Occurrence, Genetic Diversity, and

Persistence of Enterococci in a Lake

Superior Watershed
Qinghong Ran, Brian D. Badgley, Nicholas Dillon, Gary M.
Dunny and Michael J. Sadowsky
Appl. Environ. Microbiol. 2013, 79(9):3067. DOI:
10.1128/AEM.03908-12.
Published Ahead of Print 1 March 2013.

These include:
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Occurrence, Genetic Diversity, and Persistence of Enterococci in a

Lake Superior Watershed
Qinghong Ran,a Brian D. Badgley,a* Nicholas Dillon,c Gary M. Dunny,c Michael J. Sadowskya,b
Biotechnology Institutea and Department of Soil, Water, and Climate,b University of Minnesota, St. Paul, Minnesota, USA; Department of Microbiology, University of
Minnesota, Minneapolis, Minnesota, USAc

ecal contamination of recreational waters is a widespread

problem across the Great Lakes region of the United States.
Because of difficulties and high costs associated with detection and
quantitation of fecal pathogens (1), fecal indicator bacteria (FIB)
were chosen to assess the potential presence of pathogens. Traditionally, Escherichia coli and fecal coliforms have been used as FIB
in freshwater systems, while enterococci were initially used as indicators in marine waters. Previous studies have shown that E. coli
can become naturalized to the microbial community in tropical,
subtropical, and temperate soil and sand (24). This likely limits
the use of this bacterium as an indicator of water quality. Moreover, these culture-based methods cannot differentiate among
sources of fecal bacteria (5).
The U.S. Environmental Protection Agency (EPA) has suggested that coastal and Great Lakes states adopt enterococci as an
alternative indicator of fecal contamination (6). Epidemiological
studies have shown that the enterococcal concentration has a
strong positive correlation with the risk of gastroenteritis associated with swimming in contaminated freshwater (7). However,
the potential advantages of enterococci over E. coli as the indicator
of choice to detect fecal contamination of waterways and the environment warrant further investigation (8), especially in the cold
climate associated with the Lake Superior watershed.
It has been suggested that some Enterococcus species are primarily of environmental origin (5). Some species, including Enterococcus faecalis, E. faecium, E. casseliflavus, E. hirae, E. mundtii,
E. gallinarum, E. durans, E. avium, and E. sulfureus, have been
repeatedly isolated from sand, sediment, water, or plants (912).
In addition, evidence of the persistence of enterococci in municipal oxidation ponds and in microcosms simulating environmental conditions has been previously documented (13, 14). The high
level of fecal bacteria in sand, sediment, soil, and submerged vegetation has raised public health concerns (2, 15, 16). Since matrices harboring enterococci may protect these bacteria from inactivation by sunlight and from protozoan predation or offer a range

May 2013 Volume 79 Number 9

of surfaces for attachment and nutrient acquisition (1720), it has

been suggested that they may serve as reservoirs for FIB (21).
However, the majority of studies on the ecology of enterococci
have been done in tropical and subtropical environments or in
warmer Great Lakes states.
Enterococci are nearly ubiquitous and have been isolated from
a variety of substrates (4, 10, 15). Species recovered from marine
environments included mainly E. faecalis, E. faecium, E. casseliflavus, E. hirae, and E. mundtii (10, 15, 22). Of these species, E. casseliflavus has been shown to replicate and persist in vegetationcontaining microcosms (23). Since the ecology of enterococci in
water, sand, and soil may be affected by abiotic and biotic factors
(21), it is likely that the concentration and species composition of
enterococci in temperate freshwater environments differ from
those found in other environments. In the few studies carried out
in temperate freshwater environments, enterococci were recovered from backshore beach sand in Lake Michigan from early fall
to early summer of the next year, and the dominant species (92%)
recovered was E. faecium (9). Another study carried out at Lake
Huron found that the density of enterococci in wet sand was
higher than that in water (16). But none of the previous studies
examined the species composition over time and in different substrates at the study sites.
The bacterium E. faecalis has received much attention because
of its ability to survive and grow under a variety of harsh condi-

Received 17 December 2012 Accepted 22 February 2013

Published ahead of print 1 March 2013
Address correspondence to Michael J. Sadowsky, Sadowsky@umn.edu.
* Present address: Brian D. Badgley, Department of Crop and Soil Environmental
Sciences, Virginia Tech, Blacksburg, Virginia, USA.
Copyright 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03908-12

Applied and Environmental Microbiology

p. 30673075

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In 2012, the U.S. EPA suggested that coastal and Great Lakes states adopt enterococci as an alternative indicator for the monitoring of recreational water quality. Limited information, however, is available about the presence and persistence of enterococci in
Lake Superior. In this study, the density, species composition, and persistence of enterococci in sand, sediment, water, and soil
samples were examined at two sites in a Lake Superior watershed from May to September over a 2-year period. The genetic diversity of Enterococcus faecalis isolates collected from environmental samples was also studied by using the horizontal, fluorophore-enhanced repetitive PCR DNA fingerprinting technique. Results obtained by most-probable-number analyses indicated
that enterococci were present in 149 (94%) of 159 samples and their densities were generally higher in the summer than in the
other months examined. The Enterococcus species composition displayed spatial and temporal changes, with the dominant species being E. hirae, E. faecalis, E. faecium, E. mundtii, and E. casseliflavus. DNA fingerprint analyses indicated that the E. faecalis
population in the watershed was genetically diverse and changed spatially and temporally. Moreover, some DNA fingerprints
reoccurred over multiple sampling events. Taken together, these results suggest that some enterococci are able to persist and
grow in the Lake Superior watershed, especially in soil, for a prolonged time after being introduced.

Ran et al.

MATERIALS AND METHODS

Sampling site description. Two sampling sites in a Lake Superior watershed were used in these studies: the new DBC beach in the Duluth-Superior Harbor and the Kingsbury Creek bank where it intersects Stark Road
(KS) in Proctor, MN (Fig. 1A). The two sites were previously described (2,
32). At the DBC site, samples were collected from submerged sediment
located 5 m from the waterline (S5), wet sand at the shoreline (SL), wet
sand located 1 m upshore from the SL (NS), and dry sand located 8 m
upshore from the waterline (US) (Fig. 1B). The Minnesota Lake Superior
Beach Monitoring Program reported a high number of beach advisory
days based on E. coli concentrations at the DBC in the summer of 2011
(33). At the KS site, samples were collected 5 m (KS5) and 14 m (KS14)
from the creek (Fig. 1C). On 23 May 2011, three exclosure boxes (referred
to as KS14I), made from 32-gallon trash cans (50-cm diameter) as described previously (34), were buried at the KS14 location in order to
exclude external enterococcal sources: runoff and feces deposition from
animals. Four mesh-covered windows were placed into the exclosure
boxes to facilitate air exchange. The exclosure boxes were buried in the soil
at a depth of 10 cm, and the original trash can lids covered the tops of the
boxes.
Sample collection. Triplicate samples, located 1 m apart, were collected from the top 10-cm layer of sand, submerged sediment, and soil.
Samples were taken by a shovel or with core tubes disinfected with 70%
ethanol. Samples were stored in Whirl-Pak bags at 4C until they were
processed in the lab. In 2011, water (W) and dry sand (US) samples were
also collected from the DBC. All samples were processed by the day after
sampling. Sample temperature was measured in 2011 by inserting a thermometer to a depth of 10 cm in the sampling area. Sampling stopped at
the KS14 site in August 2011 because of road construction that resulted in
the destruction of the sampling areas. While two of the exclosure boxes
were damaged in August, one was not damaged severely and samples were
collected on 24 August 2011. The soil in the very top layer in the exclosure
boxes became dry over time, so it was removed when sampling. In addition, runoff flowed through a recently installed drainage culvert into our
sampling area (KS5) in September 2011.
Enumeration and isolation of cultivable enterococci. Samples were
thoroughly mixed before processing. A 10-g aliquot of each sample and
100 ml of extraction buffer (35) amended with 0.01% Tween 20 were
added to a 160-ml wide-mouth milk dilution bottle (Corning, Tewksbury,

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FIG 1 Study sites at Lake Superior. (A) Sampling sites in the Lake Superior
watershed. (B) Sampling areas at the DBC. (C) Sampling areas at the KS site.
Legend: DBC Duluth Boat Club beach; KS Kingsbury Creek bank. Panels
A and C are modified from reference 2.

MA) containing 10 g of 3-mm glass beads (Fisher Scientific, Pittsburgh,

PA). The mixture was shaken at 280 oscillations/min for 40 min on a
horizontal, reciprocating shaker. The bottle contents were allowed to settle for 30 min. The supernatants from the May 2010 samples were filtered
through 0.45-m membranes that were placed onto the surface of m-EI
Agar by the U.S. EPA method 1600 membrane filtration (MF) technique
(36). However, because of the low enterococcal densities in samples and
the high turbidity of the extractant, no enterococci were recovered and
observed on m-EI Agar in May 2010 (detection limit, 1 CFU/2 g of original
sample). Thereafter, standard method 9230 B, a multiple-tube mostprobable-number (MPN) method, was used (37). Aliquots consisting of
10 ml, 1 ml, and 0.1 ml of the supernatants of May, June, and September
samples (detection limit, 1.8 MPN/10 g of original sample) or 1 ml, 0.1 ml,
and 0.01 ml of the supernatants of July and August samples (detection
limit, 1.8 MPN/1 g of original sample) were inoculated into azide dextrose
broth. Other steps were followed according to method 9230 B. To isolate
enterococci, 20- to 25-g samples were treated as described above. The
appropriate amount of extractant was filtered or directly spread onto
m-EI Agar plates. The MF method was used to enumerate enterococci in
water samples. Colonies on m-EI Agar with a blue halo were picked and
cultivated in 150 l Enterococcosel broth (Difco-BBL, Franklin Lakes,
NJ) in 96-well microtiter plates. After growth, 40 l of 80% glycerol was
added to the wells exhibiting a brownish black color and plates were stored
at 80C until used.
Enterococcus isolate identification to the species level. Enterococcus
isolates were stamped from glycerol stocks onto LB agar or Trypticase soy
agar plates with a 48-pin replicator (Boekel Scientific, Feasterville, PA).
Overnight colonies were suspended in 100 l of double-distilled H2O
(ddH2O). Multiplex PCR was used to identify eight species of enterococci:

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tions (24). This bacterium is the primary Enterococcus species inhabiting the intestinal tracts of humans and some animals (25)
and is the predominant species causing hospital-acquired infections (26). The majority of studies on the survival of E. faecalis
have been done in medical fields (2729), and many environmental studies have examined the survival and genetic diversity of a
limited number of Enterococcus species (15, 30). Therefore, there
is very limited information on the survival of E. faecalis in the
environment. One of the few studies done under lab conditions
found that E. faecalis survived longer than E. coli did in sand microcosms (31). Moreover, there is currently little information on
the genetic relatedness among E. faecalis strains isolated from different habitats.
The objective of the studies reported here was to examine the
occurrence of enterococci in sand, sediment, water, and soil in a
Lake Superior watershed. Two different sites were included: the
new Duluth Boat Club (DBC) beach site and the Kingsbury Creek
bank site. Moreover, the studies also examined changes in Enterococcus species composition over considerable spatial and temporal
ranges. The E. faecalis isolates identified were genotyped to examine their genetic diversity, and since naturalized E. coli strains have
been isolated from both sites (2, 32), we further determined if
enterococci could also persist in these two habitats.

Enterococci in a Lake Superior Watershed

May 2013 Volume 79 Number 9

The correlation between the sample temperature and the enterococcal

density in 2011 was studied by using the Pearson product-moment correlation coefficient. HFERP banding patterns were analyzed by using
Bionumerics software (version 3.0; Applied Math, Inc.) as described previously (43).

RESULTS

Enterococcal density. Enterococci were not detected in May 2010

at the DBC and KS sites by the MF technique (detection limit, 1
CFU/2 g of original sample). However, enterococci were ubiquitous in samples analyzed by the MPN technique. This is probably
due to the different detection limits of these two methods. By the
MPN technique, enterococci were detected in 94% of the samples,
with concentrations ranging from 3 105 to 5.6 105 MPN/100
g of sample (dry weight of sand, sediment, and soil samples and ml
of water samples) (Fig. 2). Even in upshore sand samples at the
DBC, where the moisture ranged from 0.3 to 4.5%, 14 of 15 samples (93%) contained enterococci, with concentrations ranging
from 2 101 to 1.6 104 MPN/100 g of sample. The enterococci
were not found in some of the May, June, and September samples
(detection limit, 1.8 MPN/10 g of original sample). Generally, the
densities were greater when the temperatures were higher. This is
supported by the positive correlations between enterococcal densities and sample temperatures in 2011 (DBC, r 0.57, P 9.4
108; KS, r 0.51, P 0.01).
At the DBC, the comparisons of monthly enterococcal densities across different matrices showed that the nearshore sand samples harbored 7 to 87 times more enterococci than did water
throughout the sampling period (all P values, 0.05). Moreover,
the enterococcal density in water was positively correlated with
those in SL sand samples (r 0.62, P 0.01) and submerged
sediment samples (r 0.64, P 0.01), suggesting that the bacteria
might be transported among these three matrices.
The density of enterococci varied from 7.5 101 to 5.6 105
MPN/100 g of oven-dried sample at the KS site over the study
period (Fig. 2, KS5 and KS14). The data obtained in 2010 showed
no significant difference in the overall enterococcal concentration
between the KS5 and KS14 samples at 0.05 (t test, P 0.30).
In 2011, however, the overall enterococcal density at the KS5 site
was significantly greater than that at the KS14 site (t test, P 4.0
103). This may be due in part to the fewer samples taken at the
KS14 site. When monthly comparisons of the KS and DBC sites over
the 2-year period were carried out, the overall mean enterococcal
densities in soil at the KS site (combining KS5 and KS14 samples)
were greater than those at the DBC site (combining sand and sediment samples) in July and August (both P values are 0.05).
Diversity of Enterococcus species composition. The species
status of 2,441 enterococcal isolates was determined by multiplex
PCR and biochemical analyses. The majority of the isolates
(97.8%) could be assigned to one of eight species: E. faecalis, E.
faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. durans, or E. avium. The most abundant species at the DBC were E.
hirae (36.4%), E. faecium (27.6%), E. faecalis (14.5%), and E.
mundtii (12.0%). In contrast, the dominant species at the KS site
were E. faecalis (48.8%), E. mundtii (20.0%), E. casseliflavus
(14.2%), and E. faecium (10.8%). Further analyses showed that
the Enterococcus species composition varied both spatially and
temporally (Fig. 3).
Genetic diversity of E. faecalis in the Lake Superior watershed. Over the 2-year study period, 309 and 227 E. faecalis isolates

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E. faecalis (FL), E. faecium (FM), E. casseliflavus (CA), E. hirae (HI), E.

mundtii (MU), E. gallinarum (GA), E. durans (DU), and E. avium (AV)
(38). American Type Culture Collection strains E. faecalis ATCC 19433, E.
faecium ATCC 19434, E. casseliflavus ATCC 25788, E. hirae ATCC 8043, E.
gallinarum ATCC 49573, E. durans ATCC 19432, and E. avium ATCC
14025 and an E. mundtii strain previously isolated from the environment
by the lab were used as positive controls. A tube not containing any template DNA served as the negative control. The primers and PCR parameters used were described previously (38).
HFERP DNA fingerprinting. Isolates identified as E. faecalis by multiplex PCR were subjected to biochemical tests for verification. The biochemical tests included arginine hydrolysis and arabinose, raffinose, and
sorbitol fermentation with the basal medium as previously described (25).
Isolates that exhibited arginine hydrolysis and were sorbitol fermentation
positive and arabinose and raffinose fermentation negative were verified
as E. faecalis. The E. faecalis isolates were transferred into new 96-well
microplates for horizontal, fluorophore-enhanced repetitive PCR
(HFERP) DNA fingerprinting. This technique is similar to repetitive-sequence PCR-based DNA fingerprinting but uses fluorescently labeled
primers and size markers to more adequately examine genetic population
structures among bacteria (39).
DNA, extracted using a GenElute bacterial genomics DNA kit (SigmaAldrich), was used as the template for HFERP DNA fingerprinting of the
2010 E. faecalis isolates. DNA was extracted with the GenElute Bacterial
Genomics DNA kit (Sigma-Aldrich). For isolates obtained in 2011, however, a new rapid colony method was developed. Approximately 0.5 l of
each colony was suspended in 100 l of ddH2O and frozen at 20C
overnight, and 2 l of the thawed suspension was used directly as the PCR
template. Both methods were tested on the same strains and found to yield
identical results. The BOXA2R primer (5=-ACGTGGTTTGAAGAGATT
TTCG-3=) was used for DNA fingerprinting (40). The PCR protocol was
modified on the basis of previous reports (23, 41). The master mixture for
96 reaction mixtures was prepared first. Each reaction mixture contained
5 l of 5 Gitschier buffer (42), 0.25 l of 100% dimethyl sulfoxide, 1 l
of 50 M BOXA2R primers (50% unlabeled primers and 50% 6-carboxyfluorescein-labeled primers), 0.625 l of 25 mM deoxynucleoside
triphosphate, 0.2 l of 20 mg/ml bovine serum albumin, 0.4 l of 5-U/l
Taq (Denville Choice Taq), and 15.525 l of nuclease-free H2O. Two
microliters of template DNA (100 ng of DNA for 2010 isolates) was added
to each reaction mixture in a final volume of 25 l. PCR was carried out in
a PTC 100 or PTC 200 thermal cycler (Bio-Rad MJ Research, Hercules,
CA) by the protocol described previously (41), except that 30 cycles was
used instead of 35 cycles. E. faecalis strain OG1RF and ddH2O served as
positive and negative controls, respectively. DNA fingerprint data were
analyzed as previously described (39).
Statistical analysis. MPN values were determined as described previously (37). When all tubes were negative (the MPN index was 1.8 MPN/
100 ml), 0.1 MPN/100 ml was used for log transformation and statistical
analysis. When all tubes were positive (the MPN index was 1,600 MPN/
100 ml), a value of 1,600 MPN/100 ml was used. Enterococcal density was
expressed as MPN/100 g of oven-dried sample (sand, submerged sediment, and soil samples) or CFU/100 ml (water samples). Sample moisture
was expressed as the ratio of the mass of water to the mass of the original
sample.
To satisfy the assumption of a normal distribution, the density data
were log transformed for all statistical analyses. Multiple comparisons of
densities were carried out by using unprotected Fishers least significant
difference at 0.05 (R software). The precipitation data for the KS site
were obtained from the Lake Superior-Duluth streams website (www
.lakesuperiorstreams.org/). The data recorded at the Duluth Lift Bridge
were used for the DBC site because of its close proximity (a straight-line
distance of about 1,100 m). The daily mean temperatures at the KS and
DBC sites in 2010 were obtained at the Thompson Hill I-35 mile post 248
(a straight-line distance of about 3,600 m) and Duluth Sky Harbor (a
straight-line distance of about 5,500 m) weather stations, respectively.

Ran et al.

represent standard errors. The same letter in more than one bar indicates that there is no significant difference (P 0.05). BDL1 indicates that enterococcal
densities were below the detection limit (1 CFU/2 g of original sample) of the MF technique. The BDL2 indicates that the enterococcal densities were below the
detection limit (1.8 MPN/10 g of original sample) of the MPN technique. The numbers (5, 6, 7, 8, and 9) on the x axis represent sampling months (May, June,
July, August, and September, respectively). Samples: S5, submerged sediment located 5 m from the waterline; SL, wet sand located at the waterline; NS, wet sand
located 1 m upshore from the waterline; US, dry sand located 8 m upshore from the waterline; W, water; KS5, soil located 5 m from the creek water; KS14, soil
located 14 m from the creek water; KS14I, soil in exclosure boxes.

from the DBC and KS sites, respectively, were subjected to HFERP

DNA fingerprinting. Since the fingerprints of positive-control
strain E. faecalis OG1RF had a minimum similarity of 95% over
repeated analyses (including multiple PCR analyses using DNA
and colonies as templates), fingerprints with 95% similarity
were regarded as the same genotype (data not shown).
The E. faecalis population was diverse in the Lake Superior
watershed over the 2-year period and was composed of unique
isolates, groups of a few isolates, and large groups of clonal isolates
(Fig. 4). Further examination of the dendrogram showed that
large groups usually contained a large proportion of isolates collected in July and August. Among the 536 isolates examined, 148
genotypes were identified. Their similarity values ranged from 9.8
to 94.9%, with the majority of isolates being 60% similar to each

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other. The Shannon diversity index value was 4.08, suggesting a

high level of diversity within the total population.
The genetic diversity of E. faecalis isolates at the DBC site was
greater than that at the KS site. At the DBC site, 108 genotypes
were identified among the 309 isolates examined. Seven of the
genotypes contained 10 isolates. However, the majority of DNA
fingerprint patterns were unique. In contrast, 46 genotypes were
detected among the 227 isolates obtained from the KS site. The
Shannon diversity indices of E. faecalis at the KS and DBC sites
were 2.87 and 3.84, respectively. Further examination of the dendrogram generated with isolates from the KS site revealed that two
large groups, accounting for 38% of the isolates, contained 52 and
34 isolates, respectively. The total population diversity was also
examined by discriminant analyses. As shown in Fig. 5, multivar-

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FIG 2 Density of enterococci in the Lake Superior watershed. The densities and temperatures are shown as bar and scatter-line plots, respectively. Error bars

Enterococci in a Lake Superior Watershed

strains isolated from the Lake Superior watershed. A cutoff value of 85% was
selected in order to display the dendrogram. The number next to a cluster is the
number of isolates in that cluster.

watershed. *, the number of isolates analyzed was less than 24; ND, no data
available as densities were below the detection limit; NA, data not accessible;
S5, submerged sediment located 5 m from the waterline; SL, wet sand located
at the waterline; NS, wet sand located 1 m upshore from the waterline; US, dry
sand located 8 m upshore from the waterline; W, water; KS5, soil located 5 m
from the creek water; KS14, soil located 14 m from the creek water; KS14I, soil
in exclosure boxes.

iate analysis of variance (MANOVA) indicated that the E. faecalis

population exhibited spatial and temporal variability, although
there was some overlap because of the relative relatedness of different groups. This finding was further supported by Jackknife
analysis ( Table 1). Jaccard similarity values ranging from 1.8 to
6.8% suggested that very few genotypes were shared by the different groups.
Recurrence of some E. faecalis fingerprints. Further examination of the dendrogram showed that some E. faecalis DNA fingerprints occurred over multiple (2) sampling events at each sampling site. For example, 21 of 25 KS5 isolates in August 2010, 21 of
25 KS14 isolates in August 2010, and 8 of 10 KS14 isolates in
September 2010 clustered together with similarity values of 97%
(Fig. 6). These isolates were considered to be of the same genotype.
In total, 12 and 8 genotypes obtained from the DBC and KS sites,
respectively, recurred over multiple sampling times, accounting
for 25 and 52% of the isolates collected at the two sites, respec-

May 2013 Volume 79 Number 9

Second Discrimin
nant 24%

FIG 3 Diversity of Enterococcus species composition in the Lake Superior

tively. Moreover, MANOVA (Fig. 7A) showed that the majority of

these isolates, especially those isolated from KS soil, clustered and
were separate from the others, suggesting that some of these isolates likely persist in these environments.
Enterococci in exclosure boxes. The enterococcal density in
the exclosure boxes at the KS14 site initially decreased below the
detection limit (1.8 MPN/10 g of original sample) in June 2011 but
later increased to as much as 3.7 103 MPN/100 g of oven-dried
sample as the temperature increased in August 2011 (Fig. 2,

2010 DBC
2011 DBC

2010 KS
2011 KS

First Discriminant 60%

FIG 5 MANOVA of all HFERP DNA fingerprints generated from environmental E. faecalis isolates grouped by year and site. The discriminants are
shown by the distance along the x and y axes. The percentage of variation each
discriminant accounts for is shown, and the number before the site name
indicates the year the isolates were collected.

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FIG 4 Partial dendrogram generated from DNA fingerprints of E. faecalis

Ran et al.

TABLE 1 Jackknife analyses of DNA fingerprints from E. faecalis strains

grouped by site and year

2011

Yr and site

DBC

KS

DBC

KS

2010
DBC
KS

71.4
13.0

5.7
84.3

12.3
7.7

16.1
8.0

2011
DBC
KS

13.6
1.9

7.1
2.9

72.9
7.1

DISCUSSION

The goals of this study were to examine the population structure

of enterococci in a Lake Superior watershed and to determine if
these bacteria can persist in the extraintestinal environment, as
was reported for E. coli at the same sites (2, 32). The main finding
of this study was that enterococci can persist in this Great Lakes
environment for a prolonged time after being introduced and

Percent Similarity
97

100

1/25-KS14-July 2010
21/25-KS5-August
21/2
S A
2010
21/25-KS14-August 2010
1/7-KS5-September 2010
8/10-KS14-September 2010

FIG 6 Partial dendrogram generated from HFERP DNA fingerprints of some

E. faecalis isolates collected at the KS site. The terms on the right of the dendrogram indicate the number of strains clustered (for example, 1/25 is 1 out of
25 strains), the sampling site, and the sampling time.

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Recurrent DBC
Unique DBC

Recurrent KS
Unique KS
KS14I

First
i Discriminant
i i i
82%
2%

FIG 7 MANOVA of HFERP DNA fingerprints generated from environmental

E. faecalis isolates grouped by site and their frequency. Graph A contains all of
the isolates from the DBC and KS sites; graph B contains only the isolates from
the KS site. Recurrent indicates that the genotype appeared over multiple
sampling events, unique indicates that the genotype only appeared once.
KS14I indicates the isolates collected from the exclosure boxes at the KS14 site.

likely grow when environmental conditions (moisture and temperature) become favorable.
The densities of enterococci were positively correlated with
sample temperatures (DBC, r 0.57, P 9.4 108; KS, r
0.51, P 0.01). Since our samples were taken from the top layer of
the sampling areas around noon, the sample temperature was
quite close to the air temperature, which could reach around 33C
in July. This is quite close to the optimum growth temperature for
enterococci (35C). Moreover, nutrients, including sea grass debris carried onto the beach sand by wave action and natural grass
at the KS site, might also favor the persistence and growth of these
bacteria in the summer months.
To test if enterococci could persist for a prolonged time or
become naturalized (2) to the environment examined, we covered the sampling area at the KS14 site to avoid direct contamination from external sources. We consistently isolated enterococci
within exclosure boxes. Since the sampling areas were covered, the
isolates were likely independent of recent contamination events
and represented persistent enterococci in the environment. The
disappearance and reappearance of enterococci and the inconsistent Enterococcus species composition in our study might be due
to the heterogeneous character of the microbial community in the
soil environment and the limited number of culturable enterococci studied.
The presence of persistent enterococci at the study site was also
supported by the presence of recurrent E. faecalis DNA fingerprints over multiple sampling events, especially those strains
isolated at the KS site. There is limited human activity at the KS
site, and there was not consistent precipitation before sampling

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KS14I-2011). As expected, the moisture of the samples in the exclosures was slightly lower than that of soil exposed to the environment (June, KS14, 11%, KS14I, 10%; July, KS14, 17%, KS14I,
15%). Isolates of E. faecalis, E. casseliflavus, E. mundtii, and E. hirae
were consistently isolated from exclosure boxes from July to September, after the sampling areas were protected (Fig. 3, KS14I2011).
Since the exclosure boxes limited direct enterococcal input
from external sources, it was of interest to examine the genetic
diversity of the E. faecalis isolates in the exclosure boxes at the
KS14 site. The population of E. faecalis in the exclosure boxes was
relatively diverse, and 17 genotypes, with similarity values ranging
from 49.0 to 92.5%, were identified among 47 isolates. One large
group contained 18 isolates that were collected in August 2011. No
fingerprints appeared over multiple sampling events. Moreover,
MANOVA showed that these isolates tended to be separate from
other isolates from the KS site (Fig. 7B). Because of disruption of
the sampling area by road construction, the isolates collected in
September 2011 were excluded from the MANOVA.

Recurrent KS
Unique KS

First Discriminant 59%

16.1
59.8
Second Discriminant 18 %

2010

Second Diiscriminant 24%

Maximum similarity (%)

Enterococci in a Lake Superior Watershed

May 2013 Volume 79 Number 9

submerged sediment, SL, and nearshore sand samples at the DBC

were related to antecedent precipitation 24 h prior to sampling
(submerged sediment, r 0.67, P 0.05; SL sand, r 0.55, P
0.12; nearshore sand, r 0.44, P 0.24). This suggested that
runoff from rain events might contain E. faecalis and transport
this bacterium from nearshore sand into water or into matrices
having contact with water. The Enterococcus species composition
shifted dramatically at the KS site in 2011, perhaps because of
disruption from road construction. The species composition at
this site is also likely influenced by vegetation. For example, some
yellow-pigmented enterococci, including E. casseliflavus and E.
mundtii, are considered to associate mainly with plants (4850),
and the KS site contained extensive vegetation. A previous study
also suggested that rainfall and gravity could transport the bacteria
from plant leaves to soil (48). Since this study aimed to uncover
the species composition of these bacteria at the sites, instead of
tracking their source, further studies on the possible sources of
enterococci at these sites are needed.
At the DBC site, the positive correlation of enterococcal densities in water, submerged sediment, and SL sand samples; the
diverse Enterococcus species composition in water and sand samples; and the co-occurrence of some E. faecalis fingerprints in different matrices support the hypothesis that external forces are
involved in the transport of these bacteria. Previous research reported seiche tides in the Duluth-Superior Harbor occur every
several hours, with amplitudes ranging from 3 to 25 cm (51, 52). It
is possible that seiche mixed the enterococci among SL sand, submerged sediments, and water. Considering that nearshore sand
samples were wet throughout all of the sampling events, seiche
might also transport enterococci from nearshore sand into water.
Wave action and runoff from rain events were suggested to transport bacteria from sand or soil to adjacent water (21), elevating
their populations in water and confounding their use as fecal indicators.
The E. faecalis strains isolated in the Lake Superior watershed
were genetically diverse, with a Shannon diversity index of 3.84 at
the DBC and 2.87 at the KS site. Brownell et al. (30) reported that
the Shannon diversity indexes of enterococci ranged from 1.88 to
2.69 in raw sewage, pristine river water, storm water-impacted
sediments, and water on the basis of repetitive-sequence PCRbased DNA fingerprinting with BOXA2R primers. The discriminant analyses done here showed that the genetic diversity of all of
our E. faecalis isolates varied spatially and temporally and was
likely influenced by the diverse sources of these bacteria, similar to
what was seen in a previous study of E. coli (53).
Understanding of the occurrence and persistence of enterococci in freshwater environments is important before all Great
Lakes and coastal states decide to use enterococci as the fecal indicator bacteria for the assessment of recreational water quality.
Moreover, since Lake Superior watersheds are different in temperature and beach composition from those of the other Great Lakes,
it is also important to examine the presence and species distribution of enterococci near Lake Superior. In contrast to some previous studies, our results provide in-depth information on the distribution, genetic diversity, and persistence of enterococci in
freshwater environments. Our results also showed the seasonal
change in the enterococcal concentration in the watershed, which
was partially due to the persistence and growth of enterococci in
the environment after their introduction. The diversity of Enterococcus species composition and the genetic diversity of E. faecalis

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(www.lakesuperiorstreams.org/). This indicated that at least some

of these isolates were not related to direct input from human or
animal feces and recent runoff from rain events. Moreover, some
of these recurrent isolates were very abundant in samples at the KS
site. For example, 21 (84%) of 25 E. faecalis isolates collected at the
KS14 site in August 2010 had the same fingerprint. In addition,
isolates collected in July and August usually clustered as large
groups. Since we did not observe any animals or feces in the sampling area at the KS site over the sampling period, our data suggest
that these E. faecalis isolates were persistent after being introduced
into the extraintestinal environment. Moreover, these isolates
likely grew in the summer months, especially in July and August,
when environmental conditions, including temperature, moisture, and nutrients, become favorable and support microbial
growth (8). This likely partially explained the elevated enterococcal concentration and high percentage of E. faecalis bacteria in the
summer of 2010 at the KS site. We also found that only a very
limited number of strains could be repeatedly isolated over the 2
years. Taken together, these results suggested that some enterococci are able to persist in the Lake Superior environment, especially in soil, but because of extreme cold temperatures and nutrient depravation, they might not become naturalized to the
environments examined.
We also found that enterococcal densities in sand, sediment,
and soil samples were high. On the basis of a mass unit calculation,
the enterococcal concentration in a majority of the samples exceeded the standard of 35 CFU/100 g of sample (6). Exceedances
are expected if we express the concentration as MPN/100 ml of
interstitial water, since the moisture of a majority of the samples
was below 50%. If the high density of enterococci at the sites we
examined was due mainly to their persistence and growth, it may
lead to unnecessary beach closures. Therefore, assessment of the
public health risks of illnesses associated with exposure to the
matrices examined at the DBC site is suggested.
Monthly enterococcal densities in nearshore sand samples at
the DBC site were 7 to 87 times greater than those found in water
throughout the sampling period on the basis of a mass unit. A
similar phenomenon was also observed at some Lake Huron
beaches and marine beaches (16, 44). Compared with water, sand
and soil provide relatively favorable environments for the bacteria
to survive (18, 21). Considering that our sampling time was generally near noon, when water was exposed to strong sunlight, a
lower enterococcal concentration in water samples than in sand
may be responsible for some of the noted disparity in values.
Consistent with previous research (10, 15, 45), the abundant
species identified in sand, sediment, and soil samples of the freshwater environments examined included E. hirae, E. faecalis, E.
faecium, E. mundtii, and E. casseliflavus. However, unexpectedly,
there was a very low percentage of E. faecalis and a high percentage
of E. faecium in the water column at the DBC site. Previous research reported relatively high percentages of both E. faecalis and
E. faecium in marine water in the United States (10, 22) and in lake
water in Russia (45), and some studies reported that the most
abundant species in sewage was E. faecium (46, 47). Further studies using microbial source tracking techniques need to be carried
out in order to better understand the source of enterococci in
water.
The spatial and temporal dynamics of Enterococcus species
composition reflected the effects of environmental factors, such as
runoff from rain events. The percentages of E. faecalis bacteria in

Ran et al.

isolates likely reflect diverse input sources and multiple environmental factors influencing the growth and distribution of enterococci.
ACKNOWLEDGMENTS
This work was funded in part by the Minnesota Sea Grant College Program, supported by the NOAA Office of Sea Grant, United States Department of Commerce, under grant no. R/CC-02-10. This paper is journal
reprint no. JR 604 of the Minnesota Sea Grant College Program.
We thank Matthew Hamilton and John Ferguson for help with
HFERP DNA fingerprint analyses and Jessica Eichmiller for her assistance
with sampling. We also thank Alexandria Boehm and Dawn Manias for
providing strains and Charlene Jackson for multiplex PCR support.

REFERENCES

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aem.asm.org

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on June 1, 2014 by guest

1. Leclerc H, Mossel D, Edberg S, Struijk C. 2001. Advances in the bacteriology of the coliform group: their suitability as markers of microbial
water safety. Annu. Rev. Microbiol. 55:201234.
2. Ishii S, Ksoll W, Hicks R, Sadowsky M. 2006. Presence and growth of
naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl. Environ. Microbiol. 72:612 621.
3. Byappanahalli MN, Yan T, Hamilton MJ, Ishii S, Fujioka RS, Whitman
RL, Sadowsky MJ. 2012. The population structure of Escherichia coli
isolated from subtropical and temperate soils. Sci. Total Environ. 417-418:
273279.
4. Fujioka R, Sian-Denton C, Borja M, Castro J, Morphew K. 1998. Soil:
the environmental source of Escherichia coli and enterococci in Guams
streams. J. Appl. Microbiol. 85:83S 89S.
5. Ferguson D, Signoretto C. 2011. Environmental persistence and naturalization of fecal indicator organisms, p 379 397. In Hagedorn C, Blanch
AR, Harwood VJ (ed), Microbial source tracking: methods, applications,
and case studies. Springer, New York, N.Y.
6. US EPA. 2012. Recreational water quality criteria. 820-F-12-058. Office of
Water, US Environmental Protection Agency, Washington DC.
7. Cabelli VJ, Dufour AP, McCabe L, Levin M. 1982. Swimmingassociation gastroenteritis and water quality. Am. J. Epidemiol. 115:606
616.
8. Byappanahalli MN, Nevers MB, Korajkic A, Staley ZR, Harwood VJ.
2012. Enterococci in the environment. Microbiol. Mol. Biol. Rev. 76:685
706.
9. Byappanahalli MN, Whitman RL, Shively DA, Ting WTE, Tseng CC,
Nevers MB. 2006. Seasonal persistence and population characteristics of
Escherichia coli and enterococci in deep backshore sand of two freshwater
beaches. J. Water Health 4:313320.
10. Ferguson D, Moore D, Getrich M, Zhowandai M. 2005. Enumeration
and speciation of enterococci found in marine and intertidal sediments
and coastal water in southern California. J. Appl. Microbiol. 99:598 608.
11. Ott EM, Mller T, Mller M, Franz C, Ulrich A, Gabel M, Seyfarth W.
2001. Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses. J. Appl. Microbiol. 91:54 66.
12. Lata P, Ram S, Agrawal M, Shanker R. 2009. Enterococci in River Ganga
surface waters: propensity of species distribution, dissemination of antimicrobial-resistance and virulence-markers among species along landscape. BMC Microbiol. 9:140 150.
13. Moriarty E, Nourozi F, Robson B, Wood D, Gilpin B. 2008. Evidence for
growth of enterococci in municipal oxidation ponds, obtained using antibiotic resistance analysis. Appl. Environ. Microbiol. 74:7204 7210.
14. Yamahara K, Walters S, Boehm A. 2009. Growth of enterococci in
unaltered, unseeded beach sands subjected to tidal wetting. Appl. Environ.
Microbiol. 75:15171524.
15. Badgley BD, Nayak BS, Harwood VJ. 2010. The importance of sediment
and submerged aquatic vegetation as potential habitats for persistent
strains of enterococci in a subtropical watershed. Water Res. 44:5857
5866.
16. Wheeler Alm E, Burke J, Spain A. 2003. Fecal indicator bacteria are
abundant in wet sand at freshwater beaches. Water Res. 37:3978 3982.
17. Davies-Colley R, Donnison A, Speed D, Ross C, Nagels J. 1999. Inactivation of faecal indicator micro-organisms in waste stabilisation ponds:
interactions of environmental factors with sunlight. Water Res. 33:1220
1230.

18. Davies CM, Long J, Donald M, Ashbolt NJ. 1995. Survival of fecal
microorganisms in marine and freshwater sediments. Appl. Environ. Microbiol. 61:1888 1896.
19. Villar C, de Cabo L, Vaithiyanathan P, Bonetto C. 1999. Pore water N
and P concentration in a floodplain marsh of the Lower Paran River.
Hydrobiologia 392:6571.
20. Whitman RL, Shively DA, Pawlik H, Nevers MB, Byappanahalli MN.
2003. Occurrence of Escherichia coli and enterococci in Cladophora (Chlorophyta) in nearshore water and beach sand of Lake Michigan. Appl. Environ. Microbiol. 69:4714 4719.
21. Halliday E, Gast RJ. 2011. Bacteria in beach sands: an emerging challenge
in protecting coastal water quality and bather health. Environ. Sci. Technol. 45:370 379.
22. Bonilla T, Nowosielski K, Esiobu N, McCorquodale D, Rogerson A.
2006. Species assemblages of Enterococcus indicate potential sources of
fecal bacteria at a South Florida recreational beach. Mar. Pollut. Bull.
52:807 810.
23. Badgley B, Thomas F, Harwood V. 2010. The effects of submerged
aquatic vegetation on the persistence of environmental populations of
Enterococcus spp. Environ. Microbiol. 12:12711281.
24. Facklam RR, Carvalho M, Teixeira LM. 2002. History, taxonomy, biochemical characteristics, and antibiotic susceptibility testing of enterococci, p 154. In Gilmore MS (ed), The enterococci: pathogenesis, molecular biology, and antibiotic resistance. ASM Press, Washington, DC.
25. Wheeler A, Hartel P, Godfrey D, Hill J, Segars W. 2002. Potential of
Enterococcus faecalis as a human fecal indicator for microbial source tracking. J. Environ. Qual. 31:1286 1293.
26. Huycke MM, Sahm DF, Gilmore MS. 1998. Multiple-drug resistant
enterococci: the nature of the problem and an agenda for the future.
Emerg. Infect. Dis. 4:239 249.
27. Gentry-Weeks CR, Karkhoff-Schweizer RA, Pikis A, Estay M, Keith JM.
1999. Survival of Enterococcus faecalis in mouse peritoneal macrophages.
Infect. Immun. 67:2160 2165.
28. Ruiz-Garbajosa P, Del Campo R, Coque TM, Asensio A, Bonten M,
Willems R, Baquero F, Cantn, R. 2009. Longer intestinal persistence of
Enterococcus faecalis compared to Enterococcus faecium clones in intensivecare-unit patients. J. Clin. Microbiol. 47:345351.
29. Sedgley C, Lennan S, Appelbe O. 2005. Survival of Enterococcus faecalis in
root canals ex vivo. Int. Endod. J. 38:735742.
30. Brownell M, Harwood V, Kurz R, McQuaig S, Lukasik J, Scott T. 2007.
Confirmation of putative stormwater impact on water quality at a Florida
beach by microbial source tracking methods and structure of indicator
organism populations. Water Res. 41:37473757.
31. Feng F, Goto D, Yan T. 2010. Effects of autochthonous microbial community on the die off of fecal indicators in tropical beach sand. FEMS
Microbiol. Ecol. 74:214 225.
32. Ishii S, Hansen D, Hicks R, Sadowsky M. 2007. Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior.
Environ. Sci. Technol. 41:22032209.
33. Hakala C, Lesmeister A, Westbrook A. 2011. Minnesota Lake Superior
beach monitoring and notification program annual report. Minnesota
Department of Health, Duluth, MN. http://www.mnbeaches.org/data/20
11_Beaches_Annual_Report_MDH[1].pdf.
34. Ishii S, Yan T, Vu H, Hansen DL, Hicks RE, Sadowsky MJ. 2010. Factors
controlling long-term survival and growth of naturalized Escherichia coli
populations in temperate field soils. Microbes Environ. 25:8 14.
35. Kingsley M, Bohlool B. 1981. Release of Rhizobium spp. from tropical
soils and recovery for immunofluorescence enumeration. Appl. Environ.
Microbiol. 42:241248.
36. US EPA. 2006. Method 1600: enterococci in water by membrane filtration
using membrane-enterococcus indoxyl--D-glucoside agar (m-EI agar).
EPA-821-R-06-009. Office of Water, US Environmental Protection
Agency, Washington, DC.
37. American Public Health Association. 2005. Standard methods for the
examination of water and wastewater, 21st ed, p 9-54 9-88. American
Public Health Association, Washington DC.
38. Jackson C, Fedorka-Cray P, Barrett J. 2004. Use of a genus-and speciesspecific multiplex PCR for identification of enterococci. J. Clin. Microbiol.
42:3558 3565.
39. Johnson L, Brown M, Carruthers E, Ferguson J, Dombek P, Sadowsky
M. 2004. Sample size, library composition, and genotypic diversity among
natural populations of Escherichia coli from different animals influence

Enterococci in a Lake Superior Watershed

40.
41.
42.
43.

44.

May 2013 Volume 79 Number 9

46.
47.
48.
49.
50.
51.
52.
53.

2010. Investigation of distribution, species composition, and degree of

resistance to antibiotics of the bacteria of the Enterococcus genus in Lake
Baikal. Contemp. Prob. Ecol. 3:457 462.
Laukov A, Juris P. 1997. Distribution and characterization of Enterococcus species in municipal sewages. Microbios 89:73 80.
Moore D, Guzman J, McGee C. 2008. Species distribution and antimicrobial resistance of enterococci isolated from surface and ocean water. J.
Appl. Microbiol. 105:10171025.
Mundt JO. 1961. Occurrence of enterococci: bud, blossom, and soil studies. Appl. Microbiol. 9:541544.
Mundt JO. 1963. Occurrence of enterococci on plants in a wild environment. Appl. Microbiol. 11:141144.
Mundt JO, Graham WF, McCarty I. 1967. Spherical lactic acidproducing bacteria of southern-grown raw and processed vegetables.
Appl. Microbiol. 15:13031308.
Jordan TF, Stortz KR, Sydor M. 1981. Resonant oscillation in DuluthSuperior Harbor. Limnol. Oceanogr. 26:186 190.
Stortz K, Sydor M. 1980. Transports in the Duluth-Superior Harbor. J.
Great Lakes Res. 6:223231.
Byappanahalli MN, Whitman RL, Shively DA, Ferguson J, Ishii S,
Sadowsky MJ. 2007. Population structure of Cladophora-borne Escherichia coli in nearshore water of Lake Michigan. Water Res. 41:3649 3654.

aem.asm.org 3075

Downloaded from http://aem.asm.org/ on June 1, 2014 by guest

45.

accuracy of determining sources of fecal pollution. Appl. Environ. Microbiol. 70:4478 4485.
Koeuth T, Versalovic J, Lupski J. 1995. Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements
in diverse bacteria. Genome Res. 5:408 418.
Malathum K, Singh K, Weinstock G, Murray B. 1998. Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of
Enterococcus faecalis at the subspecies level. J. Clin. Microbiol. 36:211215.
Kogan SC, Doherty M, Gitschier J. 1987. An improved method for
prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A. N. Engl. J. Med. 317:985990.
Badgley BD, Ferguson J, Heuvel AV, Kleinheinz GT, McDermott CM,
Sandrin TR, Kinzelman J, Junion EA, Byappanahalli MN, Whitman RL.
2011. Multi-scale temporal and spatial variation in genotypic composition
of Cladophora-borne Escherichia coli populations in Lake Michigan. Water
Res. 45:721731.
Bonilla TD, Nowosielski K, Cuvelier M, Hartz A, Green M, Esiobu N,
McCorquodale DS, Fleisher JM, Rogerson A. 2007. Prevalence and
distribution of fecal indicator organisms in South Florida beach sand and
preliminary assessment of health effects associated with beach sand exposure. Mar. Pollut. Bull. 54:14721482.
Parfenova V, Pavlova O, Kravchenko O, Tulupova Y, Kostornova T.