Variability, antibiotic sensitivity, and the influence of ... · Antibiotic Resistance (ABR) Test 11 ... Bar Chart shows the correlation between E. coli concentration ... consistent

South Dakota State University

Sadia Salam¹, Rachel McDaniel¹ and Bruce Bleakley²

¹Department of Agricultural and Biosystems Engineering, SDSU;

²Department of Biology and Microbiology, SDSU

Variability, antibiotic sensitivity, and the influence of

particle size on E. coli in Eastern South Dakota’s

streambed sediment


Outline

2

• Background

• Study Site

• Methodology

• Results and Discussions

• Conclusion

• Future Work


Introduction

• Fecal Indicator Bacteria (FIB) are more persistent in sediment than water column (Garzio-Hadzick et al., 2010 and

Rehmann et al., 2009)

• E. coli, is the prime water quality impairment in South Dakota’s streams (EPA, 2015)

3


Figure 1: Sources and survival of bacteria in the water column*

*Picture Courtesy: University of Maryland,

Center for Environmental Science

4


Project Goal

Determine E.colivariability in streambed sediment to inform sediment sample collection and processing

5


Study Site

Figure 2: Location of Skunk Creek in Lower Big Sioux Watershed

6


Figure 3: Location of four monitoring sites in Skunk Creek, Big Sioux River. The monitoring sites

(Sk1, Sk2, Sk3 and Sk4) of Skunk Creek (Sk) located in Minehaha and Moody counties, SD

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cattle

crossing

site and

located in

upstream

located in

down

stream


Sediment Sample Collection and Processing

• 25 samples in 5X5 grid formation

• Used sterile wide mouth bottle

• Top 3 cm

• Used 1:11 dilutions

• Phosphate buffer solution

• Used the supernatant for samples processing

Figure 4: 25 gridded (5X5)

location for Sediment sample

collection

8


• Modified mTEC Agar and standard membrane filtration

• The sample was processed two times, within 8 hours & within 24 hours

Figure 5: E. coli grown using

membrane filtration

E. coli Enumeration

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Particle Size Analysis

• American Society for Testing and Materials (ASTM) D 422 standard test method

• Sieve Analysis test

10

Figure 6: Particle Size distribution

by sieve analysis test


Antibiotic Resistance (ABR) Test

11

• Modified Kirby-Bauer Method

• Used five Antibiotics:

i. Penicillin (10U)

ii. Erythromycin (2μg)

iii. Tetracycline (30μg)

iv. Sulfisoxazole (30μg)

v. Ampicillin (10μg)Figure 7: ABR test plates for five antibiotics


Result and Discussion


E. Coli Variability

Figure 8: Boxplot showing the E. coli variability in Sk1, Sk2,

Sk3 and Sk4 monitoring site

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Bank of the Stream

Bank of the Stream

Flow

Figure 9: Variability map of E. coli concentration in CFU/g

for site (a) Sk1 (b) Sk2

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Legend

E. coli Concentration (CFU/g)

0 - 100

100 - 200

200 - 300

300 - 400

400 - 500

500 - 600

600 - 700

700 - 800

800 - 900

900 - 1,000

(a)

(b)

South Dakota State University15

Flow

Bank of the Stream


for site (c) Sk3 and (d) Sk4

Legend


0 - 100

100 - 200

200 - 300

300 - 400

400 - 500

500 - 600

600 - 700

700 - 800

800 - 900

900 - 1,000

Bank of the Stream

Bank of the Stream

(c)

(d)


Bank of the Stream

Bank of the Stream

Flow


for site (a) Sk1 (b) Sk2

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Legend


0 - 100

100 - 200

200 - 300

300 - 400

400 - 500

500 - 600

600 - 700

700 - 800

800 - 900

900 - 1,000

(a)

(b)


E. coli concentrations ratio of stream bank vs. center:

0

8

99

4

0 20 40 60 80 100

Sk1

Sk2

Sk3

Sk4

Bank (A, E)/Center (B, C, D)

Mo

nit

ori

ng

Sit

es

Figure 11: The E. coli concentration ratio between bank

and center of the streambed sediment

• There is no significant difference found between the bank and center of

the stream

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Temporal Stability

Site p-value Hₒ

Sk1 0.337 Fail to reject

Sk2 0.0054 Reject



Table 1: No significant

difference found between 8

hours and 24 hours E. coli

concentration data except

site Sk2

• Null Hypotheses (Hₒ):

8 hours data = 24 hours data

• 95% Confidence interval

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• Sample can be processed within 24 hours

without significant differences in the

resulting sample E. coli concentrations in

the majority of cases


Grain Size Distribution

Figure 12: Summary of grain size distribution in all four sites

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116

4 2

91

77

85 93

8 7 11 5

0

20

40

60

80

100

Sk1 Sk2 Sk3 Sk4

Ga

in S

ze D

istr

ibu

tio

n (

%)

Monitoring Sites

Silt &

ClaySand

Gravel


Relationship between E. coli concentration and sediment grain size

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-0.56

0.62

-0.48

0.70

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

Sk1 Sk2 Sk3 Sk4

Corr

elati

on

Facto

r, R

Monitoring Sites

Figure 13: Bar Chart shows the correlation between E. coli concentration

and sediment grain size (<0.075 mm)

• All site shows strong correlation between particle size (< 0.075 mm)

and E. coli concentration


Sample Size Analysis

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Figure 14: Sample Size calculation for different

margin of error at 95% Confidence Interval

Margin of

Error (+/- CFU/g)

Average

Sample Size

Required

20 18

40 9

60 6

80 4

100 4

Table 2: Average sample

size for different margin of

error for all site at 95%

Confidence Interval

0

5

10

15

20

25

30

E= 20 E= 40 E= 60 E= 80 E= 100

Sa

mp

le S

ize

Margin of Error

Sk1

Sk2

Sk3

Sk4

Average


Figure 15: ABR test result for selected isolates of the monitoring site Sk2

Antibiotic Resistance (ABR) Test

100 100

62.9

39.3

100

37.1

60.7

0

20

40

60

80

100

Penicillin Erythromycin Tetracyclin Sulfisoxazole Ampicillin

E.

coli

Isola

tes

(%)

AntibioticsResistant Susceptible

• For all the antibiotics, the proportion of isolates showed fairly

consistent results throughout the stream cross section

n = 89 n = 89 n = 89 n = 12n = 89


• E. coli variability was highest in the upstream (cattlecrossing) site and lowest in the most downstream site

• There is no significant difference in E. coli concentrationsbetween the edge and middle of the stream

• Samples can be processed within 24 hours in the majority ofcases

• The streambed sediment of all the sites are dominated bysand particles

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Conclusions


• All site shows strong correlation between particle size

(< 0.075 mm) and E. coli concentration

• Sample size analysis showed, on average, a minimum of 6 sediment samples are required to adequately represent the average E. coli concentration in the sediment for a moderate margin of error. This sample size is more than many previous studies (Garzio-Hadzick et al., 2010; Pandey et al., 2014 and Abia et al., 2015; )

• The proportion of isolates from site Sk2 showed consistentresult in ABR test throughout the stream cross section

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Future Work

• Assess the seasonal variation of E. coli concentration in the streambed sediment

• Determine the impact of storm event on streambed sediment containing E. coli

• Assess the attachment rates of E. coli to sediment particles

• ABR testing for site Sk1 to compare cattle crossing site to downstream site

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Reference• Abia, L. K. A., E. Ubomba-Jaswa, C. C. Ssemakalu, and M. N. B. Momba. 2015.

Development of a rapid approach for the enumeration of Escherichia coli in riverbedsediment: case study, the Apies River, South Africa. Journal of Soils and Sediments15(12):2425-2432

• Garzio-Hadzick, A., D. R. Shelton, R. L. Hill, Y. A. Pachepsky, A. K. Guber, and R.Rowland. 2010. Survival of manure-borne E. coli in streambed sediment: effects oftemperature and sediment properties. Water Res 44(9):2753-2762

• Pandey, P. K., and a. S. M. L. 2014. Assessing Linkages between E. coli Levels inStreambed Sediment and Overlying Water in an Agricultural Watershed in Iowaduring the First Heavy Rain Event of the Season. Transactions of the ASABE:1571-1581

• Rehmann, C. R., and M. L. Soupir. 2009. Importance of interactions between thewater column and the sediment for microbial concentrations in streams. Water Res43(18):4579-4589

• EPA. (2015). South Dakota Water Quality Assessment Report. Accessed Sept. 2015 at <http://ofmpub.epa.gov/waters10/attains_state.control?p_state=SD>

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Acknowledgments

• South Dakota Department of Environment and Natural

Resources

• Dr. Rachel McDaniel and Dr. Bruce Bleakly

• Our Research Group (Miranda Lebrun, Louis Amegbletor

and Sara Mardaninejadjouneghani)

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Variability, antibiotic sensitivity, and the influence of ... · Antibiotic Resistance (ABR) Test 11 ... Bar Chart shows the correlation between E. coli concentration ... consistent