AUSTRALIA’S
PREMIER VETERINARY
SCIENCE TEXT
WILDLIFE & ZOOS
Oxalate-degrading bacteria, including Oxalobacter formigenes,
colonise the gastrointestinal tract of healthy koalas (Phascolarctos
WILDLIFE & ZOOS
cinereus) and those with oxalate nephrosis
KN Speight,a* M Houston-Francis,a M Mohammadi-Dehcheshmeh,a E Ebrahimie,a,b S Saputraa and DJ Trotta
Background Koalas in the Mount Lofty Ranges, South Australia,
have a high prevalence of oxalate nephrosis, or calcium oxalate
kidney crystals. Gastrointestinal tract oxalate-degrading bacteria,
particularly Oxalobacter formigenes, have been identified in other
animal species and humans, and their absence or low abundance
is postulated to increase the risk of renal oxalate diseases. This
study aimed to identify oxalate-degrading bacteria in the gastro-
intestinal tract of koalas and determine their association with oxa-
late nephrosis.
Methods Caecal and faecal samples were collected at necropsy
from 22 Mount Lofty Ranges koalas that had been euthanased on
welfare grounds, with 8 koalas found to have oxalate nephrosis
by renal histopathology. Samples were analysed by PCR for the
oxc gene, which encodes oxalyl-CoA decarboxylase, and also by
Illumina sequencing of the V3–V4 region of the bacterial 16S
rRNA gene.
Results The oxc gene was detected in 100% of koala samples,
regardless of oxalate nephrosis status. Oxalobacter formigenes was
detected in all but one faecal sample, with no difference in abun-
dance between koalas affected and unaffected by oxalate
nephrosis. Other species of known oxalate-degrading bacteria
were infrequently detected.
Conclusion This is the first study to identify Oxalobacter and
other oxalate-degrading bacterial species in koalas, but an associ-
ation with oxalate nephrosis and absence or low abundance of
Oxalobacter was not found. This suggests other mechanisms
underlie the risk of oxalate nephrosis in koalas.
Keywords gastrointestinal microflora; koalas; oxalate nephrosis;
Oxalobacter
Abbreviations OUT, operational taxonomical unit; TWC, tooth
wear classification
Aust Vet J 2019;97:166–170 doi: 10.1111/avj.12799
I
n South Australia, oxalate nephrosis is recognised as a leading
disease of the Mount Lofty Ranges koala (Phascolarctos ciner-
eus) population, reported in over 50% of rescued wild koalas
between 2008 and 2010 (n = 51)1 and more recently at 32% between
2012 and 2013 (n = 85).2 This is substantially higher than is reported
for the eastern states, in which oxalate nephrosis is diagnosed at
*Corresponding author.
a
School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy,
South Australia 5371, Australia; natasha.speight@adelaide.edu.au
b
School of Medicine, The University of Adelaide, SA, Australia
166 Australian Veterinary Journal Volume 97 No 5, May 2019
necropsy in < 5% koalas.3,4 Oxalate nephrosis is characterised by the
formation of calcium oxalate crystals within the tubules of the
kidneys, which is associated with renal dysfunction caused by
inflammation, tubule obstruction and fibrosis.1
The cause of oxalate nephrosis in koalas is currently unknown and is
likely to be complex and multifactorial. The oxalate content of die-
tary eucalyptus leaves has been investigated and the majority of leaf
samples found to be < 1% on a dry weight basis, with occasional leaf
samples having a high concentration of up to 7.5% oxalate.5 These
findings were not thought to support dietary oxalate being the pri-
mary cause of oxalate nephrosis, as koalas are eucalypt specialists,
and as such are likely to have evolved mechanisms to cope with this
level of dietary oxalate. This may be similar to other herbivorous
mammals that ingest dietary oxalate, in which gastrointestinal
oxalate-degrading bacteria have been identified6–8 and include Lacto-
bacillus spp.,9 Enterococcus faecalis10 and the specialist oxalate
degrader Oxalobacter formigenes.
Oxalobacter formigenes is a strictly anaerobic, gram-negative bacte-
rium that uses oxalate as its sole source of energy.11 It breaks down
oxalate in the gastrointestinal tract12 through the action of oxalyl-
CoA decarboxylase, which catalyses the decarboxylation of oxalyl-
CoA to yield CO2 and formyl-CoA. The first O. formigenes isolates
were cultured from the rumen contents of sheep,8 with other strains
found in caecal samples from pigs, guinea pigs13 and rats14 and in
faecal samples from humans.15 Animals ingesting diets high in oxalate,
such as the white-throated woodrat (Neotoma albigula), which pri-
marily feeds on cacti, have been found to harbour high numbers of
gastrointestinal oxalate-degrading bacteria, including O. formigenes.9
Other animal species have been shown to be able to adapt to gradually
increasing levels of dietary oxalate through colonisation with these
bacteria.16
In humans who are predisposed to oxalate disease, it has been shown
that kidney calcium oxalate stone formation is more likely to occur
if their gastrointestinal tract is not colonised with O. formigenes.17–19
The presence of O. formigenes has also been shown to help reduce
urinary oxalate excretion20 and the recurrence of stone forma-
tion.17,21 Similarly in dogs, it has been found that there is a lower
prevalence of O. formigenes in the faeces of those with calcium oxa-
late kidney stones compared with those that are unaffected.22
The role of oxalate-degrading bacteria in oxalate metabolism in
koalas, particularly those with oxalate nephrosis, is yet to be investi-
gated, but Oxalobacter has been identified in two Queensland koalas
in a study characterising the entire microbiota of caecal, colon and
faecal samples using 16S rRNA gene sequencing.23 Hence, the
© 2019 Australian Veterinary Association

present project aimed to determine if there is an association between
oxalate nephrosis and the absence or low abundance of oxalate-
degrading bacteria such as Oxalobacter in caecal and faecal samples
from Mount Lofty Ranges koalas.
Materials and methods
Sample collection from koalas
Caecal and faecal samples were obtained at necropsy from 22 rescued
wild koalas that were euthanased on welfare grounds between March
and September 2016 at the Adelaide Koala and Wildlife Hospital
and other suburban veterinary clinics. Necropsy to determine health
status was performed within 0–48 h post mortem. Koalas were
assigned to age groups using tooth wear classification (TWC) by
assessing cusp wear of the first upper premolar on a scale of I–VII,
as described previously.24 The caecal contents were collected from
the midpoint of the caecal length and faecal pellets collected from
the distal colon; all samples were stored at -80
C. Kidneys were cut
in half longitudinally, examined grossly for deposition of oxalate
crystals in the cortex, medulla and pelvis and then fixed in 10% neu-
tral buffered formalin. Kidney samples were processed by routine
histological techniques and stained with haematoxylin and eosin.
Kidney sections were assessed blind with an Olympus BX51 micro-
scope and classified as affected or unaffected by oxalate nephrosis.
Calcium oxalate deposits were identified using polarisation micros-
copy to visualise crystal birefringence, as well as by presence of
crystal ‘ghosts’, which occur when protein residues remain following
loss of crystals during processing.1
DNA extraction and PCR
Whole bacterial DNA was extracted directly from faecal and caecal
samples using a MO BIO PowerFecal® DNA Isolation Kit (MO BIO
Laboratories, Inc., CA, USA). The oligonucleotides used as PCR
primers, spanning a specific 413–415-bp region of the oxc gene, were
as designed by Sidhu et al.25 The sequences of the forward and
reverse oxc primers were 50 -AATGTAGAGTTGACTGA-30 (OXFfp)
and 50 -TTGATGCTGTTGATACG-30 (OXFrp), respectively (Sigma
Aldrich, NSW, Aust). PCR was carried out in a master mix contain-
ing 5 μL of 5× MyTaq® buffer, 1 μL each of the primers OXFfp and
OXFrp (10 μmol/L), 0.2 μL taq polymerase, 2 μL of DNA template
with the rest made up of nuclease free water to 25 μL total. The opti-
mal reaction profile was 94
C for 1 min followed by 35 cycles of
94
C for 1 min for denaturation, 53
C for 1 min for annealing,
72
C for 1 min for primer extension, and 72
C for 5 min for elonga-
tion using a T100 Thermal Cycler (BioRad, CA, USA). PCR products
were size-separated by gel electrophoresis in 1.5% agarose for
60 min at 120 V using a 100-kb DNA ladder. The gel was placed in
Gel Red™ solution (Biotium, CA, USA) for 1 h, illuminated with
ultraviolet and photographed for documentation. DNA was also
extracted from sheep rumen fluid as a positive control, because
O. formigenes was first isolated from the sheep rumen.8
Microbiome profiling and analysis
Bacterial DNA was extracted from caecal and faecal samples of oxa-
late nephrosis-positive and -negative Mount Lofty Ranges koalas
DNA concentration and purity was determined using a NanoDrop
© 2019 Australian Veterinary Association
WILDLIFE & ZOOS
spectrophotometer at wavelengths of 260 nm and 280 nm. DNA was
standardised to 5 ng/μL for each sample in accordance with sample
preparation for Illumina 16S Metagenomic Sequencing Library per
WILDLIFE & ZOOS
manufacturer’s protocol. Quality control was performed on all sam-
ples to determine the presence of the V3–V4 16S rRNA gene region
before 16S amplicon library preparation and sequencing. To this
end, PCR was performed to check the presence of the V3–V4 hyper-
variable region of the 16S rRNA gene. The primer sequences (Sigma
Aldrich, NSW) were as follows:
16sv3v4F: 50 -TCGTCGGCAGCGTCAGATGTGTATAAGAGA CA
GCCTACGGGNGGCWGCAG-30
16sv3v4R: 50 -GTCTCGTGGGCTCGGAGATGTTATAAGAGACAG
GACTACHVGGGTATCTAATCC-30 .
The optimal PCR reaction conditions were 95
C for 3 min followed
by 25 cycles of 95
C for 30 s for denaturation, 55
C for 30 s for
annealing, 72
C for 30 s for primer extension and 72
C for 5 min
for elongation using a T100 Thermal Cycler (BioRad). DNA was sent
to the Australian Cancer Research Foundation for library prepara-
tion and sequencing. The V3–V4 16S rRNA gene region was PCR
amplified by 16S Amplicon Library Preparation. The resulting
amplicon was sequenced using an Illumina MiSeq V3 machine as
paired-end 250-bp reads.
Sequenced samples were received from the Foundation in Fastq for-
mat. Samples that showed low quality and poor sequencing depth
were removed from the dataset. The CLC Microbial Genomics Mod-
ule of CLC Genomics Workbench ver. 9.5 software (Qiagen, Hilden,
Germany) was used to assign taxonomy to the reads from different
samples by clustering with representative sequences of pseudo-
species operational taxonomical units (OTUs) to compute the rela-
tive abundance of each OTU. For detection of Oxalobacter, samples
were compared with OTUs that belonged to Oxalobacter spp.
Adapter trimming, fixed length trimming, merging paired reads and
filtering based on the number of reads (to remove the reads with low
coverage) were performed to obtain sequences that were comparable.
The OTU clustering tool was used to cluster the reads and reduce
the read collection in each sample to representative sequences (clus-
ter centroids) that were 97% similar to any member of the cluster
they represent that has been deposited in the Greengenes database.26
Statistical analysis
To compare the relative abundance of O. formigenes in koala caecal
and faecal samples based on oxalate nephrosis status, normality dis-
tribution of relative abundance was confirmed by Kolmogorov-
Smirnov normality test using the Minitab-18 tool (http://www.
minitab.com). The relative abundance of O. formigenes in koala cae-
cal and faecal samples based on oxalate nephrosis status was com-
pared with both Z-test and Fisher’s exact test using Minitab 18. The
cut-off for determination of high/low relative abundance was deter-
mined according to the median of relative frequency of
O. formigenes. To compare the relative abundance of O. formigenes
in caecal and faecal samples from individual koalas, a paired t-test in
Minitab 18 was used. Pearson’s correlation was used to determine
the significance of relative abundance of O. formigenes between cae-
cal and faecal samples.
Australian Veterinary Journal Volume 97 No 5, May 2019 167
 WILDLIFE & ZOOS

Results The highest relative abundance in a caecal sample was 0.00295 from
a koala that was unaffected by oxalate nephrosis and 0.00175 in a
Koalas and oxalate nephrosis faecal sample from a koala that was affected by oxalate nephrosis.
WILDLIFE & ZOOS

Of the 22 koalas presented for necropsy, 8 were female and 14 were Overall, caecal samples had significantly higher O. formigenes relative
male and they had an average TWC of 3.1  1.1. Of these 22 koalas, abundance than faecal samples (P = 0.008) (Figure 1), but there was
8 showed gross pathology consistent with oxalate nephrosis at nec- no correlation between relative abundance in caecal and faecal sam-
ropsy based on visualisation of calcium oxalate crystals within the ples from individual koalas (P = 0.455) (Figure 2).
kidneys as described by Speight et al.1 Histopathological examination
confirmed that these 8 koalas had oxalate nephrosis and the remain- The presence of other known oxalate-degrading bacteria in the cae-
ing 14 koalas did not. cal and faecal microbiome of koalas was determined, but few were
detected. Clostridium was present at the level of genus in two
Koalas affected by oxalate nephrosis consisted of 3 females and
5 males with an average TWC of 2.5  0.9 (≈ 3 years of age). The
remaining 14 koalas classified as unaffected by oxalate nephrosis
consisted of 5 females and 9 males with an average TWC of
3.5  1.1 (≈ 4 years of age).

PCR for oxc gene

PCR for the oxc gene associated with O. formigenes was performed
on faecal and caecal samples from the 22 koalas. The oxc gene was
found in all faecal and caecal samples tested (100%), regardless of
oxalate nephrosis status.

Microbiome analysis for oxalate-degrading bacteria

Microbiome analysis was performed on 19 caecal (7 affected, 12 unaf-
fected) and 19 faecal samples (7 affected, 12 unaffected) from a total
of 21 koalas. Oxalobacter formigenes was detected in all caecal sam-
ples and all but one faecal sample, which was from a koala with oxa-
late nephrosis and also the lowest caecal relative abundance
(0.000402%). There was no statistical difference in the relative abun-
dance of O. formigenes between koalas based on oxalate nephrosis Figure 1. Relative abundance of Oxalobacter formigenes in caecal and
faecal samples from Mount Lofty Ranges koalas.
status for either caecal (P = 0.479) or faecal (P = 0.964) samples
(Table 1).

Using the caecal sample median relative abundance (0.000943) as a

cut-off, there appeared to be a higher proportion of koalas affected
by oxalate nephrosis (5/10) with a relative abundance of
O. formigenes ≥ 0.000943 than those below the cut-off (2/9), but this
was not statistically significant (P = 0.35). Using the faecal sample
median relative abundance (0.000435) as a cut-off, there appeared to
be a lower proportion of koalas affected by oxalate nephrosis (2/8)
with a relative abundance of O. formigenes ≥ 0.000435 than those
below this median (5/11); however, these proportions were not sig-
nificantly different (P = 0.63).

Table 1. Relative abundance of Oxalobacter formigenes in caecal and

faecal microbiome of Mount Lofty Ranges koalas unaffected and
affected by oxalate nephrosis

Oxalate n Caecal Faecal

nephrosis
status
Mean (%) SD Mean (%) SD

Unaffected 12 0.000987 0.000641 0.000431 0.000369 Figure 2. Relative abundance of Oxalobacter formigenes in caecal and
Affected 7 0.001206 0.000624 0.000442 0.000593 faecal samples from koalas affected (positive) and unaffected (negative)
by oxalate nephrosis.

168 Australian Veterinary Journal Volume 97 No 5, May 2019 © 2019 Australian Veterinary Association

samples at low relative abundance, some samples showed Pseudomo-
nas veronii and one sample showed Streptococcus. Lactobacillus and
Lactococcus spp. were not detected.
Discussion
In this study we aimed to determine if the absence or a low abun-
dance of oxalate-degrading bacteria, including O. formigenes, was
associated with the occurrence of oxalate nephrosis in Mount Lofty
Ranges koalas. However, both PCR and microbiome analyses
showed that O. formigenes was ubiquitous in caecal and faecal sam-
ples from Mount Lofty Ranges koalas, irrespective of oxalate nephro-
sis status. There was also no significant difference found in the
relative abundance of O. formigenes between affected and unaffected
koalas, nor for other oxalate-degrading bacteria.
The microbiome analysis targeted the variable regions 3 and 4 for
delineation of the 16S rRNA gene sequence representing Oxalobacter
species compared with the total bacterial population and found that
all but one sample from the koalas, regardless of oxalate nephrosis
status, was positive for the Oxalobacter 16S rRNA gene. All of the
koalas were also positive for the O. formigenes-specific oxc gene by
PCR. The single koala that was negative for the Oxalobacter 16S
rRNA gene in faeces had the lowest caecal abundance of
O. formigenes. This may indicate that lower abundance of Oxalobac-
ter may not be readily detected by Illumina MiSeq sequencing
methodology.
Barker et al.23 were able to detect the presence of the Oxalobacter
16S rRNA gene by sequencing variable regions (V1–V3) in caecal
and faecal samples from the hindgut of two wild Queensland koalas.
They also showed a variation in bacterial abundance between the
sampled gut regions, as well as high variation between the two
koalas. However, the relative abundance results for Oxalobacter
were much higher in the two Queensland koalas for both caecal
samples (0.05% and 0.7%) and faeces (0.16% and 0.08%) (see their
supplementary material) than we found in our study (4.89E-05 -
0.00295%). This may relate to the different variable gene regions
targeted (V3–V4 in our study vs V1–V3).
However, it is possible that the lower overall abundance of Oxalobac-
ter spp. in the Mount Lofty Ranges koalas compared with the two
Queensland koalas in the previous study23 could be partly related to
the higher occurrence of oxalate nephrosis in Mount Lofty koalas.1,27
A lower abundance of oxalate-degrading bacteria could increase the
amount of oxalate absorbed from the gut into the bloodstream and
increase the risk of renal oxalate deposition. This, coupled with the
findings of a previous study in which dietary leaf oxalate content in
Mount Lofty Ranges eucalyptus leaves was approximately double
that of those in Moggill, Queensland, also supports this.5 Usually,
animals with higher oxalate diets would have a higher abundance of
oxalate-degrading bacteria;16 for example, the woodrat, which has a
relative Oxalobacter abundance of up to 0.02%.9 However, the lower
overall Oxalobacter abundance in the Mount Lofty Ranges koalas
still does not explain why no differences in abundance were seen
between those with oxalate nephrosis and those that were not
affected.
© 2019 Australian Veterinary Association
WILDLIFE & ZOOS
The lack of other species of oxalate-degrading bacteria in the gut
microbiome of all the Mount Lofty Ranges koalas was equally unex-
pected. In the woodrat, Lactobacillus was found to be the most abun-
WILDLIFE & ZOOS
dant bacterial genus with oxalate-degrading activity, with up to 13%
relative abundance in the foregut chamber that is proximal to the
stomach, but only < 0.05% in faeces and the caecum.9 Koalas lack a
foregut chamber and this absence of microbial oxalate degradation
prior to the stomach and small intestine could increase oxalate
absorption before its degradation in the hindgut. In the woodrat,
however, O. formigenes was most abundant in the caecum, showing
that the hindgut remains important for oxalate degradation activity.
The presence of oxalate-degrading bacteria throughout the gastroin-
testinal tract of the woodrat likely explains the remarkable ability of
this species to cope with its high oxalate diet28 and suggests that all
gut regions should be investigated in koalas.
If oxalate-degrading bacteria were found to play a protective role in
the development and severity of oxalate nephrosis in koalas, they
could potentially be administered as oral probiotic therapy. Human
studies have found that oral administration of O. formigenes can
result in reduced urinary and plasma oxalate20,21 and hence shows
promise for commercial probiotic development.29 However, issues of
poor gut colonisation21 and variable urinary oxalate reduction30 need
further research. As an obligate anaerobe, O. formigenes is difficult to
culture, but recently the OxcC13 strain has emerged as a good candi-
date for future probiotic trials because of its aerotolerance and ability
to persist in low oxalate environments.31,32 An O. formigenes probi-
otic could be given to koala joeys at the time that they acquire their
gastrointestinal tract symbiotic microbiota from their mother via
‘pap’ feeding, in which the mother produces a soft faeces, thought to
be derived from the caecum, for the joey to ingest.33 This usually
occurs at first emergence from the pouch, a few weeks prior to the
transition from milk to leaf diet.34
In conclusion, this study identified the presence of O. formigenes in
caecal and faecal samples of Mount Lofty Ranges koalas using PCR
and microbiome profiling. However, an association between the
presence and abundance of gastrointestinal O. formigenes and oxa-
late nephrosis status was not found. Furthermore, other oxalate-
degrading bacteria were not found to play a significant role in koalas.
The role of O. formigenes in the koala, which has a highly developed
hindgut to digest its specialist eucalypt diet, and its relation to die-
tary oxalate intake and development of the disease oxalate nephrosis,
needs further investigation.
It is likely that the pathogenesis of oxalate nephrosis in koalas is
complex, involving a combination of dietary, metabolic, environ-
mental and genetic factors. It is possible that an underlying inherited
condition of abnormal oxalate metabolism, similar to that which
occurs in humans and as has recently been reported in the
Australian native Gilbert’s potoroo,35 could be affecting the Mount
Lofty Ranges koalas. In humans this inherited disease, primary
hyperoxaluria, involves decreased activity of key metabolic liver
enzymes,36 leading to increased endogenous oxalate production and
kidney stone disease.37 If a similar underlying genetic condition
occurs in koalas, dietary oxalate and gastrointestinal oxalate-
degrading bacteria would still play an important role in the manifes-
tation of the disease, as in humans.
Australian Veterinary Journal Volume 97 No 5, May 2019 169
 WILDLIFE & ZOOS
Acknowledgments
Thanks are owed to Jessica Fabijan, Adrian Hines, the Adelaide
WILDLIFE & ZOOS
Koala and Wildlife Hospital and other Adelaide veterinary clinics.
Thanks also to Elizabeth Hickey for help with DNA extraction
and PCR.
Conflict of Interest and Funding
The authors declare no conflicts of interest or sources of funding for
the work presented here.
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(Accepted for publication 21 February 2019)
© 2019 Australian Veterinary Association