Stewardship and the Environment - Wastewater Treatment and Antibiotics

Timothy M. LaParaDepartment of Civil, Environmental,

and Geo- EngineeringUniversity of Minnesota

4 November 2015

http://www.cdc.gov/drugresistance/threat-report-2013/

Antimicrobial resistance is a complex problem driven by many

interconnected factors; single, isolated interventions have little

impact.

Pruden et al. 2013 Environ. Health Persp. 121:878-885. WHO. 2012. www.who.int/mediacentre/factsheets/fs194/en/41

How is antibiotic resistance detected/quantified?

Medical Approach:1. Isolate pathogen from a patient2. Determine which antibiotics are effective

Model Organism Approach:1. Isolate a model organism 2. Determine which antibiotics are effective

(Great Plate Count Anomaly?)

New Methodology

• Target genes quantified by real-time polymerase chain reaction

Cycle Number

Fluo

rese

nce

105 g

enes

104 g

enes

103 g

enes

102 g

enes

6.6×104 genes

3.1×102 genes

The “ARG” Cycle

http://www.cdc.gov/drugresistance/threat-report-2013/

1019 copies/day

1017 copies/day

1018 copies/day

Auerbach et al. 2007. Water Research 41:1143–1151; Smith et al. 2004. Applied and Environmental Microbiology 70:7372–7377.

My Vision: ARG Control

Source of ARGs Technology to TreatARGs

General Research Questions

• What effect does current wastewater treatment operations have on the spread of ARGs?

• How can we reduce ARGs leaving a wastewater treatment facilities?

UntreatedWastewater

BarRack Grit

Chamber

PrimaryClarifier

Cell Recycle

DischargeStream

AerationTank

SecondaryClarifier

SeasonalDisinfection

Where to look?

AnaerobicDigestion(or other)

Farmland

1

23

0

Part 1: Is treated effluent a pertinent source of antibiotic resistance genes?

Photo of treated and raw municipal wastewater from Blue Lake Wastewater Treatment Plant (2003)

Source: LaPara et al. 2015. Multiple discharges of treated municipal wastewater have a small effect on the quantities of numerous antibiotic resistance determinants in the Upper Mississippi River. Environmental Science and Technology 49:11509-11515.

St. Louis River, Duluth-Superior Harbor, WLSSD, and Lake Superior

St. Louis River Lake Superior

Source: LaPara et al. 2011. Tertiary-treated municipal wastewater is a significant point-source of antibiotic resistance genes into Duluth-Superior Harbor. Environmental Science and Technology 45:9543-9549.

Antibiotic resistance genes

Source: LaPara et al. 2015. Multiple discharges of treated municipal wastewater have a small effect on the quantities of numerous antibiotic resistance determinants in the Upper Mississippi River. Environmental Science and Technology 49:11509-11515.

Source: LaPara et al. 2015. Multiple discharges of treated municipal wastewater have a small effect on the quantities of numerous antibiotic resistance determinants in the Upper Mississippi River. Environmental Science and Technology 49:11509-11515.

Part 2: Treatment of Wastewater Solids

• The overwhelming majority of bacteria (antibiotic resistant or otherwise) in sewage end up in sewage sludge

• Treatment technologies vary substantially depending on the end goal – anaerobic digestion at 37°C is most common

Aerobic digestion and air dry slowly reduce ARG levels

Sources: Burch et al. 2013. Environmental Science and Technology 47(17):9965-9971. Burch et al. 2013. Frontiers in Microbiology – Antimicrobials, Resistance and Chemotherapy 4:17. doi: 10.3389/fmicb.2013.00017.

Higher temperature is better at eliminating ARGs during anaerobic digestion

Source: Diehl and LaPara. 2010. Effect of temperature on the fate of genes encoding tetracycline resistance and the integrase of Class 1 integrons within anaerobic and aerobic digesters treating municipal wastewater solids. Environ. Sci. Technol. 44:9128-9133.

= 22°C = 37°C = 46°C = 55°C

Time (d)0 1 2 3 4 5

102

103

104

105tet(X)

Time(d)0 1 2 3 4 5

100

101

102

103

104tet(W) intI1

Time (d)0 1 2 3 4 5

102

103

104

105

106

Part 3: Treated Wastewater Solids are Often Applied to Agricultural Soils

Antibiotic resistance genes decline when wastewater solids are applied to soil

Source: Burch, Sadowsky, and LaPara.2014. Fate of antibiotic resistance genes and class 1 integrons in soil microcosms following the application of treated residual municipal wastewater solids. Environmental Science and Technology 48:5620-5627.

Sandy Soil Silty-Loamy Soil

How do decay rates (t1/2) compare?

Gene Thermophilic Anaerobic Digestion

Aerobic Digestion Air Drying In soils

erm(B) 3.6 6.4 15

intI1 0.28 6.3 31.7 75

sul1 4.6 35.5 55

tet(A) 1.6 4.4 8.8 30

tet(W) 0.96 2.8 7.3 18

tet(X) 0.5 5.7 17.2 40

Take-home messages• Real-time PCR allows us to study the spread

of antibiotic resistance in an entirely new and more mechanistic/quantitative way

• Raw wastewater is bad, bad, bad• Treated wastewater still contains a lot of

antibiotic resistance genes• Thermophilic anaerobic digestion = Great!• Antibiotic resistance genes decay slowly in

soil

What am I really saying?

Wouldn’t it be great to collect a large fraction of antibiotic resistance genes and destroy them?

Our existing infrastructure (sewers & wastewater treatment plants) gets us almost all of the way to that goal!

Significance of My Research• Current efforts to slow the proliferation

of antibiotic resistance are focused on “resistance prevention.”

• I suggest that municipal wastewater treatment can be applied to provide “resistance control.”

• My proposed approach completely supports (does not supplant) on-going resistance prevention efforts

Acknowledgements

• Minnesota Environment and Natural Resources Trust Fund

• National Science Foundation

Acknowledgements

• David Tan• Mi Yan• Tucker Burch• Pat McNamara• Matt Madson• David Diehl• Mike Sadowsky• Kyle Sandberg

• Spencer Borchardt• Kevin Lang• Tim Johnson• Pat McNamara• Matt Madson• David Diehl• Mike Sadowsky• Kyle Sandberg

Thank You!