Chuck SomervilleMarshall UniversityHuntington, WV

In other words . . .In other words . . .Are fecal indicators (coliforms, fecal

coliforms, E. coli) sufficient for determining the safety of water resources?

Specifically, do they predict the numbers and distribution of antibiotic resistant bacteria in surface waters?

Why Study Antibiotic Resistance?Why Study Antibiotic Resistance?

http://www.fda.gov/oc/opacom/hottopics/antiresist_facts.html

Why Study Antibiotic Resistance?Why Study Antibiotic Resistance?NARMS - the National Antimicrobial Resistance

Monitoring System

USFDA, USDA & CDC collaboration formed in 1996

Monitors resistance of isolates from humans, animals, raw food products & retail meats

Does notnot monitor water supplies

http://www.fda.gov/cvm/narms_pg.html

Why Study the Ohio River?Why Study the Ohio River?

Experimental Questions . . .Experimental Questions . . .

Are antibiotic resistant bacteria present in detectable numbers in the Ohio River?

If so, how are they distributed?

Testing a common assumption . . .Testing a common assumption . . .The use of antibiotics in medicine and

agriculture selects for resistant strains in the animal gut.

Failing or absent septic systems causes the distribution of resistant bacteria to the environment.

Antibiotic resistant bacteria should be a subset of fecal-derived bacteria in environmental samples.

Total cultivable bacteria

Total Coliforms

Fecal Coliforms

Escherichia coli

Antibiotic resistant bacteria

MethodsMethods

Mid-channel, sub-surface water samples

5 mile intervals in mainstem

All major tributaries

MethodsMethodsR2A agar + antibiotic for cultivable resistant

cellsFungizone (375 ng/ml; used in all plates)Ampicillin (50 g/ml)Ciprofloxacin (4 g/ml)Erythromycin (8 g/ml)Streptomycin (25 g/ml)Sulfamethizole (128 g/ml)Tetracycline (12.5 g/ml)Virginiamycin (16 g/ml)

http://www.nsri.upd.edu.ph/mrsl/img/services1.jpg

MethodsMethodsm-FC at 44.5˚C for fecal

coliforms (2001 – 2003)

IDEXX Colilert QuantiTray/2000 for total coliforms and Escherichia coli (2004 – present)

QT/2K + antibiotic for resistant coliforms and E. coli (2005 – present)

http://ceeserver3.mit.edu/~Nepal/Mic_mFC.jpg

MethodsMethodsConcurrent determination of:

pH, DO, turbidity, conductivityChlorophyll (UC)Total N & P (UC)Particulate N & P (UC)Phytoplankton (NKU)Zebra mussel veligers (TMC)Land use patterns

Results - 2006Results - 2006

Two, 100-mile reaches of OR with complete cultivable counts (CFU/ml) and coliform data (MPN/ml)

Compared numbers of resistant cultivable cells to numbers of E. coli and resistant coliforms

198

0.26 0.00 0.00

0

50

100

150

200

250

300

Me

an

CF

U o

r M

PN

pe

r m

l

1

RM 200 to 305.2

R2A-cip E. coli cipR total coliforms cipR E. coli

* **

CiprofloxacinCiprofloxacin

75

0.26 0.01 0.00

0

50

100

150

200

250

300

Av

era

ge

CF

U o

r M

PN

pe

r m

l

1

RM 200 to 305.2

R2A-tet E. coli tetR coliforms tetR E. coli

* * *

TetracyclineTetracycline

199

0.26 0.34 0.02

0

50

100

150

200

250

300

Av

era

ge

CF

U o

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PN

pe

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l

1

RM 200 to 305.2

R2A-vir E. coli virR coliforms virR E. coli

***

VirginiamycinVirginiamycin

VirginiamycinVirginiamycin

103

0.49 0.03 0.00

0

50

100

150

200

250

300

Av

era

ge

CF

U o

r M

PN

pe

r m

l

1

RM 410 to 498.8

R2A-cip E. coli cipR coliforms cipR E. coli

***

CiprofloxacinCiprofloxacin

VirginiamycinVirginiamycinCiprofloxacinCiprofloxacin

76

0.49 1.04 0.02

0

50

100

150

200

250

300

Av

era

ge

CF

U o

r M

PN

pe

r m

l

RM 410 to 498.8

R2A-tet E. coli tetR coliforms tetR E. coli

***

TetracyclineTetracycline

289

0.49 10.29 0.29

0

50

100

150

200

250

300

Av

era

ge

CF

U o

r M

PN

pe

r m

l

RM 410 to 498.8

R2A-vir E. coli virR coliforms virR E. coli

***

VirginiamycinVirginiamycin

Results - 2006

Two, 100-mile reaches of OR with complete cultivable counts (CFU/ml) and coliform data (MPN/ml)

Compared distribution of resistant cultivable cells to distribution of E. coli and resistant coliforms

0

200

400

600

800

1000

200 225 250 275 300

CF

U p

er m

lR2A-cip R2A-tet R2A-vir

0.0

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8.0

10.0

200 225 250 275 300

MP

N p

er m

l

River Mile- - - coliforms E. coli

- - - coliforms E. coli

Total cultivable bacteria

Total Coliforms

Fecal Coliforms

Escherichia coli

Antibiotic resistant bacteria

Conclusions

The Ohio River and major tributaries are significant reservoirs for antibiotic resistant bacteria.

Antibiotic resistant bacteria are present in much greater numbers than fecal indicator bacteria.

Conclusions

The distribution of antibiotic resistant bacteria is distinct from that of fecal indicator bacteria.

Antibiotic resistant bacteria are not a subset of fecal indicator bacteria

ConclusionsAntibiotic resistant bacteria in surface waters

may represent a significant public health issue.

Their concentration and distribution can not be predicted by monitoring fecal-indicator organisms.

Additional work is needed to determine how resistant cells are distributed and their impacts on health.

Implications & new questionsTracking known sources of fecal bacteria

does not reliably track ARB. So what?

Can resistance genes be transmitted on sand bed filters?

What types of genetic elements are involved in resistance?

CF

U/m

l

Flow Volume (liters)

Total bacteria

Amp-R bacteria

Thanks . . .Thanks . . .MU Students/StaffMU Students/Staff

Lisa SmithLisa Smith

Andy JohnsonAndy Johnson

Tim DotsonTim Dotson

Robert NagyRobert Nagy

FundingFundingNSF-EPSCoRNSF-EPSCoR

MU Graduate CollegeMU Graduate College

Ms. Shelba PewMs. Shelba Pew

CollaboratorsCollaboratorsMiriam Kannan (NKU)Miriam Kannan (NKU)

Rebecca Evans-Kelly Rebecca Evans-Kelly (NKU)(NKU)

Michael Miller (UC)Michael Miller (UC)

John Hageman (TMC)John Hageman (TMC)