Hilary J. Worgan

A comparison of the microbiome and the metabolome of different regions of the equine hindgut

The microbiome and associated metabolome of faecal samples were compared to those from the caecum and right dorsal colon of horses and ponies euthanised for nonresearch purposes by investigating the microbial population community structure as well as their functional metabolic products. Through the use of 16S rRNA gene dendrograms, the caecum microbiome was shown to cluster separately from the other gut regions. 16S rRNA gene-based quantitative PCR (q-PCR) also demonstrated differences between the caecum and the other gut regions. Metabolites as identified by Fourier transform infrared clustered in a similar way and specific metabolic products (volatile fatty acids and ammonia) also varied by region. Protozoal 18S rDNA concentration and archaeal mcrA gene concentration quantified by q-PCR were found in higher numbers in the colon than the other gut regions. Diversity calculations using Simpson and Shannon-Wiener indices demonstrated higher diversity in the right dorsal colon and faeces than in the caecum. All findings of this study suggest that faecal samples are likely to represent the microbial population of the right dorsal colon to some extent but not that of the caecum, indicating careful consideration is required when planning microbial investigations of the hindgut of the horse.

Contrasts between the cryoconite and ice-marginal bacterial communities of Svalbard glaciers

Cryoconite holes are foci of unusually high microbial diversity and activity on glacier surfaces worldwide, comprising melt-holes formed by the darkening of ice by biogenic granular debris. Despite recent studies linking cryoconite microbial community structure to the functionality of cryoconite habitats, little is known of the processes shaping the cryoconite bacterial community. In particular, the assertions that the community is strongly influenced by aeolian transfer of biota from ice-marginal habitats and the potential for cryoconite microbes to inoculate proglacial habitats are poorly quantified despite their longevity in the literature. Therefore, the bacterial community structures of cryoconite holes on three High-Arctic glaciers were compared to bacterial communities in adjacent moraines and tundra using terminal-restriction fragment length polymorphism. Distinct community structures for cryoconite and ice-marginal communities were observed. Only a minority of phylotypes are present in both habitat types, implying that cryoconite habitats comprise distinctive niches for bacterial taxa when compared to ice-marginal habitats. Curiously, phylotype abundance distributions for both cryoconite and ice-marginal sites best fit models relating to succession. Our analyses demonstrate clearly that cryoconites have their own, distinct functional microbial communities despite significant inputs of cells from other habitats.

Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).

Strong Stability and Host Specific Bacterial Community in Faeces of Ponies

The horse, as a hindgut fermenter, is reliant on its intestinal bacterial population for efficient diet utilisation. However, sudden disturbance of this population can result in severe colic or laminitis, both of which may require euthanasia. This study therefore aimed to determine the temporal stability of the bacterial population of faecal samples from six ponies maintained on a formulated high fibre diet. Bacterial 16S rRNA terminal restriction fragment length polymorphism (TRFLP) analyses of 10 faecal samples collected from 6 ponies at regular intervals over 72 hour trial periods identified a significant pony-specific profile (P<0.001) with strong stability. Within each pony, a significantly different population was found after 11 weeks on the same diet (P<0.001) and with greater intra-individual similarity. Total short chain fatty acid (SCFA) concentration increased in all ponies, but other changes (such as bacterial population diversity measures, individual major SCFA concentration) were significant and dependent on the individual. This study is the first to report the extent of stability of microbes resident in the intestinal tract as represented with such depth and frequency of faecal sampling. In doing so, this provides a baseline from which future trials can be planned and the extent to which results may be interpreted.

A ring test of a wireless in vitro gas production system

The in vitro gas production (GP) technique has been widely used for feed evaluation. However, variability in results limits useful comparisons. Results from a ring test undertaken in four laboratories (Italy – IT, Spain – SP, Wales – WA and Denmark – DK) using the same wireless equipment (ANKOM Technology), same substrates and same laboratory protocol are presented, including calculation of repeatability and reproducibility according to ISO 5725-2. Hay, maize starch and straw samples and units without sample (blanks) were incubated in five repetitions using rumen inoculum from cows (DK, IT and WA) or sheep (SP). Curves, corrected for blanks, were fitted using an exponential regression model with a lag time. The following variables were considered: (i) GP24 and GP48: raw values at 24 and 48 h (mL/g DM), corrected for blanks; (ii) A: asymptotic GP (mL/g DM); (iii) T1/2: time when half A is produced (h); (iv) GPMR: maximum predicted GP rate (mL/h); (v) L: lag time (h). A mixed model including laboratories as random effect was used. A significant interaction between substrate and laboratories was found for all variables except A. The most repeatable and reproducible results were observed for A and GP48. The results from this ring test suggest the need for more standardisation, particularly in the procedures that occur outside the laboratory.

Use of earthworm casts to validate FT-IR spectroscopy as a 'sentinel' technology for high-throughput monitoring of global changes in microbial ecology

Summary This study aimed to evaluate metabolic fingerprinting by Fourier transform infrared (FT-IR) spectroscopy as a technique for investigating microbial communities and their activities in soil. FT-IR spectra from earthworm casts, and other ‘biosamples’, were compared using multivariate cluster analyses. The work formed part of a wider study to quantify the risk of horizontal gene flow and to assess ecological impacts associated with the release of GM crops or recombinant micro-organisms. A range of samples, including pure cultures of similar soil bacteria, plant materials and earthworm casts of various ages and feeding regimes were analysed. A subset of the cast FT-IR data was compared with DGGE analysis of extracted DNA/RNA. Cluster analysis of FT-IR spectra was capable of differentiating between different bacterial, litter and cast samples. There was congruence between FT-IR and DGGE clustering for food type but not for cast age. Further detailed work on the microbial populations will be needed to investigate relationships between microbial and spectroscopy data.

Food quality and microbial succession in ageing earthworm casts: standard microbial indices and metabolic fingerprinting

Scullion, J., Goodacre, R., Elliott, G., Wei, H., Worgan, H., Gwynn-Jones, D., Griffith, G.W., Darby, R., Bailey, M., Clegg, C., Draper, J. (2003). Food quality and microbial succession in ageing earthworm casts: standard microbial indices and metabolic fingerprinting. Pedobiologia, 47, (5-6), 888-894. 7th International Symposium on Earthworm Ecology, Cardiff, Wales, 2002. Sponsorship: BBSRC/ NERC

Changes in the bacterial population of the caecum and stomach of the rabbit in response to addition of dietary caprylic acid.

The effect of caprylic acid, either in its pure form, or as Akomed R, on the microbial community of the stomach and caecum of farmed rabbits was investigated. This fatty acid, which is often added to the diet of farmed rabbits to reduce mortality rates was shown to reduce the number of coliforms isolated from both the stomach and the caecum. Moreover, it led to a reduction in the total number of anaerobic bacteria isolated from the caecum, but not for those isolated from the stomach. Its mode of action remains unclear, but here it is shown by use of both DGGE and TRFLP analysis that these changes are not confined to one specific group of bacteria, but rather affects a number of species.

Molecular Phylogeny of Spirodinium equi, Triadinium caudatum and Blepharocorys sp. from the Equine Hindgut

Single cell morphotypes of the species Triadinium caudatum and Spirodinium equi, together with a representative of the genus Blepharocorys (Blepharocorys sp.) were used for phylogenetic analysis based on their 18S rRNA genes. Spirodinium equi clustered with sequences already described for the entodiniomorphs isolated from horses and the Blepharocorys sp. also grouped within the Entodiniomorphida clade, although both sequences were distinct from those described from rumen ciliates. Triadinium caudatum clustered within the Vestibuliferida, and most closely to that of Paraisotricha, only other member of this order which has been described in the horse. It was concluded that although members of the orders Entodiniomorphida and Vestibuliferida are present in the equine gut, and that they share an ancient linage with their rumen counterparts, they are ancestrally different groups.

Comparison of the microbial population in rabbits and guinea pigs by next generation sequencing

This study aimed to determine the microbial composition of faeces from two groups of caecotrophagic animals; rabbits and guinea pigs. In addition the study aimed to determine the community present in the different organs in the rabbit. DNA was extracted from seven of the organs in wild rabbits (n = 5) and from faecal samples from domesticated rabbits (n = 6) and guinea pigs (n = 6). Partial regions of the small ribosomal sub-unit were amplified by PCR and then the sequences present in each sample were determined by next generation sequencing. Differences were detected between samples from rabbit and guinea pig faeces, suggesting that there is not a microbial community common to caecotrophagic animals. Differences were also detected in the different regions of the rabbits’ digestive tracts. As with previous work, many of the organisms detected were Firmicutes or unclassified species and there was a lack of Fibrobacteres, but for the first time we observed a high number of Bacteroidetes in rabbit samples. This work re-iterates high levels of Firmicutes and unclassified species are present in the rabbit gut, together with low number of Fibrobacteres. This suggests that in the rabbit gut, organisms other than the Fibrobacteres must be responsible for fibre digestion. However observation of high numbers of Bacteroidetes suggests that this phylum may indeed have a role to play in digestion in the rabbit gut.