Microbial composition and stability in a serially sampled cohort of in-training

The major taxa at phyla level were Firmicutes and Bacteroidetes at 47% and 46%, respectively and these were found to be inversely correlated with one another. The inverse correlation between Firmicutes and Bacteroidetes levels within the human gut microbiome

PhD Thesis Ebony Ellen Escalona 176 has been previously documented in studies exploring the effect of diet (Kelder et al., 2014). These major phyla have long been known to be dominant features in the mammalian gut microbiome and the percentage of these phyla vary according to study type and are influenced by factors such as diet and disease (Willing et al., 2009; Costa et al., 2012; Daly et

al., 2012; Steelman et al., 2014). However, a predominance of the Firmicutes phyla is noted

in the majority of studies assessing the equine intestinal microbiome. Shifts in phyla dominance have been documented in colitis and laminitic patients when compared to healthy horses, with rises in bacterial members of Bacteroides (Costa et al., 2012; Steelman et al., 2012). A study by O’Donnell et al., 2013 assessed a similar cohort of horses as the work presented here and revealed a Firmicutes to Bacteroidetes ratio of greater than 2:1 with a Firmicutes range between 47-74%. This is a larger range than documented in this study of 36- 55% and the Firmicutes to Bacteroidetes ratio was also found to be lower at 1:1. A number of research papers have noticed an association with certain clinical phenotypes and the Firmicutes to Bacteroidetes ratio. The lean phenotype in rodents has been found to be associated with increased Bacteroidetes: Firmicutes ratio when compared to an obese phenotype (Ley, Turnbaugh, et al., 2006; Turnbaugh et al., 2006). Human weight loss studies and dietary fibre supplementation have also shown an increase in Bacteroidetes (Ley, Turnbaugh, et al., 2006; Holscher et al., 2015). A total fibre diet in horses has also been shown to significantly increase the number of cellulolytic Bacteroidetes bacteria when compared to a diet with up to 50% barley (Julliand et al., 2001). The body mass phenotype of these racehorses compared to O’Donnell, et al., 2013 would have been similar but their diets may have differed with regards to fibre content. Detailed dietary information is not available but this is a likely reason for the difference in Firmicutes and Bacteroidetes percentages found in faeces.

PhD Thesis Ebony Ellen Escalona 177 Despite the four horses that were serially sampled coming from different yards and different dietary regimes their microbial composition were remarkably similar to one another and stable over time. Human oral microbiomes have shown high levels of inter-individual stability and did not vary with distinctly different dietary habits. However, metabonomics profiles from the same samples did reveal diet related biomarkers (De Filippis et al., 2014). The most abundant three bacterial orders in this cohort of healthy Tbs were found in relative abundance of between 5-60%. The percentage variability of the two most abundant orders:

Firmicutes Clostridia Clostridiales and Bacteroidetes Bacteroidia Bacteroidales were stable

at between 15-23%. This stability was seen in all four horses over the 10 week sample period. There has been minimal research undertaken examining the normal stability or fluctuations of faecal bacterial output in horses maintained on a uniform diet. A paper by Blackmore et al., 2013 found a stable pony-specific faecal bacterial output from 6 ponies when sampled regularly over a 72 hour sample period. However, this sample period is short and does not take into account long term stability over weeks, months or years (Blackmore TM, Dugdale A, Argo CM, Curtis G, Pinloche E, et al., 2013). The same study also sampled the same 6 ponies at the beginning and after 11 weeks on a high fibre diet. Over this sample period the population structure (species richness and evenness) was different between the two study periods. However, this is unsurprising as sampling commenced only after a week of acclimatisation to the diet. The work here also demonstrates horse specific profiles when observed via PCoA plots (see Figure 4.20) and through discriminant analysis (see Figure 4.24). Greater inter-horse bacterial population variation is seen in comparison to intra-horse variation and this phenomenon is well documented in previous equine studies (Costa et al., 2012; Dougal et al., 2012). There is a scarcity in data relating to long term stability of the intestinal microbiota with one study by Goachet et al.,2010 assessing apparent digestibility of dry matter, solid and liquid retention time and the faecal microbial ecosystem over a two year

PhD Thesis Ebony Ellen Escalona 178 period. However, next generation sequencing was not employed and microbial ecosystem measurements were made using culture techniques for total viable anaerobic bacteria and lactic-acid utilising counts (Goachet et al., 2010).

Researchers have suggested that the identification of a core microbiome within a healthy cohort of a species may help to provide a baseline of consistent bacterial components that in turn can be used as a platform for disease diagnosis and potential therapeutic targets (Jalanka- Tuovinen et al., 2011; Shade and Handelsman, 2012) . With specific regards to horses, the identification of a core microbiome may help in dietary management and pre/probiotic supplementation to minimise disease risk. However, the presence of absence of certain bacterial communities does not take into account the core functions of these bacteria as bacterial actions may be shared across a number of different community members (Turnbaugh and Gordon, 2009). For instance, the identification of a core microbiome may be useless if only a low abundance of key bacterial communities are required for digestive function and pathogen control.

A number of studies have explored the equine core intestinal microbiome (Dougal et al., 2013; O'Donnell et al., 2013; Dougal et al., 2014). However, it is difficult to compare findings due to differing sample location (intestinal segments versus faeces), sample numbers and case selection. Dougal et al., 2013 found that samples taken from the distal large intestine (right dorsal colon, small colon and faeces) demonstrated a core microbiome which was defined as OTU clustering at 97% similarity and present at greater than 0.1% of the community and this was made up of 9 families. This is comparable with the work presented here which isolated 15 core family members in Thoroughbred faeces however, only 3 family members were identified as the same as Dougal et al, 2013 work. These were Bacteroidales

Prevotellaceae, Clostridiales Lachnospiraceae and Clostridiales Ruminococcaceae.

PhD Thesis Ebony Ellen Escalona 179 these core microbiome profiles may reflect dietary differences of grass versus concentrate and hay diets. The predominance of the phyla Firmicutes in this study and the study by Dougal et al., 2013 is demonstrated at 53% and 46% of the total core phyla respectively (Dougal et al., 2013).

A study with greater relevance to this work was carried out on 6 Thoroughbred racehorses to identify a core microbiome (O'Donnell et al., 2013). 34 core genera were identified and this is 12 more than identified in this work. However, the cut off parameters set in this study were less stringent as core was described as the presence of microbiota in at least 4 out of the 6 study animals and only one sample time point was taken. The increased number of core communities is reflected higher up the taxonomic tree also (12 core phyla compared to 4 in this study). Despite these discrepancies between studies, the majority of genera in both studies belonged to the Clostridiales order, which is also true of human studies investigating the core microbiome (Tap et al., 2009; Jalanka-Tuovinen et al., 2011; Sekelja et al., 2011). Members of the Lachnospiraceace family are the most abundant core component in this Thoroughbred study and this is unsurprising as Lachnospiraceace are known butyrate producers which is an important energy source for mammalian colonocytes and the equine host (Pryde et al., 2002; Jalanka-Tuovinen et al., 2011).

Regardless of the methods used to define core and the communities identified, the faecal core is repeatedly made up of many low abundance OTUs. This may be a factor into the vulnerability of the equine hind gut to dysbiosis, altered fermentation patterns which may lead to potentially devastating metabolic or intestinal disease (Dougal et al., 2013). Some bacteria are biomarkers for a number of diverse environments suggesting species adaptation, whereas others are more specific. These findings highlight a gap in our knowledge in relation to the functional role of these key bacteria and to what extent these activities can be shared amongst communities before disease occurs.

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