Progress 12/01/08 to 11/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This study aimed to determine if biotic contaminants originating from pig production farms are disseminated into soil and groundwater microbial communities. A spatial and temporal sampling of soil and groundwater in proximity to pig production farms was conducted, and quantitative PCR (Q-PCR) was utilized to determine the abundances of tetracycline resistance genes (i.e., tetQ and tetZ) and integrase genes (i.e., intI1 and intI2). We observed that the abundances of tetZ, tetQ, intI1, and intI2 in the soils increased at least 6-fold after manure application, and their abundances remained elevated above the background for up to 16 months. Q-PCR further determined total abundances of up to 5.88 × 10(9) copies/ng DNA for tetZ, tetQ, intI1, and intI2 in some of the groundwater wells that were situated next to the manure lagoon and in the facility well used to supply water for one of the farms. We further utilized 16S rRNA-based pyrosequencing to assess the microbial communities, and our comparative analyses suggest that most of the soil samples collected before and after manure application did not change significantly, sharing a high Bray-Curtis similarity of 78.5%. In contrast, an increase in Bacteroidetes and sulfur-oxidizing bacterial populations was observed in the groundwaters collected from lagoon-associated groundwater wells. Genera associated with opportunistic human and animal pathogens, such as Acinetobacter, Arcobacter, Yersinia, and Coxiella, were detected in some of the manure-treated soils and affected groundwater wells. Feces-associated bacteria such as Streptococcus, Erysipelothrix, and Bacteroides were detected in the manure, soil, and groundwater ecosystems, suggesting a perturbation of the soil and groundwater environments by invader species from pig production activities.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Hong, PY, Li, X, Yang, X, Zhang, Y, Wang, X and Mackie, RI. 2012. Monitoring airborne biotic contaminants in the indoor environment of pig and poultry confinement buildings. Environ. Microbiol. 14:1420-1431.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Hong, PY, Yannarell AC, Dai Q, Ekizoglu M and Mackie, RI. 2013. Monitoring the perturbation of soil and groundwater microbial communities due to pig production activities. Appl. Environ. Microbiol. 79:2620-2629.
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Progress 12/01/11 to 11/30/12
Outputs
OUTPUTS: This study aims to determine if biotic contaminants originating from pig production farms are disseminated into soil and groundwater microbial communities. A spatial and temporal sampling of soil and groundwater was conducted, and Q-PCR was utilized to determine the abundance of tetracycline resistance genes (i.e., TetQ and TetZ) and integrase genes (i.e., IntI1 and IntI2). We observed that the abundance of TetZ, TetQ, IntI1 and IntI2 in the soils increased by at least six-fold after manure application, and their abundance remained elevated above background for up to 16 months. Q-PCR further determined a total abundance of up to 5.88 x 109 copies/ng DNA of TetZ, TetQ, IntI1 and IntI2 in some of the groundwater wells that were situated next to the manure lagoon and in the facility well used to supply water for the farm. We further utilized 16S rRNA-based pyrosequencing to assess the microbial communities, and our comparative analyses suggest that most of the soil samples collected before and after manure application did not change significantly, sharing a high Bray-Curtis similarity of 78.5%. In contrast, an increase in Bacteroidetes and sulfide-oxidizing bacterial populations was observed in the groundwaters collected from lagoon-associated groundwater wells. Genera associated with opportunistic human and animal pathogens like Acinetobacter, Arcobacter, Yersinia and Coxiella were detected in some of the manure-applied soils and impacted groundwater wells. Fecal-associated bacteria like Streptococcus, Erysipelothrix and Bacteroides were detected in the manure, soil and groundwater ecosystems, suggesting perturbation of invader species from pig production activities to the soil and groundwater environment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The use of antibiotics in livestock production is a ubiquitous practice. Swine, poultry and cattle production have reported the use of antibiotics for treatment (i.e., therapeutic), prevention of animal diseases (i.e., prophylactic), and for growth promotion. As such, water bodies in close proximity to animal operations are often at risk of biotic (manure) and abiotic (antibiotic resistance gene) contamination. Groundwater constitutes 50% of the drinking water supply for the U.S. population, and over 97% of the drinking water sources for rural communities. It remains of high priority to maintain the quality of our water resources. The project concerns fundamental examination of the microbial communities, the antibiotic resistance genes, and the mobile gene pools that are associated with swine production facilities and evaluates if these contaminants will impose a potential adverse risk to nearby water bodies. To achieve this long term goal, this project aims to first establish the presence of biotic contaminants and the diversity of the mobile gene pool that are associated along a contamination gradient in a swine production environment. Subsequently, bacterial isolates will be isolated from soil samples obtained within the swine production facilities. The bacterial isolates will be well-characterized. Specifically, we aim to look for bacterial isolates that are resistant to tetracycline, and evaluate the genes that confer its tetracycline resistant traits. We also aim to look for genotypic traits like mobile genetic elements, and determine if the phenotypic traits encoded by the mobile genetic elements are transferrable to other bacterial isolates endemic to a non-contaminated water system, as well as the human and animal commensal microbiota. Overall, these specific aims would allow us to assess the extent of microbial risk imposed on the well-being of the environment, humans and animals. The regulatory bodies and scientific community are the major stakeholders of this work. The knowledge gained from this work would enable regulatory bodies to better monitor and assess the biotic and abiotic risk imposed by animal operations. This work addresses a priority of USDA-NRI Program 26.0 to 'understand the sources, fate, the transport of pathogens, such as bacteria, protozoa and viruses in soil, surface and groundwater, and irrigation systems of agricultural and rural watersheds to reduce zoonotic pathogens in the environment.' Our work would help to enhance the long-range sustainability of U.S. agricultural systems by providing the data necessary for the proper assessment of microbial risk. This will subsequently allow for better design and implementation of informed management decisions about antibiotic usage in agriculture, treatment of animal waste, and the use of animal manure as fertilizer.
Publications
- Hong, PY, Li, X, Yang, X, Zhang, Y, Wang, X and Mackie, RI. 2012. Monitoring airborne biotic contaminants in the indoor environment of pig and poultry confinement buildings. Environ. Microbiol. 14:1420-1431.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: Given the growing concerns over human and animal health issues related to confined animal feeding operations, an in-depth examination is required to monitor for airborne bacteria and the associated antibiotic resistance genes. Our 16S rRNA-based pyrosequencing revealed that the airborne microbial community skewed towards a higher abundance of Firmicutes (>59.2%) and Bacteroidetes (4.2-31.4%) within the confinement buildings, while the office environment was predominated by Proteobacteria (55.2%). Furthermore, bioaerosols in the confinement buildings were sporadically associated with genera of potential pathogens, and these genera were more frequently observed in the bioaerosols of pig and layer hen confinement than the turkey confinement buildings and office environment. High abundances of tetracycline resistance genes (9.55 x 102 to 1.69 x 106 copies/ng DNA) were also detected in the bioaerosols sampled from confinement buildings. Bacterial lineages present in the poultry bioaerosols clustered apart from those present in the pig bioaerosols and among the different phases of pig production, suggesting that different livestock as well as production phase were associated with a distinct airborne microbial community. By understanding the diversity of biotic contaminants associated with the different confinement buildings, this study facilitates the implementation of better management strategies to minimize potential health impacts on both livestock and humans working in this environment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The use of antibiotics in livestock production is a ubiquitous practice. Swine, poultry and cattle production have reported the use of antibiotics for treating (i.e., therapeutic), prevention of animal diseases (i.e., prophylactic), and for growth promotion. As such, water bodies in close proximity to animal operations are often at risk of biotic (manure) and abiotic (antibiotic resistance gene) contamination. Groundwater constitutes 50% of the drinking water supply for the U.S. population, and over 97% of the drinking water sources for rural communities. It remains of high priority to maintain the quality of our water resources. The project concerns fundamental examination of the microbial communities, the antibiotic resistance genes, and the mobile gene pools that are associated with swine production facilities and evaluates if these contaminants will impose a potential adverse risk to nearby water bodies. To achieve this long term goal, this project aims to first establish the presence of biotic contaminants and the diversity of the mobile gene pool that are associated along a contamination gradient in a swine production environment. Subsequently, bacterial isolates will be isolated from soil samples obtained within the swine production facilities. The bacterial isolates will be well-characterized. Specifically, we aim to look for bacterial isolates that are resistant to tetracycline, and evaluate the genes that confer its tetracycline resistant traits. We also aim to look for genotypic traits like mobile genetic elements, and determine if the phenotypic traits encoded by the mobile genetic elements are transferrable to other bacterial isolates endemic to a non-contaminated water system, as well as the human and animal commensal microbiota. Overall, these specific aims would allow us to assess the extent of microbial risk imposed to the well-being of the environment, humans and animals. The regulatory bodies and scientific community are the major stakeholders of this work. The knowledge gained from this work would enable regulatory bodies to better monitor and assess the biotic and abiotic risk imposed by animal operations. This work addresses a priority of USDA-NRI Program 26.0 to "understand the sources, fate, the transport of pathogens, such as bacteria, protozoa and viruses in soil, surface and groundwater, and irrigation systems of agricultural and rural watersheds to reduce zoonotic pathogens in the environment." Our work would help to enhance the long-range sustainability of U.S. agricultural systems by providing the data necessary for the proper assessment of microbial risk. This will subsequently allow for better design and implementation of informed management decisions about antibiotic usage in agriculture, treatment of animal waste, and the use of animal manure as fertilizer.
Publications
- Gronvold, A.M., L'abee-Lund, T.M., Sorum, H., Skancke, E., Yannarell, A.C. and Mackie, R.I. 2010. Changes in fecal microbiota of healthy dogs administered amoxicillin. FEMS Microbiol. Ecol. 71:313-326.
- Gronvold, A.M., L'Abee-Lund, T.M., Strand, E., Sorum, H., Yannarell, A.C. and Mackie, R.I. 2010. Fecal microbiota of horses in the clinical setting: Potential effects of penicillin and general anesthesia. Vet. Microbiol. 145:366-372.
- Gronvold, A.M., Mao, Y., L'Abee-Lund, T.M., Sorum, H., Sivertsen, T., Yannarell, A.C. and Mackie, R.I. 2011. Fecal microbiota of calves in the clinical setting: effect of penicillin treatment. Vet. Microbiol. 153:354-360.
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: We have made two sampling trips to one commercial swine confinement facility designated as Site E, a 2,300-hog finishing facility that began operations in July 1998. At this site, the sandstone is used by local residents to draw water from wells in the underlying sandstone, with many wells at depths less than 30m. Local geology indicates that groundwater at this site is vulnerable to contamination. To first establish the contamination gradient at Site E, we utilized primers targeting at tetracycline resistance genes to establish the copy number of antibiotic resistance genes (TetQ and TetZ) in the manure and groundwater sampled from different wells (Facility well, E1, E4, E6 and E7). Our Q-PCR results showed that tetracycline resistance genes were present at barely detectable concentration in background wells (E1 and facility well), which were located upstream of the groundwater flow. A contamination gradient was also observed. For example, tetracycline resistance genes were detected in E6 and E7 wells. Both wells were in close proximity to the waste pit. TetQ gene but not TetZ was detected in E6 well, at abundance of ~136 copy number/ng DNA. Both TetQ and TetZ genes were present in E7 wells at abundance ranging from 28-122 copy numbers/ng DNA. The highest abundance of tetracycline resistance genes were detected in E4 well, which was located downstream of the waste pit along the direction of groundwater flow. The detected abundance of tetracycline resistance genes was up to 11,462 copy number/ng DNA. Although this abundance is significantly lower than that detected in the waste pit (5x105 copy number/ng DNA), it was nevertheless more than 88-fold higher than that detected in the less impacted E6 well. Therefore, a contamination gradient was established at site E, with decreasing contamination from the manure at waste pit, to E4 well, E7 well, E6 well and subsequently E1 and the facility well. Next, we determined the microbial community using 16S rRNA-based pyrosequencing, and asked if the pattern correlated with the contamination gradient. Our findings showed that in the background wells (i.e., facility well and E1), the microbial community was comprised predominantly of Proteobacteria (~95% of total microbial community). However, the microbial community in the groundwater shifted towards increasing abundance of Firmicutes and Bacteroidetes (from ~2% to ~10%) with increasing contamination. A dendrogram depicting the clustering of microbial communities further showed differentiation of microbiota at the different sampling sites, and that the clustering correlates to the extent of contamination. Furthermore, the microbial communities sampled from the same site shared high similarity between duplicates, suggesting a consistent clustering pattern and reproducible shift of the microbial community with contamination. In summary, our findings indicated an increase in the abundance of tetracycline resistance genes and a corresponding shift in the microbial community as contamination occurs. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The use of antibiotics in livestock production is a ubiquitous practice. Swine, poultry and cattle production have reported the use of antibiotics for treating (i.e., therapeutic), prevention of animal diseases (i.e., prophylactic) and for growth promotion. As such, water bodies in close proximity to animal operations are often at risk of biotic (manure) and abiotic (antibiotic resistance gene) contamination. Groundwater constitutes 50% of the drinking water supply for the U.S. population, and over 97% of the drinking water sources for rural communities. It remains of high priority to maintain the quality of our water resources. The project concerns fundamental examination of the microbial communities, the antibiotic resistance genes and the mobile gene pools that are associated with swine production facilities, and evaluates if these contaminants will impose a potential adverse risk to nearby water bodies. To achieve this long term goal, this project aims to first establish the presence of biotic contaminants and the diversity of the mobile gene pool that are associated along a contamination gradient in a swine production environment. Subsequently, bacterial isolates will be isolated from soil samples obtained within the swine production facilities. The bacterial isolates will be well-characterized. Specifically, we aim to look for bacterial isolates that are resistant to tetracycline, and evaluate the genes that confer its tetracycline resistant traits. We also aim to look for genotypic traits like mobile genetic elements, and determine if the phenotypic traits encoded by the mobile genetic elements are transferrable to other bacterial isolates endemic to a non-contaminated water system, as well as the human and animal commensal microbiota. Overall, these specific aims would allow us to assess the extent of microbial risk imposed to the well-being of environment, human and animals. The regulatory bodies and scientific community are the major stakeholders of this work. The knowledge gained from this work would enable regulatory bodies to better monitor and assess the biotic and abiotic risk imposed by animal operations. This work addresses a priority of USDA-NRI Program 26.0 to "understand the sources, fate, the transport of pathogens, such as bacteria, protozoa and viruses in soil, surface and groundwater, and irrigation systems of agricultural and rural watersheds to reduce zoonotic pathogens in the environment." Our work would help to enhance the long-range sustainability of U.S. agricultural systems by providing the data necessary for the proper assessment of microbial risk. This will subsequently allow for better design and implementation of informed management decisions about antibiotic usage in agriculture, treatment of animal waste, and the use of animal manure as fertilizer.
Publications
- Hong, P., Yannarell, A.C. and Mackie, R.I. 2010. The contribution of antibiotic residues and antibiotic resistance genes from livestock operations to antibiotic resistance in the environment and food chain. Chapter 6 In: Zoonotic Pathogens in the Food Chain. Editors Krause D.O and Hendrick, S. CABI, UK.
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Progress 12/01/08 to 11/30/09
Outputs
OUTPUTS: RNA methylase genes are common antibiotic resistance determinants for multiple drugs of the macrolide, lincosamide, and streptogramin B (MLSB) families. We used molecular methods to investigate the diversity, distribution, and abundance of MLSB methylases in waste lagoons and groundwater wells at two swine farms with a history of tylosin (a macrolide antibiotic structurally related to erythromycin) and tetracycline usage. Phylogenetic analysis guided primer design for quantification of MLSB resistance genes found in tylosin-producing Streptomyces (tlr(B), tlr(D)) and commensal/pathogenic bacteria (erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q)). The near absence of tlr genes at these sites suggested a lack of native antibiotic-producing organisms. The gene combination erm(ABCF) was found in all lagoon samples analyzed. These four genes were also detected with high frequency in wells previously found to be contaminated by lagoon leakage. A weak correlation was found between the distribution of erm genes and previously reported patterns of tetracycline resistance determinants, suggesting that dissemination of these genes into the environment is not necessarily linked. Considerations of gene origins in history (i.e. phylogeny) and gene distributions in the landscape provide a useful "molecular ecology" framework for studying environmental spread of antibiotic resistance. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Using a cultivation-independent, PCR-based approach, we have been able to detect a number of different classes of tetracycline- and erythromycin-resistance genes (tet and erm, respectively) in the groundwater adjacent to hog waste treatment lagoons. This suggests that treatment lagoons at animal production facilities can serve as reservoirs of antibiotic resistance. We have also found these genes in soils that have been amended with pit- or lagoon-treated manure as fertilizer. Positive detections of these genes have come from background "control" wells that are upgradient of the source lagoons, as well as from background soil samples collected from farm fields prior to manure injection (that is, fields that have been unmanured for at least three years). These results highlight the difficulty of establishing proper "negative controls" for environmental antibiotic resistance work, and, more importantly, they point to the existence of a "native" antibiotic resistance gene pool within the environmental microbiota. However, the spatial and temporal patterns of antibiotic resistance genes at these three sites suggests that exposure to hog waste is an important factor in the spread of antibiotic resistance. Different genes have differential abilities to persist in soils and waters, which suggests that a "gene ecology" perspective, which includes the recognition that genes may differ in their capacity to find new hosts via horizontal gene transfer, will be important for assessing the impact of agricultural activities on antibiotic resistance.
Publications
- Chee-Sanford, J.C., Mackie, R.I., Koike, S., Krapac, I.G., Lin, Y.F., Yannarell, A.C., Maxwell, S. and Aminov, R.I. 2009. Fate and transport of antibiotic residues and antibiotic resistance genes following land application of manure waste. J. Environ. Qual. 38:1086-108.
- Koike, S., Aminov, R.I., Yannarell, A.C., Gans, H.D., Krapac, I.G., Chee-Sanford, J.C. and Mackie, R.I. 2009. Molecular ecology of Macrolide-Lincosamide-Streptogramin B Methylases in waste lagoons and subsurface waters associated with swine production. Microb Ecol. 2009 Nov 19. [Epub ahead of print].
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