Transport of antibiotic resistant bacteria into tile drainage systems

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2 11 th Annual Drainage Research Forum Owatonna, Minnesota November 23 rd, 21 Transport of antibiotic resistant bacteria into tile drainage systems Michelle Soupir Agricultural & Biosystems Engineering, Iowa State University Collaborators: Trang Hoang, Alok Bhandari and Tom Moorman

3 Image by Christina Goeddel

Microbial Indicator Fecal coliform http://greenerloudoun.wordpress.

4 Pathogens are a leading cause of water quality impairments in streams and rivers Agricultural practices Leading source of all impairments Causes of waterborne disease outbreaks resulting in gastroenteritis Enterococci are a potential indicator of pathogens Microbial Indicator Fecal coliform /2/16/thanks-for-the-free-cow-urine-in-mydrinking-water/ Federal Standard for primary contact 2 CFU/1 ml Developing resistance to many antibiotics E. coli 126 CFU/1 ml Enterococci 33 CFU/1 ml

administered at subtherapeutic levels Up to 9% excreted 1. 2.

5 Factors we considered in this study: Representative scenario 1/3 of land area in Iowa is drained Bacteria move into tiles via macropores Consider only no-till Tylosin commonly administered at subtherapeutic levels Up to 9% excreted Relationships between sources and resistant organisms typically inferred swine farm What is lacking? surface transport Image by Alok Bhandari

6 Factors we considered in this study: 3. What is causing the resistance? 5

7 Objectives of the study: 1. To detect and quantify the occurrence of tylosin-resistant enterococci in: manure from swine facilities feeding tylosin at sub-therapeutic doses; soils amended with swine waste and tile drain flow from swine waste amended agricultural fields; 2. To assess the effects of hydrology and solids transport on the enterococci in tile water; 3. To test enterococci isolates for known macrolide resistant genes

8 Approach Field Study: Nashua, Iowa Manure & Soil Sampling ISU Northeast Research Farm 36 plots (.4 ha each) Soil type: moderate to poorly drained Tile drainage: 1979 Corn-soybean rotation

Approach Rainfall Simulation ISU Northeast

rotation Water Sampling Spring: sampling

9 Approach Rainfall Simulation ISU Northeast Research Farm 36 plots (.4 ha each) Soil type: moderate to poorly drained Tile drainage: 1979 Corn-soybean rotation Water Sampling Spring: sampling started after flow started Fall: sampling started after rainfall simulation started 8

10 Analysis for Enterococci Membrane Filtration Technique (APHA, 1998) menterococcus agar with tylosin at 35 mg/l (resistant) and without tylosin (control) Approach Confirmation EPA Method 16 Transfer Incubate Transfer Incubate Transfer Incubate BEA Incubate at 35 o C for 48 hrs Gram stain Transfer Incubate BHI Broth BHI Broth Transfer Incubate BHI Broth 6.5% NaCl Total Suspended Solids

11 Select isolates DNA extraction PCR Amplification (Denaturation, annealing, and extension) Positive controls: Dr. Roberts, University of Washington Negative controls: ATCC Approach Resistant Gene Detection by PCR

Results Enterococci in manure and soil samples 1.E+7 Total 1.E+6 1.2x1 6 1.4x1 6 5.7x1 5 3.8x1 5 1.3x1 5 1.x1 5 Resistant 1.E+5 6.7x1 4 5.x1 4 1.E+4 1.E+3 1.E+2 3.3x1 1 1.

12 Results Enterococci in manure and soil samples 1.E+7 Total 1.E+6 1.2x x x x x1 5 1.x1 5 Resistant 1.E+5 6.7x1 4 5.x1 4 1.E+4 1.E+3 1.E+2 3.3x1 1 1.E+1 1.E+ Swine manure Soil after manure application Soil before manure application Swine manure Soil after manure application Soil before manure application Spring Fall 11

13 Enterococci concentration (cfu/1ml) Flow(L/h) Enterococci and tile flow Spring 29 Total : 33 to 4967 CFU/1 ml Resistant: 67 to 1167 CFU/1 ml min Total Resistant Flow min Elapsed time since flow started (min) 12

14 Enterococci concentration (cfu/1 ml) Enterococci concentration and tile drained flow Flow (L/h) Fall 29 Total : up to 2567 cfu/1 ml Resistant: up to 1433 cfu/1 ml min Total Resistant Flow min Elapsed time since rainfall simulation started (min) 13

Enterococci concentration (log1 cfu/ha) Enterococci concentration (log1 cfu/ha) Results: Enterococci loading cfu/ha Spring 29 1.E+12 1.E+11 1.E+1 1.E+9 1.

15 Enterococci concentration (log1 cfu/ha) Enterococci concentration (log1 cfu/ha) Results: Enterococci loading cfu/ha Spring 29 1.E+12 1.E+11 1.E+1 1.E+9 1.E+8 Total Resistant Resistant/Total = 23% p-value <<.5 4.6x x1 1 Fall 29 1.E+12 1.E+11 1.E+1 1.E+9 1.E+8 1.E+7 Total Resistant Resistant/Total = 73% p-value <<.5 1.E x x Elapsed time since flow started (min) Elapsed time since rain fall simulation started (min) 14

16 TSS (mg/l) Enterococci concentration (cfu/1ml) TSS (mg/l) Enterococci concentration and TSS: Spring Simulation Total Resistant TSS Elapsed time since drain-flow started (minutes) Poor regression because sediment was deposited in the tile line Total enterococi concentration (cfu/1 ml) 15

17 TSS (mg/l) Enterococci concentration (cfu/1 ml) TSS (mg/l) Enterococci concentration and TSS: Fall Simulation Total Resistant TSS Elapsed time since rainfall simulation started (mins) 4 3 Good correlation because of base -flow 2 1 R² = Total enterococci concentration (cfu/1 ml)

18 Gene detection frequency (%) Resistant gene detection Manure Soil Tile water msra ErmF ErmB ErmT ermc msra ErmF ErmB ErmT ermc Spring Fall

19 Conclusions 1. This study has provided the first data on tracking the transport pathway of tylosin-resistant enterococci from a specified source through a subsurface drainage system Transport of resistant enterococci from swine manure through soil and into tile drainage was confirmed Enterococci control and tylosin resistant concentrations were significantly different 2. In the presence of base-flow, good correlation between TSS and enterococci was observed. 3. Tylosin resistant genes were detected in enterococci isolates from manure, soil and tile water: msra, ermb, ermf, ermt, erma, ermx. Both resistance mechanisms to tylosin occurred in enterococci isolates. 18

20 Acknowledgements Multi-university USDA-NRI grant on Role of Directly Connected Macropores in Pathogen Transport to Subsurface Drainage VOSP scholarship from Vietnamese government Graduate & undergraduate students: Chi Hoang, Pramod Pandey, Martha Zwonitzer, Ping Liu, Rachel McDaniel, Christina Goeddel and Bridgette Huss 19

Occurrence of Tylosin-Resistant Enterococci in Swine Manure and Tile Drainage Systems under No-Till Management

Agricultural and Biosystems Engineering Conference Proceedings and Presentations Agricultural and Biosystems Engineering 6-2010 Occurrence of Tylosin-Resistant Enterococci in Swine Manure and Tile Drainage