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Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Creek, Cow Creek, and Boomer Creek Shiping Deng Department of Plant and Soil Sciences,

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Presentation on theme: "Distribution and Occurrence of Indicator Fecal Bacteria in Stillwater Creek, Cow Creek, and Boomer Creek Shiping Deng Department of Plant and Soil Sciences,"— Presentation transcript:

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 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 Impact of Cultivation on the Nature of Organic Matter and Sorption Behavior of Nonionic Pesticide in Soil Linking Soil Enzyme Activities to Ecosystem Functions Linking Soil Enzyme Activities to Ecosystem Functions Phyllosphere Bacterial Endophytes and Phosphorus Nutrition in Corn 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.” “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.” “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 Fecal Coliform  < 200 CFU/100ml E. coli  < 126 CFU/100ml E. coli  < 126 CFU/100ml Fecal Strep (Enterococci)  < 33 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 GuidelinesGeometric MeanSingle Sample E. Coli Enterococcoci (freshwater)3361 _______________________________________________________

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

10 Enterococcus sp. infection in pulmonary tissuepulmonary Genus:Enterococcus ( established in 1984, >34 species ) Family:Enterococcaceae Order:Lactobacillales Class:Bacilli Division: Firmicutes Kingdom: Bacteria 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 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. To examine whether urban population and farming activities contribute to fecal bacterial contamination in water.

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

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

14 Method of analysis Quantifying Enterococci using IDEXX Enterolert TM 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 41 o C for 24 h. 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 41 o C 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. 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 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 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. 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 catalase negative. Confirmed that the isolate is gram positive. Confirmed that the isolate is gram positive. Confirmed to be spherical or ovoid cells. Confirmed to be spherical or ovoid cells.

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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). 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 ). 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. 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. 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. 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. 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. 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. 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. Of the evaluated 276 isolates obtained during base flow period, 187 were positive.

22 Enterococci Isolated and Evaluated Sample evaluatedconfirmedevaluatedconfirmed % S 1 Surface Surface S S S 4 Surface S S S Sum S 1 Sediments Sediments 2300 S 2 Sediments S S 4 Sediments S S 6 Sediments S 7 Sediments Sum Isolates % + Enterococci of IDEXX counts Base-FlowHigh-Flow Isolates % + Enterococci of IDEXX counts

23 Enterococci Evaluation IDEXX Enterolert TM test IDEXX Enterolert TM test Black colonies on Enterococcusel agar plates (Esculin hydrolysis) Black colonies on Enterococcusel agar plates (Esculin hydrolysis) BHIA with 6.5% NaCl BHIA with 6.5% NaCl Catalase negative Catalase negative Gram positive Gram positive Spherical or ovoid cells 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. 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). 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 enterolert TM quantification (lost esculin hydrolysis ability; enzymes responsible for this function were not produced)? Do Enterococci adapt to the environment and become false positive based on IDEXX enterolert TM quantification (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? 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 Sampled at all seven sites plus one additional site at the source stream for boomer lake

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

28 Hypothesis Enterococci quantity 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 genetic diversity of the isolated enterococci To evaluate enterococci diversity in relation to sites and potential sources To evaluate enterococci diversity in relation to sites and potential sources To detect and quantify Escherichia coli in Stillwater Creek watershed To detect and quantify Escherichia coli in Stillwater Creek watershed

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

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

32 Enterococcus specific (Ke et al., J Clinic Microbiol. 37: ) Ent 15’-TACTGACAAACCATTCATGATG-3’ Ent 25’-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. 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-RBSL15’-CAAGGCATCCACCGT-3’ G15’-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: ) but applied to differentiate enterococcus species by Tyrrell et al. (1997 J Clinic Microbiol. 35: ).

34 Base flow pH Surface water SM WN Sediments SedimentsSM WN Temperature (ºC) Surface water SM WN Turbidity (A 595 nm) Surface water SM – WN – High flow pH Temperature (ºC) Turbidity (A 595 nm) – 0.48 Ranges of pH, Temperature, and Turbidity in Samples Taken from Eight Sites in 2008 Across Stillwater Creek Watershed

35 Enterococci and E. coli detected Base Flow SurfaceSedimentSurfaceSediment S ,209, S24532,421, S31701,504, S45511,911, S570822,351, S63766,020, S73023,166, S801,921,

36 Enterococci and E. coli detected High Flow Surface S185,29212,075 S229,07216,279 S3216,365159,839 S471,32727,211 S520,1619,699 S619,25026,369 S710,26312,464 S83411,574

37 Mean Percentages of IDEXX Counts that were Positive Enterococci 2008 Samples Summer 2008 Base flow, surface water 99.0n=271 Base flow, sediments 93.0n=225 High flow, surface water 97.0n=210 Winter 2008 Base flow, surface water 89.0n=252 Base flow, sediment 83.0n=252

38 Correlation coefficients between Enterococci or Escherichia 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 High flow, surface water 0.70*n=80.70*n=24 Base flow, surface water 0.84*n=80.83*n=24 Base flow, sediments 0.66*n=80.35*n=24

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40 Seven Enterococci species 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. 200 bp 300 bp 100 bp L A B C D E F G L 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.

41 Enterococci species detected in isolates obtained at base flow in surface water (n=213) ABCDEF G S nd S nd 2 S323153nd 1 S nd 1 S5697nd S nd S nd S8nd Total nd 31

42 Enterococci species detected in isolates obtained at base flow in sediments (n=176) ABCDEF G S11125nd S210313nd 1 S36542nd 1 S48nd34 S5513nd 1 S nd S nd S81322 nd Total

43 Enterococci species detected in isolates obtained at high flow in surface water (n=313) ABCDEF G S122122nd S nd S nd 1 S nd S nd 1 S nd S nd S8nd310nd Total nd

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45 Conclusions Quantity of Enterococci varies in space (Sites 1 to 8) and time (baseflow and highflow periods). 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). 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. High Enterococci counts are found in sediment samples.

46 Conclusions Enteroccoccus faecalis was the most dominant of all detected species Enteroccoccus faecalis was the most dominant of all detected species Seven species were found across all sites in this watershed. Seven species were found across all sites in this watershed. Enterococcus faecium and E. avium showed some site specificity. Enterococcus faecium and E. avium showed some site specificity.

47 ACKLEDGEMENT Donna Ria Caasi Donna Ria Caasi Chor Tee Tan Chor Tee Tan Melissa Molzahn Melissa Molzahn


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