Occurrence, Genetic Diversity, And Persistence of Enterococci in A

Published Ahead of Print 1 March 2013. 10.1128/AEM.03908-12.

2013, 79(9):3067. DOI:Appl. Environ. Microbiol. Dunny and Michael J. SadowskyQinghong Ran, Brian D. Badgley, Nicholas Dillon, Gary M.

Superior WatershedPersistence of Enterococci in a Lake Occurrence, Genetic Diversity, and

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Occurrence, Genetic Diversity, and Persistence of Enterococci in aLake 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 ofMinnesota, Minneapolis, Minnesota, USAc

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

Fecal contamination of recreational waters is a widespreadproblem across the Great Lakes region of the United States.Because of difficulties and high costs associatedwith detection andquantitation of fecal pathogens (1), fecal indicator bacteria (FIB)were chosen to assess the potential presence of pathogens. Tradi-tionally, Escherichia coli and fecal coliforms have been used as FIBin freshwater systems, while enterococci were initially used as in-dicators inmarine waters. Previous studies have shown that E. colican become naturalized to the microbial community in tropical,subtropical, and temperate soil and sand (24). This likely limitsthe use of this bacterium as an indicator of water quality. More-over, these culture-based methods cannot differentiate amongsources of fecal bacteria (5).

The U.S. Environmental Protection Agency (EPA) has sug-gested that coastal and Great Lakes states adopt enterococci as analternative indicator of fecal contamination (6). Epidemiologicalstudies have shown that the enterococcal concentration has astrong positive correlation with the risk of gastroenteritis associ-ated with swimming in contaminated freshwater (7). However,the potential advantages of enterococci over E. coli as the indicatorof choice to detect fecal contamination of waterways and the en-vironment warrant further investigation (8), especially in the coldclimate associated with the Lake Superior watershed.

It has been suggested that some Enterococcus species are pri-marily of environmental origin (5). Some species, including En-terococcus faecalis, E. faecium, E. casseliflavus, E. hirae, E.mundtii,E. gallinarum, E. durans, E. avium, and E. sulfureus, have beenrepeatedly isolated from sand, sediment, water, or plants (912).In addition, evidence of the persistence of enterococci in munici-pal oxidation ponds and in microcosms simulating environmen-tal conditions has been previously documented (13, 14). The highlevel of fecal bacteria in sand, sediment, soil, and submerged veg-etation has raised public health concerns (2, 15, 16). Since matri-ces harboring enterococci may protect these bacteria from inacti-vation by sunlight and from protozoan predation or offer a range

of surfaces for attachment and nutrient acquisition (1720), it hasbeen suggested that they may serve as reservoirs for FIB (21).However, the majority of studies on the ecology of enterococcihave been done in tropical and subtropical environments or inwarmer Great Lakes states.

Enterococci are nearly ubiquitous and have been isolated froma variety of substrates (4, 10, 15). Species recovered from marineenvironments included mainly E. faecalis, E. faecium, E. casselifla-vus, E. hirae, and E. mundtii (10, 15, 22). Of these species, E. cas-seliflavus has been shown to replicate and persist in vegetation-containing microcosms (23). Since the ecology of enterococci inwater, sand, and soil may be affected by abiotic and biotic factors(21), it is likely that the concentration and species composition ofenterococci in temperate freshwater environments differ fromthose found in other environments. In the few studies carried outin temperate freshwater environments, enterococci were recov-ered from backshore beach sand in Lake Michigan from early fallto early summer of the next year, and the dominant species (92%)recovered was E. faecium (9). Another study carried out at LakeHuron found that the density of enterococci in wet sand washigher than that in water (16). But none of the previous studiesexamined the species composition over time and in different sub-strates at the study sites.

The bacterium E. faecalis has received much attention becauseof 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, [email protected].

* Present address: Brian D. Badgley, Department of Crop and Soil EnvironmentalSciences, Virginia Tech, Blacksburg, Virginia, USA.

Copyright 2013, American Society for Microbiology. All Rights Reserved.

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tions (24). This bacterium is the primary Enterococcus species in-habiting the intestinal tracts of humans and some animals (25)and is the predominant species causing hospital-acquired infec-tions (26). The majority of studies on the survival of E. faecalishave been done inmedical fields (2729), andmany environmen-tal studies have examined the survival and genetic diversity of alimited number of Enterococcus species (15, 30). Therefore, thereis very limited information on the survival of E. faecalis in theenvironment. One of the few studies done under lab conditionsfound that E. faecalis survived longer than E. coli did in sand mi-crocosms (31). Moreover, there is currently little information onthe genetic relatedness among E. faecalis strains isolated from dif-ferent habitats.

The objective of the studies reported here was to examine theoccurrence of enterococci in sand, sediment, water, and soil in aLake Superior watershed. Two different sites were included: thenewDuluth Boat Club (DBC) beach site and the Kingsbury Creekbank site. Moreover, the studies also examined changes in Entero-coccus species composition over considerable spatial and temporalranges. The E. faecalis isolates identified were genotyped to exam-ine their genetic diversity, and since naturalized E. coli strains havebeen isolated from both sites (2, 32), we further determined ifenterococci could also persist in these two habitats.

MATERIALS AND METHODSSampling site description. Two sampling sites in a Lake Superior water-shed were used in these studies: the new DBC beach in the Duluth-Supe-rior 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 sedimentlocated 5 m from the waterline (S5), wet sand at the shoreline (SL), wetsand located 1 m upshore from the SL (NS), and dry sand located 8 mupshore from the waterline (US) (Fig. 1B). The Minnesota Lake SuperiorBeach Monitoring Program reported a high number of beach advisorydays 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 23May 2011, three exclosure boxes (referredto as KS14I), made from 32-gallon trash cans (50-cm diameter) as de-scribed previously (34), were buried at the KS14 location in order toexclude external enterococcal sources: runoff and feces deposition fromanimals. Four mesh-covered windows were placed into the exclosureboxes to facilitate air exchange. The exclosure boxeswere buried in the soilat a depth of 10 cm, and the original trash can lids covered the tops of theboxes.

Sample collection. Triplicate samples, located 1 m apart, were col-lected 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 wereprocessed in the lab. In 2011, water (W) and dry sand (US) samples werealso collected from the DBC. All samples were processed by the day aftersampling. Sample temperature was measured in 2011 by inserting a ther-mometer to a depth of 10 cm in the sampling area. Sampling stopped atthe KS14 site in August 2011 because of road construction that resulted inthe destruction of the sampling areas. While two of the exclosure boxeswere damaged in August, one was not damaged severely and samples werecollected on 24 August 2011. The soil in the very top layer in the exclosureboxes became dry over time, so it was removed when sampling. In addi-tion, runoff flowed through a recently installed drainage culvert into oursampling area (KS5) in September 2011.

Enumeration and isolation of cultivable enterococci. Samples werethoroughly mixed before processing. A 10-g aliquot of each sample and100 ml of extraction buffer (35) amended with 0.01% Tween 20 wereadded to a 160-mlwide-mouthmilk dilution bottle (Corning, Tewksbury,

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 ahorizontal, reciprocating shaker. The bottle contents were allowed to set-tle for 30 min. The supernatants from theMay 2010 samples were filteredthrough 0.45-m membranes that were placed onto the surface of m-EIAgar by the U.S. EPA method 1600 membrane filtration (MF) technique(36). However, because of the low enterococcal densities in samples andthe high turbidity of the extractant, no enterococci were recovered andobserved onm-EIAgar inMay 2010 (detection limit, 1CFU/2 g of originalsample). Thereafter, standard method 9230 B, a multiple-tube most-probable-number (MPN) method, was used (37). Aliquots consisting of10 ml, 1 ml, and 0.1 ml of the supernatants of May, June, and Septembersamples (detection limit, 1.8MPN/10 g of original sample) or 1ml, 0.1ml,and 0.01 ml of the supernatants of July and August samples (detectionlimit, 1.8MPN/1 g of original sample) were inoculated into azide dextrosebroth. Other steps were followed according to method 9230 B. To isolateenterococci, 20- to 25-g samples were treated as described above. Theappropriate amount of extractant was filtered or directly spread ontom-EI Agar plates. The MF method was used to enumerate enterococci inwater samples. Colonies on m-EI Agar with a blue halo were picked andcultivated in 150 l Enterococcosel broth (Difco-BBL, Franklin Lakes,NJ) in 96-well microtiter plates. After growth, 40 l of 80% glycerol wasadded to thewells exhibiting a brownish black color andplateswere storedat80C until used.

Enterococcus isolate identification to the species level. Enterococcusisolates were stamped from glycerol stocks onto LB agar or Trypticase soyagar plates with a 48-pin replicator (Boekel Scientific, Feasterville, PA).Overnight colonies were suspended in 100 l of double-distilled H2O(ddH2O).Multiplex PCRwas used to identify eight species of enterococci:

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

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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 TypeCulture Collection strains E. faecalisATCC19433, E.faeciumATCC19434,E. casseliflavusATCC25788,E. hiraeATCC8043,E.gallinarum ATCC 49573, E. durans ATCC 19432, and E. avium ATCC14025 and an E.mundtii strain previously isolated from the environmentby the lab were used as positive controls. A tube not containing any tem-plate DNA served as the negative control. The primers and PCR parame-ters used were described previously (38).

HFERP DNA fingerprinting. Isolates identified as E. faecalis by mul-tiplex PCR were subjected to biochemical tests for verification. The bio-chemical tests included arginine hydrolysis and arabinose, raffinose, andsorbitol fermentationwith the basalmediumas previously described (25).Isolates that exhibited arginine hydrolysis and were sorbitol fermentationpositive and arabinose and raffinose fermentation negative were verifiedas E. faecalis. The E. faecalis isolates were transferred into new 96-wellmicroplates for horizontal, fluorophore-enhanced repetitive PCR(HFERP) DNA fingerprinting. This technique is similar to repetitive-se-quence PCR-based DNA fingerprinting but uses fluorescently labeledprimers and size markers tomore adequately examine genetic populationstructures among bacteria (39).

DNA, extracted using a GenElute bacterial genomics DNA kit (Sigma-Aldrich), was used as the template for HFERP DNA fingerprinting of the2010 E. faecalis isolates. DNA was extracted with the GenElute BacterialGenomics DNA kit (Sigma-Aldrich). For isolates obtained in 2011, how-ever, a new rapid colony method was developed. Approximately 0.5 l ofeach colony was suspended in 100 l of ddH2O and frozen at 20Covernight, and 2l of the thawed suspension was used directly as the PCRtemplate. Bothmethodswere tested on the same strains and found to yieldidentical results. The BOXA2R primer (5=-ACGTGGTTTGAAGAGATTTTCG-3=) was used for DNA fingerprinting (40). The PCR protocol wasmodified on the basis of previous reports (23, 41). Themastermixture for96 reaction mixtures was prepared first. Each reaction mixture contained5 l of 5Gitschier buffer (42), 0.25 l of 100% dimethyl sulfoxide, 1 lof 50MBOXA2R primers (50% unlabeled primers and 50% 6-carboxy-fluorescein-labeled primers), 0.625 l of 25 mM deoxynucleosidetriphosphate, 0.2 l of 20 mg/ml bovine serum albumin, 0.4 l of 5-U/lTaq (Denville Choice Taq), and 15.525 l of nuclease-free H2O. Twomicroliters of template DNA (100 ng of DNA for 2010 isolates) was addedto each reactionmixture in a final volume of 25l. PCRwas carried out ina PTC 100 or PTC 200 thermal cycler (Bio-Rad MJ Research, Hercules,CA) by the protocol described previously (41), except that 30 cycles wasused instead of 35 cycles. E. faecalis strain OG1RF and ddH2O served aspositive and negative controls, respectively. DNA fingerprint data wereanalyzed as previously described (39).

Statistical analysis.MPN values were determined as described previ-ously (37).When all tubes were negative (theMPN index was1.8MPN/100 ml), 0.1 MPN/100 ml was used for log transformation and statisticalanalysis. When all tubes were positive (theMPN index was1,600MPN/100ml), a value of 1,600MPN/100ml was used. Enterococcal density wasexpressed as MPN/100 g of oven-dried sample (sand, submerged sedi-ment, and soil samples) or CFU/100ml (water samples). Samplemoisturewas expressed as the ratio of the mass of water to the mass of the originalsample.

To satisfy the assumption of a normal distribution, the density datawere log transformed for all statistical analyses. Multiple comparisons ofdensities were carried out by using unprotected Fishers least significantdifference at 0.05 (R software). The precipitation data for the KS sitewere obtained from the Lake Superior-Duluth streams website (www.lakesuperiorstreams.org/). The data recorded at the Duluth Lift Bridgewere used for the DBC site because of its close proximity (a straight-linedistance of about 1,100 m). The daily mean temperatures at the KS andDBC 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 (astraight-line distance of about 5,500 m) weather stations, respectively.

The correlation between the sample temperature and the enterococcaldensity in 2011 was studied by using the Pearson product-moment cor-relation coefficient. HFERP banding patterns were analyzed by usingBionumerics software (version 3.0; Applied Math, Inc.) as described pre-viously (43).

RESULTSEnterococcal density. Enterococci were not detected inMay 2010at the DBC and KS sites by the MF technique (detection limit, 1CFU/2 g of original sample). However, enterococci were ubiqui-tous in samples analyzed by the MPN technique. This is probablydue to the different detection limits of these two methods. By theMPN technique, enterococci were detected in 94%of the samples,with concentrations ranging from 3 105 to 5.6 105 MPN/100g of sample (dryweight of sand, sediment, and soil samples andmlof water samples) (Fig. 2). Even in upshore sand samples at theDBC, where the moisture ranged from 0.3 to 4.5%, 14 of 15 sam-ples (93%) contained enterococci, with concentrations rangingfrom 2 101 to 1.6 104 MPN/100 g of sample. The enterococciwere not found in some of theMay, June, and September samples(detection limit, 1.8 MPN/10 g of original sample). Generally, thedensities were greater when the temperatures were higher. This issupported by the positive correlations between enterococcal den-sities and sample temperatures in 2011 (DBC, r 0.57, P 9.4108; KS, r 0.51, P 0.01).

At the DBC, the comparisons of monthly enterococcal densi-ties across differentmatrices showed that the nearshore sand sam-ples harbored 7 to 87 times more enterococci than did waterthroughout the sampling period (all P values,0.05). Moreover,the enterococcal density in water was positively correlated withthose in SL sand samples (r 0.62, P 0.01) and submergedsediment samples (r 0.64,P 0.01), suggesting that the bacteriamight 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 studyperiod (Fig. 2, KS5 and KS14). The data obtained in 2010 showedno significant difference in the overall enterococcal concentrationbetween the KS5 and KS14 samples at 0.05 (t test, P 0.30).In 2011, however, the overall enterococcal density at the KS5 sitewas significantly greater than that at theKS14 site (t test,P 4.0103). This may be due in part to the fewer samples taken at theKS14 site.Whenmonthly comparisons of the KS andDBC sites overthe 2-year period were carried out, the overall mean enterococcaldensities in soil at the KS site (combining KS5 and KS14 samples)were greater than those at the DBC site (combining sand and sedi-ment samples) in July and August (both P values are0.05).

Diversity of Enterococcus species composition. The speciesstatus of 2,441 enterococcal isolates was determined by multiplexPCR 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. du-rans, 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 sitewere E. faecalis (48.8%), E. mundtii (20.0%), E. casseliflavus(14.2%), and E. faecium (10.8%). Further analyses showed thatthe Enterococcus species composition varied both spatially andtemporally (Fig. 3).

Genetic diversity of E. faecalis in the Lake Superior water-shed.Over the 2-year study period, 309 and 227 E. faecalis isolates

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from theDBC andKS sites, respectively, were subjected toHFERPDNA fingerprinting. Since the fingerprints of positive-controlstrain E. faecalis OG1RF had a minimum similarity of 95% overrepeated analyses (including multiple PCR analyses using DNAand colonies as templates), fingerprints with 95% similaritywere regarded as the same genotype (data not shown).

The E. faecalis population was diverse in the Lake Superiorwatershed over the 2-year period and was composed of uniqueisolates, groups of a few isolates, and large groups of clonal isolates(Fig. 4). Further examination of the dendrogram showed thatlarge groups usually contained a large proportion of isolates col-lected in July and August. Among the 536 isolates examined, 148genotypes were identified. Their similarity values ranged from 9.8to 94.9%, with themajority of isolates being60% similar to each

other. The Shannon diversity index value was 4.08, suggesting ahigh level of diversity within the total population.

The genetic diversity of E. faecalis isolates at the DBC site wasgreater than that at the KS site. At the DBC site, 108 genotypeswere identified among the 309 isolates examined. Seven of thegenotypes contained10 isolates. However, the majority of DNAfingerprint patterns were unique. In contrast, 46 genotypes weredetected among the 227 isolates obtained from the KS site. TheShannon diversity indices of E. faecalis at the KS and DBC siteswere 2.87 and 3.84, respectively. Further examination of the den-drogram generatedwith isolates from theKS site revealed that twolarge groups, accounting for 38% of the isolates, contained 52 and34 isolates, respectively. The total population diversity was alsoexamined by discriminant analyses. As shown in Fig. 5, multivar-

FIG 2 Density of enterococci in the Lake Superior watershed. The densities and temperatures are shown as bar and scatter-line plots, respectively. Error barsrepresent standard errors. The same letter in more than one bar indicates that there is no significant difference (P 0.05). BDL1 indicates that enterococcaldensities 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 thedetection 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 5m from the waterline; SL, wet sand located at the waterline; NS, wet sandlocated 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, soillocated 14 m from the creek water; KS14I, soil in exclosure boxes.

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iate analysis of variance (MANOVA) indicated that the E. faecalispopulation exhibited spatial and temporal variability, althoughthere was some overlap because of the relative relatedness of dif-ferent groups. This finding was further supported by Jackknifeanalysis ( Table 1). Jaccard similarity values ranging from 1.8 to6.8% suggested that very few genotypes were shared by the differ-ent groups.

Recurrence of someE. faecalisfingerprints.Further examina-tion of the dendrogram showed that some E. faecalisDNA finger-prints occurred over multiple (2) sampling events at each sam-pling site. For example, 21 of 25 KS5 isolates in August 2010, 21 of25 KS14 isolates in August 2010, and 8 of 10 KS14 isolates inSeptember 2010 clustered togetherwith similarity values of97%(Fig. 6). These isolateswere 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, accountingfor 25 and 52% of the isolates collected at the two sites, respec-

tively.Moreover,MANOVA (Fig. 7A) showed that themajority ofthese isolates, especially those isolated from KS soil, clustered andwere separate from the others, suggesting that some of these iso-lates likely persist in these environments.

Enterococci in exclosure boxes. The enterococcal density inthe exclosure boxes at the KS14 site initially decreased below thedetection limit (1.8MPN/10 g of original sample) in June 2011 butlater increased to as much as 3.7 103 MPN/100 g of oven-driedsample as the temperature increased in August 2011 (Fig. 2,

FIG 3 Diversity of Enterococcus species composition in the Lake Superiorwatershed. *, the number of isolates analyzed was less than 24; ND, no dataavailable as densities were below the detection limit; NA, data not accessible;S5, submerged sediment located 5 m from the waterline; SL, wet sand locatedat the waterline; NS, wet sand located 1m upshore from the waterline; US, drysand located 8 m upshore from the waterline; W, water; KS5, soil located 5 mfrom the creek water; KS14, soil located 14m from the creek water; KS14I, soilin exclosure boxes.

FIG 4 Partial dendrogram generated from DNA fingerprints of E. faecalisstrains isolated from the Lake Superior watershed. A cutoff value of 85% wasselected in order to display the dendrogram.The number next to a cluster is thenumber of isolates in that cluster.

nant

24%

2010 DBC 2010 KS

Seco

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iscr

imin

2010 DBC2011 DBC

2010 KS2011 KS

First Discriminant 60%

FIG 5 MANOVA of all HFERP DNA fingerprints generated from environ-mental E. faecalis isolates grouped by year and site. The discriminants areshown by the distance along the x and y axes. The percentage of variation eachdiscriminant accounts for is shown, and the number before the site nameindicates the year the isolates were collected.




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KS14I-2011). As expected, the moisture of the samples in the ex-closures was slightly lower than that of soil exposed to the envi-ronment (June, KS14, 11%, KS14I, 10%; July, KS14, 17%, KS14I,15%). Isolates ofE. faecalis,E. casseliflavus,E.mundtii, andE. hiraewere consistently isolated from exclosure boxes from July to Sep-tember, after the sampling areas were protected (Fig. 3, KS14I-2011).

Since the exclosure boxes limited direct enterococcal inputfrom external sources, it was of interest to examine the geneticdiversity of the E. faecalis isolates in the exclosure boxes at theKS14 site. The population of E. faecalis in the exclosure boxes wasrelatively diverse, and 17 genotypes, with similarity values rangingfrom 49.0 to 92.5%, were identified among 47 isolates. One largegroup contained 18 isolates thatwere collected inAugust 2011.Nofingerprints appeared over multiple sampling events. Moreover,MANOVA showed that these isolates tended to be separate fromother isolates from the KS site (Fig. 7B). Because of disruption ofthe sampling area by road construction, the isolates collected inSeptember 2011 were excluded from the MANOVA.

DISCUSSION

The goals of this study were to examine the population structureof enterococci in a Lake Superior watershed and to determine ifthese bacteria can persist in the extraintestinal environment, aswas reported for E. coli at the same sites (2, 32). The main findingof this study was that enterococci can persist in this Great Lakesenvironment for a prolonged time after being introduced and

likely grow when environmental conditions (moisture and tem-perature) become favorable.

The densities of enterococci were positively correlated withsample 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 ofthe sampling areas around noon, the sample temperature wasquite close to the air temperature, which could reach around 33Cin July. This is quite close to the optimum growth temperature forenterococci (35C). Moreover, nutrients, including sea grass de-bris carried onto the beach sand by wave action and natural grassat the KS site, might also favor the persistence and growth of thesebacteria in the summer months.

To test if enterococci could persist for a prolonged time orbecome naturalized (2) to the environment examined, we cov-ered the sampling area at the KS14 site to avoid direct contamina-tion from external sources. We consistently isolated enterococciwithin exclosure boxes. Since the sampling areas were covered, theisolates were likely independent of recent contamination eventsand represented persistent enterococci in the environment. Thedisappearance and reappearance of enterococci and the inconsis-tent Enterococcus species composition in our study might be dueto the heterogeneous character of themicrobial community in thesoil environment and the limited number of culturable entero-cocci studied.

The presence of persistent enterococci at the study site was alsosupported by the presence of recurrent E. faecalis DNA finger-prints over multiple sampling events, especially those strainsisolated at the KS site. There is limited human activity at the KSsite, and there was not consistent precipitation before sampling

TABLE 1 Jackknife analyses of DNA fingerprints from E. faecalis strainsgrouped by site and year

Yr and site

Maximum similarity (%)

2010 2011

DBC KS DBC KS

2010DBC 71.4 5.7 12.3 16.1KS 13.0 84.3 7.7 8.0

2011DBC 13.6 7.1 72.9 16.1KS 1.9 2.9 7.1 59.8

Percent SimilarityPercent Similarity

97 100

1/25-KS14-July 201021/2 S A 201021/25-KS5-August 201021/25-KS14-August 20101/7-KS5-September 20108/10-KS14-September 2010

FIG 6 Partial dendrogram generated fromHFERP DNA fingerprints of someE. faecalis isolates collected at the KS site. The terms on the right of the den-drogram indicate the number of strains clustered (for example, 1/25 is 1 out of25 strains), the sampling site, and the sampling time.

A

iscr

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ant 2

4%


Seco

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i

Recurrent KSUnique KS

Recurrent DBCUnique DBC

crim

inan

t 18

%

B

i i i i 2%

Seco

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is

Recurrent KSUnique KSKS14I


FIG 7 MANOVAofHFERPDNAfingerprints generated from environmentalE. faecalis isolates grouped by site and their frequency. Graph A contains all ofthe isolates from the DBC and KS sites; graph B contains only the isolates fromthe KS site. Recurrent indicates that the genotype appeared over multiplesampling events, unique indicates that the genotype only appeared once.KS14I indicates the isolates collected from the exclosure boxes at the KS14 site.

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(www.lakesuperiorstreams.org/). This indicated that at least someof these isolates were not related to direct input from human oranimal feces and recent runoff from rain events. Moreover, someof these recurrent isolates were very abundant in samples at the KSsite. For example, 21 (84%) of 25 E. faecalis isolates collected at theKS14 site in August 2010 had the same fingerprint. In addition,isolates collected in July and August usually clustered as largegroups. Since we did not observe any animals or feces in the sam-pling area at the KS site over the sampling period, our data suggestthat these E. faecalis isolates were persistent after being introducedinto the extraintestinal environment. Moreover, these isolateslikely grew in the summer months, especially in July and August,when environmental conditions, including temperature, mois-ture, and nutrients, become favorable and support microbialgrowth (8). This likely partially explained the elevated enterococ-cal concentration and high percentage of E. faecalis bacteria in thesummer of 2010 at the KS site. We also found that only a verylimited number of strains could be repeatedly isolated over the 2years. Taken together, these results suggested that some entero-cocci are able to persist in the Lake Superior environment, espe-cially in soil, but because of extreme cold temperatures and nutri-ent depravation, they might not become naturalized to theenvironments examined.

We also found that enterococcal densities in sand, sediment,and soil samples were high.On the basis of amass unit calculation,the enterococcal concentration in a majority of the samples ex-ceeded the standard of 35 CFU/100 g of sample (6). Exceedancesare expected if we express the concentration as MPN/100 ml ofinterstitial water, since the moisture of a majority of the sampleswas below 50%. If the high density of enterococci at the sites weexamined was due mainly to their persistence and growth, it maylead to unnecessary beach closures. Therefore, assessment of thepublic health risks of illnesses associated with exposure to thematrices examined at the DBC site is suggested.

Monthly enterococcal densities in nearshore sand samples atthe DBC site were 7 to 87 times greater than those found in waterthroughout the sampling period on the basis of a mass unit. Asimilar phenomenon was also observed at some Lake Huronbeaches and marine beaches (16, 44). Compared with water, sandand soil provide relatively favorable environments for the bacteriato survive (18, 21). Considering that our sampling time was gen-erally near noon, when water was exposed to strong sunlight, alower enterococcal concentration in water samples than in sandmay be responsible for some of the noted disparity in values.

Consistent with previous research (10, 15, 45), the abundantspecies identified in sand, sediment, and soil samples of the fresh-water 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 percentageof E. faecium in the water column at the DBC site. Previous re-search reported relatively high percentages of both E. faecalis andE. faecium inmarine water in theUnited States (10, 22) and in lakewater in Russia (45), and some studies reported that the mostabundant species in sewage was E. faecium (46, 47). Further stud-ies using microbial source tracking techniques need to be carriedout in order to better understand the source of enterococci inwater.

The spatial and temporal dynamics of Enterococcus speciescomposition reflected the effects of environmental factors, such asrunoff from rain events. The percentages of E. faecalis bacteria in

submerged sediment, SL, and nearshore sand samples at the DBCwere related to antecedent precipitation 24 h prior to sampling(submerged sediment, r 0.67, P 0.05; SL sand, r 0.55, P0.12; nearshore sand, r 0.44, P 0.24). This suggested thatrunoff from rain events might contain E. faecalis and transportthis bacterium from nearshore sand into water or into matriceshaving contact with water. The Enterococcus species compositionshifted dramatically at the KS site in 2011, perhaps because ofdisruption from road construction. The species composition atthis site is also likely influenced by vegetation. For example, someyellow-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 studyalso suggested that rainfall and gravity could transport the bacteriafrom plant leaves to soil (48). Since this study aimed to uncoverthe species composition of these bacteria at the sites, instead oftracking their source, further studies on the possible sources ofenterococci at these sites are needed.

At the DBC site, the positive correlation of enterococcal den-sities in water, submerged sediment, and SL sand samples; thediverse Enterococcus species composition in water and sand sam-ples; and the co-occurrence of some E. faecalis fingerprints in dif-ferent matrices support the hypothesis that external forces areinvolved in the transport of these bacteria. Previous research re-ported seiche tides in the Duluth-Superior Harbor occur everyseveral hours, with amplitudes ranging from 3 to 25 cm (51, 52). Itis possible that seiche mixed the enterococci among SL sand, sub-merged sediments, and water. Considering that nearshore sandsamples were wet throughout all of the sampling events, seichemight also transport enterococci from nearshore sand into water.Wave action and runoff from rain events were suggested to trans-port bacteria from sand or soil to adjacent water (21), elevatingtheir populations in water and confounding their use as fecal in-dicators.

The E. faecalis strains isolated in the Lake Superior watershedwere genetically diverse, with a Shannon diversity index of 3.84 atthe DBC and 2.87 at the KS site. Brownell et al. (30) reported thatthe Shannon diversity indexes of enterococci ranged from 1.88 to2.69 in raw sewage, pristine river water, storm water-impactedsediments, and water on the basis of repetitive-sequence PCR-based DNA fingerprinting with BOXA2R primers. The discrimi-nant analyses done here showed that the genetic diversity of all ofour E. faecalis isolates varied spatially and temporally and waslikely influenced by the diverse sources of these bacteria, similar towhat was seen in a previous study of E. coli (53).

Understanding of the occurrence and persistence of entero-cocci in freshwater environments is important before all GreatLakes and coastal states decide to use enterococci as the fecal in-dicator bacteria for the assessment of recreational water quality.Moreover, since Lake Superiorwatersheds are different in temper-ature and beach composition from those of the other Great Lakes,it is also important to examine the presence and species distribu-tion of enterococci near Lake Superior. In contrast to some previ-ous studies, our results provide in-depth information on the dis-tribution, genetic diversity, and persistence of enterococci infreshwater environments. Our results also showed the seasonalchange in the enterococcal concentration in the watershed, whichwas partially due to the persistence and growth of enterococci inthe environment after their introduction. The diversity of Entero-coccus species composition and the genetic diversity of E. faecalis




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isolates likely reflect diverse input sources and multiple environ-mental factors influencing the growth and distribution of entero-cocci.

ACKNOWLEDGMENTS

This work was funded in part by the Minnesota Sea Grant College Pro-gram, supported by the NOAAOffice of Sea Grant, United States Depart-ment of Commerce, under grant no. R/CC-02-10. This paper is journalreprint no. JR 604 of the Minnesota Sea Grant College Program.

We thank Matthew Hamilton and John Ferguson for help withHFERPDNAfingerprint analyses and Jessica Eichmiller for her assistancewith sampling. We also thank Alexandria Boehm and Dawn Manias forproviding strains and Charlene Jackson for multiplex PCR support.

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Occurrence, Genetic Diversity, and Persistence of Enterococci in a Lake Superior WatershedMATERIALS AND METHODSSampling site description.Sample collection.Enumeration and isolation of cultivable enterococci.Enterococcus isolate identification to the species level.HFERP DNA fingerprinting.Statistical analysis.

RESULTSEnterococcal density.Diversity of Enterococcus species composition.Genetic diversity of E. faecalis in the Lake Superior watershed.Recurrence of some E. faecalis fingerprints.Enterococci in exclosure boxes.

DISCUSSIONACKNOWLEDGMENTSREFERENCES

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Occurrence, Genetic Diversity, And Persistence of Enterococci in A