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Whole Genome Sequencing and Function Prediction of 133 Gut Medvecky et al. BMC Genomics (2018) 19:561 https://doi.org/10.1186/s12864-018-4959-4 RESEARCH ARTICLE Open Access Whole genome sequencing and function prediction of 133 gut anaerobes isolated from chicken caecum in pure cultures Matej Medvecky1, Darina Cejkova1, Ondrej Polansky1, Daniela Karasova1, Tereza Kubasova1, Alois Cizek2,3 and Ivan Rychlik1* Abstract Background: In order to start to understand the function of individual members of gut microbiota, we cultured, sequenced and analysed bacterial anaerobes from chicken caecum. Results: Altogether 204 isolates from chicken caecum were obtained in pure cultures using Wilkins-Chalgren anaerobe agar and anaerobic growth conditions. Genomes of all the isolates were determined using the NextSeq platform and subjected to bioinformatic analysis. Among 204 sequenced isolates we identified 133 different strains belonging to seven different phyla - Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes. Genome sizes ranged from 1.51 Mb in Elusimicrobium minutum to 6.70 Mb in Bacteroides ovatus. Clustering based on the presence of protein coding genes showed that isolates from phyla Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes did not cluster with the remaining isolates. Firmicutes split into families Lactobacillaceae, Enterococcaceae, Veillonellaceae and order Clostridiales from which the Clostridium perfringens isolates formed a distinct sub-cluster. All Bacteroidetes isolates formed a separate cluster showing similar genetic composition in all isolates but distinct from the rest of the gut anaerobes. The majority of Actinobacteria clustered closely together except for the representatives of genus Gordonibacter showing that the genome of this genus differs from the rest of Actinobacteria sequenced in this study. Representatives of Bacteroidetes commonly encoded proteins (collagenase, hemagglutinin, hemolysin, hyaluronidase, heparinases, chondroitinase, mucin- desulfating sulfatase or glutamate decarboxylase) that may enable them to interact with their host. Aerotolerance was recorded in Akkermansia and Cloacibacillus and was also common among representatives of Bacteroidetes.On the other hand, Elusimicrobium and the majority of Clostridiales were highly sensitive to air exposure despite their potential for spore formation. Conclusions: Major gut microbiota members utilise different strategies for gut colonisation. High oxygen sensitivity of Firmicutes may explain their commonly reported decrease after oxidative burst during gut inflammation. Keywords: Microbiota, Microbiome, Chicken, Whole genome sequencing, Metabolic pathway, Host-microbiota interactions, Anaerobe, Butyrate, Propionate * Correspondence: [email protected] 1Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic Full list of author information is available at the end of the article © The Author(s). 2018 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. Medvecky et al. BMC Genomics (2018) 19:561 Page 2 of 15 Background expressed by the main microbiota members [7]and Characterisation of gut microbiota is nowadays relatively verified that gut microbiota may protect chickens against simple due to recent advances in next generation se- Salmonella Enteritidis infection [9]. However, which quencing. However, though DNA sequencing is useful bacterial species are the protective ones is not known and for monitoring dynamic changes in microbiota compos- more detailed knowledge of individual microbiota mem- ition, it does not enable the understanding of biological bers is needed. One way forward is to obtain functions of individual microbiota members. Shotgun well-characterised pure cultures of gut anaerobes [11]. In sequencing of total DNA or RNA/cDNA sequencing can this study we therefore cultured hundreds of isolates of partly indicate the metabolic potential of microbial com- anaerobes from chicken caecum and sequenced more munities but is limited in addressing biological functions than 200 of them. Analysis of their genomic sequences of individual microbiota members [1–3]. Even in the showed that we collected isolates from 7 different phyla. cases of analysis of microbial communities with low The aims of the subsequent comparisons focused mainly complexity when long DNA contigs can be assembled on the representatives from phyla Bacteroidetes and and associated with a particular bacterium, such Firmicutes was to reveal differences in (poly)saccharide approaches do not allow for subsequent experimental utilisation, propionate and butyrate fermentation and verification of observed data due to the unavailability of interactions with the host including motility or the ability pure bacterial cultures. Isolation of gut anaerobes in to degrade host derived proteins. pure cultures is therefore the best way to examine the characteristics of individual microbiota members experi- Results mentally [4]. Altogether 204 isolates were obtained in pure culture Isolation of bacterial gut microbiota members in pure and sequenced. Since in several cases we sequenced cultures is usually an issue since the majority of bacterial isolates exhibiting higher than 99% sequence similarity isolates colonising the intestinal tract are strict anaer- between their genomes, the final number of different obes. Although it is commonly reported that between 10 isolates decreased to 133 (Table 1, Fig. 1). The lowest se- and 50% of bacterial species colonising the intestinal quencing coverage was 43 fold for Drancourtella massi- tract can be grown in vitro, recent studies proposed that liensis An12, the highest 1526 fold for Lactobacillus up to 90% of major gut colonisers may be grown in vitro gasseri An197, and median coverage over all sequenced if multiple culture conditions are tested [5, 6]. Despite isolates was 312 fold (see Additional file 1 for all details). this, the isolation of a particular gut anaerobe may re- The isolates belonged to 7 different phyla – Firmicutes main an issue since even the most abundant microbiota (84 isolates), Bacteroidetes (29 isolates), Actinobacteria members at species level only rarely form more than 1% (15 isolates), Proteobacteria (1 isolate each of Escherichia of the total population [7, 8]. This means that the and Desulfovibrio), Verrucomicrobia (1 isolate of Akker- desired bacterial species may be represented by a single mansia), Elusimicrobia (1 isolate of Elusimicrobium) and colony growing on an agar plate together with hundreds Synergistetes (1 isolate of Cloacibacillus). The similarity or thousands of others and the likelihood of picking up of whole sequences of 16S rRNA to the to the most the particular species is indeed rather low [6]. homologous GenBank entries was lower than 94% for 15 Chickens represent a specific case for studies focused isolates. Considering taxonomic recommendations [12], on host - microbiota interactions. Chickens in commer- these isolates represent candidates for new genera and cial production are hatched in a clean environment of two Muribaculum-like isolates might belong to a novel hatcheries without any contact with adult hens and their bacterial family or even an order. Alternative analysis colonisation is dependent on environmental sources. based on alignment of RpoB amino acid sequences [13] Moreover, newly hatched chickens can be colonised by yielded similar clustering of individual isolates as that microbiota of wide a range of compositions [7] and col- achieved by 16S rRNA comparison (Additional file 2). onisation of the intestinal tract of commercially hatched When we compared the sequence of 16S rRNA genes chickens may therefore differ from the colonisation of of all 133 isolates with the operational taxonomic units intestinal tract of chickens which would hatch in a nest. (OTUs) combined from our previous studies [7, 8], ra- Perhaps not surprisingly, commercially hatched chickens ther unexpectedly, 7 isolates were excluded from the are highly sensitive to colonisation with different patho- analysis by QIIME at the chimera removal step. Three gens, e.g. Salmonella, nevertheless, their resistance can isolates formed OTUs which we did not detect among be increased by providing them with a complex micro- the microbiota of the two studies. The rest of the isolates biota of adult hens [9, 10]. were assigned into particular OTUs. Nineteen isolates In our recent studies, we characterised the development were assigned to 11 OTUs from the 100 most frequent of gut microbiota in commercially hatched chickens OTUs, and out of the 1000 most common OTUs, we throughout their whole life [8], identified proteins obtained 76 isolates belonging to 42 OTUs. Exact Medvecky et al. BMC Genomics (2018) 19:561 Page 3 of 15 Table 1 List of strains isolated, sequenced and analysed in this study Phylum Family Species ID rRNA % sim.a genome (bp) GC (%) Actinobacteria Coriobacteriaceae Collinsella intestinalis An268 94 2,375,164 65.28 Actinobacteria Coriobacteriaceae Collinsella intestinalis
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