Dietary Supplementation of Usnic Acid, an Antimicrobial Compound in Lichens, Does Not Affect Rumen...
Transcript of Dietary Supplementation of Usnic Acid, an Antimicrobial Compound in Lichens, Does Not Affect Rumen...
Dietary Supplementation of Usnic Acid, an AntimicrobialCompound in Lichens, Does Not Affect Rumen Bacterial Diversityor Density in Reindeer
Trine Glad • Perry Barboza • Roderick I. Mackie •
Andre-Denis G. Wright • Lorenzo Brusetti •
Svein D. Mathiesen • Monica A. Sundset
Received: 14 June 2013 / Accepted: 17 December 2013
� Springer Science+Business Media New York 2014
Abstract Reindeer (Rangifer tarandus tarandus) may
include large proportions of lichens in their winter diet.
These dietary lichens are rich in phenolic secondary com-
pounds, the most well-known being the antimicrobial usnic
acid. Previous studies have shown that reindeer host rumen
bacteria resistant to usnic acid and that usnic acid is
quickly detoxified in their rumen. In the present study,
reindeer (n = 3) were sampled before, during, and after
usnic acid supplementation to determine the effect on their
rumen microbial ecology. Ad libitum intake of usnic acid
averaged up to 278 mg/kg body mass. Population densities
of rumen bacteria and methanogenic archaea determined
by real-time PCR, ranged from 1.36 9 109 to 11.8 9 109
and 9.0 9 105 to 1.35 9 108 cells/g wet weight, respec-
tively, and the two populations did not change significantly
during usnic acid supplementation (repeated measures
ANOVA) or vary significantly between the rumen liquid
and particle fraction (paired t test). Rumen bacterial com-
munity structure determined by denaturing gradient gel
electrophoresis did not change in response to intake of
usnic acid. Firmicutes (38.7 %) and Bacteriodetes
(27.4 %) were prevalent among the 16S rRNA gene
sequences (n = 62) from the DGGE gels, but representa-
tives of the phyla Verrucomicrobia (14.5 %) and Proteo-
bacteria (1.6 %) were also detected. Rapid detoxification
of the usnic acid or resistance to usnic acid may explain
why the diversity of the dominant bacterial populations and
the bacterial density in the reindeer rumen does not change
during usnic acid supplementation.
Introduction
Usnic acid is a phenolic secondary compound used as a
defense in lichens against bacteria, viruses, fungi, protozoa,
insects, herbivores, and UV-radiation [2]. Reindeer utilize
lichens as an important source of energy and other nutrients
in winter, whereas toxic reactions toward lichens/usnic acid
are reported in other ruminants such as sheep and elk [4,
15]. Ruminants rely on symbiotic anaerobic rumen
microorganisms to digest their herbivorous diet, but rumen
foregut fermentation also allows microbial detoxification
[11]. Recent studies suggest that reindeer harbor rumen
bacteria resistant to usnic acid [19] and that usnic acid and
other phenolic compounds from lichens are rapidly
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00284-014-0534-7) contains supplementarymaterial, which is available to authorized users.
T. Glad � M. A. Sundset (&)
Department of Arctic and Marine Biology, UiT The Arctic
University of Norway, 9037 Tromsø, Norway
e-mail: [email protected]
P. Barboza
Institute of Arctic Biology and Department of Biology and
Wildlife, University of Alaska, Fairbanks, AK 99775, USA
R. I. Mackie
Department of Animal Sciences, University of Illinois,
Urbana-Champaign, IL 61801, USA
A.-D. G. Wright
Department of Animal Science, University of Vermont,
Burlington, VT 05405, USA
L. Brusetti
Faculty of Science and Technology, Free University of Bozen,
39100 Bolzano, Bolzano, Italy
S. D. Mathiesen
The Norwegian School of Veterinary Science, Sjøgata 39,
9000 Tromsø, Norway
123
Curr Microbiol
DOI 10.1007/s00284-014-0534-7
detoxified in their rumen [22]. Presuming that the rumen
microbiome is the key site for usnic acid detoxification, we
hypothesize that exposure to usnic acid may select for
bacteria able to tolerate and detoxify this lichen secondary
metabolite. The current study examined the effect of usnic
acid supplementation on the density of ruminal bacteria
and methanogens, and the diversity of rumen bacteria in
reindeer.
Materials and Methods
Three female rumen-fistulated reindeer (88 ± 7.8 kg)
(Rangifer tarandus tarandus) were given ad libitum access
to a pelleted feed (2.7 % N and 34 % neutral detergent
fiber on dry matter (DM) basis). The experimental feed was
coated with a solution of 3 % w/v gelatin (Kraft Foods
Tarrytown, NY) to allow a uniform adherence of usnic acid
(Catalog #026169, Indofine Chemicals, Hillsborough, NJ).
It contained 1 % w/w usnic acid and 0.35 % w/w dry
gelatin (0.05 % N from gelatin), while the control feed was
coated with gelatin only. Animals were acclimated to the
control feed (16 days), fed usnic acid feed (17 days), and
returned to the control feed (8 days). Average daily food
intake was 17 ± 5 g/kg body mass during pre-treatment,
15 ± 6 g/kg the first 10 days of treatment, 28 ± 6 g/kg the
last 7 days of treatment, and 21 ± 6 g/kg post-treatment.
Rumen samples were collected at the end of pre-treatment,
at 9 and 17 days of usnic acid treatment, and day 8 post-
treatment. Samples were taken at the same time of day
(10:06–10:26) to minimize diurnal effects. Whole rumen
contents were collected in prewarmed thermos bottles
(37 �C) and transferred to a glove bag (VWR, Radnor,
USA) filled with CO2. Whole digesta (100 g) was trans-
ferred to 200 mL glass beakers and pressed through clean
stainless-steel filters (Model #1543, Bodum, Zurich, Swit-
zerland) to retain large particulates and extrude fluids.
Fluid-phase microbes were collected from 20 g of extrusa,
and particulate-phase microbes from 20 g of large partic-
ulate digesta, combined with 25 mL anaerobic dilution
solution (ADS) containing buffers with a detergent (Tween
80; 0.15 % w/v) for microbial detachment, then cooled to
10–15 �C in closed bottles under anaerobic conditions.
Fluid collections were centrifuged (5009g for 15 min at
4 �C) under aerobic conditions to remove large particles,
the supernatant subsequently centrifuged (18,0009g for
15 min at 4 �C) to precipitate microbial cells. Microbes
detached from particulate digesta fractions in ADS by
incubation at 4 �C for 2.5 h before centrifugation
(5009g for 15 min, 18,0009g for 15 min) to separate
microbial cells. Microbial cells in centrifuged pellets from
fluid and particulate phases were redissolved in 1.4 mL
stool lysis buffer with a pH of 5.40–5.60 (ASL Buffer;
#19802 Qiagen, Valencia CA, USA) and stored at -80 �C
until further analysis.
Microbial densities were analyzed in triplicate using
real-time PCR [20] with bacterial 16S rRNA gene primers
1114F and 1257R [5] and methanogenic archaea primers
qmcrA-F and qmcrA-R [6]. Statsistical comparisons to
detect any changes in the microbial population (bacteria
and methanogens) densities due to treatment (before, dur-
ing, and after usnic acid supplementation) (repeated mea-
sures ANOVA) or between populations associated with the
two different rumen fractions (liquid and particle fraction)
(paired t-test) was run in STATA 12.0 (StatCorp, College
Station, TX, USA).
Effect of usnic acid on rumen bacterial community
structure was analyzed using DGGE profiling, PCR
amplifying the 16S rRNA gene V3 region with primers 1
and 2 (with GC clamps) [12] generating 220 bp amplicons.
PCR reactions were carried out as described by Simpson
et al. [16, 17]. DGGE was performed using a Bio-Rad
D-Code System (Hercules, CA) and gels were silver-
stained and scanned using a GS-710 calibrated imaging
densitometer (BioRad Inc., Hercules, CA). Principal com-
ponent analysis (primer-E software www.primer-e.com)
was used to analyze the DGGE profiles, and Bray-Curtis
dissimilarity to quantify the compositional dissimilarity
between different samples (liquid vs. particle), sampling
time, and animal.
Dominant bacterial phylotypes were identified using
DGGE and 16S rRNA gene sequencing. Total DNA from the
samples was extracted using FastDNA� SPIN Kit for soil
(Qbiogene, Irvine, California). The V6–V8 region was PCR
amplified using primers F968 with a GC clamp and R1401
[24]. The gels were stained with SYBR� Green I nucleic acid
gel-stain (Invitrogen, California, USA), DGGE bands
excised and reamplified with the primers F968 without the
GC clamp and R1401 as described above, with addition of
2 ll dimethyl sulfoxide (DMSO) in the reaction mixture.
PCR reamplification was initiated by hotstart (95 �C for
3 min), 30 cycles (94 �C 30 s, 64 �C 1 min, and 72 �C
1 min), and a final extension at 72 �C for 10 min. Amplifi-
cation products were purified with QIAquick PCR Purifica-
tion Kit (Qiagen, Solna, Sweden) and cloned using the
pGEM�-T Easy Vector System (Promega, Madison, USA).
Recombinant plasmids were sequenced on a Genetic ana-
lyzer (Applied Biosystems, Foster City, USA) using ABI
BigDye Terminator chemistry using sequencing primers
M13 forward and reverse (Invitrogen). Sequences were
assembled using LasergeneTM Seqman v. 7.1.0. (DNA-
STAR, Madison, USA) and chimeras removed [8]. Sequence
data were deposited in GenBank (Accession numbers
GQ449392—GQ449453). Sequences were identified
using BLAST [1], and assigned to operational taxonomic
units (OTUs) based on a 97 % sequence identity criterion
T. Glad et al.: Usnic acid and reindeer rumen bacteria
123
using the FastGroupII platform [23]. The liquid and particle
fraction clone libraries were compared using The Ribosomal
Database Project [3].
Results and Discussion
Rumen bacterial density did not change significantly, and
diversity remained unchanged during supplementation of
usnic acid (Table 1, Fig. 1), suggesting that usnic acid is
rapidly degraded and detoxified by rumen bacteria in
reindeer, as previously reported by Sundset et al. [22], and
consequently does not influence dominant bacterial popu-
lations. Previous studies by our group have also shown that
reindeer host bacteria resistant to lichen antimicrobial
compounds such as usnic acid [19], and this may offer an
alternative explanation why usnic acid supplementation did
not appear to affect rumen bacterial numbers and diversity
in this current project (Table 1; Fig. 1; Table S1 and S2).
Numbers of rumen methanogens were lower than those
previously reported in Norwegian reindeer on natural
pastures [21], and did not change significantly during usnic
acid treatment or vary significantly between the different
rumen fractions investigated (Table 1). Similarly, bacterial
densities associated with the rumen liquid and particle
fractions (Table 1) did not differ either. However, popu-
lation profiling using DGGE gels demonstrated a variation
in the bacterial diversity between the different fractions,
and samples from each animal grouped together when
analyzing the DGGE profiles using cluster analysis and
principal component analysis (Fig. 1). Nine bands from the
DGGE gel of the liquid fraction generated 2–3 different
clones each (25 in total), while 13 DGGE bands from the
particle fraction generated 1–4 different clones each (37 in
total). Among sequences obtained (n = 62), 58 distinct
OTUs were identified. Only one (L9-5) showed a sequence
identity of 97 % to its nearest valid taxon (Selenomonas
ruminantium), while the remaining were novel with only
80–96 % identity to their nearest relative. The high pro-
portion of novel 16S rRNA gene sequences in the rumen of
reindeer is consistent with previous findings [13, 14, 18]. A
meta-analysis of all publicly available 16S rRNA genes of
rumen origin (n = 13,478 bacterial sequences) revealed 19
bacterial phyla, with Firmicutes (57.8 %), Bacteroidetes
(26.7 %), and Proteobacteria (6.9 %) being the most
Table 1 Density (cell numbers/
g wet weight) of bacteria and
methanogenic archaea in the
rumen liquid and particle
fraction of reindeer (Rangifer
tarandus tarandus) pre-
treatment, during usnic acid
supplementation (day 9 and 17),
and 8 days post-treatment
Animal and treatment Bacteria Methanogenic archaea
Liquid Particle Liquid Particle
Prior to treatment
Mean (n = 3) 5.16 9 109 6.08 9 109 1.03 9 108 3.35 9 107
SD 0.79 9 109 3.25 9 109 0.37 9 108 0.96 9 107
Day 9
Mean (n = 3) 2.83 9 109 7.98 9 109 4.48 9 107 2.21 9 107
SD 1.66 9 109 3.09 9 109 4.32 9 107 0.56 9 107
Day 17
Mean (n = 3) 4.27 9 109 5.48 9 109 9.36 9 107 3.06 9 107
SD 1.97 9 109 0.34 9 109 4.64 9 107 2.58 9 107
8 days after treatment
Mean (n = 3) 4.20 9 109 6.50 9 109 5.83 9 107 7.66 9 106
SD 1.24 9 109 3.34 9 109 2.86 9 107 4.83 9 106
-20-10
010
-30-20
-100
10-10
-5
0
5
10
17
PC1 (31%)
1
16
2
13
15
6
10
8
14
129
54
3
7
1119
20
18
21
PC2 (17%)
22
24
23PC
3 (9
%)
Fig. 1 Principal-component analysis of the DGGE profiles (V3
region of the 16S rRNA gene) showing that there is no pattern
related to treatment (pre-, during, and post-dosing of usnic acid).
upper triangle animal no. 151, black circle animal no. 533; asterisk
animal no. 539. Numbering: 1–4 animal no 151 particle fraction; 5–8
animal no 151 liquid fraction; 9–12 animal no 533 particle fraction;
13–16 animal no 533 liquid fraction; 17–20 animal no 539 particle
fraction; 21–24 animal no 539 liquid fraction. For all number series,
the pre-treatment sample has the lowest number, then 9 and 17 days
of usnic acid treatment, and the 8 days post treatment sample has the
highest number
T. Glad et al.: Usnic acid and reindeer rumen bacteria
123
dominant ones [9]. Similarly in this (Table 2), and previous
studies of the reindeer rumen [13, 14, 18], Firmicutes and
Bacteroidetes were found to be the dominant phyla. Liquid
and particle libraries were significantly different for both
Firmicutes and Bacteroidetes clones (Table 2). The large
Clostridial cluster XIVa (Firmicutes) includes typical ru-
minal strains in addition to the novel usnic acid resistant
Eubacterium rangiferina isolated from reindeer [19]. Sev-
eral clones belonged to the Bacteroidetes, clustering with
Prevotella species such as Prevotella ruminocola and
Prevotella salivae, and only 2.7 % of the particle fraction
sequences were Proteobacteria. A notable finding was the
large number of novel clones affiliated with the phylum
Verrucomicrobia, which are poorly represented among
cultivated organisms and little is consequently known
about their phenotypic properties. Only a few Verrucomi-
crobia sequences have been reported in the rumen so far
[9] comprising only 2.2 % of the fecal microbiota in
humans and 59 other mammalian species investigated by
Ley et al. [10]. However, analysis of human intestinal
mucosa biopsies revealed Verrucomicrobia clones ranging
from 5–9 %, and they comprised as much as 17.2 % of the
fecal bacterial community in on wild gorilla examined [7].
In conclusion, real-time PCR and DGGE profiling
revealed no changes in rumen microbial densities or bac-
terial diversity in the presence of usnic acid, suggesting
that the microbes are either resistant to the antimicrobial
properties of the compound usnic acid or that this lichen
secondary compound is rapidly degraded and consequently
do not influence the dominant microbial populations in
reindeer.
Acknowledgments This project is funded by The Reindeer Hus-
bandry Research Fund as part of the International Polar Year con-
sortium # 399 EALAT: Climate change and reindeer husbandry. We
thank A. Falk, J. Edwards, A. Yannarell, and J.-N. Kim for technical
assistance, and R. J. Forster, M. Morrison, and Dr. Zhongtang Yu for
advice on the rumen sampling protocol.
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Phylum Library
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(Number of clones) (25) (37) (62)
Verrucomicrobia 12.0 13.5 14.5
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Proteobacteria 0 2.7 1.6
Firmicutes 60.0 27.0 38.7
Unclassified 16.0 18.9 17.7
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