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Shiping Deng Department of Plant and Soil Sciences, Oklahoma State University

Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Creek, Cow Creek, and Boomer Creek. Shiping Deng Department of Plant and Soil Sciences, Oklahoma State University . Outline. Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Watershed

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Shiping Deng Department of Plant and Soil Sciences, Oklahoma State University

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  1. Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Creek, Cow Creek, and Boomer Creek Shiping Deng Department of Plant and Soil Sciences, Oklahoma State University

  2. Outline • Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Watershed • Impact of Cultivation on the Nature of Organic Matter and Sorption Behavior of Nonionic Pesticide in Soil • Linking Soil Enzyme Activities to Ecosystem Functions • Phyllosphere Bacterial Endophytes and Phosphorus Nutrition in Corn

  3. “Keystone beach is closed”Tulsa World--5/30/2003 • “The Corps of Engineers has closed the beach at Keystone Lake’s busiest park because E. coli bacteria in the water has reached dangerous levels.” • “Two water samples showed greater than 2,400 E. coli colonies per 100 ml of water, and one sample measured 1,300. Each sample grossly exceeded the 120 units deemed acceptable for human contact.”

  4. OKLAHOMA STANDARDS • Fecal Coliform  < 200 CFU/100ml • E. coli < 126 CFU/100ml • Fecal Strep (Enterococci)  < 33 CFU/100ml Only one of these conditions must be met.

  5. EPA Bacterial Guidelines Federal Register, July, 2004, EPA proposed rules: Escherichia coli or enterococci for fresh recreation water and enterococci for marine recreation water. ______________________________________________ EPA Guidelines Geometric Mean Single Sample E. Coli 126 235 Enterococcoci (freshwater) 33 61 _______________________________________________________

  6. Methods in detecting fecal contamination in water • Multiple-tube Fermentation • Membrane Filtration • Biochemical Tests • Chromagenic and Fluorogenic Substances • Presence-Absence (PA) Test • Chemical Indicators such as Caffeine • DNA (-PCR, -hybridization)

  7. BASIS OF STANDARDS • Fecal Coliform standard is empirically based, and an indicator of the presence of conditions that are likely to have pathogens present (not recommended in the 2004 proposed rules based on epidemiological studies conducted by EPA). • E. coli and Enterococci standards are statistically based to give low illness rate with full body contact, reflecting swimming-associated rate of illness of 8 illnesses per 1000 swimmers at freshwater beaches (USEPA).

  8. ASM RESPONSES TO THE 2004 RECOMMENDATIONS • Issues relate to this study: • Pathogen concentrations in contaminated water, • Pathogen survival in receiving waters, • pathogen transport, • Seasonal variations, • Pathogen accumulation in sediment, and • Pathogen distribution and dynamics.

  9. Are inhabitant in the intestinal tract of warm blooded animals. The release of these microbes and human pathogens through the feces of human and animals to the environment have caused concerns for public health. Therefore, Enterococci have been used as an indicator bacteria of fecal contamination in drinking and recreation water. Enterococci

  10. Kingdom: Bacteria Division: Firmicutes Enterococcus sp. infection in pulmonary tissue Class: Bacilli Order: Lactobacillales Family: Enterococcaceae Genus: Enterococcus (established in 1984, >34 species) Species: E. avium, E. durans, E. faecalis, E. faecium

  11. Objectives • To quantify the seasonal (base-flow and high-flow event) and spatial (surface water and sediments) variations of Enterococci in Stillwater creek, Cow creek, and Boomer creek; and • To examine whether urban population and farming activities contribute to fecal bacterial contamination in water.

  12. Site 2 Site 5 Cow creek Boomer creek Site 1 Site 3 Stillwater creek Site 6 Site 4 Site 7

  13. Brief description of each site • All sample locations were coordinated with a global positioning system (GPS). • Location coordinates, temperature and water depth were recorded at each sampling site. • Turbidity and pH were determined immediately in the laboratory following sampling.

  14. Method of analysis Quantifying Enterococci using IDEXX EnterolertTM system • Took 100 ml water sampling, mixed with IDEXX Enterolert reagent, and transfer the mixture to an IDEXX Quanti-tray  seal the tray and incubate at 41oC for 24 h. • Record the Enterolert results after 24 hours. Fluorescence, due to the activity of β-Glucosidase, was interpreted under UV light at 365 nm. Determine the number of Enterococci per 100 mL by referring to the IDEXX Quanti-Tray MPN Table.

  15. Method of analysis Isolation and Verification • Isolated Enterococci from the Quanti-trays and striked on Enterococcosel agar plates • Following incubating the plates at 37°C for 24 h, observe for black colonies and record results. Positive plates showed esculin hydrolysis with a dark brown to black color in the medium

  16. Method of analysis Isolation and Verification • Streak one colony from each positive Enterococcosel plate on to Brain Heart Infusion Agar (BHIA) with 6.5% NaCl and incubate plates at 37°C for 24 h. Observe and record the results. • Confirmed that the isolate is catalase negative. • Confirmed that the isolate is gram positive. • Confirmed to be spherical or ovoid cells.

  17. EnterococciCounts in Stillwater Creek, Cow Creek, and Boomer Creek During Base-Flow Period Isolates Sample Enterococci counts by % Positive Enterococci Confirmed of IDEXX counts Enterococci IDEXX Enterolert™ evaluated confirmed % -1 -1 MPN 100 mL or 100 g MPN 100-mL-1 or 100 g-1 S Surface 294 25 21 83.0 244 1 S Sediments 365,556 23 0 0 0 1 S Surface 531 25 23 92.0 504 2 S Sediments 226,667 18 2 7.4 16,773 2 S Surface 2,463 25 23 92.0 2,266 3 6 2.5 x 10 1,500,000 S Sediments 19 9 58.3 3 S Surface 279 25 19 75.5 209 4 S Sediments 600,000 9 4 44.4 266,400 4 S Surface 821 25 25 100.0 821 5 S Sediments 673,333 3 1 33.3 224,220 5 S Surface 513 25 24 99.2 492 6 6 1.0 x 10 S Sediments 17 9 52.4 524,000 6 S Surface 154 25 24 95.8 148 7 S Sediments 710,000 11 2 11.1 78,810 7 Total 276 187

  18. SUMMARY • Counts of Enterococci ranged from 2.17 to 3.36 log (MPN 100 mL-1) in surface water and 4.22 to 6.18 log (MPN 100 mL-1) in sediments (with the exception of site 1 which did not show a positive Enterococci count). • All surface water showed Enterococci counts exceeding the recommendation made for recreation water by EPA and Oklahoma Department of Environmental Quality (DEQ) (<33 counts per 100 mL-1).

  19. SUMMARY • Counts of Enterococci were significantly higher in the creeks following passing through the OSU Animal Science Arena (S2 VS S3), suggesting potential impact of farming activity on fecal bacterial contamination in watersheds. • The impact from the urban population was not detectable in the surface water. • Prolonged transportation of fecal microbial contamination to distant water bodies were not evident.

  20. SUMMARY • Counts of Enterococci in the surface water during high-flow period were approximately 100 times higher than those sampled during base-flow period. • Therefore, effective management of storm water is critical in maintaining water quality in the environment.

  21. SUMMARY • Based on detection by the IDEXX Enterolert™ System alone, significant portion of Enterococci counts in sediments were false positive, while the false positive Enterococci counts in surface water was low. • Of the evaluated 276 isolates obtained during base flow period, 187 were positive.

  22. Enterococci Isolated and Evaluated Base-Flow High-Flow Isolates Isolates % + Enterococci % + Enterococci Sample evaluated confirmed of IDEXX counts evaluated confirmed of IDEXX counts % % S Surface 25 21 83 25 20 83 1 S Surface 25 23 92 25 21 92 2 S Surface 25 23 92 25 20 92 3 S Surface 25 19 75.5 25 25 75.5 4 S Surface 25 25 100 25 25 100 5 S Surface 25 24 99.2 25 24 99.2 6 S Surface 25 24 95.8 25 25 95.8 7 Sum 175 159 90.9 175 160 91.4 S Sediments 23 0 0 1 S Sediments 18 2 7.4 2 S Sediments 19 9 58.3 3 S Sediments 9 4 44.4 4 S Sediments 3 1 33.3 5 S Sediments 17 9 52.4 6 S Sediments 11 2 11.1 7 Sum 89 25 28.1

  23. Enterococci Evaluation • IDEXX EnterolertTM test • Black colonies on Enterococcusel agar plates (Esculin hydrolysis) • BHIA with 6.5% NaCl • Catalase negative • Gram positive • Spherical or ovoid cells

  24. Esculin Hydrolysis Hydrolysis By Enterococcus Glycoside esculin ----------------> Dextrose + Esculetin • Esculetin forms an olive green to black complex with iron(III) ions. • Hydrolysis enzymes are constitutive for this test (but can be induced, i.e. E. coli).

  25. QUESTIONS • Do Enterococci adapt to the environment and become false positive based on IDEXX enterolertTMquantification (lost esculin hydrolysis ability; enzymes responsible for this function were not produced)? • Can esculysis hydrolysis of the false positive isolates be induced under a suitable growth conditions?

  26. Repeat the experiment • Sampled at all seven sites plus one additional site at the source stream for boomer lake

  27. Site 2 Site 5 Cow creek Boomer creek Site 1 Site 3 Stillwater creek Site 6 Site 4 Site 7 Site 8

  28. Hypothesis Enterococciquantity and diversity in the water environment is directly linked to its potential sources and vary in space and time

  29. Additional Objectives • To evaluate genetic diversity of the isolated enterococci • To evaluate enterococci diversity in relation to sites and potential sources • To detect and quantify Escherichia coli in Stillwater Creek watershed

  30. Experimental Design (General procedure) IDEXX Enterolert UV Transilluminator Site selection Sampling (Variations: space, season, & water flow) Spectrophotometry pH measurement Data: location coordinates, Temp. & water depth Data: Turbidity & pH Data: Most Probable Number (based on no. of positive wells) MPN positive DATA ANALYSIS Determine species of Enterococcus Confirmation/ Isolation in Enterocossel Agar PCR (Primer: ITS-PCR, Jensen et al., 1993 & Tyrrell et al., 1997) Polymerase Chain Reaction/ PCR (Primers: Ent-1 and Ent-2, Ke et al. 1999) Confirm as Enterococcus isolate

  31. To confirm isolates as Enterococcus: primers are Ent-1 and Ent-2 (Ke et al., 1999) • expected result - 112 bp and 252 bp • To identify enterococcus at species level: PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes • expected result - 300 to 600 bp and large minor bands (Jensen et al., 1993 and Tyrell et al., 1997)

  32. Enterococcus specific (Ke et al., 1999. J Clinic Microbiol. 37:3497-3503) Ent 1 5’-TACTGACAAACCATTCATGATG-3’ Ent 2 5’-AACTTCGTCACCAACGCGAAC-3’ The expected size of PCR products is 112 bp. This was used to confirm all enterococcus isolates obtained.

  33. Intergenic Ribosomal PCR • PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes can produce amplicon profiles characteristic of the enterococcus examined. ITS-RBS L1 5’-CAAGGCATCCACCGT-3’ G1 5’-GAAGTCGTAACAAGG-3’ The expected size of the amplicons varies from 300 to 600 bp and large minor bands. Often two major bands between 300 to 500 bp were observed. Primers were designed by Jensen et al. (1993 AEM 59:945-952) but applied to differentiate enterococcus species by Tyrrell et al. (1997 J Clinic Microbiol. 35:1054-1060).

  34. Ranges of pH, Temperature, and Turbidity in Samples Taken from Eight Sites in 2008 Across Stillwater Creek Watershed

  35. Enterococciand E. coli detected

  36. Enterococci and E. coli detected

  37. Mean Percentages of IDEXX Counts that were Positive Enterococci2008 Samples

  38. Correlation coefficients between EnterococciorEscherichia coli concentrations and percentages of respective isolates that were positive for esculin hydrolysis (PPE)* Significant at 0.05; n/a = not applicable; samples were taken in 2008

  39. L A B C D E F G L 300 bp 200 bp 100 bp • Seven Enterococcispecies banding patterns in 8% Polyacrylamide gel A – 1.0*, E. Faecalis B – 0.70*, E. Faecalis C – 1.0*,Ent. faecium, D –0.90*, E. gallinarum, E – 0.97*, E. hirae, F – 0.80*, E. avium, G – 0.80*, E. dispar. • *SD = 2C/ N1 + N2 • where C is the no. of positive band matches, N1 is the total no. of bands in standard, and N2 is the total no. of bands in sample. • These banding patterns were developed using 8% Polyacrylamide gel and PCR amplicons of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes using primers ITS-RBS-L1 and ITS-RBS-G1. The PCR amplicons were digested with Sau3AI to obtain characteristic banding patterns that are species specific. These seven banding patterns were observed among 702 Enterococci isolates.

  40. Enterococcispecies detected in isolates obtained at base flow in surface water (n=213)

  41. Enterococcispecies detected in isolates obtained at base flow in sediments (n=176)

  42. Enterococcispecies detected in isolates obtained at high flow in surface water (n=313)

  43. Conclusions • Quantity of Enterococci varies in space (Sites 1 to 8) and time (baseflow and highflow periods). • Highest Enterococci counts are found in creek water following passing through areas nearby animal production units (Site 3), and heavy wildlife activities (Site 6). • High Enterococci counts are found in sediment samples.

  44. Conclusions • Enteroccoccusfaecaliswas the most dominant of all detected species • Seven species were found across all sites in this watershed. • Enterococcusfaeciumand E. aviumshowed some site specificity.

  45. ACKLEDGEMENT • Donna Ria Caasi • Chor Tee Tan • Melissa Molzahn

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