Metagenomic investigation of the intestinal microbiome in healthy and diarrheic horses

1. Metagenomic investigation of the intestinal microbiome in healthy and diarrheic horses M. costa1, A. Sturgeon1, L. G. Arroyo2, H. R. STAEMPFLI2, J S. Weese1 1Dept. of Pathobiology, 2 Dept. of Clinical Studies, University of Guelph, Guelph, Ontario, Canada • Introduction • The intestinal tract contains one of the most dense, dynamic and complex bacterial populations (microbiomes) of any environment on the planet. It has been called the ‘2nd genome’ in testament to its size and complexity. In humans, it is believed that the intestinal microbiome contains up to 1000 different species and approximately 1012 bacteria/g. • The intestinal microbiome acts as a barrier to establishment and overgrowth of enteropathogens, interacts with the immune system, and plays a crucial role in digestion. It also participates in many other critical functions that are poorly understood. In horses, disruption of the intestinal microflora is thought to be associated with a wide range of problems, most notably colitis and laminitis. Yet, despite the clear importance of the intestinal microflora, our understanding of what constitutes ‘normal’ and ‘abnormal’ is woefully inadequate. Most investigations of the equine microflora have typically involved bacterial culture, which only allows for superficial assessment of parts of the cultivable component on the microflora, a significant limitation since a large component on the microflora is thought to consist of unknown or unculturable microorganisms. The development of culture-independent methods has led to a revolution in characterization of complex microbial populations. • The objectives of this study were to characterize the fecal microbiome in healthy horses and compare that to horses with diarrhea of varying etiologies. • Materials and Methods • Fecal samples were collected from 6 healthy and 10 diarrheic horses presented to the Ontario Veterinary College for treatment of idiopathic colitis. Healthy horses had no recent (90d) history of gastrointestinal disease, antibiotic treatment or probiotic treatment. • DNA was extracted and purified. 16s rRNA gene (V3-V5 region) PCR was performed, followed by next generation sequencing (Roche GS Junior 454 Sequencer). Standard quality control filters were applied. • Sequence data were uploaded to the MG-RAST server (http://metagenomics.anl.gov) for analysis using the SILVA Small Subunit rRNA database, with a maximum e-value of 1e-30, minimum identify of 97%, and minimum alignment length of 75 base pairs (bp) as cutoff values. A convenience selection of sequences were loaded into NCBI BLAST using the nucleotide collection (nr/nt) database to confirm sequence identity determinations. • Descriptive data were generated. Principal component analysis was performed. Comparisons between diarrheic and normal horses were performing by t-test or Mann-Whitney test, as appropriate. Relative abundance comparisons were based on normalized data. • Results • Six healthy and 10 diarrheic horses with idiopathic diarrhea were enrolled. The number of sequences that passed all quality control filters ranged from 6579 and 21600 per horse, 12901 + 6491 (mean + SD) in healthy horses and 11834 +8550 in diarrheic horses (P=0.63) (Table 1). • 99.5% of sequences from healthy horses and 99.7% from diarrheic horses were characterized to at least the Phylum level. The remaining sequences could indicate novel Phyla. • In healthy horses, the Firmicutes Phylum predominated, accounting from 49-73% (mean 65%, SD 12%) of sequences, while Bacteroidetes were most abundant in diarrheic horses (Figure 1). Diarrheic horses had significantly more Fusobacteria and Spirochaetes and fewer Actinobacteria (all P<0.0003). • 54-119 different Genera were identified in healthy horses, corresponding to 100-239 different species, compared to 38-104 Genera and 70-196 Species in diarrheic horses (P=0.14 and 0.08, respectively). • Numerous different Clostridium species were identified. In healthy horses, 16.3 + 4.4 species were present, compared to 11.5 + 5.6 in diarrheic horses (P=0.11). • Organisms typically associated with the oral microflora were common, such as Capnocytophaga spp, (5/6 normal and 6/10 diarrheic, at up to 3.9% of sequences) and Porphyromonasspp (5/6 normal and 10/10 diarrheic, up to 9.8% of sequences). • 31 different Lactobacillus spp were identified, but there was no difference in relative abundance between groups (P=0.35). Between 1-5 (mean 3.2) Lactobacillus spp were present in healthy horses and 2-11 (mean 5.3) in diarrheic horses (P=0.19). • Clostridium difficile was detected in 3/10 diarrheic and 1/6 healthy horses. Escherichia coli was detected in 8/10 diarrheic but 0/6 healthy horses. Clostridium sordellii was detected in 2 diarrheic horses.  • Various organisms that are recognized enteropathogens in other species but of unknown relevance in horses were detected, such as Yersiniaand Shigellaspp. • Discussion • The species richness indicates the complexity of the equine intestinal microbiome and this study provides the most comprehensive indication of this important and complex microbiome. •   Numerous differences between normal and diarrheic horses were identified, including changes at a high (Phylum) level. Differentiating cause versus effect is impossible, but identification of organisms disproportionately present in diarrheic horses can lead to investigation of their potential role as causative agents. •   The predominance of Clostridia and related organisms in healthy horses demonstrates the importance of this much-maligned group of bacteria. •   The decreased abundance of Firmicutes suggests that efforts at therapeutically manipulating the intestinal microbiome may need to concentrate on this group, rather than the typical approach using lactic acid bacteria. •   The abundance of Fusobacteria in diarrheic horses was interesting as the role of this Phylum in equine colitis has not been reported. •   Various unculturable (e.g. Clostridium sordellii) and previously unidentified organismswere identified, indicating the need for non-culture-dependent methods. Similarly, some organisms that are uncommonly isolated from healthy horses (e.g. Clostridium perfringens from 3/6 healthy horses) were detected. •   While culture independent methods and next generation sequencing eliminate many biases from culture or cloning-based methods, there can be some PCR amplification bias, so certain groups (e.g. Bifidobacteriumspp) might be underestimated. Evaluation of other target genes is indicated for further comprehensive study of this microbiome. • The marked differences in the microbiome between healthy and diarrheic horses indicate that colitis needs to be considered a population disease, rather than one that occurs simply through overgrowth of an individual pathogen. Diarrheic Normal Figure 2: Principle component analysis of healthy (red) and diarrheic (green). Figure 1: Comparison of the fecal microbiome of healthy and diarrheic horses at the Phylum level. Figure 2: Relative frequency of Phyla in healthy and diarrheic horses Acknowledgements This study was supported by Equine Guelph. Table 1: Sequence data summary.