Occurrence, genetic diversity, and persistence of enterococci in a Lake Superior watershed

Abstract

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.

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 .

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 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.

Fig 3

Diversity of Enterococcus species composition in the Lake Superior 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.

Fig 4

Partial dendrogram generated from DNA fingerprints of E. faecalis 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.

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.

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.

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.