Foregut digesta from five feral dromedary camels were inoculated into three different enrichment media: cotton thread, filter paper, and neutral detergent fiber. A total of 283 16S rRNA gene sequences were assigned to 33 operational taxonomic units by using 99% species-level identity. LIBSHUFF revealed significant differences in the community composition across all three libraries

Bryant MP

Dehority BA

Halliwell G

Iqbal A

Lane DJ

Mann SO

Nelson KE

Pei C-X

Philips A

Samsudin AA

Shane BS

Shannon CE

Singleton DR

Tajima K

van Gylswyk NO

Whitford MF

Wright ADG

ABSTRACT
          Foregut digesta from five feral dromedary camels were inoculated into three different enrichment media: cotton thread, filter paper, and neutral detergent fiber. A total of 283 16S rRNA gene sequences were assigned to 33 operational taxonomic units by using 99% species-level identity. LIBSHUFF revealed significant differences in the community composition across all three libraries.</jats:p

Anjas A. Samsudin

André-Denis G. Wright

Rafat Al Jassim

Crossref

Applied and Environmental Microbiology

Cellulolytic Bacteria in the Foregut of the Dromedary Camel (Camelus dromedarius)

Samsudin, Anjas A.

Wright, Andre-Denis G.

Al Jassim, Rafat

University of Queensland eSpace

Cellulolytic Bacteria in the Foregut of the Dromedary Camel (Camelus
dromedarius)
Anjas A. Samsudin,a,b André-Denis G. Wright,c and Rafat Al Jassima
The University of Queensland, School of Agriculture and Food Sciences, Gatton, Queensland, Australiaa; Department of Animal Science, Faculty of Agriculture, Universiti
Putra Malaysia, Serdang, Selangor, Malaysiab; and Department of Animal Science, University of Vermont, Burlington, Vermont, USAc
Foregut digesta from five feral dromedary camels were inoculated into three different enrichment media: cotton thread, filter
paper, and neutral detergent fiber. A total of 283 16S rRNA gene sequences were assigned to 33 operational taxonomic units by
using 99% species-level identity. LIBSHUFF revealed significant differences in the community composition across all three
libraries.
In semiarid and arid lands in Australia, feral dromedary camels(Camelus dromedarius) have solitary access to feed supplies be-
cause they have adapted to harsh environmental conditions,
which are unfavorable to other species of animals, especially do-
mesticated livestock. Camels can utilize the abundance of low-
quality shrubs and trees, such as mulga (Acacia aneura), broad-
leaved whitewood (Atalaya hemiglauca), capper bushes (Capparis
spinosa), and emu bush (Eremophila sp.) (14). In addition, camels
can often browse on a broad spectrum of plants like thorny
bushes, halophytes, and aromatic types, which aremostly avoided
by domestic ruminants (7). The high content of lignocellulose in
the cell wall, combined with the antinutritional factors of these
shrubs and trees, has prevented cattle and sheep from using these
plants as a feed source.
Like ruminants, camels have a complex gut microbiome that
includes bacteria, archaea, protozoa, and fungi to coordinate
plant biomass breakdown. The aim of the present work was to
carry out 16S rRNA gene sequence analysis to identify fiber-di-
gesting bacteria from the foregut of the dromedary camel that
were able to utilize the different fiber types supplied in the enrich-
ment medium as a carbon source.
Adult dromedary camels that were browsing native plants in
the Queensland outback were taken to a commercial slaughter-
house, where digesta contents (500 ml) were collected from the
foreguts of five random animals. This study was conducted ac-
cording to the animal ethics guidelines set by The University of
Queensland Animal Ethics Committee (AEC approval number
SAS/069/08/UQ).
One milliliter of the rumen digesta was used to inoculate cul-
ture tubes containing prereduced basal medium 10 (1) that was
enriched with one of three different fiber types, either filter paper
(FP) (10), cotton thread (CT), or neutral detergent fiber (NDF)
from Lucerne hay (22). The medium was prepared under anaer-
obic conditions and dispensed into Hungate tubes containing the
fiber source in triplicate under carbon dioxide. The tubes were
incubated at 37°C and subsampled into a fresh fiber enrichment
medium every 7 days. After 21 days of incubation, approximately
1.5ml from each enrichmentmediumwas collected, and genomic
DNA was extracted using an established protocol (25).
The bacterial 16S rRNA genes from the different enrichment
media were amplified with the primer pairs 27f and 1492r (8)
using the protocol of Samsudin et al. (15). Amplicons from
the same enrichment media were pooled and cloned using a
TOPO TA cloning kit according to the manufacturer’s instruc-
tions (Invitrogen Corporation, San Diego, CA). A total of 300 16S
rRNA clones were sequenced using the M13 forward and reverse
primers and the BigDye Terminator apparatus and a 48-capillary
ABI 3730 genetic analyzer (Applied Biosystems). All sequences
were manually edited using CHROMAS Lite (v. 2.0) and assem-
bled using SEQMAN (v. 3.34; DNASTAR). Chimeric sequences
were identified by using the program Bellerophon (6) and were
then excluded from further analyses. The 16S rRNA gene se-
quenceswere aligned usingGreengenes (4), and the nearest neigh-
bor sequences were imported into the ARB software package (9)
for further alignment. MOTHUR (16) was used to assign se-
quences to operational taxonomic units (OTUs) based on 99%
sequence identity criterion and to estimate bacterial diversity and
population composition between clone libraries by using Shan-
non index (18) and LIBSHUFF (19) analyses, respectively.
In the CT 16S rRNA gene clone library, 100 clones were se-
quenced and analyzed, revealing 17 chimeras. The remaining 83
sequences were assigned to 11 OTUs (Table 1). Of these, eight
OTUs (CAMbact152 to -155, 157 to -159, and -161)were from the
phylum Firmicutes and were affiliated with Butyrivibrio, Clostrid-
ium, Eubacterium, Pseudobutyrivibrio, Schwartzia, Selenomonas,
and uncultured bacteria from the bovine rumen and the hoatzin
crop. Two OTUs (CAMbact160 and CAMbact162) were from the
phylum Proteobacteria and were affiliated with Anaerobiospiril-
lum, Shigella, Succinivibrio, and an uncultured bovine rumen
clone. The remaining OTU (CAMbact156) was from the phylum
Synergistetes and grouped with Pyramidobacter.
In the FP 16S rRNA gene clone library, 100 sequences were
assigned to 18 OTUs (Table 1). Of these, 13 OTUs (CAMbact152,
-153, -158, -163, -164, -165 to -167, -169, -172, -173, -175, and
-176) represented the phylum Firmicutes, with sequences from
Anaerovorax, Butyrivibrio, Clostridium, Eubacterium, Mogibacte-
rium, Oscillobacter, Pseudobutyrivibrio, Schwartzia, Streptococcus,
Succiniclasticum, uncultured rumen bacteria from domesticated
Received 2 August 2012 Accepted 1 October 2012
Published ahead of print 5 October 2012
Address correspondence to Rafat Al Jassim, r.aljassim@uq.edu.au.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.02420-12
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ruminants, uncultured oral bacteria, and bacteria from nonrumi-
nant animals. Two OTUs (CAMbact171 and CAMbact174) be-
longed to the genus Fibrobacter, and the remaining three OTUs
(CAMbact 156, CAMbact168, and CAMbact170) belonged to the
genera Pyramidobacter, Prevotella, and Succinivibrio, respectively.
In the NDF 16S rRNA gene clone library, 100 sequences were
assigned to 11OTUs (Table 1). Eight OTUs (CAMbact162, CAM-
baact177 to -180 andCAMbact182 to -184)were from the phylum
Proteobacteria and were affiliated with Citrobacter, Escherichia,
Ruminobacter, Shigella, and two uncultured clones from the por-
cine intestine and feces of the rodent family Caviidae. The remain-
ing three OTUs (CAMbact158, CAMbact163, and CAMbact181)
were from the phylum Firmicutes andwere affiliatedwithClostrid-
ium, Streptococcus, and Enterococcus. The presence of Escherichia
coli in the NDF medium was unclear. It is speculated that E. coli
probably used the fermentation products from the other bacteria
for its growth and survival in that medium. The 21 days of incu-
bation, with subculturing every 7 days, may have enriched mono-
mer-utilizing bacteria, rather than the polymer utilizers, which
may explain the detection of E. coli as one of the dominant organ-
isms in these media.
In total, 283 sequences were assigned to 33OTUs (Table 1). Six
OTUswere unique only to the CTmedium, 13OTUswere unique
only to the FPmedium, and 8OTUs were unique only to the NDF
medium. Thirteen of the 33 OTUs had 98% identity to their
nearest relatives, indicating 13 possible new species, while 4 OTUs
had 92% identity, indicating four possible new genera. Firmic-
utes was the most abundant phylum in the FP and CT media,
whereas the phylum Proteobacteria was prevalent in the NDFme-
dium. According to the Shannon indices, the FP library (2.36 
0.19 [mean standard error of themean]) was significantlymore
diverse (P  0.05) than either the CT (1.73  0.20) or NDF
(1.68 0.16) libraries. The solubility of degradable cellulose in the
filter paper along with the increased susceptibility to biological
attack due to the large surface area may explain why the bacterial
community was more diverse in the FPmedium (5). Comparison
of the community compositions across the three 16S rRNA gene
sequence clone libraries using the LIBSHUFF algorithm also indi-
cated significant differences (P 0.0001).
At the species level, Pseudobutyrivibrio ruminis was the most
abundant species in the CT enrichment medium (38.5%), Eubac-
terium sp. was the most abundant species in the FP medium
(32%), and Ruminobacter amylophilus was the most abundant
species in the NDFmedium (36%). The reason for the presence of
TABLE 1 Bacterial 16S rRNA sequences from the foregut of dromedary camels, cultured from three different fiber enrichment media
16S rRNA gene OTU
No. of
clonesa
GenBank
accession no.
Fiber
enrichment(s)b Nearest valid taxon
% sequence
similarity
CAMbact152 48 HQ008599 CT/FP Eubacterium sp. F1 98.3
CAMbact153 42 HQ008600 CT/FP Pseudobutyrivibrio ruminisM80 98.9
CAMbact154 4 HQ008601 CT Selenomonas ruminantium S4 99.2
CAMbact155 4 HQ008602 CT Selenomonas ruminantium L1 98.8
CAMbact156 14 HQ008603 CT/FP Pyramidobacter piscolensW5455 95.6
CAMbact157 2 HQ008604 CT Selenomonas ruminantium L14 97.8
CAMbact158 19 HQ008605 CT/FP/NDF Clostridium bifermentans JCM 7832 99.9
CAMbact159 6 HQ008606 CT Selenomonas ruminantium S36 98.4
CAMbact160 1 HQ008607 CT Anaerobiospirillum thomasii DSM11806 92.0
CAMbact161 3 HQ008608 CT Schwatrzia succinivorans S1-1 99.8
CAMbact162 3 HQ008609 CT/NDF Shigella boydii 5216-70 100.0
CAMbact163 22 HQ008610 FP/NDF Streptococcus bovis 100.0
CAMbact164 3 HQ008611 FP Succiniclasticum ruminis SE10 92.6
CAMbact165 1 HQ008614 FP Butyrivibrio hungatei Su6 97.1
CAMbact166 3 HQ008617 FP Anaerovorax sp. 93.7
CAMbact167 8 HQ008618 FP Clostridium sp. AP81 93.0
CAMbact168 5 HQ008620 FP Prevotella ruminicola 23 97.0
CAMbact169 1 HQ008621 FP Clostridium sp. P6 91.0
CAMbact170 1 HQ008622 FP Succinivibrio dextrinosolvens 0544 99.7
CAMbact171 6 HQ008623 FP Fibrobacter succinogenes RS223 99.5
CAMbact172 1 HQ008624 FP Schwartzia succinivorans DSM10502T 90.0
CAMbact173 1 HQ008625 FP Mogibacterium neglectum P9a-h 92.8
CAMbact174 1 HQ008626 FP Fibrobacter intestinalis JG1 99.2
CAMbact175 6 HQ008627 FP Clostridium butyricum IDCC 5101 99.8
CAMbact176 1 HQ008628 FP Oscillobacter valerigenes Sjm18-20 91.0
CAMbact177 36 HQ008631 NDF Ruminobacter amylophilusH18 97.4
CAMbact178 3 HQ008633 NDF Citrobacter amalonaticus 99.8
CAMbact179 18 HQ008634 NDF Escherichia coli O111:H-11128 99.9
CAMbact180 4 HQ008635 NDF Shigella sp. 29 (2010) 99.9
CAMbact181 6 HQ008637 NDF Enterococcus faecium L118 99.9
CAMbact182 4 HQ008640 NDF Escherichia coli 4106 98.9
CAMbact183 2 HQ008643 NDF Citrobacter koseri CDC8132-86 98.1
CAMbact184 4 HQ008645 NDF Citrobacter freundii JCM24066 99.5
a A total of 283 clones were examined.
b Some of the OTU groups were able to grow on fiber-enriched medium of more than one type.
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a high number of P. ruminis bacteria in the CT medium remains
unclear, as P. ruminis is a butyrate-producing bacterium that is
nonxylanolytic, nonamylolytic, and nonproteolytic (22). It is
likely that Pseudobutyrivibrio survives on other components of the
medium. In contrast, Eubacterium cellulosolvens and members of
the genus Clostridium are reported to possess cellulolytic activity
but are usually present in relatively low numbers in the rumen (2,
3, 17). In the present study, Clostridium bifermentans, a species
common in feces, sewage, and soil, was recovered in all three me-
dia (Fig. 1), and Eubacteriumwas themost abundant species in the
FPmedium and was also present in the CTmedium. Eubacterium
was also present in large numbers in foregut samples of the drom-
edary camel (15) and in alpacas and sheep fed with alfalfa (13).
In the rumen, Fibrobacter succinogenes, Ruminococcus albus,
Ruminococcus flavefaciens, and Butyrivibrio sp. are actively in-
volved in degrading plant polymers, like cellulose and hemicellu-
lose.However, in the present study, a very lownumber of clones of
Fibrobacter succinogenes were detected in the FP enrichment me-
dium, and no cellulolytic ruminococci were detected from the
three media. In previous studies, sequences from F. succinogenes
were not recovered from the rumen when cattle were fed hay or
hay plus concentrate (21, 24) or from clone libraries of wild Afri-
can ruminants and domesticated zebu cattle (11). In the present
study, competition among the fibrolytic bacteria to utilize the
available substrate in the culturing medium could also have
caused the low number of clones. For example, when fibrolytic
bacteria are cultured together in enrichment medium with cellu-
lose, R. flavefaciens is inhibited, while R. albus increases in num-
bers during 12 to 48 h of degradation, before becoming dominated
by the massive growth of F. succinogenes after 60 to 70 h of degra-
dation (12).
In conclusion, fiber type influences bacterial species that grow
in fiber-enriched medium. The bacterial growth can therefore in-
dicate the bacterium’s fiber type preference. However, common
species of bacteria that share more than one type of fiber enrich-
ment were also identified. The identification of new bacterial spe-
cies and possibly new genera reveals the significance of the results
of this study. This provides an insight into the poorly researched
bacterial ecosystemof the camel and lays the foundation for future
studies.
ACKNOWLEDGMENTS
We thank Korinne Northwood (CSIRO Livestock Industries [CLI]) and
Paul Evans (CLI) for their technical assistance and advice.
Anjas A. Samsudin was a scholarship recipient from the Malaysian
Ministry of Higher Education and Universiti Putra Malaysia.
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Cellulolytic Bacteria in the foregut of the dromedary camel (Camelus dromedarius)

http://espace.library.uq.edu.au/view/UQ:287795/UQ287795_OA.pdf

Cellulolytic Bacteria in the foregut of the dromedary camel (Camelus dromedarius)

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