What are the sources of bacteria in your watershed? They may not be what you expect

Why Study Bacteria in Water?

According to the 305(b) report, the majority of waterbodies in the US do not meet established water quality standards. Over half (51%) of river miles assessed in 2010 were impaired. Pathogens are the leading cause of water quality impairment in rivers and streams of the US, impairing 16% of river/stream miles assessed. Computer models used to assess bacteria sources and loads in impaired watersheds are generally able to attribute loadings to specific land uses or in some cases specific animal categories based on known or estimated animal population numbers and fecal production rates. To provide better data on the predominant animal sources of bacterial impairments, Texas initiated a bacterial source tracking (BST) program in 2003.

What did we do?

Texas BST program uses a combination of two methods, ERIC PCR and riboprinting. To support this program, Texas assembled an E. coli BST library consisting of more than 1,600 E. coli isolates collected from over 1,400 different samples and representing in excess of 50 animal classes. Using this library, comprehensive BST has been conducted in dozens of watersheds across the state (Figure 1) to date.

Figure 1. Locations of BST projects in Texas

Figure 1. Locations of BST projects in Texas.

What have we learned?

Throughout these studies, wildlife contributions have been found to be the predominant source of bacteria (Figure 2) with non-avian wildlife being a primary contributor. Similarly, recent evaluations of small watershed and edge-of-field runoff from grazed and ungrazed pasture and range land have found background loading – loadings from wildlife and naturalized soilborne E. coli – to be significant.

Figure 2. Summary of ten Texas BST study findings.

Figure 2. Summary of ten Texas BST study findings.

This background loading is not currently adequately addressed in most water quality models, total maximum daily loads, or other water quality management efforts. This can have serious implications to application of water quality standards, particularly when applied to storm events where background runoff naturally exceeds water quality standards, as well as to TMDLs and other watershed based plans where ignoring background concentrations may lead to inaccurate load allocations and reductions as well as incongruence of modeling and BST results.

Future plans:

Future plans include working to identify the “unidentified” by continuing to expand the species in the BST library. Additionally, work is ongoing to evaluate naturalized soil borne E. coli and better evaluate wildlife populations in research watersheds. The BST team is also working to improve library independent BST methods.

Authors:

  • Dr. Kevin Wagner, Associate Director, Texas Water Resources Institute, klwagner@ag.tamu.edu
  • Dr. Terry Gentry, Associate Professor, Texas A&M Department of Soil & Crop Sciences, tgentry@ag.tamu.edu
  • Dr. Daren Harmel, Supervisory Agricultural Engineer, USDA-Agricultural Research Service, daren.harmel@ars.usda.gov
  • Dr. George Di Giovanni, Professor, Environmental and Occupational Health Sciences, University of Texas Health Science Center at Houston School of Public Health, El Paso Regional Campus, George.d.digiovanni@uth.tmc.edu
  • Lucas Gregory, Project Specialist & Quality Assurance Officer, Texas Water Resources Institute, lfgregory@ag.tamu.edu
  • Dr. Elizabeth Casarez, Research Associate, University of Texas Health Science Center at Houston School of Public Health, El Paso Regional Campus, Elizabeth.A.Casarez@uth.tmc.edu
  • Dr. Karthikeyan, Associate Professor, Texas A&M Department of Biological and Agricultural Engineering, karthi@tamu.edu

Additional information:

  • Di Giovanni, G.D., E. Casarez, T. Gentry, E. Martin, L. Gregory, K. Wagner. 2013. Support Analytical Infrastructure and Further Development of a Statewide Bacterial Source Tracking Library. Texas Water Resources Institute Technical Report TR-448. College Station, TX: Texas A&M University. (http://twri.tamu.edu/reports/2013/tr448.pdf)
  • TWRI Bacteria Fate and Transport website- http://bft.tamu.edu/
  • Texas Bacterial Source Tracking Library website – http://texasbst.tamu.edu/

Acknowledgements:

Thanks to the Texas State Soil and Water Conservation Board for providing continued funding and support for the Texas Bacterial Source Tracking Program.

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2015. Title of presentation. Waste to Worth: Spreading Science and Solutions. Seattle, WA. March 31-April 3, 2015. URL of this page. Accessed on: today’s date.

Analyses of Microbial Populations and Antibiotic Resistance Present in Stored Swine Manure from Underground Storage Pits


Why Study Antibiotic Resistance in Manure?

Antimicrobial compounds have been commonly used as feed additives for domestic animals to reduce infection and promote growth. Recent concerns have suggested such feeding practices may result in increased microbial resistance to antibiotics, which can have an impact on human health. As part of our research project we have been studying the commensal microbial populations present in stored swine manure and the swine GI tract. We have extended this work to include studies on the antibiotic resistance present in these populations.

What did we do?          

Predominant microbial populations were identified by both pure culture isolations and direct 16S rDNA sequencing of total DNA from swine feces and stored manure samples. Antibiotic resistance was analyzed using similar pure culture isolation methods. Pure cultures were isolated following plating on anaerobic and aerobic media containing tetracycline, tylosin, or erythromycin. Polymerase chain reaction (PCR) analyses using primers based on a variety of antibiotic resistance genes was carries out with both pure culture isolates and total DNA from swine feces and stored manure.

What have we learned? 

Results of pure culture isolation and direct 16S rDNA gene sequence analyses indicate that the bacterial populations of the swine GI tract (feces) and stored manure ecosystems are predominantly composed of anaerobic, low mole %G+C, Gram-positive bacteria, most of which represent novel genera and species. Results of antibiotic resistance gene PCR studies demonstrated the presence of a variety of tet (e.g., tetK, tetO) and erm (e.g., ermA, ermC) resistance gene classes in both anaerobic and aerobic pure cultures and total DNA from both swine feces and stored manure, as well as the identification of novel bacteria containing new resistance genes. Comparison of DNA sequences suggests that horizontal transfer of resistance genes between bacterial strains has also occurred. The data indicate that both the swine gastrointestinal (GI) tract and stored swine manure may serve as reservoirs of known and novel antibiotic resistant bacteria and resistan ce genes.

Future Plans    

We are interested in developing methods to reduce antibiotic resistance in the swine GI tract and stored manure, and to determine if antibiotic resistance genes present in these ecosystems can be transferred to bacteria that may affect human health (e.g., E. coli, Salmonella, Campylobacter).

Authors

Terence R. Whitehead, Research Microbiologist, USDA-ARS- National Center for Agricultural Utililzation Research, Peoria, IL 61604 terry.whitehead@ars.usda.gov

Michael A. Cotta, USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL 61604

Additional information 

Terence R. Whitehead, NCAUR, 1815 N. University St., Peoria, IL 61615 309-681-6272

USDA-ARS-NCAUR-Bioenergy Research Unit: http://ars.usda.gov/main/site_main.htm?modecode=50-10-05-20

Cotta, M.A., Whitehead, T.R., and Zeltwanger, R.L. Isolation, Characterization, and Comparison of Bacteria from Swine Faeces and Manure Storage Pits. (2003) Env. Microbiol. 5:737-745. http://onlinelibrary.wiley.com/doi/10.1046/j.1467-2920.2003.00467.x/pdf

Whittle, G., Whitehead, T.R., Hamburger, N., Shoemaker, N.B., Cotta, M.A., and Salyers, A.A. Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits . (2003) Appl. Environ. Microbiol. 69:4151-4158. http://aem.asm.org/content/69/7/4151.full

Cotta, M.A., Whitehead, T.R., Falsen, E., Moore, E. and Lawson, P.A. Robinsonella peoriae gen.nov., sp. nov., isolated from a swine-manure storage pit and a human clinical source. (2009) Int. J. System. Evol. Microbiol. 59:150-155. https://pubmed.ncbi.nlm.nih.gov/19126740/

Whitehead, T.R. and Cotta, M.A. Stored Swine Manure and Swine Feces as Reservoirs of Antibiotic Resistance Genes. (2013) Lett. Appl. Microbiol. 56:264-267. http://onlinelibrary.wiley.com/enhanced/doi/10.1111/lam.12043/

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2015. Title of presentation. Waste to Worth: Spreading Science and Solutions. Seattle, WA. March 31-April 3, 2015. URL of this page. Accessed on: today’s date.

 

Composting Swine Slurry to Reduce Indicators and Antibiotic Resistance Genes


Purpose 

Over the last twenty years there have been considerable increases in the incidence of human infections with bacteria that are resistant to commonly used antibiotics. This has precipitated concerns about the use of antibiotics in livestock production. Composting of swine manure has several advantages, liquid slurries are converted to solid, the total volume of material is reduced and the stabilized product is more easily transported off-site. The goal of this study was to determine if composting can also be used to reduce the concentration of indicators and bacteria containing genes for antibiotic resistance (AR) in swine manure.

What did we do? 

Sample Analysis:

Compost trials were conducted in either fall (FT) or spring (ST) and piles were turned once, three times or upon reaching 65 ºC. Microbial indicators and populations with AR genes for tetracycline, erythromycin and sulfonamide resistance were quantified by culture and/or quantitative, real-time (qPCR) analysis.

Compost materials and conditions:

Decomposed materials (a mixture of swine slurry and woodchips) were obtained on two separate occasions from swine high-rise finishing facilities (HRFF) located in western Kentucky. The HRFF houses between 4,000 and 4,800 swine which are placed in the facility at 18 to 20 kg and are removed after three months (weighing about 105 kg). The high-rise floor raises the living area 3.7 m above the ground. Manure, excess feed, water and wastewater drop through slatted floors into 2.5 cm screened woodchips (average size 1.9 ± 0.9 cm). The slurry-woodchip material was turned up to three times per week while under the HRFF. When the material was visibly moist, reducing its ability to absorb additional waste materials, it was removed from the facility for finishing in windrows. In fall 2011 (FT) and Spring 2012 (ST), HRFF slurry-woodchip mix (approximately 60 m3 weighing 48.4 Mg) was brought by semi-trailer trucks to the Western Kentucky University Agricultural complex where ma terials were divided into three or four windrow piles. In the FT, swine slurry-woodchip mixes having a bulk density of 849.6 kg m-3 and consisting of around 19.6 m3 of material were formed into three piles of approximately 10.4 m x 2.1 m x 0.9 m (L x W x H). In the ST, swine slurry-woodchip mixes having a bulk density of 778.4 kg m-3 and consisting of around 18.8 m3 of material were formed into three piles of approximately 5.8 m x 2.7 m x 1.2 m (L x W x H) and a fourth batch (unturned) was left piled at the side (0X; 3.6 m3). In each study, piles were turned using a windrow compost turner either once per week (1X), three times per week (3X) or upon the internal compost temperature reaching 65 ºC (@65). Compost for the FT @65 treatment heated to 65 ºC by day 14 and was turned 11 times over the course of the trial. However, during the ST, the @65 pile did not heat for the first 63 days (mean temperature 27 ± 8 ºC) therefore weekly turning was initiated at that time. Samples were taken on days 0 and three and then weekly for the first 12 weeks and bi-weekly until composting was stopped at day 112 for the FT and day 142 for the ST.

What have we learned? 

In the FT, concentrations of enterococci decreased below culturable detection within 21 days, corresponding with a 99% decrease in detection by qPCR (Fig. 1). Similar decreases in qPCR detection in the ST took longer (day 49 or day 77 of composting). Changes in the concentration of bacteria with AR genes varied by antibiotic type (erythromycin (36% – 97%), tetracycline (94% to 99%) and sulfonamide (53% to 84%) and compost season (greater decreases in ST). There were few differences based on turning regime. Even the unturned compost pile had 90%, 98% and 56% reduction in bacteria resistant to erythromycin, tetracycline and sulfonamide, respectively.

Results suggest that composting effectively decreases the concentration of indicators and AR genes in swine manure. As concerns over antibiotic resistance and pathogens increase, composting provides a valuable manure management tool for decreasing contaminants and improving the value of this material as a soil conditioner.

Future Plans    

Volume reduction, low moisture and low readily degradable organic matter suggest that the finished compost would have lower transportation costs and should provide value as a soil conditioner. Studies are warranted to evaluate its agronomic value as an alternative source of plant nutrients. Future studies will be conducted to evaluate the nutrient value this compost as an organic fertilizer for row crop production.

Authors       

Kimberly Cook, Research Microbiologist, USDA ARS kim.cook@ars.usda.gov

Carl Bolster, USDA ARS; Karamat Sistani, USDA ARS

Additional information                

http://www.ars.usda.gov/main/site_main.htm?modecode=50-40-05-00

Acknowledgements      

This research was conducted as part of USDA-ARS National Program 214: Agricultural and Industrial By-products: CRIS 6445-12630-004-00D. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

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The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2015. Title of presentation. Waste to Worth: Spreading Science and Solutions. Seattle, WA. March 31-April 3, 2015. URL of this page. Accessed on: today’s date.

Assessment of Bioaerosol Transport at a Large Dairy Operation

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Abstract

In an effort to assess the off-site transport of bioaerosols, airborne bacteria, fungi, and endotoxin were collected at a 10,000 cow dairy operation. Compared to background locations, the general trend was that bioaerosol concentrations were higher immediately downwind, then decreased with distance from the animal housing. While bioaerosol concentrations did not follow a seasonal trend, they did significantly correlate with meteorological factors such as temperature and solar radiation. Bioaerosol concentrations were also found to be greatest at night, which can be attributed to changes in animal activity and wind speed and reduced exposure of the microorganisms to UV radiation. An analysis of clones generated from air samples collected downwind from the animal housing and pivots spraying dairy wastewater revealed that none of sequence matches were affiliated with bacteria known to be pathogenic to otherwise healthy humans. Results from ongoing research to better understand bioaerosol formation and drift losses during spray irrigation events of dairy wastewater will also be discussed.

Using glass impingers to capture airborne bacteria at a downwind location from a dairy.

Why Study Bioaerosols at Dairies?

Because confinement of cattle increases the microbioal load at dairy production facilities, there are concerns about on-site and off-site exposures to airborne microorganisms and microbial byproducts. The purpose of this study was to monitor concentrations of airborne bacteria, fungi, and endotoxin at a 10,000 cow open-freestall dairy and fields being irrigated with wastewater to assess their potential to be transported off site. This information is important, as inhalation or ingestion of some bioaerosols can be detrimental to health through infection, allergy, or toxicosis.

Open-face filters for capture of airborne endotoxin.

What Did We Do?

Over a one-year period at the dairy, bioaerosols were collected at upwind (background) and downwind sites using glass impingers, direct impaction on media, and a wetted-wall cyclone. Bacteria and fungi were quantified using culture-dependent techniques, while bacteria were also characterized to the genus and species levels by analyzing a region of the 16S ribosomal RNA gene. Airborne endotoxin were captured on filters, then extracted and subsequenetly quantified using the Limulus amebocyte lysate assay.

Wetted-wall cyclone being used to capture bioaerosols for subsequent identification using PCR-based approach.

What Have We Learned?

Compared to background sites, the general trend was that concentrations of airborne bacteria and  endotoxin were higher immediately downwind, then decreased with distance from the animal housing. While bioaerosol concentrations did not follow a seasonal trend, they did significantly correlate with meteorological factors such as temperature, wind speed, and solar radiation. Bacteria and endotoxin concentrations were also found to be greatest at night, which can be attributed to changes in animal activity and wind speed and reduced exposure of the microorganisms to UV radiation. Analysis of cloned 16S rRNA genes generated from air samples collected downwind from the animal housing and pivots spraying dairy wastewater revealed that none of sequences were affiliated with bacteria known to be pathogenic to healthy humans.

Future Plans

Conduct a quantitative microbial risk assessment for zoonotic bacterial pathogens in dairy wastewaters that are land applied using center pivot irrigation systems.  

Authors

Robert Dungan, Research Microbiologist, USDA-ARS Northwest Irrigation & Soils Research Laboratory, Kimberly, Idaho, robert.dungan@ars.usda.gov

April Leytem, Soil Chemist, USDA-ARS, Kimberly, Idaho

David Bjorenberg, Agricultural Engineer, USDA-ARS, Kimberly, Idaho

Additional Information

Dungan, R.S. and A.B. Leytem. 2009. Airborne endotoxin concentrations at a large open-lot dairy in southern Idaho. J. Environ. Qual. 38:1919-1923.

Dungan, R.S., A.B. Leytem, and D.L. Bjorneberg. 2010. Year-long assessment of airborne endotoxin at a concentrated dairy operation. Aerobiologia. 26:141-148.

Dungan, R.S., A.B. Leytem, S.A. Verwey, and D.L. Bjorneberg. 2010. Assessment of bioaerosols at a concentrated dairy operation. Aerobiologia. 26:171-184.

Dungan, R.S. and A.B. Leytem. 2011. Ambient endotoxin concentrations and assessment of transport at an open-lot and open-freestall dairy. J. Environ. Qual. 40:462-467.

Dungan, R.S., A.B. Leytem, and D.L. Bjorneberg. 2011.  Concentrations of airborne endotoxin and microorganisms at a 10,000 cow open-freestall dairy. J. Anim. Sci. 176:426-434.

Dungan, R.S. 2012. Use of a culture-independent approach to characterize aerosolized bacteria at an open-freestall dairy operation. Environ. Int. 41:8-14.

Acknowledgements

Independent Dairy Environmental Action League (IDEAL)

 

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2013. Title of presentation. Waste to Worth: Spreading Science and Solutions. Denver, CO. April 1-5, 2013. URL of this page. Accessed on: today’s date.