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Diet Is a Major Factor Governing the Fecal Butyrate-Producing Community Structure Across Mammalia, Aves and Reptilia

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Diet Is a Major Factor Governing the Fecal Butyrate-Producing Community Structure Across Mammalia, Aves and Reptilia The ISME Journal (2015) 9, 832–843 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Diet is a major factor governing the fecal butyrate-producing community structure across Mammalia, Aves and Reptilia Marius Vital1, Jiarong Gao1, Mike Rizzo1, Tara Harrison2 and James M Tiedje1 1Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA and 2Potter Park Zoo, Lansing, MI, and College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA Butyrate-producing bacteria have an important role in maintaining host health. They are well studied in human and medically associated animal models; however, much less is known for other Vertebrata. We investigated the butyrate-producing community in hindgut-fermenting Mammalia (n ¼ 38), Aves (n ¼ 8) and Reptilia (n ¼ 8) using a gene-targeted pyrosequencing approach of the terminal genes of the main butyrate-synthesis pathways, namely butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). Most animals exhibit high gene abundances, and clear diet-specific signatures were detected with but genes significantly enriched in omnivores and herbivores compared with carnivores. But dominated the butyrate-producing community in these two groups, whereas buk was more abundant in many carnivorous animals. Clustering of protein sequences (5% cutoff) of the combined communities (but and buk) placed carnivores apart from other diet groups, except for noncarnivorous Carnivora, which clustered together with carnivores. The majority of clusters (but: 5141 and buk: 2924) did not show close relation to any reference sequences from public databases (identity o90%) demonstrating a large ‘unknown diversity’. Each diet group had abundant signature taxa, where buk genes linked to Clostridium perfringens dominated in carnivores and but genes associated with Ruminococcaceae bacterium D16 were specific for herbivores and omnivores. Whereas 16S rRNA gene analysis showed similar overall patterns, it was unable to reveal communities at the same depth and resolution as the functional gene-targeted approach. This study demonstrates that butyrate producers are abundant across vertebrates exhibiting great functional redundancy and that diet is the primary determinant governing the composition of the butyrate-producing guild. The ISME Journal (2015) 9, 832–843; doi:10.1038/ismej.2014.179; published online 24 October 2014 Introduction sequences, Ley et al. (2008) found an overall relationship in mammals between the structure of Many studies investigating host–microbiota interac- the fecal microbiota, diet and host phylogeny. tions have demonstrated that the intestinal micro- Whereas studies based on traditional bacterial biota has a critical role in maintaining gut taxonomic markers can provide first insights into homeostasis and host health. A mutualistic relation- host and microbiota inter-relationships, functional ship between the host and its microbiota exists, investigations are essential in order to understand where bacteria provide essential substrates such as the key processes. Several studies focused on vitamins or short-chain fatty acids, whereas the host microbial functions for specific animals (c.f. delivers nutrients and a stable growth environment Lavery et al., 2012; Tun et al., 2012), and a global (Nicholson et al., 2012). Most investigations have metagenomic screen across Mammalia demon- been performed with humans and a few model strated that the bacterial functional repertoire is organisms (mouse, rat and pig), but much less is primarily governed by diet and does not reflect host known for other mammals or distinct vertebrate phylogeny (Muegge et al., 2011). That study classes. In a pioneer study using 16S rRNA gene revealed that the microbiota in carnivores and herbivores are characterized by different trade-offs in amino-acid pathways and by an opposite direc- Correspondence: JM Tiedje, Center for Microbial Ecology, Michi- tionality in the central phosphoenolpyruvate-pyru- gan State University, 540 Plant and Soil Sciences Building, East vate-oxaloacetate cycle. Similar differences were Lansing, MI 48823, USA. E-mail: [email protected] observed in human subjects who were exposed to Received 10 March 2014; revised 22 August 2014; accepted 3 diets mirroring those of carnivores or herbivores September 2014; published online 24 October 2014 demonstrating that broad functional differences can The butyrate-producing community in vertebrates M Vital et al 833 be achieved with distinct nourishments even in the diet-related signals. All samples were collected same host indicating that direct host-specific selec- within 24 h after defecation and were stored at tion processes are limited (at least in humans) À 80 1C. Animals were categorized into three distinct (David et al., 2013). diet groups based on feeding protocols provided by The divergence between overall bacterial function individual sources (Supplementary Table S2). Cate- and host phylogeny raises the question whether gorization into carnivores and herbivores was based specific physiological processes of the host that are on the animal’s predominant diet, and some taxa in linked to certain microbial functions, such as those groups were additionally fed minor amounts of components of the immune system or energy require- substances that were not strictly associated with the ments of the colonic epithelium, evolved with diet. If corresponding diet. Zoo animals were fed diets this is not the case and those processes are conserved researched to be appropriate for the taxon and used throughout hosts, or coevolved with certain phyloge- by most US zoos. DNA extraction was performed netic groups (for example, all mammals), we would using the PowerSoil kit (MoBio, Jefferson City, MO, expect functional redundancy of the microbiota that USA) according to the manufacturer. is adapted to the individual diets. In this context, the production of butyrate is of specific interest as it is considered essential for maintaining health in Amplification of functional genes humans and usual animal models, where it serves as Primers used were described previously (Vital et al., the main energy source for colonocytes, shows 2013). Amplification of DNA samples was achieved immunomodulatory effects and influences local with the AccuPrime PCR system (Life Technologies, gene expression (Hamer et al., 2008; Furusawa Grand Island, NY, USA), where 5 ng of template et al., 2013). Whereas butyrate is detected in feces DNA was combined with all individual forward and of various vertebrates (Stevens and Hume, 1998), reverse primers containing the same barcode (total indicating that it serves important roles throughout final concentration of 0.4 mM). The PCR protocol (35 this taxon, it is currently unknown how butyrate- cycles) was carried out according to the manufac- producing communities evolved with distinct diets. turer, with annealing temperatures and MgSO4 In particular, carnivores are interesting as butyrate concentrations of 51 1C, 3 mM and 48 1C, 2 mM for production is primarily linked to bacteria that feed butyryl-CoA:acetate CoA-transferase (but) and buty- on carbohydrates such as starch or xylan, converting rate kinase (buk), respectively. Harvesting of target pyruvate to butyrate using the ‘acetyl-CoA pathway’ bands and re-amplification are described in (Vital (Bennett and Rudolph, 1995). However, this path- et al., 2013). Sequencing was performed on the 454 way can also be fed by amino acids and additional FLX system at the Utah State University (Logan, UT, amino-acid-based butyrate-synthesis pathways are USA) using the Lib-A kit according to the manu- recognized as well (Buckel and Barker, 1974; Barker facturer (454 Life Sciences, Branford, CT, USA). et al., 1982; Gerhardt et al., 2000). The few existing studies specifically focusing on butyrate producers in different animal taxa demon- Functional genes’ data analysis strate that butyrate-producing communities are To estimate gene abundances of but and buk (as omnipresent; however, their composition can be percentage of total bacteria exhibiting the gene), considerably distinct from that of humans (c.f. target bands after the first PCR amplification were Eeckhaut et al., 2011; Levine et al., 2013). Detailed categorized into six distinct brightness groups investigations on these important functional com- (5: above 20%. 4: 10%–20%, 3: 4%–10%, 2: 1%–4%, munities across vertebrates are missing and it is 1: below 1% and 0: not detectable; category 1 was currently unknown how they have adapted to omitted for but gene analysis because of a higher distinct hosts. detection threshold compared with buk) based on standard curves. The following concentrations from reference organisms (but: mix of Roseburia inulini- Materials and methods vorans/Faecalibacterium prausnitzii and buk: Clos- tridium perfringens) were used to establish standard Samples and DNA extraction curves: 5 ng (100%), 2 ng (40%), 1 ng (20%), 0.5 ng An overview of all collected fecal samples is given (10%), 0.2 ng (4%), 0.1 ng (2%), 0.05 ng (1%), 0.02 ng in Table 1. The majority of samples were derived (0.4%) and 0.01 ng (0.2%). Band brightness was from Potter Park Zoo (Lansing, MI, USA), except for measured using the software imageJ (http://imagej. dogs (private), giant panda (Ningbo, China), guinea- nih.gov/ij/). Two independent experiments were pig, mouse, rat (commercial pet-store) and grizzly performed and average values were used as the bear (Detroit
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