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a Department of Crop and Soil Sciences, 3111 Plant Sciences, University of Georgia, Athens, GA 30602-7272
b Marine Advisory and Technological Transfer Center, 715 Bay Street, Brunswick, GA 31520-4601
* Corresponding author (pghartel{at}uga.edu).
Received for publication November 6, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: ATCC, American Type Culture Collection • BST, bacterial source tracking • CFU, colony-forming units
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In the case of geography, the evidence suggests that E. coli subspecies are variable. In Australia, geography accounted for 2% of the genetic variations in E. coli isolated from two populations of Australian feral house mice 15 km apart (Gordon, 1997) and 5% of the genetic variation in E. coli from other Australian mammals (Gordon and Lee, 1999). When expanded to a worldwide basis, geographic origin was one of the more important factors to differentiate E. coli (Souza et al., 1999). Hartel et al. (2002) also noted that ribotype sharing of E. coli subspecies within an animal species decreased with increased distance for some animals (i.e., cattle and horses), but not for others (i.e., chicken and swine). Finally, Buchan et al. (2001) observed that none of the 45 DNA banding patterns obtained with denaturing gradient gel electrophoresis (DGGE) of the 16S to 23S intergenic spacer region from 51 E. coli isolates in two water sources 11 km apart overlapped with each other. In the case of changes with time, Jenkins et al. (2003) observed that over a 9-mo period, only 20 of 240 ribotypes (8.3%) were shared at two or more sampling times and were considered resident ribotypes for six randomly selected cattle. Similar findings were observed for the clonal composition of E. coli isolates obtained from feral house mice (Gordon, 1997).
In the case of rainfall, Hartel et al. (2001) obtained E. coli isolates from the Chattahoochee River and its tributaries in Georgia during baseflow and stormflow conditions. They observed 162 ribotypes among 239 isolates obtained during baseflow conditions and 86 ribotypes among 107 isolates obtained during stormflow conditions. When a unique ribotype was defined as being observed in only one location during baseflow and stormflow, 110 of the combined 149 unique ribotypes (74%) remained unique. Finally, in the case of primary versus secondary habitats, evidence suggests that the clonal composition of E. coli changes substantially during the transition from the host to the external environment (Gordon, 2001). Whittam (1989) observed that only 10% of the 113 distinct E. coli clones were recovered from both chickens and their litter. A later study by Gordon et al. (2002) of two households and their associated septic tanks showed that E. coli diversity, as determined by multilocus enzyme electrophoresis, was high in one household and low in another. Thus, differences in E. coli clonal composition may exist between primary and secondary habitats.
Although the data are limited, the results are discouraging because they suggest, at least for E. coli, that geography, time, rainfall, and habitat affect bacterial subspecies composition, and therefore, in the case of BST methods requiring a permanent host origin database, a commensurately large host origin database will be required to encompass these compositional changes. This is a major disadvantage for BST because considerable time and expense will be needed to establish this permanent database.
There may be an inexpensive, timesaving alternative to this permanent database: sampling during either baseflow or stormflow conditions over an ever-decreasing geographic area to determine specific locations with persistent high fecal contamination. Each round of sampling is conducted over a 1-d period. In this manner, subspecies changes with geography, time, rainfall, and habitat are minimized. If BST is required, then environmental isolates are compared with potential host sources directly at the site. No permanent host origin database is required, reducing laboratory costs and time.
Our objective was to test this targeted sampling protocol as a prelude to BST. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. The indicator bacteria were fecal enterococci, recommended by USEPA for use in marine waters (USEPA, 2002). The BST method was ribotyping. Because this method requires a single bacterial species, and the primary concern on the river was human fecal contamination, Enterococcus faecalis was the bacterium selected. The host range of this bacterium is limited to humans and birds when the bacterium is isolated with specific phenotypic tests (Wheeler et al., 2002).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The results of the general sampling suggested a targeted sampling of two areas, a portion of the White Chimney River and the headwaters of the Sapelo River. The first targeted sampling was conducted on Thursday, 25 Apr. 2002, with 10 samples from the White Chimney River and 13 samples from the headwaters of the Sapelo River. The local riverkeeper suggested that optimal results were likely to be obtained on the weekend or a Monday when large numbers of tourists were either in the area or had just left. Therefore, a second sampling was conducted on the White Chimney River on Sunday, 2 June 2002, and in the headwaters of the Sapelo River on Monday, 3 June 2002. A total of 51 samples were obtained from the White Chimney River and 53 samples from the Sapelo River headwaters during this sampling series.
Bacterial source tracking was conducted on Sunday, 30 June 2002, at the headwaters of the Sapelo River and a private wastewater treatment facility. Four samples were obtained: raw sewage coming into the facility, treated sewage in a wastewater lagoon, effluent from a pipe connecting the lagoon to the Sapelo River, and the nearby river. Raw sewage came from nearby businesses only a few hundred meters away. The river sample was taken about 10 m from the pipe.
Fecal Enterococcal Sampling
At each sampling site, a grab sample was obtained 10 cm beneath the water surface with a sterile 250-mL Nalgene bottle (Nalge Nunc International, Rochester, NY). The location was determined with a GPSMAP 175 system (Garmin International, Olathe, KS). All samples were stored on ice in a cooler and processed within 6 h. Water samples were processed with the Enterolert system (IDEXX Laboratories, Westbrook, ME) because the system requires significantly less time per sample for setup, reading, and recording the results than other available methods (Budnick et al., 1996). River samples were serially diluted with sterile distilled water to 10-1 and 10-2 in sterile manufacturer-supplied polystyrene bottles. A minimum 10-fold dilution is required for marine waters with the Enterolert system because the salt in the water affects the medium. Wastewater samples were either left undiluted (100) or were serially diluted with sterile distilled water to 10-2 and 10-4 in sterile polystyrene bottles. A package of powdered Enterolert reagent was added to each polystyrene bottle. After the reagent was dissolved in the sample, the contents of each bottle were added to a Quanti-tray, a sterile disposable panel containing 97 wells. Each Quanti-tray was mechanically sealed. The sealing distributed the sample uniformly into the wells. Each Quanti-tray was incubated for 24 h at 41 ± 0.5°C. The Enterolert system uses Defined Substrate Technology (Edberg and Edberg, 1988). The nutrient indicator substrate, 4-methylumbelliferyl-ß-D-glucoside, is cleaved by enterococci with the enzyme ß-glucosidase, yielding ß-D-glucoside and a fluorescent product, 4-methylumbelliferone (Fricker and Fricker, 1996). Fluorescing (positive) wells were counted under a 365-nm UV light (Model EA-160; Spectronics Corp., Westbury, NY). The number of positive wells was converted to a most probable number (MPN) value based on the dilution factor and manufacturer-supplied MPN tables.
To obtain fecal enterococcal isolates, positive wells of the Quanti-trays were labeled with an acetate marker. The back of each Quanti-tray was surface-disinfected with 70% ethanol and each positive well was slit open with a sterile scalpel. A 10-µL portion was removed from each positive well with a sterile plastic loop and was streaked onto a 5-cm Petri plate of Enterococcosel agar (Becton Dickinson and Co., Sparks, MD). Plates were incubated in Ziploc bags (DowBrands, Indianapolis, IN) at 37°C for 48 h.
Speciation of Enterococcal Isolates
One black isolate (indicating esculin hydrolysis) was picked from each Enterococcosel agar plate with a sterile plastic stab. Each isolate was suspended in 125 µL of saline–phosphate buffer (0.35 g KH2PO4, 0.65 g K2HPO4, and 8.5 g NaCl L-1; pH 7.0) contained in each well of a 96-well microtiter plate. Three wells of the 96-well plate were reserved for American Type Culture Collection (ATCC, Manassas, VA) controls (Ent. faecalis ATCC #19433, Ent. faecium ATCC #19434, and Ent. gallinarum ATCC #49573), and three wells were reserved for randomly placed uninoculated controls. Each isolate was inoculated with a replicator (Sigma, St. Louis, MO) into separate microtiter plates containing Brain Heart Infusion (BHI) broth (Difco, Sparks, MD) with 6.5% NaCl, arginine hydrolysis medium (Difco) with and without arginine, and modified pyruvate, arabinose, and raffinose carbon utilization media (all Sigma) as described by Wheeler et al. (2002). Plates were incubated at 37°C and results were recorded after 24 and 48 h. A catalase test with 8.82 M H2O2 was performed (MacFaddin, 1976) in the remaining saline phosphate–cell suspension to ensure that each isolate was catalase negative. Isolates identified as Ent. faecalis were placed in a cryoprotectant mixture of saline–phosphate buffer (700 µL), glycerol (100 µL), and dimethyl sulfoxide (100 µL), and kept for long-term storage at -70°C.
Restriction Enzyme
Testing Ent. faecalis isolates on the RiboPrinter (DuPont Qualicon, Wilmington, DE) required determining the best restriction enzyme to discriminate among the isolates. The Ent. faecalis isolates were the same human and chicken isolates tested by Wheeler et al. (2002). Fourteen different restriction enzymes were tested: AseI, BamHI, BglI, BglII, EcoRI, EcoRV, HaeIII, HindIII, HinfI, PstI, PvuII, SalI, XbaI, and XhoI. These enzymes were recommended by the Qualicon Corporation (E. Cole, personal communication, 2002) because they were commercially available in high specific activity (
40 units µL-1), fully active at 37°C, compatible with the high ionic strength of the RiboPrinter DNA preparation buffers, and produced DNA fragments in the 0.6- to 50-kb range. Each enzyme was tested in a single digestion. In addition, two enzymes, AseI and BamHI, were tested in a double digestion because of previous success in obtaining good discriminatory patterns with these enzymes and Ent. faecium (Turlak et al., 2001). Finally, a double digestion with EcoRI and PvuII was tested because these enzymes, when tested separately, yielded a combined pattern that provided good discrimination among Ent. faecalis isolates (Wheeler et al., 2002). All enzymes were initially tested against five randomly selected Ent. faecalis isolates, three from humans and two from chickens. The restriction enzymes that yielded the most bands in the 1- to 50-kb range recognized by the RiboPrinter, and, upon analysis, discriminated the best between humans and chickens, were tested against an additional five Ent. faecalis isolates, four from humans and one from a chicken. The best restriction enzyme was tested against all 26 Ent. faecalis isolates obtained from humans and chickens as described by Wheeler et al. (2002).
Ribotyping
Bacterial isolates were processed on a RiboPrinter as described by Wheeler et al. (2002). Briefly, each Ent. faecalis isolate was streaked onto a Petri plate containing Brain Heart Infusion agar (Difco) and incubated at 37°C for 24 h. Each RiboPrinter sample was obtained by touching the end of a sterile colony pick to a solid lawn of growth, followed by mixing the sample with 40 µL of buffer. This step was repeated once before 30 µL of the buffer was transferred to a sample carrier. After the samples were inactivated in a heat treatment station, 5 µL of two lysing agents were added to each sample before placing the sample carrier into the RiboPrinter system. The DNA of each sample was digested by the restriction enzyme PstI. The DNA fragments were size-separated by electrophoresis on a precast agarose gel and transferred to a nylon membrane. The membrane was exposed to a series of chemical and enzymatic treatments to produce chemiluminescent DNA fragments. The resultant banding pattern was captured by a CCD camera and stored as a TXT file. The files were imported into GelCompar II (Version 3.0; Applied Maths, 2003) for final analysis. Intra- and inter-gel variations were assessed with the Ent. faecalis ATCC #19433 strain. Optimization (shift between any two patterns) was set at 1.56% and tolerance (maximum distance between two band positions on different patterns) was set at 1.00%. Both the optimization and tolerance settings were the default settings for the software program. Similarity indices were determined using Dice's coincidence index (Dice, 1945) and the distance among clusters calculated using the unweighted pair-group method using arithmetic averages (UPGMA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Bacterial source tracking was conducted at the wastewater treatment facility near the Sapelo River on Sunday, 30 June 2002. Fecal enterococci (CFU per 100 mL) were 550000 from raw sewage, 860 from the lagoon, 200 from the pipe, and 740 from the river. Of 216 total raw sewage, wastewater lagoon, and Sapelo River isolates, a majority (106 isolates or 51%) were Ent. faecalis (Table 1). Except for Ent. faecium in raw sewage (39 isolates or 58%), numbers of Ent. faecium, Ent. gallinarum, and non-enterococci were low (<9 isolates per site). Numbers of other enterococci ranged from 4 to 30%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Gordon (2001) suggests that bacterial clonal composition changes during the transition from host to external environment. In this study, raw sewage for the wastewater treatment facility came from local businesses, only a few hundred meters away, so the sewage should represent a fresh fecal sample from humans. When raw sewage was matched with the river, three shared ribotypes representing 40% of the 65 Ent. faecalis isolates were observed; when the wastewater lagoon was matched with the river, five shared ribotypes representing 47% of the 83 Ent. faecalis isolates were observed. Therefore, the BST results were similar. When Ent. faecalis isolates from raw sewage were compared with isolates from the lagoon, only three ribotypes were shared (10%), but these three ribotypes represented 49% of the 68 Ent. faecalis isolates. The data suggest that there was little ribotype sharing between raw sewage and the lagoon, but this lack of sharing was ameliorated because the few ribotypes that were shared represented almost a majority of the isolates. Our results support the idea that clonal composition may change from host to secondary habitat (Gordon et al., 2002), but under the conditions of targeted sampling, the change had little impact because the results suggested the same conclusion (that the source of fecal contamination for the river was the wastewater treatment facility).
In the case of targeted sampling, low counts probably resulted from transient sources of fecal contamination, whereas high counts were more likely from persistent sources. The general sampling suggested that a portion of the White Chimney River, with a count of 689 fecal enterococci per 100 mL, was contaminated, but targeted sampling showed consistently low numbers (ranged from <10 to 41 CFU per 100 mL). The source of fecal contamination was probably transient. In contrast, counts on the headwaters of the Sapelo River were consistently high and were considered a persistent source.
Using the RiboPrinter to ribotype the bacterial isolates worked well. Not only did the RiboPrinter automate ribotyping, save time, and eliminate tediousness, it also increased precision and accuracy by matching Ent. faecalis isolates to a 100% similarity index. Thirty-nine of 83 Ent. faecalis isolates from the wastewater lagoon and the Sapelo River, almost a majority (47%) of the isolates, matched at a 100% similarity index. In contrast, obtaining a 100% similarity index with manual ribotyping is difficult. With manual ribotyping, the banding pattern of the inter-gel control results in a similarity index of approximately 90% (Hartel et al., 2002; Wheeler et al., 2002); here the banding pattern of the inter-gel control resulted in a similarity index of 100%. The major disadvantage of the RiboPrinter was its cost. Targeted sampling does not necessarily require ribotyping; there are a number of other phenotypic and genotypic BST methods that an investigator may consider depending on the reproducibility, discriminatory power, ease of interpretation, and ease of performance required. Nevertheless, it is important to note that targeted sampling reduced the cost of BST required because no permanent host origin database was needed.
Bacterial source tracking suggested that almost half the contribution of fecal contamination to the Sapelo River came from the wastewater treatment facility. It is important to note that the Sapelo River has other potential sources of fecal contamination. Some wild birds are known sources of Ent. faecalis (Wheeler et al., 2002), and we have observed birds, mainly snowy egrets (Egretta thula) and wood storks (Mycteria americana), on this portion of the river. Direct fecal sampling of these shy birds was not done because such a sampling is difficult.
The best restriction enzyme for ribotyping Ent. faecalis was PstI. The restriction enzymes PvuII and XbaI were also good choices. These enzymes should be considered for BST in instances where one restriction enzyme fails to discriminate among the host sources of Ent. faecalis isolates. Neither the double digest of AseI and BamHI (Turlak et al., 2001) nor the double digest of EcoRI and PvuII (Wheeler et al., 2002) discriminated well among the Ent. faecalis isolates obtained from humans and chickens.
Targeted sampling may be a useful tool for water managers responsible for completing total maximum daily load (TMDL) implementation plans. There is also no reason why targeted sampling would not work for other organisms (e.g., E. coli). We are currently testing targeted sampling with this bacterium on the Yagüez River in Puerto Rico.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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