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Comparing Microbiotas in the Upper Aerodigestive and Lower Respiratory Tracts of Lambs Laura Glendinning1* , David Collie1, Steven Wright1, Kenny M

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Comparing Microbiotas in the Upper Aerodigestive and Lower Respiratory Tracts of Lambs Laura Glendinning1* , David Collie1, Steven Wright1, Kenny M Glendinning et al. Microbiome (2017) 5:145 DOI 10.1186/s40168-017-0364-5 RESEARCH Open Access Comparing microbiotas in the upper aerodigestive and lower respiratory tracts of lambs Laura Glendinning1* , David Collie1, Steven Wright1, Kenny M. D. Rutherford2 and Gerry McLachlan1 Abstract Background: Recently, the importance of the lung microbiota during health and disease has been examined in humans and in small animal models. Whilst sheep have been proposed as an appropriate large animal model for studying the pathophysiology of a number of important human respiratory diseases, it is clearly important to continually define the limits of agreement between these systems as new concepts emerge. In humans, it has recently been established that the lung microbiota is seeded by microbes from the oral cavity. We sought to determine whether the same was true in sheep. Results: We took lung fluid and upper aerodigestive tract (oropharyngeal) swab samples from 40 lambs (7 weeks old). DNA extraction was performed, and the V2-V3 region of the 16S rRNA gene was amplified by PCR then sequenced via Illumina Miseq. Oropharyngeal swabs were either dominated by bacteria commonly associated with the rumen or by bacteria commonly associated with the upper aerodigestive tract. Lung microbiota samples did not resemble either the upper aerodigestive tract samples or reagent-only controls. Some rumen-associated bacteria were found in lung fluids, indicating that inhalation of ruminal bacteria does occur. We also identified several bacteria which were significantly more abundant in lung fluids than in the upper aerodigestive tract swabs, the most predominant of which was classified as Staphylococcus equorum. Conclusions: In contrast to humans, we found that the lung microbiota of lambs is dissimilar to that of the upper aerodigestive tract, and we suggest that this may be related to physiological and anatomical differences between sheep and humans. Understanding the comparative physiology and anatomy underlying differences in lung microbiota between species will provide a foundation upon which to interpret changes associated with disease and/or environment. Keywords: Lung, Microbiota, Sheep, Lambs, Oropharynx, Rumen, 16S Background achieve this, the majority of previous studies have relied The use of 16S ribosomal RNA (rRNA) gene sequencing on human volunteers. has facilitated the study of difficult to culture, low However, many individuals are hesitant to participate biomass microbial communities present in the lower re- in research bronchoscopy due to the perceived incon- spiratory tract. The impact of the lung microbiota on venience and a fear of complications [1], despite the low human health is a rapidly growing area of research. In risk involved. Mice and rats have been used to explore order to understand this impact, it is important to also the relationship between the lung microbiota and airway understand the lung microbiota dynamics during health inflammation [2–4], microbiota at different body sites and to include healthy controls in disease studies. To [5], the environment [6], acute lung injury [7] and antibiotic [8] and corticosteroid exposure [9]. However, rodents are of limited use when exploring spatial or lon- * Correspondence: [email protected] gitudinal lung microbiota dynamics due to their small 1The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, Midlothian EH25 9RG, UK lung size. Recognising the utility of large animal models Full list of author information is available at the end of the article in this regard, and the anatomical and immunological © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Glendinning et al. Microbiome (2017) 5:145 Page 2 of 9 relevance of sheep as models [10–13], our group has (PBS) was injected through the seared section into the previously used this species to explore the changes in tracheal lumen. The head and neck were oriented such the lung microbiota upon Pseudomonas aeruginosa in- that the PBS would flow caudally down the thorax. A fection [14] and to explore the spatial variability present second clamp was immediately placed caudal to the site within the healthy lung [15]. of injection to prevent backflow, leakage and potential Subclinical microaspiration of pharyngeal secretions is contamination. The lamb cadavers were then tipped so a feature of health and this can contribute to the lung that the PBS would run caudally into their lungs and microbiota composition [16], and the microbiome of the then tipped back again so that the fluid would collect in human lungs more closely resembles that of the mouth the tracheal lumen immediately caudal to the position of than that of the nose or the lower gastrointestinal tract the second clamp. A sampling site identified on the [17]. It is not yet known whether the same relationship ventral surface of the trachea was seared, and a needle holds for species other than humans. In ruminating and syringe were used to collect the pooled fluid. On sheep, where the oropharynx is exposed to ruminal con- average, 4 ± 1.7 ml (mean ± SD) of lung fluid was tents on a frequent basis, one would anticipate that lung collected per animal. Lung fluid was stored on ice until microbiota would similarly reflect this influence. In this further processing. Oropharyngeal swabs were sterilely paper, we find that the presence of rumen-like bacteria cut into 500 μl PBS. Lung fluids were centrifuged at in the upper aerodigestive tract is correlated with 13,000g for 5 min. The supernatant was removed, and changes in the lung microbiota and rumen-type bacteria the pellets were resuspended in 500 μl PBS. The oropha- are present in lamb lungs. We also identify bacteria ryngeal swabs and lung fluids were stored at −80 °C until which are more indicative of the lungs than the orophar- DNA extraction. ynx, indicating that the presence of the sheep lung microbiota is not merely due to passive diffusion of DNA extraction, amplification and sequencing microbes from the upper aerodigestive tract. DNA extractions using the PowerSoil DNA isolation Kit (Mo Bio, Carlsbad, USA) and quantitative PCR (qPCR) Methods using the 16S rRNA gene qPCR primers UniF340 (5- Animals and sampling ACTCCTACGGGAGGCAGCAGT-3) and UniR514 (5- Scottish Mule X Suffolk lambs (20 males and 20 females, ATTACCGCGGCTGCTGGC-3) were performed as de- unweaned), raised on pasture from 48 h after birth, were scribed previously [15]. Extraction kit reagent controls, used in this study. These lambs were part of a study on consisting of reagent-only extractions, were produced the animal welfare implications of prenatal stress which for every day DNA extractions were performed. PBS was approved by Scotland’s Rural College’s (SRUC) Ani- controls were created by extracting DNA from 500 μlof mal Experiments Committee and was conducted under the PBS which had also been used to wash out the lamb Home Office licence. All lambs were raised by their lungs. A mock community control was included which dams, and prior to euthanasia, their only food sources has been described previously [15]. were ewe’s milk and pasture. At 7 weeks old (mean A nested PCR reaction was used to produce amplicons age = 48.8 days ± 0.8 standard deviation (SD); mean for sequencing; this technique was chosen to reduce PCR weight ± SD = 20.6 kg ± 2.6 kg), the lambs were eutha- bias caused by barcoded primers [18]. The first round of nised by barbiturate overdose then the cadavers were PCR amplified the V1-V4 16S hypervariable regions using transported from the farm to the dissecting room (~ the primers 28F (5-GAGTTTGATCNTGGCTCAG-3) 5 min). Oropharyngeal swabs were taken using cotton- and 805R (5-GACTACCAGGGTATCTAATC-3). The tipped swabs (Swab Plain Wood Cotton Tip Sterile (710- conditions were 94 °C for 2 min followed by 20 cycles of 0181), Copan, Italy). To prevent oral contamination, the 94 °C for 1 min, 55 °C for 45 s and 72 °C for 1.5 min swabs were stored in protective plastic sheaths from followed by a final extension step of 72 °C for 20 min. which the swab could be advanced and retracted once it Clean-up was performed using the AMPure XP PCR puri- was positioned at the sampling site. Swabs were then fication system (Beckman Coulter, Brea, USA). transferred into a new plastic sheath and stored on ice. In a previous study, we found that PCR bias in high The ventral aspect of the neck was shaved, and a template samples could be reduced by diluting amplicons sterile scalpel used to incise through the skin and sub- from the first round of PCR to a similar concentration to cutaneous tissues to expose the ventral surface of the those of lung fluid samples [15]. Therefore, in this study, trachea. A sampling site was identified on the exposed we used our qPCR values to calculate the dilutions needed ventral surface, and the trachea cranial to this site was to achieve this. The second round of PCR used the bar- completely closed off by both string ligature and clamp coded V2-V3 primers 104F (5-GGCGVACGGGTGAG- placement. The selected sampling site was then heat TAA-3) and 519R (5-GTNTTACNGCGGCKGCTG-3). seared, and 50 ml of sterile phosphate-buffered saline The dilutions and barcoded primers used for each sample Glendinning et al. Microbiome (2017) 5:145 Page 3 of 9 can be found in Additional file 1.
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