Report from Trine Glad, PhD student, Department of Arctic and Marine Biology and Department of Pharmacy, University of Tromsø
Project 2009/1415-12
Financial support to participate at the conference on gastrointestinal function, CGIF2009, in Chicago 20-22 April 2009
Fate and effect of usnic acid in lichen on the bacterial population in the reindeer rumen
Background
Reindeer (Rangifer tarandus tarandus) are able to utilize lichens as an important source of energy and nutrients. Lichens are symbiotic organisms consisting of a fungus and an algae and/or cyanobacteria. The fungal components synthesise and accumulate a wide variety of phenolic pigments including usnic acid (UA, which protects against UV-radiation, herbivory by animals and insects, as well as pathogenic microbes (Ingolfsdottir 2002). Reindeer and their symbiotic rumen microbiota have interacted with lichens through millions of years of evolution, and the recent report on a novel reindeer rumen bacterial species able to grow in the presence of UA indicates that the rumen microorganisms of reindeer may have evolved mechanisms to resist the antibiotic effects of UA (Sundset et al 2008, 2009).
Objectives
The objectives of this study was (1) to determine if UA is degraded by rumen microbes in reindeer, (2) to isolate and identify UA-resistant rumen bacteria, and (3) to examine the effect of UA on rumen bacterial diversity and identify predominant phylotypes in reindeer supplemented with UA.
Results
1) UA disappearance was calculated to be 56-70% in C.stellaris and 38-61% in C. nivalis 2) UA and lobaric acid showed a potent antimicrobial effect on 63 of the isolates belonging to the genera Bacillus, Clostridium, Enterococcus and Streptococcus. Three Clostridial isolates were found to be resistant to all lichen acids tested, one showing a 98.1% 16S rRNA gene sequence identity to Butyrivibrio hungatei, and the other two 99.5-100% 16S rRNA gene identity to Pseudobutyrivibrio ruminis. The resistant isolates were all motile, curved rods and stained Gram negative although they are phylogenetically within Clostridium cluster XIVa. 3) Fourteen bands were excised from the DGGE gel of the particle and liquid fraction, and between two and five different clones generated from each band were sequenced. Most of the sequences were novel, indicating a high percentage of uncultivated bacteria in the reindeer rumen as previously shown by Sundset et al (2007).
Conclusions
This study has for the first time proved that UA is degraded by the rumen microflora. We have also identified two bacterial isolates that are resistant to UA. DNA sequencing of gel bands from the DGGE profiling revealed a high number of uncultivated bacteria in the particle and liquid ruminal fractions from reindeer.
Evaluation/presentation
The results were presented in a poster at the Conference on gastrointestinal function, CGIF2009, in Chicago 20-22 April 2009. The abstract is accepted for presentation at CGIF2009 and publication in Microbial Ecology, an international per-review journal (Glad et al. 2009).
References
Ingolfsdottir K (2002). Usnic Acid. Phytochemistry (Amsterdam) 61:729-736.
Glad T, Falk A, Barboza P, Kohn A, Brusetti L, Mathiesen SD, Mackie RI, Sundset MA (2009) Fate and effect of usnic acid in lichen on the bacterial population in the reindeer rumen. Accepted to CGIF2009, Chicago 20-22 April 2009, conference abstracts submitted to Microbial Ecology.
Sundset MA, Prestæng KE, Cann IKO, Mathiesen SD, Mackie RI (2007). Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer. Microbial Ecol. 54:424-438
Sundset MA, Kohn A, Mathiesen SD, Præsteng KE (2008). Eubacterium rangiferina, a Novel Usnic Acid Resistant Bacterium From The Reindeer Rumen. Naturwissenschaften 95:741-749.
Sundset MA, Edwards JE, Cheng YF, Senosiain RS, Fraile MN, Northwood KS, Præsteng KE, Glad T, Mathiesen SD, Wright AD (2009). Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture. Microbial ecology, 57:335-348.
