Genotypic Characterization of Streptococcus infantarius subsp. coli
Isolates from Sea Otters with Infective Endocarditis and/or Septicemia
and from Environmental Mussel Samples
Katrina L. Counihan-Edgar,a* Verena A. Gill,bAngela M. Doroff,cKathleen A. Burek,dWoutrina A. Miller,aPatricia L. Shewmaker,e
Spencer Jang,hCaroline E. C. Goertz,fPamela A. Tuomi,fMelissa A. Miller,gDavid A. Jessup,gand Barbara A. Byrnea
University of California, Davis, Veterinary Medicine: Pathology, Microbiology and Immunology, Davis, California, USAa; U.S. Fish and Wildlife Service Marine Mammals
Management, Anchorage, Alaska, USAb; Kachemak Bay Research Reserve, Homer, Alaska, USAc; Alaska Veterinary Pathology Services, Eagle River, Alaska, USAd;
Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia, USAe; Alaska SeaLife Center, Seward, Alaska, USAf; Marine Wildlife Veterinary Care
and Research Center, California Department of Fish and Game, Santa Cruz, California, USAg; and University of California, Davis, Microbiology Laboratory, William R.
Pritchard Veterinary Medical Teaching Hospital, Davis, California, USAh
Pulsed-fieldgelelectrophoresis(PFGE)wasusedtotype128 Streptococcusinfantarius subsp.coliisolatesfromseaottersand
bers are gastrointestinal tract commensals in several mammals
and some of which are also associated with bacteremia, septice-
mia, and endocarditis in humans and animals, including pigeons,
mink, and ruminants (3, 8, 10, 15).
S. infantarius subsp. coli is a significant problem in the north-
ern sea otter (Enhydra lutris kenyoni) population along coastal
this agent was the cause of death in approximately 30% of 613
carcasses collected and necropsied between 2004 and 2010 (V.
Gill, personal communication). Further, death due to IE and/or
septicemia with isolation of S. infantarius subsp. coli has also oc-
curred in 4 of 281 southern sea otter (Enhydra lutris nereis) car-
casses collected and necropsied along the central California coast
between 2004 and 2008 (M. Miller, personal communication). S.
infantarius subsp. coli in sea otters has not been examined previ-
The aims of the present study were to determine the genetic
relatedness of S. infantarius subsp. coli isolates by using pulsed-
field gel electrophoresis (PFGE), to evaluate wild mussels as a po-
this bacterium to sea otters, and to examine the temporal and
spatial distributions of PFGE types. Cultivation and preliminary
identification of isolates from sea otter tissues as SBEC members
were performed at the Microbiology Laboratory of the University
of California, Davis, William R. Pritchard Veterinary Medical
Teaching Hospital, and confirmation of S. infantarius subsp. coli
isolates was performed at the Centers for Disease Control and
Prevention as previously described (2, 4, 5, 7).
Wild blue mussels (Mytilus spp.) that serve as sea otter prey
and water quality sentinels were collected from sites within the
northern and southern sea otter ranges and screened for S. infan-
(1). Thirty mussels collected at each site at each time point were
shucked and collectively homogenized with an equal volume of
plated on Edwards modified medium supplemented with colistin
treptococcus infantarius subsp. coli is a member of the Strepto-
coccus bovis-equinus complex (SBEC), many of whose mem-
sulfate and oxolinic acid (EMCO) to select for Streptococcus spp.
lates as S. infantarius subsp. coli by using previously described
methods (2, 4, 5, 7). Mussels sampled from Monterey Bay, CA
Mussels collected from Monterey Bay, CA (Scott Creek, Carmel
River, and Elkhorn Slough), and Estero Bay, CA (Cayucos, Motel
Point, and Morro Bay), in April 2007 yielded three isolates from
pling yielded no isolates. In Alaska, mussels collected from Is-
mailof Island, Kasitsna Bay, and Bear Cove in June 2007 and
Homer, Ninilchik, Bishop Beach, and Homer Harbor in August
2007 provided no S. infantarius subsp. coli isolates.
PFGE of 128 S. infantarius subsp. coli isolates, 111 from 58
northern sea otters, 11 from 4 southern sea otters, and 6 from
mussels, was performed. Agarose plugs were prepared, digested
with SmaI restriction enzyme, and subjected to PFGE as previ-
ously described (14). Banding pattern analysis was performed us-
ing BioNumerics, version 4.6 (Applied Maths, Austin, TX), un-
weighted-pair group analysis with arithmetic means and Dice’s
coefficient (1% optimization and 2% position tolerance) (7, 9).
ered closely related and constituted a PFGE group (9). Isolates
with 90% or greater PFGE pattern similarity were considered
Northern and southern sea otter and mussel S. infantarius
Received 27 September 2012 Accepted 28 September 2012
Published ahead of print 10 October 2012
Address correspondence to Barbara A. Byrne, firstname.lastname@example.org.
*Present address: Katrina L. Counihan-Edgar, Alaska SeaLife Center, Seward,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
December 2012 Volume 50 Number 12Journal of Clinical Microbiologyp. 4131–4133 jcm.asm.org
subsp. coli isolates generated six major PFGE groups, with 57% of
the isolates clustering in group I (Table 1). Unexpectedly, group I
contained a mixture of northern and southern sea otter and mus-
sel isolates. The high degree of relatedness among 128 S. infan-
tarius subsp. coli isolates obtained from sea otters and their prey
over 5 years was notable. This contrasts with prior studies that
examined the PFGE types of SBEC isolates from human cases of
IE, which demonstrated considerable diversity. For example,
PFGE characterization of seven human S. bovis isolates collected
from blood samples of endocarditis patients at a hospital over a
1-year period indicated that all were unrelated (8). The high de-
gree of relatedness among S. infantarius subsp. coli isolates in this
study suggests that there may be something unique about these
bacteria related to their pathogenicity, their ability to survive in
the marine environment, or the susceptibility of sea otters to in-
fection. Alternatively, there could be common infection sources
due to human activity or animals in the regions.
The temporal and spatial distributions of the PFGE groups
the yearly prevalence of each group, which fluctuated over time.
Group I was generally the predominant strain and was the only
group present each year. Additionally, the majority of isolates
concentrated around Homer, AK, where there is both a larger
human population and a more intensive sampling effort for sea
otter carcass recovery. However, isolates were also obtained from
more distant geographical regions, such as the Aleutian Islands,
Glacier Bay National Park, Resurrection Bay, and Ninilchik. The
from carcasses collected in 2008 near Nordyke Island, on the west
side of Cook Inlet. This was the only location where a specific
PFGE type was isolated in the absence of all other types. The
sources of S. infantarius subsp. coli in that area and whether iso-
lates of this PFGE type are more or less virulent are unknown.
Surprisingly, both northern and southern sea otter S. infan-
tarius subsp. coli isolates were grouped together (groups I and II)
despite the large geographical separation of the two populations.
ing personnel or animals, and an analogous situation seems to be
occurring with the two sea otter populations (10). This could in-
dicate a lack of genetic diversity within S. infantarius subsp. coli,
in the discriminatory power of PFGE.
The isolation of S. infantarius subsp. coli from mussels in
could acquire the bacteria from contaminated water or prey such
as mussels. Many marine organisms accumulate pathogens, but
bivalves are especially adept at concentrating bacteria through fil-
ter feeding (12). In our study, an S. infantarius subsp. coli mussel
fell into group I. Another S. infantarius subsp. coli isolate from
mussels in Moss Landing, CA, was placed in group II along with
isolates from three southern sea otters. These results indicate
otters and suggest that sea otters may be infected by environmen-
Sea otters that died because of S. infantarius subsp. coli IE
and/or septicemia were generally septic, and isolates were ob-
coli isolated from two or more anatomic locations. When isolates
from different tissues within the same animal were compared,
69% of the sea otters had all of their isolates fall in the same PFGE
group and 59% of the animals had all of their isolates produce
currently infected with multiple S. infantarius subsp. coli strains,
multiple isolates from the same animal. This is similar to Campy-
found that 5 to 10% of humans were coinfected in sporadic Cam-
pylobacter infections, and another study determined that 50% of
humans were infected by multiple strains in Campylobacter out-
breaks (6, 11).
The S. infantarius subsp. coli isolates evaluated in this study
exhibited a high degree of relatedness, with one PFGE type pre-
dominating. Notably, several sea otters were infected by multiple
in coastal environments. Additional research is needed to deter-
mine transmission routes, as well as for identification and reme-
diation of specific sources of S. infantarius subsp. coli that could
FIG 1 Temporal distribution of PFGE groups generated by the 111 Alaskan
northern sea otter S. infantarius subsp. coli isolates.
TABLE 1 PFGE groups generated by S. infantarius subsp. coli isolates
from northern sea otters, southern sea otters, and mussels
Total no. of
isolates % Relatednessa
No. of isolates from:
Total 12852.9 111116
aPercent relatedness was determined by BioNumerics unweighted-pair group analysis
with arithmetic means and Dice’s coefficient (1% optimization and 2% position
bNSO, northern sea otters.
cSSO, southern sea otters.
Counihan-Edgar et al.
jcm.asm.org Journal of Clinical Microbiology
lead to a reduction of the exposure of sea otters and humans uti- Download full-text
lizing the coastal environment. Further exploration of the patho-
genic properties of S. infantarius subsp. coli will elucidate why
mental, host, and pathogen interactions.
This project was supported by the California Department of Fish and
Game’s Oil Spill Response Trust Fund through the Oiled Wildlife Care
Network at the Wildlife Health Center, School of Veterinary Medicine,
University of California, Davis. The U.S. Fish and Wildlife Region 7 Ma-
rine Mammals program funded the health and disease monitoring pro-
from California sea otters were obtained through collaborations with the
California Department of Fish and Game, Office of Spill Prevention and
Response, and other members of the Southern Sea Otter Alliance.
ing Network members who collected sea otter carcasses for testing and
helped with necropsy and sample collection. Thanks also to Dana Jenski
for all of her hard work in the necropsy laboratory and for tirelessly ship-
ping samples to the University of California, Davis.
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S. infantarius subsp. coli Genotypic Characterization
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