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• 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

“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.”

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.

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_______________________________________________________

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)

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).

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.

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.

Enterococcus sp. infection in pulmonary tissue

Genus: Enterococcus (established in 1984, >34 species)Family: EnterococcaceaeOrder:Lactobacillales

Class: BacilliDivision: FirmicutesKingdom: Bacteria

Species: E. avium, E. durans, E. faecalis, E. faecium

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.

Stillwater creek

Cow

cre

ek

Boom

er c

reek

Site 1

Site 2

Site 3

Site 4

Site 5

Site 6

Site 7

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.

Method of analysisQuantifying 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.

Method of analysisIsolation 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

Method of analysisIsolation 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.

Sample Enterococci counts by IDEXX Enterolert™ evaluated confirmedMPN 100 mL-1 or 100 g-1 % MPN 100-mL-1 or 100 g-1

S1 Surface 294 25 21 83.0 244S1 Sediments 365,556 23 0 0 0S2 Surface 531 25 23 92.0 504S2 Sediments 226,667 18 2 7.4 16,773S3 Surface 2,463 25 23 92.0 2,266S3 Sediments 2.5 x 106 19 9 58.3 1,500,000S4 Surface 279 25 19 75.5 209S4 Sediments 600,000 9 4 44.4 266,400S5 Surface 821 25 25 100.0 821S5 Sediments 673,333 3 1 33.3 224,220S6 Surface 513 25 24 99.2 492S6 Sediments 1.0 x 106 17 9 52.4 524,000S7 Surface 154 25 24 95.8 148S7 Sediments 710,000 11 2 11.1 78,810

Total 276 187

Isolates

Enterococci Counts in Stillwater Creek, Cow Creek, and Boomer Creek During Base-Flow Period

% Positive Enterococciof IDEXX counts

Confirmed Enterococci

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).

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.

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.

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.

Enterococci Isolated and Evaluated

Sampleevaluated confirmed evaluated confirmed

% %S1 Surface 25 21 83 25 20 83S2 Surface 25 23 92 25 21 92S3 Surface 25 23 92 25 20 92S4 Surface 25 19 75.5 25 25 75.5S5 Surface 25 25 100 25 25 100S6 Surface 25 24 99.2 25 24 99.2S7 Surface 25 24 95.8 25 25 95.8

Sum 175 159 90.9 175 160 91.4S1 Sediments 23 0 0S2 Sediments 18 2 7.4S3 Sediments 19 9 58.3S4 Sediments 9 4 44.4S5 Sediments 3 1 33.3S6 Sediments 17 9 52.4S7 Sediments 11 2 11.1

Sum 89 25 28.1

Isolates % +Enterococci of IDEXX counts

Base-Flow High-FlowIsolates % +Enterococci

of IDEXX counts

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

Esculin Hydrolysis

Hydrolysis By EnterococcusGlycoside 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).

QUESTIONS• Do Enterococci adapt to the environment

and become false positive based on IDEXX enterolertTM 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?

Repeat the experiment• Sampled at all seven sites plus one

additional site at the source stream for boomer lake

Stillwater creek

Cow

cre

ek

Boom

er c

reek

Site 1

Site 2

Site 3

Site 4

Site 5

Site 6

Site 7

Site 8

Hypothesis

Enterococci quantity and diversity in the water environment is directly linked to its potential sources and vary in space and time

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

Experimental Design (General procedure)

Site selectionSampling(Variations: space, season, & water flow)

Data: location coordinates, Temp. & water depth

SpectrophotometrypH measurement

Data: Turbidity & pH

IDEXX EnterolertUV 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 asEnterococcus

PCR (Primer: ITS-PCR, Jensen et al., 1993 & Tyrrell et al., 1997)

Determine species ofEnterococcus

DATA ANALYSIS

MPN

• 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)

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.

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).

Base flowpH Surface water SM 7.8 - 8.1

WN 7.6 - 8.0 Sediments SM 7.6 - 8.1

WN 8.0 - 8.3 Temperature (ºC)

Surface water

SM 23.0 - 28.0

WN 5.2 - 8.0 Turbidity (A595nm)

Surface water

SM 0.018 – 0.068

WN 0.023 – 0.047 High flow

pH 7.4 - 7.9 Temperature (ºC) 22.8 - 24.7Turbidity (A595nm) 0.061 – 0.48

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

Creek Watershed

Enterococci and E. coli detected

Base FlowEnterococcus E. coli

Site Surface Sediment Surface Sediment

S1 1055 70,209,876 63 187S2 453 2,421,195 82 351S3 170 1,504,565 24 323S4 551 1,911,082 149 380S5 708 22,351,184 391 2422S6 376 6,020,545 90 607S7 302 3,166,894 139 510S8 0 1,921,282 3 239

Enterococci and E. coli detectedHigh Flow

Enterococcus E. coli

Site Surface Surface

S1 85,292 12,075S2 29,072 16,279S3 216,365 159,839S4 71,327 27,211S5 20,161 9,699S6 19,250 26,369S7 10,263 12,464S8 341 1,574

Mean Percentages of IDEXX Counts that were Positive

Enterococci 2008 Samples

Sampling condition% Positive

Enterococcus

Summer 2008Base flow, surface water 99.0 n=271Base flow, sediments 93.0 n=225

High flow, surface water 97.0 n=210Winter 2008Base flow, surface water 89.0 n=252Base flow, sediment 83.0 n=252

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

Sampling condition E. Coli (MPN/ 100 ml)

Enterococcus (MPN/100 ml)

High flow, surface

water

0.70* n=8 0.70* n=24

Base flow, surface water 0.84* n=8 0.83* n=24

Base flow, sediments 0.66* n=8 0.35* n=24

Baseflow Condition (Summer 2005)

0

20

40

60

80

100 Baseflow Condition (Summer 2008)

Baseflow Condition (Winter 2008)

Days before sampling

0 4 8 12 16 20 24 28

Dai

ly R

ainf

all (

mm

)

0

20

40

60

80

100 Highflow Condition (Summer 2008)

0 4 8 12 16 20 24 28

Seven Enterococci species banding patterns in 8% Polyacrylamide gel

A – 1.0*, E. FaecalisB – 0.70*, E. FaecalisC – 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 + N2where 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.

Enterococci species detected in isolates obtained at base flow in surface water

(n=213)A B C D E F G1.0*

E. faecalis

0.70* E.

faecalis

0.70* E.

faecium

0.90*E.

gallinarum

0.97*E.

hirae

0.80*E.

avium

0.80* E.

dispar

S1 12 11 3 1nd

nd ndS2 18 13 2 1 nd 2 ndS3 23 15 3 nd nd 1 ndS4 11 2 1 5 nd nd 1S5 6 9 7 nd nd nd ndS6 10 17 1 1 nd nd ndS7 8 8 19 2 nd nd ndS8 nd nd nd nd nd nd nd

Total 88 75 36 10 nd 3 1

Enterococci species detected in isolates obtained at base flow in sediments

(n=176)A B C D E F G1.0*

E. faecalis

0.70* E.

faecalis

0.70* E.

faecium

0.90*E.

gallinarum

0.97*E.

hirae

0.80*E.

avium0.80*

E. dispar

S1 11 2 5 nd nd nd ndS2 10 3 1 3 nd nd 1S3 6 5 4 2 nd nd 1S4 8 nd 3 4 nd nd ndS5 5 1 3 nd nd 1 ndS6 17 6 6 1 8 nd ndS7 9 2 15 1 2 nd ndS8 13 2 2 13 nd nd nd

Total 79 21 39 24 10 1 2

Enterococci species detected in isolates obtained at high flow in surface water

(n=313)A B C D E F G1.0*

E. Faecali

s

0.70* E.

Faecalis

0.70* E.

Faecium

0.90*E.

gallinarum

0.97*E.

hirae

0.80*E.

avium0.80*

E. dispar

S1 22 12 2 nd nd nd ndS2 19 17 1 3 nd nd ndS3 16 12 3 2 nd 1 ndS4 16 12 10 6 nd nd ndS5 12 15 12 12 nd 1 ndS6 17 13 9 7 4 1 ndS7 13 17 3 2 8 nd ndS8 nd 3 10 nd nd nd nd

Total 115 101 50 32 12 3 nd

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.

Conclusions• Enteroccoccus faecalis was the most

dominant of all detected species• Seven species were found across all sites

in this watershed.• Enterococcus faecium and E. avium

showed some site specificity.

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