Veterinary Microbiology 166 (2013) 429–437

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Detection of Helicobacter species in the gastrointestinal tract

of ringtail possum and koala: Possible influence of diet, on the
gut microbiota
Thosaporn Coldham a, Karrie Rose b, Jani O’Rourke a, Brett A. Neilan a,
Helen Dalton a, Adrian Lee a, Hazel Mitchell a,*
a
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia
b
Veterinary & Quarantine Centre, Taronga Zoo, Australia

A R T I C L E I N F O A B S T R A C T

Article history: The presence of Helicobacter spp. was examined in the liver and in different regions of the
Received 7 October 2012 gastrointestinal tract (GIT) including the stomach, 3 cm above ileum, ileum, caecum, colon
Received in revised form 21 May 2013 and rectum of 10 ringtail possums (RTPs) and 3 koalas using a combination of microscopy,
Accepted 19 June 2013 culture and PCR. Helicobacter was detected in the distal end of the GIT of 7 of 10 RTPs by
direct PCR and in all (10/10) RTPs by nested PCR. Five ‘S’ shaped isolates with bipolar
Keywords: sheathed flagella were isolated from the lower bowel of 3 of the 10 RTPs. 16S rRNA
Ringtail possum sequence analysis of these 5 isolates confirmed them as potentially novel Helicobacter
Koala
species. No Helicobacter species were cultured from the koalas, however Helicobacter DNA
Helicobacter species
was detected, in the majority of liver and/or stomach samples of the three koalas and in the
Phylogenetic study
Transmission electron microscopy
colonic region of one koala, using nested PCR. The 16S rRNA gene was sequenced directly
from DNA extracted from the homogenised livers and mucus scrapings of the stomach
from koala 1 and were confirmed to be Helicobacter species. Based on histopathological
examination of sections from the liver and intestine no evidence of infection could be
related to the presence of helicobacters in either the RTP or koala. Based on our results, it is
possible that diet may influence the detection of Helicobacter species; however this
required further investigation.
ß 2013 Elsevier B.V. All rights reserved.

1. Introduction differ significantly to the microorganisms found in the

lumen of GIT (Lee, 1991). In addition the composition of
One important component of the gastrointestinal these microorganisms has been reported to not only vary
ecosystem is the subset of microbiota that colonise the at different locations within the GIT but also among
surface of the gastrointestinal mucosa of mammals. To be different animal species. The major bacterial genera that
able to colonise the gastrointestinal tract (GIT) this compose the actively motile, spiral-shaped mucosa-
complex community of organisms must be able to thrive associated microbiota include Borrelia, Spirillum, Helico-
in the environment and utilise nutritional conditions that bacter and Campylobacter (Solnick and Schauer, 2001). To
exist there. Often these mucosa-associated microorgan- date members of the genus Helicobacter have been cultured
isms are actively motile, spiral-shaped organisms that from the GIT of humans as well as from a range of placental
mammals and birds (Solnick and Schauer, 2001; Fox,
2002). In addition a recent study by our group has reported
up to three morphologically different – comma, fusiform
* Corresponding author. Tel.: +61 02 9385 2040; fax: +61 02 9385 1483.
E-mail address: [email protected] (H. Mitchell).
and spiral shaped Helicobacter species colonising mucus

0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2013.06.026
430 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437

layer of the lower bowel of an Australian marsupial, the Australia. All RTPs were non-caged animals while the
common brushtail possum (Trichosurus vulpecula, BTP) koalas were zoo animals. These marsupials had been
(Coldham et al., 2011). injured or were in ill health and had subsequently died or
Only four tree-dwelling Australian marsupial herbi- had been euthanised for compassionate reasons. For each
vores that feed mainly on eucalypt leaves have been animal, three samples of tissue were collected from each of
described, the common brushtail possum, the common the following sites: the liver, stomach, mid ileum, ileum at
ringtail possum (Pseudocheirus peregrinus, RTP), the koala 3 cm above the caecum (3-ileum), caecum, colon and
(Phascolarctos cinereus) and the greater glider (Petauroides rectum. The first sample from a particular location was
volans) (Kerle, 2001). All of these are caecum fermenters frozen at 70 8C for DNA extraction and PCR amplification.
thus the majority of plant cell wall digestion takes place in The second sample was frozen in Brain heart Infusion–
a greatly expanded caecum (Hume, 1999). Glycerol medium (BHIG) and kept at 70 8C until cultured.
The aim of the current study was to determine if, given The third sample was fixed in formalin for histology.
the high abundance of tannins and phenols obtained from
ingestion of eucalypt leaves in the RTP and koalas, 2.2. Bacterial isolation and biochemical characterisation
Helicobacter spp., are present in the GIT and the liver of
these marsupials. To answer this question the presence of Homogenised liver samples and scrapings of gastro-
Helicobacter spp., was determined in different regions of the intestinal mucus were cultured on horse blood agar plate
GIT of RTPs and koalas using a combination of three (HBA-composition per 100 ml: sterile defibrinated horse
methods: microscopy, culture and PCR. This approach is blood 5 ml, Amphotericin 25 mg and blood agar base No. 2
essential as no individual method is suitable for the (Oxoid) 3.5 g) and on campylobacter selective agar plates
detection of Helicobacter species, as firstly bacterial mor- [CSA: 100 ml of HBA plus Skirrow’s selective supplement
phology (from fresh smear or silver stain section) can only (Polymyxin B (Sigma) 2.5 mg/ml, Vancomycin (Eli Lilly &
serve as a guide for the detection of helicobacters, secondly Co., Australia) 10 mg/ml and Trimethoprim (Sigma) 5 mg/
some Helicobacter species are likely to be non-cultivable ml)]. Both the direct inoculation method and a selective
using current methodologies and thirdly Helicobacter genus filtration method (Robertson et al., 2001; Coldham et al.,
specific PCR based on a selected primer set might not detect 2011) were used in this study.
all Helicobacter species/strains, including unknown Helico- The phenotypic characteristics of all isolates were
bacter spp. that might present in Australian marsupials. determined using standardised methods recommended in
Furthermore PCR inhibitors may be present in the DNA an extensive identification scheme designed for Campylo-
extracted from the tissue samples. To reduce any effect of bacter, Helicobacter and related bacteria (On et al., 1996;
inhibitors that could be present in the samples, nested PCR Coldham et al., 2011). Nitrate reduction and gamma-
was performed in parallel to direct PCR. In addition, the glutamyl transferase activity were examined using the
colonisation location of Helicobacter spp. in the GIT was API-Campy Identification System (BioMe’rieux, Marcy-I’
examined using Fluorescent in situ hybridisation (FISH) on Etoile, France). Indoxyl acetate hydrolysis was examined
tissue sections in which Helicobacter spp. had been detected using the disc method (Hodge et al., 1990). Alkaline
by all three methods. To investigate signs of any pathology phosphatase and hippurate hydrolysis was examined
associated with Helicobacter spp. as has been observed in using Rosco diagnostic tablets (UTEC diagnostics, Den-
other animals histopathological analysis was conducted on mark). Susceptibility to nalidixic acid (30 mg, NA 30,
all specimens. Oxoid), cephalothin (30 mg, KF 30, Oxoid) and metronida-
The detection and isolation rates of these bacteria in the zole (5 mg, MTZ 5, Oxoid) was determined by disc diffusion
RTP were then compared with that in koalas to determine on HBA plates. Strains were determined as sensitive if
any possible impact of diet, feeding strategies or the type of there was a zone of inhibition and resistant if there was no
digestive system on the colonisation of the GIT by zone (Karmali et al., 1980; Coldham et al., 2011).
Helicobacter spp. Currently only three of four tree-dwelling Freshly grown bacteria from HBA plates were negatively
Australian marsupial herbivores have been studied the RTP stained with 2% uranyl acetate on a colloidal/carbon grid
and koala in this study and the BTP in a previous study by (483 or 400 mesh), and were examined using transmission
our group (Coldham et al., 2011). Given that the same electron microscopy (H7000-Hitachi, Tokyo, Japan).
methodologies were used in both studies we compared the
results obtained in the RTP and koala with those previously 2.3. DNA extraction and PCR
reported in the BTP.
DNA from the liver and GIT mucus scrapings of all 10
RTPs and 3 koalas was extracted using the phenol
2. Materials and methods
chloroform method. Briefly, DNA was extracted using
2.1. Animal and collection of specimens proteinase K digestion, followed by phenol, phenol–
chloroform–isoamyl alcohol (25:24:1) and chloroform
Liver and GIT specimens from 10 ringtail possums: 6 extraction and finally ethanol precipitation. DNA from
female and 2 male adults, 1 unspecified sex juvenile and 1 bacterial cells was extracted using the puregene DNA
unspecified sex and age (named RTP 1–RTP 10) and 3 isolation kit (Gentra systems, QIAGEN, Australia).
koalas: 1 male juvenile, 1 female adult and 1 male adult, Helicobacter genus-specific PCR (direct PCR) was
(named koala 1 – koala 3) were collected from the performed using primers H276f (50 -CTATGACGGG-
Veterinary & Quarantine centre, Taronga Zoo, Sydney, TATCCGGC-30 , E. coli position 276–293) and H676r (50 -
 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 431

ATTCCACCTACCTCTCCCA-30 , E. coli position 676–658) (Riley (Amann et al., 1992) labelled with fluorescein-isothiocya-
et al., 1996) as described previously by Coldham et al. nate (FITC)] and HRh [Helicobacter genus specific probe, 50 -
(2011). The PCR reactions underwent an initial denaturation TCTCAGGCCGGATACCCGTCATAGCCT-30 (Fox et al., 1992),
period at 94 8C for 5 min followed by 35 cycles of labelled with tetramethyl-rhodamine-isothiocyanate
denaturation at 94 8C for 5 s, annealing at 57 8C for 5 s and (TRITC) (GENSET Pacific Pty. Ltd., Lismore, Australia]. The
extension at 72 8C for 30 s followed by a final extension at cell control suspensions were fixed in 4% paraformaldehyde.
72 8C for 2 min. The PCR was conducted in a Sprint thermal Fixed H. pylori strain SS1 (positive control) and Psychrobacter
cycler (Hybaid, Ashford, Middlesex, United Kingdom). spp. strain SW5 (negative control) were used as bacterial cell
Nested PCR was conducted by amplifying the prokar- controls in every test. A full description of the FISH
yote 16S RNA gene using the universal bacterial primers, technique has been described previously in the paper by
F27 (50 -AGAGTTGATCCTGGCTCAG-30 , E. coli position 8–27) Coldham et al. (2011).
and R1494 (50 -TACGGCTACCTTGTTACGAC-30 , E. coli posi-
tion 1494–1513) (Neilan et al., 1997; Weisburg et al., 2.6. Histopathology
1991). Reactions in the first round of the PCR underwent an
initial denaturation period at 94 8C for 5 min, followed by Four-micron sections of paraffin embedded samples
30 cycles of 94 8C for 10 s, 50 8C for 15 s and 72 8C for 2 min were cut and stained using a modified Steiner silver stain
and a final extension at 72 8C for 7 min. The PCR product and a haematoxylin & eosin (H&E) stain. The silver stained
(1:10 dilution) of this first round PCR was then used as a sections were examined for the presences of spiral/helical
template to amplify Helicobacter genus specific DNA (direct shaped bacteria and the H&E stained sections for
PCR) in the second round PCR, using primers H276f and histopathological changes.
H676r as described above.

3. Results
2.4. Sequence analysis
3.1. Bacterial cultivation
PCR amplification of the 16S rRNA gene was conducted
using the universal bacterial primers F27 and R1494 Following bacterial culture, phase contrast microscopy
(Neilan et al., 1997; Weisburg et al., 1991). The PCR was conducted on suspect colonies, and any bacteria with a
product obtained from this reaction was then used as DNA thin spiral or fusiform shape that were actively motile
template for the sequencing reaction, which employed were selected for confirmation using a Helicobacter genus
each of the following primers: H276f, H676r (Riley et al., specific PCR. In general these bacteria appeared as a thin
1996), F27, R1494, 341R, 530F, 1115F and 1220R (Neilan smear or very small and clear colonies on both HBA and
et al., 1997; Weisburg et al., 1991). 16S rRNA sequences CSA (Fig. 1). In cases where no typical colonies showing
were assembled and compared with the sequences of other actively motile thin spiral or fusiform shape bacteria were
bacteria in the GenBank. Sequences were aligned using observed, at least one to two isolates of curved to spiral
alignment tools in the ClustalX package (Thompson et al., shaped bacteria were collected for confirmation from each
1997) and the phylogenetic tree was constructed by the animal. Of the 10 RTPs examined, 5 of 13 individual
neighbour-joining method of Saitou and Nei (1987) as cultures of spiral to curved shaped bacteria were identified
described in the paper by Coldham et al. (2011). as Helicobacter species using the Helicobacter genus specific
PCR. These isolates cultured from 3 different RTPs (RTP1,
2.5. Fluorescent in situ hybridisation (FISH) RTP5 and RTP 9) were further examined using transmis-
sion electron microscopy (TEM). The negative stains
The oligonucleotide probes used were EUB338-FITC showed that they were all ‘S’ shaped; measuring 0.3 by
[Eubacterial 16S rRNA probe, 50 -GCTGCCTCCCGTAGGAGT-30 2.5 mm in size; with bipolar sheathed flagella and no

Fig. 1. The appearance of tiny pinpoint colonies (A) and a thin water-like film (B) on the HBA or CSA plates.
432 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437

and compared to other sequences in the GenBank

database. The 16S rRNA sequences have been deposited
in GenBank and the accession numbers for RTP 1 to RTP 5
are AY554130–AY554134 respectively. The sequences
obtained from these 5 isolates showed 95% similarity to
Helicobacter sp. MIT 99-5507 isolated from rhesus monkey
2 (accession number AF33334) (Fox et al., 2001) and 94%
identical to H. canis and H. hepaticus. Given this low level of
homology, it is likely that the RTP isolates constitute a new
species. The similarity among the 16S rRNA gene
sequences of the 5 isolates was 99.6–99.9%, indicating
that the five isolates potentially are the same species.
Helicobacter species DNA was detected by nested PCR in
the homogenised livers of all 3 koalas as well as from the
stomach of koala 1 and koala 3. To confirm that these were
truly Helicobacter species we amplified and sequenced the
Fig. 2. A transmission electron micrograph of an ‘S’ shaped Helicobacter 16S rRNA gene directly from DNA extracted from the
isolate, RTP5S. The bacterium measured 0.3 mm  2.5 mm and had
homogenised liver of koala 1 (1318 bp) and mucus
bipolar-sheathed flagella.
scrapings of the stomach of koala 1 (1071 bp). Comparison
of these sequences to other sequences in the GenBank
periplasmic fibres (Fig. 2). These were designated as database showed the DNA from the liver of koala 1 to have
isolates RTP1S to RTP5S. 99% similarity to Helicobacter bilis and the DNA from the
From the 3 koalas, no bacteria having a typical thin stomach of koala 1 to have 99% similarity to Helicobacter
spiral or fusiform shape were observed on microscopy and felis. The 16S rRNA accession numbers in GenBank for koala
no thin smears or any typical small and clear colonies were 1 liver is AY583532 and for koala 1 stomach is AY583533.
found on HBA or CSA. Although six individual isolates Based on our results we constructed a phylogenetic tree
having a curved rod shape appearance were investigated, using the 5 RTP isolates, the 18 BTP isolates obtained from
none were identified as Helicobacter species using the our previous study on brushtail possums (Coldham et al.,
Helicobacter genus specific PCR. Based on their non-typical 2011), 35 members of the genera Helicobacter (including
colony appearance, bacterial morphology and the results of 32 validly published species) and three closely related
PCR we concluded that these isolates were not Helicobacter species; Wolinella succinogenes, Arcobacter butzleri and
species. As this study aimed to determine the presence of Campylobacter jejuni (Fig. 3). The sequences obtained from
Helicobacter spp., we did not undertake any further ‘BTP1C to BTP9C’ (comma shaped), ‘BTP1F to BTP6F’
identification of these isolates. (fusiform shaped), ‘BTP 1S to BTP3S’ (S shaped) and ‘RTP1S
The results of culture, the location from which the isolates to RTP5S’ (S shaped) isolates were shown to cluster into 4
were cultivated and the direct and nested PCR results groups, supporting the view that these 4 groups constitute
obtained from the 10 RTP’s and 3 koalas are shown in Table 1. 4 different species.
As 16S rRNA analysis alone does not always provide
3.2. 16S rRNA gene sequencing analysis and biochemical conclusive evidence for species level identification and
characteristics may prove highly misleading (Vandamme et al., 2000), all 5
RTP isolates were further tested for biochemical char-
Sequencing of the near complete 16S rRNA gene acteristics and susceptibly to nalidixic acid, cephalothin
(1500 bp) of the 5 isolates from the RTPs was undertaken and metronidazole. The results of these tests and a

Table 1
The results of Helicobacter culture, direct and nested PCR obtained from the 10 RTPs and 3 koalas.

Animals Regions

Liver Stomach Mid ileum 3-Ileum Caecum Colon Rectum

RTP 1 _ N N C, N N C, dP, N dP, N

RTP 2 _ _ _ _ _ _ N
RTP 3 _ _ _ _ _ N N
RTP 4 _ _ _ _ _ dP, N dP, N
RTP 5 _ _ _ N N C, N dP, N
RTP 6 _ _ _ _ _ N N
RTP 7 _ _ _ _ _ N dP, N
RTP 8 _ _ _ _ _ N dP, N
RTP 9 _ _ _ _ N C, dP, N C, dP, N
RTP 10 _ _ _ _ _ _ dP, N
Koala 1 N N N _ _ N _
Koala 2 N _ _ _ _ _ _
Koala 3 N N _ _ _ _ _

C = Helicobacter culture positive; dP = direct PCR positive; N = nested PCR positive; – = Helicobacter culture, direct and nested PCR, all negative.
 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 433

Fig. 3. Phylogenetic tree for RTP1S-RTP5S isolates, 18 BTP isolates (Coldham et al., 2011), 34 members of the genera Helicobacter (include 32 validly
published species), and three closely related species: Wolinella succinogenes, Arcobacter butzleri and Campylobacter jejuni. The scale bar represents 0.01
expected substitutes per site.

comparison with other validly published Helicobacter caecum and colonic regions of koala 1 are shown in
species is shown in Table 2. All 5 RTP isolates were found Fig. 4.
to possess characteristics common to Helicobacter species, As isolation and detection of helicobacters using culture
including catalase positivity, hippurate hydrolysis nega- and direct PCR was unsuccessful in the koala the spatial
tive, urease activity negative, sensitive to cephalothin and distribution of bacteria belonging to the genus Helicobac-
resistance to nalidixic acid. ter, using FISH, was not performed in any of the koalas as
we considered that the numbers of helicobacters if present,
3.3. The spatial distribution of mucus-associated would be too low for microscopic detection. The FISH
microorganisms in fixed sections of the liver and different technique was however performed to determine colonisa-
regions of the GIT of RTPs and koalas tion location in a formalin-fixed colonic section of RTP 9 in
which Helicobacter species had been isolated as well as
No microorganisms were observed in any of the silver detected by PCR. Bacteria belonging to the Helicobacter
stained sections of the liver of RTPs or koalas using light genus were observed predominantly colonising the mucus
microscopy. Due to a large number of clumps of layer lining the epithelium and in the crypts of the colon
bacteria-like particles present in the luminal contents, (Fig. 5).
detection of mucus-associated microorganisms in the
silver stained sections from different regions of the 3.4. Histopathology
ringtail possum GIT was problematic. However small
numbers of long, curved to spiral shaped organisms were Histopathological examination of the liver sections
observed in the mucus layer overlying the epithelium from the RTP showed all to be within the normal range. No
and in the crypts of the caecum, colon and rectum of RTP significant lesions were observed in the stomachs. The
1, RTP 5 and RTP 9. Examples of the distribution of these lower bowel of all RTPs include RTP 1, RTP 5 and RTP 9 from
mucus-associated bacteria in the colonic region of RTP 9 which Helicobacter species were isolated, was normal
are shown in Fig. 4. Microscopic examination of silver showing only a few infiltrations of mononuclear cells and
stained sections obtained from the 3 koalas showed 2 eosinophils in the intestinal mucosa.
predominant morphological types of microorganisms, No significant change in histopathology was observed
cocci and filamentous bacillary forms. Small numbers of in the liver, stomachs or the lower bowel of any of the
long, curved to spiral shaped organisms observed in the koalas.
434 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437

+, 100% strains positive; , 100% strains negative; V, variable results; (), 7–38% strains negative; S, sensitive; R, resistant; I, intermediate; ND, not determined; NA, not applicable; Bp, bipolar; Mp, monopolar; Pt,
4. Discussion
Distribution
of flagella In the current study we used a combination of

Mp
NA
Bp

Bp
Bp
Bp
Bp
Bp
Bp
Bp
Bp

Bp

Bp
microscopic examination, culture and PCR to detect

Pt
Helicobacter species in the RTP’s and koalas as we
flagella

10–14
No. of

considered that none of these methods alone was sufficient

6–12

3–14
7–10

1–2

5–7

4–8
4–8
NA for the detection of new or unknown Helicobacter species.
2

2
2
2

1
As spiral or fusiform shape is not limited to Helicobacter
Periplasmic

species, microscopic examination can only be used as a

(0/5)

+(5/5)

guideline for the detection of Helicobacter species. Among

fibres

NA

the three methods used in this study, culture is the gold








+
+

+
standard and PCR, and in particular nested PCR, is the most
With 1%
glycine

sensitive detection method. In the current study a

+(5/5)
+(7/7)
+(5/5)

Helicobacter genus specific PCR using H276f and H676r

ND

ND









+

primers was employed for the detection of Helicobacter

At 42 8c
Growth

spp., in DNA extracted from the liver and tissue mucus

(0/5)
(0/7)
(0/5)

()
scrapings, as well as for confirmation of the identity of




V
+
+
+
+

+
+

bacterial isolates. As this primer pair had previously been

Nalidxic acid

shown to amplify some Wolinella species, a genus closely

(30 mg disc)

related to Helicobacter spp. (Oxley et al., 2004), we

R(5/5)
R(7/7)
R(5/5)

confirmed the results of the Helicobacter genus specific

PCR positive samples by sequencing the 16S rRNA gene and
Susceptibility to

R
R

R
R
R
R

R
S

S
S

comparing the sequence with those sequences in GenBank.

Cephalothin
(30 mg disc)

In any study where primers based on current knowl-

edge of a particular genus are used to detect unknown
R(7/7)
R(5/5)
S(5/5)

species/strains it is impossible to predict the possibility of

R
R
R

R
R
R

R
R
S

S
I

mismatches. This proved to be so in the current study, as a

hydrolysis

single internal mismatch between the Helicobacter genus

Indoxyl
acetate

V (4/7)
(0/5)

(0/5)

specific primers we employed and the Helicobacter species

ND

isolated from the RTP. (C:T (H676r primer 50 -ATTCCACC-




+

+
+

TACCTCTCCCA-30 : 5 RTP isolates 50 -ATTCCACT-

transferase

TACCTCTCCCA-30 ) was observed. However, given the

Glutamyl

activity

(5/5)
(0/7)

internal location of this mismatch it is unlikely to have

+(5/5)

ND

had any significant effect on the PCR product yield (Kwok





+
+

+
+
+

et al., 1990), a view supported by the fact that H276f and

phosphatase
Characteristics that differentiate ‘S’ shaped RTP isolates from other Helicobacter species.

H676r successfully amplified all 5 RTP isolates.

Alkaline

As shown in Table 1, Helicobacter species were found

activity

(0/5)
(0/7)
(0/5)

colonising the distal end (colon and rectum) of the GIT of

Helicobacter species isolated from Brushtail possum (Coldham et al., 2011).








V
+

+
+
+

all RTP’s, except RTP1 in which one Helicobacter spp. was

isolated from the ileum at 3 cm above the caecum. In this
hydrolysis
Hippurate

animal, Helicobacter DNA was detected in all regions of the

(0/5)
(0/7)
+(5/5)

GIT except for the liver. According to this animals’ history,

ND

ND











it was found dead, and thus it is impossible to rule out the

activity

possibility that bacteria from the distal end of the GIT may
Urease

(0/5)
(0/7)
+(5/5)

have translocated to other sites (the stomach and small






+
+

+
+

intestine) following death, prior to sample collection. In

reduction

RTP 2, 3 and 6, Helicobacter DNA was detected only at the

Nitrate

(0/5)
+(7/7)
v(3/5)

distal end of the GIT using nested PCR, suggesting that

Helicobacters were present in very low numbers in these






V
+

+
+
+

animals. The colonisation of Helicobacter species appeared

production

to have no impact on the histopathology of the liver or GIT

Catalase

+(5/5)
+(7/7)
+(5/5)

of the RTPs.
()

The cultivation rate of Helicobacter species, as compared


+
+
+

+
+
+
+
+
+

with detection using PCR, was low. In the rectal region of

BTP ‘fusiform’ shapeda
BTP ‘comma’ shapeda

the RTP, only one Helicobacter isolate was cultured (RTP 9)

while Helicobacter DNA was detected in the rectal region of
RTP ‘S’ shaped

7 RTPs using direct PCR and in 10 using nested PCR. This

H. muridarum
H. canadensis

H. trogontum
H. hepaticus

H. pullorum

H. mustelae
H. ganmani

peritrichous.

low cultivation rate of Helicobacter species in the RTP’s

H. aurati

H. pylori
H. canis

might relate to the fact that specific nutritional require-

H. bilis
Taxon
Table 2

ments were absent in the media used for their culture. For
a

example, it is possible that they may require specific plant

 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 435

Fig. 4. Photomicrographs of silver stained GIT sections obtained from (A) the caecum of koala 1, (B and C) the colon of koala 1 and (D–F) the colon of RTP 9.
The arrow indicated curve to spiral shaped bacteria. Magnification = 1000.

secondary metabolites that are present in their GIT for the possibility that the koalas diet, which consists of
growth, or it may be that some Helicobacter species in the mainly eucalypt leaves could limit colonisation by
RTP are sensitive to the aerobic conditions created during Helicobacter in their lower bowel. Although the koalas in
sample collection. While no Helicobacter species were this study were zoo animals while the RTPs were wild, in
isolated from the 3 koalas investigated, Helicobacter DNA terms of their diet, this is unlikely to have impacted on the
was detected using nested PCR in the liver of all 3 koalas, study, as the koalas spent the majority of their life in trees
the stomach of 2 koalas (koalas 1 and 3) and the ileum and within the zoo and feeding mainly on fresh eucalypt leaves.
colon of koala 1. Sequencing analysis of DNA extracted The cultivation rate of Helicobacter spp. in the wild RTPs (3
from the liver and stomach mucus scrapings of koala 1 of 10 animals) and in the koalas (0 of 3 animals) was
confirmed that Helicobacter species were present in this considerably lower than that previously reported in wild
koala. BTP’s (11 of 11 animals) (Coldham et al., 2011).
The higher cultivation and detection rates of Helico- Given that RTP and BTP are very closely related species,
bacter spp. in the RTP’s as compared with the koalas raises believed to have diverged approximately 55 million years
436 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437

entirely on leaves, shoots or flowers, from only one or

two Eucalyptus species and are coprophagic, the diet of
BTP’s consists not only of eucalypt leaves but also of fruits,
grasses, herbs, insects and meat (Kerle, 2001; Hume, 1999;
Strahan, 1983). Further, unlike BTP who have four pointed
cusps molars, RTP have crescent shaped molars which
increase their ability to grind leaves into very fine particles,
resulting in the release of high amounts of anti-nutrients or
plant secondary metabolites present in eucalypt leaves
(Kerle, 2001; Hume, 1999). Furthermore, RTP possesses a
specific mechanism for controlling the flow of different
sized particles between the caecum and the colon.
Separation of different sized food particles occurs in the
proximal colon, with small particles (including plant
secondary metabolites) being pushed back into the
caecum, whilst large particles continue their passage
through the colon (Kerle, 2001; Hume, 1999). The low
detection levels of Helicobacter in the caecum of the RTP
suggests that Helicobacter species may not have the ability
to utilise the solutes and fine particles, or tolerate the plant
secondary metabolites, implying that they may not be
involved with fermentation. In contrast there is no
selective retention of digesta in the GIT of the BTP, thus
different sized particles do not move through the gut at
different rates.
Interestingly the caecum and proximal colon of koalas
contained a higher amount of small particles than either
the stomach or the distal colon, which may relate to the
selective retention of solutes and fine particles in the
caecum and the proximal colon, which are believed to be
important for microbial fermentation (Hume, 1999). Thus,
in the caecum and proximal colon of the koala conditions
may be unsuitable for Helicobacter spp.
In conclusion the presence of Helicobacter spp. in koalas
and RTPs provides further evidence that Helicobacter
species can colonise the GIT of Australian Marsupials.
The three marsupials studied to date belong to the order
Diprotodontia and are ‘hindgut fermenting’ herbivorous
marsupials. Australian Marsupials can however, based on
their dietary requirements and the structure of their GIT,
be classified into three feeding types, carnivores, omni-
vores and herbivores. Future studies, which investigate the
presence of Helicobacter species in carnivores and omni-
Fig. 5. Confocal-fluorescent photomicrographs (A and B) of a colonic vores, as well as herbivores not covered in this study, will
sample taken from RTP9 showing that the bacteria colonising the crypt not only provide important insights into the specific
and the mucus layer lining the epithelium respectively belong to the
natural niche of Helicobacter species, but will also increase
Helicobacter genus. (A) Bacteria hybridised with the Eu338-FITC probe
showing a green colour. (B) Bacteria hybridised with the HRh probe our understanding of the ecology of Helicobacter species.
showing a red colour. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
References

Amann, R.I., Stromley, J., Devereux, R., Key, R., Stahl, D.A., 1992. Molecular
ago (Flannery, 1994), the low cultivation and detection and microscopic identification of sulfate-reducing bacteria in multi-
species biofilms. Appl. Environ. Microbiol. 58, 614–623.
rates of Helicobacter spp. in the RTP’s was somewhat
Coldham, T., Rose, K., O’Rourke, J., Neilan, B.A., Dalton, H., Lee, A., Mitchell,
surprising, given the relatively high detection rates we H., 2011. The detection, isolation and characterisation of Helicobacter
observed in BTP’s. It is possible however that the difference species from the gastrointestinal tract of brushtail possum. Appl.
Environ. Microbiol. 77, 1581–1587.
in the detection rates of Helicobacter species in the GIT of
Flannery, T.F., 1994. Possum of the World. Geo Production, Sydney.
RTPs and BTPs may relate to differences in the ability of Fox, J.G., Paster, B.J., Dewhirst, F.E., Taylor, N.S., Yan, L.L., Macuch, P.J.,
Helicobacter species to utilise available nutrients and/or to Chmura, L.M., 1992. Helicobacter mustelae isolation from feces of
tolerate toxins produced within the GIT of these two ferrets: evidence to support fecal–oral transmission of a gastric
Helicobacter. Infect. Immun. 60, 606–611.
marsupials, or to differences in nutrients available in each Fox, J.G., Handt, L., Xu, S., Shen, Z., Dewhirst, F.E., Paster, B.J., Dangler, C.A.,
individual host. For example, while RTP feed almost Lodge, K., Motzel, S., Klein, H., 2001. Novel Helicobacter species iso-
 T. Coldham et al. / Veterinary Microbiology 166 (2013) 429–437 437

lated from rhesus monkeys with chronic idiopathic colitis. J. Med. among toxic and nontoxic Cyanobacteria of the genus Microcystis. Int.
Microbiol. 50, 420–429. J. Syst. Bacteriol. 47, 693–697.
Fox, J.G., 2002. The non-H. pylori helicobacters: their expanding role in On, S.L., Holmes, W.B., Sackin, M.J., 1996. A possibility matrix for the
gastrointestinal and systemic diseases. Gut 50, 273–283. identification of Campylobacter, Helicobacter and allied taxa. J. Appl.
Hume, I.D., 1999. Hindgut fermenters, The arboreal folivores. In: Marsu- Bacteriol. 81, 425–432.
pial Nutrition. Cambridge University Press, Cambridge, United King- Riley, L.K., Franklin, C.L., Hook Jr., R.R., Besch-Williford, C., 1996. Identi-
dom, pp. 149–204, ISBN 0 521 59406 5 (hb). fication of murine helicobacters by PCR and restriction enzyme
Hodge, D.S., Borczyk, A., Wat, L.L., 1990. Evaluation of the indoxyl acetate analyses. J. Clin. Microbiol. 34, 942–946.
hydrolysis test for the differentiation of Campylobacters. J. Clin. Robertson, B.R., O’Rourke, J.L., Vandamme, P., On, S.L., Lee, A., 2001. Heli-
Microbiol. 28, 1482–1483. cobacter ganmani sp. nov., a urease-negative anaerobe isolated from the
Karmali, M.A., De Grandis, S., Fleming, P.C., 1980. Antimicrobial suscept- intestines of laboratory mice. Int. J. Syst. Evol. Microbiol. 51, 1881–1889.
ibility of Campylobacter jejuni and Campylobacter fetus subsp. fetus to Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for
eight cephalosporins with special reference to species differentiation. reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Antimicrob. Agents Chemother. 18, 948–951. Solnick, J.V., Schauer, D.B., 2001. Emergence of diverse Helicobacter spe-
Kerle, A., 2001. Possums: the brushtails, ringtail and greater glider. In: cies in the pathogenesis of gastric and enterohepatic diseases. Clin.
Dawson, T.J. (Ed.), Australian Natural History Series. UNSW Press, Microbiol. Rev. 14, 59–97.
UNSW, Sydney, pp. 62–74. Strahan, R., 1983. The Australian Museum: Complete Book of Australian
Kwok, S., Kellogg, D.E., McKinney, N., Spasic1, D., Godal, L., Levenson1, C., Mammals, The National Photographic Index of Australian Wildlife.
Sninsky, J.J., 1990. Effects of primer–template mismatches on the Angus and Robertson Publishers, Australia ISBN 0 207 14454 0.
polymerase chain reaction: human immunodeficiency virus type 1 Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,
model studies. Nucleic Acids Res. 18 (4) 999–1005. 1997. The CLUSTAL_X windows interface: flexible strategies for mul-
Lee, A., 1991. Spiral organisms: what are they? A microbiologic introduc- tiple sequence alignment aided by quality analysis tools. Nucleic
tion to Helicobacter pylori. Scand. J. Gastroenterol. 187, 9–22. Acids Res. 25, 4876–4882.
Oxley, A.P.A., Powell, M., Mckay, D.B., 2004. Species of the family Helico- Vandamme, P., Harrington, C.S., Jalava, K., On, S.L., 2000. Misidentifying
bacteraceae detected in an Australian sea lion (Neophoca cinerea) with helicobacters: the Helicobacter cinaedi example. J. Clin. Microbiol. 38
chronic gastritis. J. Clin. Microbiol. 42 (8) 3505–3512. (June (6)) 2261–2266.
Neilan, B.A., Jacobs, D., Del Dot, T., Blackall, L.L., Hawkins, P.R., Cox, P.T., Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal
Goodman, A.E., 1997. rRNA sequences and evolutionary relationships DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703.