry

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17 0.05. The data were analyzed using the statistical package program SAS (SAS Institute, ,
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18 Cary, NC, USA).
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20 Nucleotide sequence accession numbers s
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21 The nucleotide sequence data reported in this paper appears in GenBank
22 nucleotide sequence database under the accession numbers FJ001234 to FJ001326.
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1 Results
2 Total archaeal counts in the SCB
3 The primer set ARC787F-ARC1059R and the probe ARC915F were successfully
4 used to quantify the archaeal community in the air (Efficiency = 88.8%, R2 = 0.996), with
5 a standard curve being linear from 101 to 106 genes copy per reaction. The quantitative D
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6 PCR results expressed in 16S rRNA gene copy number were considered representative of n
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7 the airborne archaeal community concentrations. No PCR inhibition effect was observed
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8 in diluted reactions of the environmental samples (data not shown). fr
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9 The real-time PCR counts revealed a high 16S rRNA gene copy number of h
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10 archaea in the air, corresponding to 106 to 108 16S rRNA gene copies m-3 air. There was :/
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11 no significant difference between the two winter visits for the concentration of archaea in m
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12 the air (Fig. 1A). However, there was a significant decrease of 16S rRNA gene copy m
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13 number of archaea during the summer period (3.5-fold less than in winter, P < 0.01) (Fig. rg
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14 1B). n

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16 DGGE profiles analysis y

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17 Diversity within the SCB archaeal bioaerosol populations was determined by 16S ,
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18 rRNA gene-targeted PCR-DGGE analysis. A denaturing gradient 20-50% (Fig. 2) was 9

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19 performed on each sample.
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20 Six different bands were observed among all DGGE profiles. The presence of two s
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21 similarly migrating bands (bands 1 & 2) suggests that some species were present in all
22 samples. Even though each sampled SCB aerosol had a similar DGGE profile, some
23 slight differences were observed since a number of DNA bands were not always detected
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