Veterinary Microbiology 133 (2009) 287–291

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

Short communication

Detection of Helicobacter and Campylobacter spp. from the aquatic

environment of marine mammals
C.G. Goldman a,*, M.J. Matteo b, J.D. Loureiro c,d, J. Degrossi e, S. Teves f,
S. Rodriguez Heredia c, K. Alvarez c, A. Beltrán González a, M. Catalano b,
J. Boccio a, G. Cremaschi a, J.V. Solnick g,h, M.B. Zubillaga a
a
Physics Department, School of Pharmacy and Biochemistry, University of Buenos Aires, Junı´n 956, 1113 Buenos Aires, Argentina
b
Microbiology, Parasitology and Immunology Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina
c
Mundo Marino Foundation, San Clemente del Tuyú, Buenos Aires, Argentina
d
Mundo Marino Oceanarium, San Clemente del Tuyú, Buenos Aires, Argentina
e
Public Health Laboratory, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
f
Microbiology Laboratory, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
g
Departments of Medicine and Medical Microbiology & Immunology, University of California, Davis, CA, USA
h
Center for Comparative Medicine, University of California, Davis, CA, USA

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

Article history: The mechanism by which Helicobacter species are transmitted remains unclear. To
Received 18 March 2008 examine the possible role of environmental transmission in marine mammals, we sought
Received in revised form 17 June 2008 the presence of Helicobacter spp. and non-Helicobacter bacteria within the order
Accepted 26 June 2008 Campylobacterales in water from the aquatic environment of marine mammals, and in
fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited
Keywords: by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the
Helicobacter bottom of dolphins’ pools. Samples were evaluated by culture, PCR and DNA sequence
Marine mammals analysis. Sequences from dolphins’ water and from regurgitated otoliths clustered with
Water 99.8–100% homology with sequences from gastric fluids, dental plaque and saliva from
Otoliths
dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from
seals’ water clustered with 99.5% homology with a sequence amplified from a Northern
sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected
from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic
environment suggests that contaminated water from regurgitated fish otoliths and
perhaps other tissues may play a role in Helicobacter transmission among marine
mammals.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction could be implicated in transmission of the infection

In spite of being a widespread infection in humans and
animals, the mode by which Helicobacter species are
transmitted remains unclear. Oral–oral and fecal–oral
modes of transmission have been suggested for human H.
pylori infection (Feldman et al., 1998). Contaminated water
* Corresponding author. Tel.: +54 11 4964 8202;
fax: +54 11 4964 8202x31.
E-mail address: cgold@ffyb.uba.ar (C.G. Goldman).
(Hulten et al., 1996; Azevedo et al., 2008), although it
does not seem to be the main route of transmission
(Feldman et al., 1998). Waterborne environmental trans-
mission of H. pylori infection might be related to a coccoid
form, a viable but non-cultivable bacterial stage that has
been widely described for other bacteria in aquatic
environments (Byrd et al., 1991; Engstrand, 2001).
Oral–oral and fecal–oral routes may also be implicated
in transmission of Helicobacter infection in marine
mammals, since Helicobacter spp. have been isolated from
the feces of dolphins (Harper et al., 2002), and detected in

0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.06.023
288 C.G. Goldman et al. / Veterinary Microbiology 133 (2009) 287–291
their dental plaque (Goldman et al., 2002) and in the feces
of seals (Oxley and McKay, 2004). However, transmission
via contaminated water is an alternative possibility that
has not been investigated. Captive pinnipeds and ceta-
ceans typically eat two fish species, corvina (Micropogonias
furnieri) and small hake (Macrodon ancylodon), and then
regurgitate fish otoliths, which are calcium carbonate
crystals from the inner ear of the fish (Popper et al., 2005).
These otoliths might contaminate the water environment
and serve as a vehicle for transmission of Helicobacter or
Campylobacter spp.
To address this hypothesis, we sought to identify
Helicobacter spp. and non-Helicobacter bacteria within the
order Campylobacterales, in water samples and in fish
otoliths regurgitated by captive marine mammals.
2. Materials and methods
2.1. Sample collection
Water samples were collected from two connected
pools that were inhabited by ten dolphins (Tursiops
gephyreus) and a killer whale (Orcinus orca), one pool that
was inhabited by two elephant seals (Mirounga leonina),
and three pools that were inhabited by seven sea lions
(Otaria flavescens) and one fur seal (Arctocephalus australis).
Samples were designated as dolphin water 1 and 2, and
seal water 1–4, respectively. Each dolphin pool contained
3  106 L of water, whereas each seal pool contained
between 2  105 and 4  105 L of water. All the pools were
filled from the same water source, which was sampled as a
control.
Three samples of otoliths were also collected from the
bottom of the two pools inhabited by the dolphins
(designated otolith 1 and 2), and another one from a
third pool that connects them (otolith 3). As a control,
otoliths, gastrointestinal tract, muscle, and branchiae
samples were dissected from forty specimens each of
corvina (M. furnieri) and small hake (M. ancylodon).
Samples from the same type and fish species were pooled
and transported on dry ice for further analysis at the
laboratory in Buenos Aires.
Five liters of water were collected from each pool in
sterile plastic containers. Chlorine was inactivated by the
addition of 5 mL of 10% (v/v) sodium thiosulfate in water.
The containers were refrigerated at 4 8C for their trans-
portation to the laboratory in Buenos Aires.
2.2. DNA extraction
Each water sample was filtered under vacuum with four
0.45 mm cellulose nitrate sterile membranes (Microclar1,
Buenos Aires, Argentina). One half of each of the four
membranes were combined in an Eppendorf tube,
mixed with proteinase K and lysis buffer, incubated at
56 8C overnight, and then processed for DNA extraction
using the QIAamp Mini Kit (QIAGEN, Inc., CA, USA)
according to the manufacturer’s directions. Approxi-
mately 25 mg of each fish tissue and otolith sample were
mixed with the same reagents and processed in the
same manner.
2.3. Culture conditions
For culture analysis, the other half of the four
membranes from each sample were laid directly on Blood
Agar Base No. 2 (Oxoid Ltd., Basingstoke, UK) containing 7%
(v/v) horse blood, pyruvic acid and MEM vitamin supple-
ment (Oxoid Ltd., Basingstoke, UK); vancomycin (10 mg/L),
polymyxin B (2500 U/L), and amphotericin B (5 mg/L). The
membranes were cultured no more than 24 h after the
collection of water samples. Otoliths and tissue samples
were mixed with 0.5 mL of sterile saline, ground under
sterile conditions, and then plated as above. Plates were
incubated at 37 8C under microaerophilic conditions (CO2
10%, O2 5%, H2 5% and N2 balance) for up to 20 days, as
described for H. cetorum by Harper et al. (2002).
Helicobacter pylori ATCC 43504 was plated as a positive
culture control.
2.4. PCR amplification
Two PCR amplifications were performed. We first
amplified with primer pair CG2 (F0 50 -GAGTTT-
GATCCTGGCTCAGAG-30 and R1 50 -ATTTTACCCCTACAC-
CAA-30 ), which amplifies a fragment of approximately
650 bp of the 16S rRNA gene of Helicobacter and non-
Helicobacter bacteria within the order Campylobacterales
(positions 9-657 of the 16S rRNA gene of H. pylori ATCC
26695, Tomb et al., 1997). Amplification was carried out in
a total volume of 50 mL containing 1 Taq polymerase
buffer, 1.5 mM MgCl2, 0.2 mM (each) deoxynucleotide, 1.0
U of Platinum1 Taq DNA Polymerase (Invitrogen Argen-
tina, Buenos Aires, Argentina), 0.1 mg each oligonucleotide
primer, and 10 mL of DNA template. PCR (94 8C for 1 min;
30 cycles of 94 8C for 30 s, 50 8C for 30 s, and 72 8C for 30 s;
72 8C for 5 min) was performed with an automatic
thermocycler (MyCycler, BioRad, CA, USA) and a 10 mL
aliquot was analyzed by electrophoresis through a 1.5%
(wt/vol) agarose gel stained with ethidium bromide. PCR
products were visualized by excitation under UV light.
Because some reactions appeared to yield product that was
of the correct size, but not sufficient for DNA sequencing,
we reamplified the material from each reaction (positive
and negative) as before, using 10 mL of amplicon from the
original PCR.
A second PCR was performed using primer pair CG1 (F1
50 -GTATCCGGCCTGAGA-30 and R1), which amplifies a
387 bp fragment of the 16S rRNA gene of the Helicobacter
genus, and corresponds to positions 271–657 of the 16S
rRNA gene of H. pylori ATCC 26695 (Tomb et al., 1997). PCR
amplification was carried out with the same conditions
described for primer pair CG2, but using an annealing
temperature of 51 8C. Material was examined by gel
electrophoresis and reamplified as described for primer
CG2. Positive PCR results with primer pairs CG1 and CG2
indicate the presence of Helicobacter spp. DNA or
Helicobacter and non-Helicobacter Campylobacterales,
while a negative PCR result with CG1 and a positive result
with CG2 indicates the presence of non-Helicobacter
bacteria within the order Campylobacterales. Positive
results with CG1 and negative results with CG2 would
be unexpected and was never observed. All PCR assays
 C.G. Goldman et al. / Veterinary Microbiology 133 (2009) 287–291
were accompanied by amplification of positive (H. cetorum
MIT 99-5656, H. pylori NYU 03-939, C. jejuni ARG 160-
2002) and negative (water) controls.
2.5. 16S rRNA sequencing
PCR products from positive samples were purified using
the QIAquick PCR Purification kit (QIAGEN Inc., CA, USA),
following the manufacturer’s instructions. Purified PCR
products were submitted to Macrogen Inc. (Seoul, Korea)
for DNA sequencing. Samples were sequenced on both
strands using forward and reverse primers on the cycle
sequencing reaction with the BigDye Terminator Cycle
Sequencing Ready Reaction kit (Applied Biosystems, Foster
City, CA, USA). Products were resolved on a 3730xl
Automated Sequencer (Applied Biosystems, Foster City,
CA, USA).
2.6. Data analysis
Sequences were edited using the ChromasPro version
1.34 (Technelysium Pty Ltd.) available on the Technely-
sium website (www.technelysium.com.au/Chroma-
sPro.html), converted into FASTA format with the
ReadSeq program (http://www-bimas.cit.nih.gov/molbio/
readseq/), and aligned using the BLAST server available at
the National Center for Biotechnology Information website
(http://www.ncbi.nlm.nih.gov/BLAST/). In order to per-
form the phylogenetic analysis, 16S rDNA sequences from
Helicobacter and Campylobacter species were retrieved
from GenBank. All the sequences were edited to a common
length, and a multiple sequence alignment was performed
by ClustalX version 1.83 (Thompson et al., 1997). A
phylogenetic tree was constructed by the neighbour-
joining method using the program MEGA version 4
(Tamura et al., 2007), and the stability of the groupings
was estimated by bootstrap analysis (10,000 replications)
using the same program.
3. Results
Positive PCR results were obtained with primer pairs
CG1 and CG2 from all water samples except that from the
pool of the elephant seals (seals water 1), which yielded a
positive result with CG2 and a negative result with CG1,
indicating the presence of Campylobacter spp. DNA in that
pool. Positive results were also obtained with both primer
pairs from the three otolith samples from the pools of
dolphins. Negative results with both primer pairs were
obtained from the control water sample and from all fish
samples, including otoliths. We were unable to culture
Helicobacter bacteria from any of the samples.
Nine DNA sequences were obtained from water and
otolith samples amplified with primer pair CG2, and were
deposited in Genbank. Sequences were also obtained from
primer pair CG1 positive samples, showing 100% homology
with their respective sequences from primer pair CG2
(data not shown). A dendrogram illustrating the phyloge-
netic position of the Helicobacter and Campylobacter spp.
DNA sequences compared to other species of these two
genera is shown in Fig. 1. Helicobacter sequences from the
289
otoliths and from water samples from the pools inhabited
by the dolphins were nearly identical (1 bp different, 99.8%
identity), and clustered with H. cetorum isolated from
dolphins and whales. Pairwise sequence comparisons also
showed 99.8–100% homology with Helicobacter identical
sequences amplified from gastric fluids, dental plaque and
saliva from dolphins in the same oceanarium (EU445533,
EU445534, EU445535, EU445536, EU445537, EU445538,
EU445539, EU445540, EU445541, unpublished data).
Helicobacter spp. amplified from water of the pools
inhabited by seals formed a second taxonomic cluster.
The sequence amplified from seal water 2 was nearly
identical to a sequence from a Northern sea lion (Harper
et al., 2003), from which it differed by 3 bp (99.5%
homology). This sequence also differed by 13 and 16 bp
(98.0 and 97.5% identity) from sequences amplified from
seal water 3 and 4, respectively, which clustered together
(11 bp difference, 98.3% identity). The sequences amplified
from the water of the seals differed by at least 30 bp (95.4%
homology) from H. cetorum, and therefore represent a
different Helicobacter spp. One sequence amplified from
water of the pool inhabited by the elephant seal (seal water
1) formed a different cluster within the genus Campylo-
bacter, differing by 4 bp (99.4% homology) with identical
sequences obtained from the dental plaque and saliva of
elephant seals from the same pool (EU596557 and
EU596560, unpublished data), and from the dental plaque
of dolphins from the same oceanarium (EU445550 and
EU445551, unpublished data). Interestingly, it differed by
more than 9% with other Campylobacter spp., including C.
insulaenigrae, which was isolated from fecal samples of
seals (Foster et al., 2004). Therefore, this sequence may
represent a novel Campylobacter sp.
4. Discussion
The detection of Helicobacter spp. from water and
otoliths in pools inhabited by dolphins, and the similarity
of these sequences to those recovered from dolphins by us
(EU445533, EU445534, EU445535, EU445536, EU445537,
EU445538, EU445539, EU445540, EU445541, unpublished
observations) and others (Harper et al., 2002), suggests
that environmental contamination is one potential cause
of Helicobacter transmission. Since neither the water
source for the pools nor the fish that were used as food
were themselves contaminated, it is possible that the
source of contamination is otoliths or other material
regurgitated by the dolphins. Likewise, the findings of a
novel Campylobacter sp. from water of the pool inhabited
by elephant seals and the high sequence identity to those
obtained from animals from the same oceanarium
suggests that these bacteria could also be transmitted
through water. Environmental transmission of Helicobacter
in the marine environment was further supported by the
identification in water inhabited by seals of Helicobacter
spp. that are closely related to sequences from a Northern
sea lion (Harper et al., 2003). Since fecal samples of the
marine mammals could not be evaluated in this study, and
due to evidence of the isolation of H. cetorum from the feces
of three cetaceans (Harper et al., 2002), fecal contamina-
tion of water and otoliths could also occur and might play a
290 C.G. Goldman et al. / Veterinary Microbiology 133 (2009) 287–291

Fig. 1. Dendrogram based on a 650 bp fragment of the 16S rRNA gene sequence, showing the phylogenetic location of Helicobacter spp. and Campylobacter
spp. from marine mammals, the water of the pools and otoliths (bold), compared to other species of these two genera. GenBank accession numbers are given
in parentheses. The scale bar represents a 1% difference in nucleotide sequences. *Sequence EU445533 is representative of eight other identical sequences
(EU445534, EU445535, EU445536, EU445537, EU445538, EU445539, EU445540, EU445541). **Sequence EU596557 is representative of three other
identical sequences (EU596560, EU445550, EU445551).
 C.G. Goldman et al. / Veterinary Microbiology 133 (2009) 287–291
role in Helicobacter transmission. Recent evidence suggests
that both gastric and enterohepatic Helicobacter species
can survive for prolonged periods in water, which also
supports the role of environmental transmission in the
marine environment (Azevedo et al., 2008). Negative
culture results could have been related to the lost of
bacterial viability during collection, transport, filtration
and culture of the samples. However, this might be also
due to a viable but non-culturable bacterial state in the
water, as was described for other Helicobacter species
(Engstrand, 2001).
Acknowledgments
We thank the team of Mundo Marino for collection of
the samples and Dr. Jose Luis Lopez for cooperation with
phylogenetic analysis. This work was supported by grant
UBACYT B041 from the University of Buenos Aires.
References
Azevedo, N.F., Almeida, C., Fernandes, I., Cerqueira, L., Dias, S., Keevil, C.W.,
Vieira, M.J., 2008. Survival of gastric and enterohepatic Helicobacter
spp. in water: implications for transmission. Appl. Environ. Microbiol.
74, 1805–1811.
Byrd, J.J., Xu, H.-S., Colwell, R.R., 1991. Viable but nonculturable bacteria in
drinking water. Appl. Environ. Microbiol. 57, 875–878.
Engstrand, L., 2001. Helicobacter in water and waterborne routes of
transmission. J. Appl. Microbiol. 30, 80S–84S.
Feldman, R.A., Eccersley, A.J.P., Hardie, J.M., 1998. Epidemiology of Heli-
cobacter pylori: acquisition, transmission, population prevalence and
disease-to-infection ratio. Br. Med. Bull. 54, 39–53.
Foster, G., Holmes, B., Steigerwalt, A.G., Lawson, P.A., Thorne, P., Byrer,
D.E., Ross, H.M., Xerry, J., Thompton, P.M., Collins, M.D., 2004. Cam-
291
pylobacter insulaenigrae sp. nov., isolated from marine mammals. Int.
J. Syst. Evol. Microbiol. 54, 2369–2373.
Goldman, C.G., Loureiro, J.D., Quse, V., Corach, D., Calderon, E., Caro, R.A.,
Boccio, J., Rodrı́guez Heredia, S., Di Carlo, M.B., Zubillaga, M.B., 2002.
Evidence of Helicobacter sp. in dental plaque of captive dolphins
(Tursiops gephyreus). J. Wildl. Dis. 38, 644–648.
Harper, C.G., Feng, Y., Xu, S., Taylor, N.S., Kinsel, M., Dewhirst, F.E., Paster,
B.J., Greenwell, M., Levine, G., Rogers, A., Fox, J.G., 2002. Helicobacter
cetorum sp. nov., a urease-positive Helicobacter species isolated from
dolphins and whales. J. Clin. Microbiol. 40, 4536–4543.
Harper, C.G., Xu, S., Rogers, A., Feng, Y., Shen, Z., Taylor, N.S., Dewhirst, F.E.,
Paster, B.J., Miller, M., Hurley, J., Fox, J.G., 2003. Isolation and char-
acterization of novel Helicobacter spp. from the gastric mucosa of harp
seals Phoca groenlandica. Dis. Aquat. Org. 57, 1–9.
Hulten, K., Han, S.W., Enroth, H., Klein, P.D., Opekun, A.R., Gilman, R.H.,
Evans, D.G., Engstrand, L., Graham, D.Y., El-Zaatari, F.A., 1996. Heli-
cobacter pylori in the drinking water in Peru. Gastroenterology 110,
1031–1035.
Oxley, A.P., McKay, D.B., 2004. Fecal shedding of Helicobacter spp. by co-
housed Australian sea lions (Neophoca cinerea) and Australian fur
seals (Arctocephalus pusillus doriferus). Vet. Microbiol. 101, 235–243.
Popper, A.N., Ramcharitar, J., Campana, S.E., 2005. Why otoliths? Insights
from inner ear physiology and fisheries biology. Mar. Freshw. Res. 56,
497–504.
Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
Biol. Evol. 24 (8), 1596–1599., http://megasoftware.net.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G.,
1997. The ClustalX windows interface: flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res.
25, 4876–4882.
Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton, G.G., Fleisch-
mann, R.D., Ketchum, K.A., Klenk, H.P., Gill, S., Dougherty, B.A., Nelson,
K., Quackenbush, J., Zhou, L., Kirkness, E.F., Peterson, S., Loftus, B.,
Richardson, D., Dodson, R., Khalak, H.G., Glodek, A., McKenney, K.,
Fitzegerald, L.M., Lee, N., Adams, M.D., Hickey, E.K., Berg, D.E.,
Gocayne, J.D., Utterback, T.R., Peterson, J.D., Kelley, J.M., Cotton,
M.D., Weidman, J.M., Fujii, C., Bowman, C., Watthey, L., Wallin, E.,
Hayes, W.S., Borodovsky, M., Karp, P.D., Smith, H.O., Fraser, C.M.,
Venter, J.C., 1997. The complete genome sequence of the gastric
pathogen Helicobacter pylori. Nature 388, 539–547.